Long-term outcomes and prognostic factors in dogs with meningoencephalitis of unknown origin and suspected necrotic lesions on magnetic resonance imaging: 37 cases (2007-2020).

OBJECTIVE: To describe the long-term outcomes, overall survival, progression-free survival, and prognostic factors in dogs with necrotizing encephalitis (NE). ANIMALS: 37 client-owned dogs clinically diagnosed with NE. METHODS: All dogs underwent MRI and CSF analysis. Cox proportional hazards regression was used to examine factors related to the risk of relapse and death, including signalment, history, diagnostic investigation results, and treatments before the first relapse. RESULTS: The medians of the overall and progression-free survival times were 639 days (IQR, 342 to 1,482 days) and 233 days (IQR, 111 to 775 days), respectively. Overall survival was highly correlated with progression-free survival. Four dogs (11%) died or were euthanized within 3 months of diagnosis. Relapse within 6 months was associated with a shorter overall survival. However, no prognostic factors for overall survival were found. The category of patients with presenting clinical signs that lasted 29 days to 6 months (OR, 3.26; 95% CI, 1.35 to 7.90) was associated with a higher risk of relapse. Seizures were presented in 75.7% of dogs, with a recurrence rate of 100%. CLINICAL RELEVANCE: This report provides comprehensive follow-up information for dogs with NE, revealing a fair prognosis and low early mortality rate. Seizure is a very common clinical sign with a high recurrence rate.

Intra-Operative Diagnosis of Benign Multicystic Peritoneal Mesothelioma: A Case Report of Rare Entity and Lessons Learned.

Benign multicystic peritoneal mesothelioma (BMPM), also known as multicystic peritoneal mesothelioma (MCPM), is a rare cystic neoplasm arising from the mesothelium lining of the abdominal and pelvic peritoneum. This entity has been disproportionately described in women of reproductive age. Both the etiology and pathogenesis of the condition are not well understood. Preoperative diagnosis is challenging as differentials are varied and include endometriosis, lymphangioma, pseudomyxoma peritonei, cystic adenomatoid tumor, and malignant peritoneal mesothelioma. Management options include cytoreductive surgery (CRS) with or without heated intraperitoneal chemotherapy (HIPEC). In this case report, we highlight the complexity of preoperative diagnosis, presentation, workup, treatment, and management of BMPM. We report the case of a female patient presenting with abdominal pain and imagining consistent with cystic intra-abdominal lesions. After an inconclusive percutaneous biopsy and a multi-disciplinary tumor board discussion, the patient was offered CRS with HIPEC. Intra-operative frozen section indicated benign epithelial lined cysts. CRS and HIPEC were performed. After a second opinion, the lesions were confirmed by pathology and immunohistochemistry to be BMPM. In this report, we discuss the gold standard of care for patients with BMPM to improve the disease control rate. This pathway is proposed in our study, and, thus, we conclude that BMPM should be considered in the differential diagnosis of patients presenting with symptomatic multiple intraperitoneal cystic lesions.

Exploration of influenza A virus PA protein-associated cellular proteins discloses its impact on mitochondrial function.

Influenza A virus can infect respiratory tracts and may cause severe illness in humans. Proteins encoded by influenza A virus can interact with cellular factors and dysregulate host biological processes to support viral replication and cause pathogenicity. The influenza viral PA protein is not only a subunit of influenza viral polymerase but also a virulence factor involved in pathogenicity during infection. To explore the role of the influenza virus PA protein in regulating host biological processes, we performed immunoprecipitation and LC‒MS/MS to globally identify cellular factors that interact with the PA proteins of the influenza A H1N1, 2009 pandemic H1N1, and H3N2 viruses. The results demonstrated that proteins located in the mitochondrion, proteasome, and nucleus are associated with the PA protein. We further discovered that the PA protein is partly located in mitochondria by immunofluorescence and mitochondrial fractionation and that overexpression of the PA protein reduces mitochondrial respiration. In addition, our results revealed the interaction between PA and the mitochondrial matrix protein PYCR2 and the antiviral role of PYCR2 during influenza A virus replication. Moreover, we found that the PA protein could also trigger autophagy and disrupt mitochondrial homeostasis. Overall, our research revealed the impacts of the influenza A virus PA protein on mitochondrial function and autophagy.

AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells.

BACKGROUND: Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis. METHODS: To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs. RESULTS: Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs. CONCLUSION: This study elucidates AGR2's pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

Robotic Resection in Succinate Dehydrogenase Subunit B (SDHB)-Mutated Hereditary Paraganglioma: A Case Report of Two Patients and A Literature Review.

Autosomal dominant hereditary paraganglioma-pheochromocytoma syndrome (HPPS) is a rare genetic disorder characterized by neuroendocrine tumor development associated with pathogenic variants in succinate dehydrogenase (SDH) enzyme complex genes. Particularly, HPPS linked to SDHB mutation poses a significant clinical challenge due to its association with aggressive tumor features and a high risk of malignancy. Our report underscores the diversity in the presentation of patients with SDHB-mutated paraganglioma and the feasibility of managing it with a minimally invasive surgical approach. In the first case, a 17-year-old female was diagnosed with a metabolically active, poorly differentiated extra-adrenal retroperitoneal paraganglioma that required challenging robotic resection. Cascade genetic testing revealed an SDHB mutation not only in her but also in three family members, pointing to the inherited nature of the syndrome. Conversely, the second case involves a 37-year-old male with an asymptomatic well-differentiated left paraaortic paraganglioma incidentally found during an unrelated medical examination. Robotic converted-to-open resection allowed the successful removal of the mass. Subsequent germline testing confirmed a deleterious SDHB mutation, initiating a process of familial cascade testing. Both patients remained symptom- and recurrence-free at 12 and six months, respectively. Through these cases, and supported by a literature review, we highlight the variable clinical presentations of HPPS, arising from the same genetic alteration. The successful application of minimally invasive surgical techniques, combined with genetic evaluation, emphasizes the necessity for a comprehensive, tailored approach to treatment and surveillance. This strategy not only addresses the immediate clinical needs but also fosters proactive management of at-risk family members, ensuring a multidisciplinary approach to this complex hereditary condition.

Integration of the cancer cell secretome and transcriptome reveals potential noninvasive diagnostic markers for bladder cancer.

PURPOSE: Bladder cancer (BLCA) is a major cancer of the genitourinary system. Although cystoscopy is the standard protocol for diagnosing BLCA clinically, this procedure is invasive and expensive. Several urine-based markers for BLCA have been identified and investigated, but none has shown sufficient sensitivity and specificity. These observations underscore the importance of discovering novel BLCA biomarkers and developing a noninvasive method for detection of BLCA. Exploring the cancer secretome is a good starting point for the development of noninvasive biomarkers for cancer diagnosis. EXPERIMENTAL DESIGN: In this study, we established a comprehensive secretome dataset of five representative BLCA cell lines, BFTC905, TSGH8301, 5637, MGH-U1, and MGH-U4, by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Expression of BLCA-specific secreted proteins at the transcription level was evaluated using the Oncomine cancer microarray database. RESULTS: The expressions of four candidates-COMT, EWSR1, FUSIP1, and TNPO2-were further validated in clinical human specimens. Immunohistochemical analyses confirmed that transportin-2 was highly expressed in tumor cells relative to adjacent noncancerous cells in clinical tissue specimens from BLCA patients, and was significantly elevated in BLCA urine compared with that in urine samples from aged-matched hernia patients (controls). CONCLUSIONS: Collectively, our findings suggest TNPO2 as a potential noninvasive tumor-stage or grade discriminator for BLCA management.

Surgical Lymph Node Biopsy for the Diagnosis of Lymphoma: A Case Report.

We report the diagnosis, treatment, and outcomes of a 52-year-old woman who originally presented to her primary care provider with adenopathy. Core needle biopsy (CNB) was inconclusive as it could not distinguish between follicular and diffuse large B-cell lymphomas (DLBCLs). A left axillary surgical lymph node biopsy was performed and demonstrated that the patient had a DLBCL arising from grade 3 follicular lymphoma. We discuss the limitations of CNB and the value of surgical lymph node biopsy in the diagnosis of lymphoma. The patient recovered from the biopsy without complications, and chemotherapy was initiated after the procedure. The patient has now remained in complete remission at 22 months.

MFI2 upregulation promotes malignant progression through EGF/FAK signaling in oral cavity squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the predominant histological type of the head and neck squamous cell carcinoma (HNSCC). By comparing the differentially expressed genes (DEGs) in OSCC-TCGA patients with copy number variations (CNVs) that we identify in OSCC-OncoScan dataset, we herein identified 37 dysregulated candidate genes. Among these potential candidate genes, 26 have been previously reported as dysregulated proteins or genes in HNSCC. Among 11 novel candidates, the overall survival analysis revealed that melanotransferrin (MFI2) is the most significant prognostic molecular in OSCC-TCGA patients. Another independent Taiwanese cohort confirmed that higher MFI2 transcript levels were significantly associated with poor prognosis. Mechanistically, we found that knockdown of MFI2 reduced cell viability, migration and invasion via modulating EGF/FAK signaling in OSCC cells. Collectively, our results support a mechanistic understanding of a novel role for MFI2 in promoting cell invasiveness in OSCC.

Identification of salivary autoantibodies as biomarkers of oral cancer with immunoglobulin A enrichment combined with affinity mass spectrometry.

Globally, oral cavity squamous cell carcinoma (OSCC) is one of the most common fatal illnesses. Its high mortality is ascribed to the fact that the disease is often diagnosed at a late stage, which indicates an urgent need for approaches for the early detection of OSCC. The use of salivary autoantibodies (autoAbs) as OSCC biomarkers has numerous advantages such as easy access to saliva samples and efficient detection of autoAbs using well-established secondary reagents. To improve OSCC screening, we identified OSCC-associated autoAbs with the enrichment of salivary autoAbs combined with affinity mass spectrometry (MS). The salivary IgA of healthy individuals and OSCC patients was purified with peptide M-conjugated beads and then applied to immunoprecipitated antigens (Ags) in OSCC cells. Using tandem MS analysis and spectral counting-based quantitation, the level of 10 Ags increased in the OSCC group compared with the control group. Moreover, salivary levels of autoAbs to the 10 Ags were determined by a multiplexed bead-based immunoassay. Among them, seven were significantly higher in early-stage OSCC patients than in healthy individuals. A marker panel consisting of autoAbs to LMAN2, PTGR1, RAB13, and UQCRC2 was further developed to improve the early diagnosis of OSCC.

Development of a salivary autoantibody biomarker panel for diagnosis of oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC) is a destructive disease with increasing incidence. OSCC is usually diagnosed at an advanced stage, which leads to poor outcomes of OSCC patients. Currently, there is a lack of biomarkers with sufficient effectiveness in early diagnosis of OSCC. To ameliorate OSCC screening, we evaluated the performances of salivary autoantibodies (auto-Abs) to nine proteins (ANXA2, CA2, ISG15, KNG1, MMP1, MMP3, PRDX2, SPARC, and HSPA5) as OSCC biomarkers. A multiplexed immunoassay using a fluorescence bead-based suspension array system was established for simultaneous assessment of the salivary levels of the above nine auto-Abs and a known OSCC-associated auto-Ab, anti-p53. Compared to healthy individuals (n = 140), the salivary levels of nine auto-Abs were significantly elevated in OSCC patients (n = 160). Notably, the salivary levels of the 10 auto-Abs in the early-stage OSCC patients (n = 102) were higher than that in the healthy group. Most importantly, utilizing a marker panel consisting of anti-MMP3, anti-PRDX2, anti-SPARC, and anti-HSPA5 for detection of early-stage OSCC achieved a sensitivity of 63.8% with a specificity of 90%. Collectively, herein we established a multiplex auto-Ab platform for OSCC screening, and demonstrated a four-auto-Ab panel which shows clinical applicability for early diagnosis of OSCC.

Development and validation of machine learning-based risk prediction models of oral squamous cell carcinoma using salivary autoantibody biomarkers.

INTRODUCTION: The incidence of oral cavity squamous cell carcinoma (OSCC) continues to rise. OSCC is associated with a low average survival rate, and most patients have a poor disease prognosis because of delayed diagnosis. We used machine learning techniques to predict high-risk cases of OSCC by using salivary autoantibody levels and demographic and behavioral data. METHODS: We collected the salivary samples of patients recruited from a teaching hospital between September 2008 and December 2012. Ten salivary autoantibodies, sex, age, smoking, alcohol consumption, and betel nut chewing were used to build prediction models for identifying patients with a high risk of OSCC. The machine learning algorithms applied in the study were logistic regression, random forest, support vector machine with the radial basis function kernel, eXtreme Gradient Boosting (XGBoost), and a stacking model. We evaluated the performance of the models by using the area under the receiver operating characteristic curve (AUC), with simulations conducted 100 times. RESULTS: A total of 337 participants were enrolled in this study. The best predictive model was constructed using a stacking algorithm with original forms of age and logarithmic levels of autoantibodies (AUC = 0.795 ± 0.055). Adding autoantibody levels as a data source significantly improved the prediction capability (from 0.698 ± 0.06 to 0.795 ± 0.055, p < 0.001). CONCLUSIONS: We successfully established a prediction model for high-risk cases of OSCC. This model can be applied clinically through an online calculator to provide additional personalized information for OSCC diagnosis, thereby reducing the disease morbidity and mortality rates.

Interactome Profiling of N-Terminus-Truncated NS1 Protein of Influenza A Virus Reveals Role of 14-3-3γ in Virus Replication.

Influenza A virus is transmitted through a respiratory route and has caused several pandemics throughout history. The NS1 protein of influenza A virus, which consists of an N-terminal RNA-binding domain and a C-terminal effector domain, is considered one of the critical virulence factors during influenza A virus infection because the viral protein can downregulate the antiviral response of the host cell and facilitate viral replication. Our previous study identified an N-terminus-truncated NS1 protein that covers the C-terminus effector domain. To comprehensively explore the role of the truncated NS1 in cells, we conducted immunoprecipitation coupled with LC-MS/MS to identify its interacting cellular proteins. There were 46 cellular proteins identified as the components of the truncated NS1 protein complex. As for our previous results for the identification of the full-length NS1-interacting host proteins, we discovered that the truncated NS1 protein interacts with the γ isoform of the 14-3-3 protein family. In addition, we found that the knockdown of 14-3-3γ in host cells reduced the replication of the influenza A/PR8 wild-type virus but not that of the PR8-NS1/1-98 mutant virus, which lacks most of the effector domain of NS1. This research highlights the role of 14-3-3γ, which interacts with the effector domain of NS1 protein, in influenza A viral replication.

Modular scaffolding by lncRNA HOXA10-AS promotes oral cancer progression.

Recent findings have implicated long noncoding RNAs (lncRNAs) as pivotal gene regulators for diverse biological processes, despite their lack of protein-coding capabilities. Accumulating evidence suggests the significance of lncRNAs in mediating cell signaling pathways, especially those associated with tumorigenesis. Consequently, lncRNAs have emerged as novel functional regulators and indicators of cancer development and malignancy. Recent transcriptomic profiling has recognized a tumor-biased expressed lncRNA, the HOXA10-AS transcript, whose expression is associated with patient survival. Functional cell-based assays show that the HOXA10-AS transcript is essential in the regulation of oral cancer growth and metastasis. LncRNA expression is also associated with drug sensitivity. In this study, we identify that HOXA10-AS serves as a modular scaffold for TP63 mRNA processing and that such involvement regulates cancer growth. These findings provide a functional interpretation of lncRNA-mediated molecular regulation, highlighting the significance of the lncRNA transcriptome in cancer biology.

Features of ophthalmic, magnetic resonance imaging, and histopathology of a feline case of idiopathic sclerosing orbital pseudotumor.

In the present report, we describe a case of sclerosing orbital pseudotumor in an 11-year-old castrated male American Shorthair cat. Ophthalmic exam showed lagophthalmos, retracted right upper eyelid, and resistant to retropulsion in his right eye. Under magnetic resonance imaging (MRI) scans, increased volume of the extraocular muscles (EOMs) of the right eye was prominent. Immunosuppressive dosage of prednisolone partially ameliorated the clinical signs, but some clinical signs were still gradually progressive or persistent. In the second MRI scan, decreased diameter of the thickened right extraocular muscles was found. After the third MRI scan, enucleation of the right eye was performed due to substantial adverse effects of systemic steroid therapy. Histopathological examination revealed no evidence of neoplastic transformation nor infection. Feline restrictive orbital myofibroblastic sarcoma (FROMS) was therefore excluded, suggesting unknown causes of extensive fibrotic changes in the right orbit of the affected cat.

Genomic and Molecular Signatures of Successful Patient-Derived Xenografts for Oral Cavity Squamous Cell Carcinoma.

Background: Oral cavity squamous cell carcinoma (OSCC) is an aggressive malignant tumor with high recurrence and poor prognosis in the advanced stage. Patient-derived xenografts (PDXs) serve as powerful preclinical platforms for drug testing and precision medicine for cancer therapy. We assess which molecular signatures affect tumor engraftment ability and tumor growth rate in OSCC PDXs. Methods: Treatment-naïve OSCC primary tumors were collected for PDX models establishment. Comprehensive genomic analysis, including whole-exome sequencing and RNA-seq, was performed on case-matched tumors and PDXs. Regulatory genes/pathways were analyzed to clarify which molecular signatures affect tumor engraftment ability and the tumor growth rate in OSCC PDXs. Results: Perineural invasion was found as an important pathological feature related to engraftment ability. Tumor microenvironment with enriched hypoxia, PI3K-Akt, and epithelial-mesenchymal transition pathways and decreased inflammatory responses had high engraftment ability and tumor growth rates in OSCC PDXs. High matrix metalloproteinase-1 (MMP1) expression was found that have a great graft advantage in xenografts and is associated with pooled disease-free survival in cancer patients. Conclusion: This study provides a panel with detailed genomic characteristics of OSCC PDXs, enabling preclinical studies on personalized therapy options for oral cancer. MMP1 could serve as a biomarker for predicting successful xenografts in OSCC patients.

The flavonoid corylin exhibits lifespan extension properties in mouse.

In the long history of traditional Chinese medicine, single herbs and complex formulas have been suggested to increase lifespan. However, the identification of single molecules responsible for lifespan extension has been challenging. Here, we collected a list of traditional Chinese medicines with potential longevity properties from pharmacopeias. By utilizing the mother enrichment program, we systematically screened these traditional Chinese medicines and identified a single herb, Psoralea corylifolia, that increases lifespan in Saccharomyces cerevisiae. Next, twenty-two pure compounds were isolated from Psoralea corylifolia. One of the compounds, corylin, was found to extend the replicative lifespan in yeast by targeting the Gtr1 protein. In human umbilical vein endothelial cells, RNA sequencing data showed that corylin ameliorates cellular senescence. We also examined an in vivo mammalian model, and found that corylin extends lifespan in mice fed a high-fat diet. Taken together, these findings suggest that corylin may promote longevity.

Quantifying Neutralizing Antibodies in Patients with COVID-19 by a Two-Variable Generalized Additive Model.

Considering the urgent demand for faster methods to quantify neutralizing antibody titers in patients with coronavirus (CoV) disease 2019 (COVID-19), developing an analytical model or method to replace the conventional virus neutralization test (NT) is essential. Moreover, a "COVID-19 immunity passport" is currently being proposed as a certification for people who travel internationally. Therefore, an enzyme-linked immunosorbent assay (ELISA) was designed to detect severe acute respiratory syndrome CoV 2 (SARS-CoV-2)-neutralizing antibodies in serum, which is based on the binding affinity of SARS-CoV-2 viral spike protein 1 (S1) and the viral spike protein receptor-binding domain (RBD) to antibodies. The RBD is considered the major binding region of neutralizing antibodies. Furthermore, S1 covers the RBD and several other regions, which are also important for neutralizing antibody binding. In this study, we assessed 144 clinical specimens, including those from patients with PCR-confirmed SARS-CoV-2 infections and healthy donors, using both the NT and ELISA. The ELISA results analyzed by spline regression and the two-variable generalized additive model precisely reflected the NT value, and the correlation between predicted and actual NT values was as high as 0.917. Therefore, our method serves as a surrogate to quantify neutralizing antibody titer. The analytic method and platform used in this study present a new perspective for serological testing of SARS-CoV-2 infection and have clinical potential to assess vaccine efficacy. IMPORTANCE Herein, we present a new approach for serological testing for SARS-CoV-2 antibodies using innovative laboratory methods that demonstrate a combination of biology and mathematics. The traditional virus neutralization test is the gold standard method; however, it is time-consuming and poses a risk to medical personnel. Thus, there is a demand for methods that rapidly quantify neutralizing antibody titers in patients with COVID-19 or examine vaccine efficacy at a biosafety level 2 containment facility. Therefore, we used a two-variable generalized additive model to analyze the results of the enzyme-linked immunosorbent assay and found the method to serve as a surrogate to quantify neutralizing antibody titers. This methodology has potential for clinical use in assessing vaccine efficacy.

Extracellular Vesicle Membrane Protein Profiling and Targeted Mass Spectrometry Unveil CD59 and Tetraspanin 9 as Novel Plasma Biomarkers for Detection of Colorectal Cancer.

Extracellular vesicles (EVs) are valuable sources for the discovery of useful cancer biomarkers. This study explores the potential usefulness of tumor cell-derived EV membrane proteins as plasma biomarkers for early detection of colorectal cancer (CRC). EVs were isolated from the culture supernatants of four CRC cell lines by ultracentrifugation, and their protein profiles were analyzed by LC-MS/MS. Bioinformatics analysis of identified proteins revealed 518 EV membrane proteins in common among at least three CRC cell lines. We next used accurate inclusion mass screening (AIMS) in parallel with iTRAQ-based quantitative proteomic analysis to highlight candidate proteins and validated their presence in pooled plasma-generated EVs from 30 healthy controls and 30 CRC patients. From these, we chose 14 potential EV-derived targets for further quantification by targeted MS assay in a separate individual cohort comprising of 73 CRC and 80 healthy subjects. Quantitative analyses revealed significant increases in ADAM10, CD59 and TSPAN9 levels (2.19- to 5.26-fold, p < 0.0001) in plasma EVs from CRC patients, with AUC values of 0.83, 0.95 and 0.87, respectively. Higher EV CD59 levels were significantly correlated with distant metastasis (p = 0.0475), and higher EV TSPAN9 levels were significantly associated with lymph node metastasis (p = 0.0011), distant metastasis at diagnosis (p = 0.0104) and higher TNM stage (p = 0.0065). A two-marker panel consisting of CD59 and TSPAN9 outperformed the conventional marker CEA in discriminating CRC and stage I/II CRC patients from healthy controls, with AUC values of 0.98 and 0.99, respectively. Our results identify EV membrane proteins in common among CRC cell lines and altered plasma EV protein profiles in CRC patients and suggest plasma EV CD59 and TSPAN9 as a novel biomarker panel for detecting early-stage CRC.

Role of the Chaperone Protein 14-3-3ε in the Regulation of Influenza A Virus-Activated Beta Interferon.

The NS1 protein of the influenza A virus plays a critical role in regulating several biological processes in cells, including the type I interferon (IFN) response. We previously profiled the cellular factors that interact with the NS1 protein of influenza A virus and found that the NS1 protein interacts with proteins involved in RNA splicing/processing, cell cycle regulation, and protein targeting processes, including 14-3-3ε. Since 14-3-3ε plays an important role in retinoic acid-inducible gene I (RIG-I) translocation to mitochondrial antiviral-signaling protein (MAVS) to activate type I IFN expression, the interaction of the NS1 and 14-3-3ε proteins may prevent the RIG-I-mediated IFN response. In this study, we confirmed that the 14-3-3ε protein interacts with the N-terminal domain of the NS1 protein and that the NS1 protein inhibits RIG-I-mediated IFN-ß promoter activation in 14-3-3ε-overexpressing cells. In addition, our results showed that knocking down 14-3-3ε can reduce IFN-ß expression elicited by influenza A virus and enhance viral replication. Furthermore, we found that threonine in the 49th amino acid position of the NS1 protein plays a role in the interaction with 14-3-3ε. Influenza A virus expressing C terminus-truncated NS1 with a T49A mutation dramatically increases IFN-ß mRNA in infected cells and causes slower replication than that of virus without the T-to-A mutation. Collectively, this study demonstrates that 14-3-3ε is involved in influenza A virus-initiated IFN-ß expression and that the interaction of the NS1 protein and 14-3-3ε may be one of the mechanisms for inhibiting type I IFN activation during influenza A virus infection. IMPORTANCE Influenza A virus is an important human pathogen causing severe respiratory disease. The virus has evolved several strategies to dysregulate the innate immune response and facilitate its replication. We demonstrate that the NS1 protein of influenza A virus interacts with the cellular chaperone protein 14-3-3ε, which plays a critical role in retinoic acid-inducible gene I (RIG-I) translocation that induces type I interferon (IFN) expression, and that NS1 protein prevents RIG-I translocation to the mitochondrial membrane. The interaction site for 14-3-3ε is the RNA-binding domain (RBD) of the NS1 protein. Therefore, this research elucidates a novel mechanism by which the NS1 RBD mediates IFN-ß suppression to facilitate influenza A viral replication. Additionally, the findings reveal the antiviral role of 14-3-3ε during influenza A virus infection.

Reducing Lung ATP Levels and Alleviating Asthmatic Airway Inflammation through Adeno-Associated Viral Vector-Mediated CD39 Expression.

Asthma is a chronic respiratory inflammatory disease. Patients usually suffer long-term symptoms and high medical expenses. Extracellular ATP (eATP) has been identified as a danger signal in innate immunity and serves as a potent inflammatory mediator for asthma. Hydrolyzing eATP in lungs might be a potential approach to alleviate asthmatic inflammation. Recombinant adeno-associated virus (rAAV) vectors that contain tissue-specific cap protein have been demonstrated to efficiently transfer exogenous genes into the lung tissues. To test anti-inflammation efficacy of rAAV-mediated CD39 gene transfer, rAAV-CD39 was generated and applied to OVA-mediated asthmatic mice. BALB/c mice were sensitized intraperitoneally and challenged intratracheally with OVA and treated with rAAV-CD39. At the end of procedure, some inflammatory features were examined. rAAV-CD39 treatment downregulated the levels of pulmonary eATP by the rescued expression of CD39. Several asthmatic features, such as airway hyperresponsiveness, eosinophilia, mucin deposition, and IL-5/IL-13 production in the lungs were decreased in the rAAV-CD39-treated mice. Reduced IL-5/IL-13 production and increased frequency of CD4+FoxP3+ regulatory T cells were detected in draining lymph nodes of rAAV-CD39 treated mice. This evidence suggested that rAAV-mediated CD39 gene transfer attenuated the asthmatic airway inflammation locally. The results suggest that rAAV-CD39 might have therapeutic potential for asthma.