Sapovirus translation requires an interaction between VPg and the cap binding protein eIF4E.

UNLABELLED: Sapoviruses of the Caliciviridae family of small RNA viruses are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture, making it a suitable model for understanding the infectious cycle of sapoviruses and the related enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus genome-encoded viral protein genome (VPg) protein, which is covalently linked to the 5' end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg, and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells and bound directly to the eIF4E protein. As has been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall, our study provides new insights into the novel mechanism of sapovirus translation, suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction. IMPORTANCE: Sapoviruses, which are members of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains noncultivable in cell culture, hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for viral translation and functions via a direct interaction with the cellular translation initiation factor eIF4E. This work provides new insights into the novel protein-primed mechanism of calicivirus VPg-dependent translation initiation.This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning (NRF-2014R1A2A2A01004292), and by the Wellcome Trust (Ref: WT097997MA). IG is a Wellcome senior fellow. The authors would like to thank Professor Jeong-Sun Kim for providing reagents and critical input into the project.This is the accepted manuscript version of the article. The final version is available from ASM at http://jvi.asm.org/content/early/2014/08/18/JVI.01650-14.abstract

Comparative analysis of multiple classification models to improve PM10 prediction performance

With the increasing requirement of high accuracy for particulate matter prediction, various attempts have been made to improve prediction accuracy by applying machine learning algorithms. However, the characteristics of particulate matter and the problem of the occurrence rate by concentration make it difficult to train prediction models, resulting in poor prediction. In order to solve this problem, in this paper, we proposed multiple classification models for predicting particulate matter concentrations required for prediction by dividing them into AQI-based classes. We designed multiple classification models using logistic regression, decision tree, SVM and ensemble among the various machine learning algorithms. The comparison results of the performance of the four classification models through error matrices confirmed the f-score of 0.82 or higher for all the models other than the logistic regression model

Porcine sapovirus replication is restricted by the type I interferon response in cell culture.

Porcine sapovirus (PSaV) of the family Caliciviridae, is the only member of the genus Sapovirus with cell culture and reverse genetics systems. When combined with the piglet model, these approaches provide a system to understand the molecular basis of sapovirus pathogenesis. The replication of PSaV in cell culture is, however, restricted, displaying an absolute requirement for bile acids and producing lower levels of infectious virus than other caliciviruses. The effect of bile acids has previously been linked to a reduction in the signal transducer and activator of transcription (STAT1)-mediated signalling pathway. In the current study, we observed that even in the presence of bile acids, PSaV replication in cell culture was restricted by soluble factors produced from infected cells. This effect was at least partially due to secreted IFN because treatment of cells with recombinant porcine IFN-β resulted in significantly reduced viral replication. Moreover, IFN-mediated signalling pathways (IFN, STAT1 and the 2',5'-oligoadenylate synthetase) were activated during PSaV infection. Characterization of PSaV growth in cell lines deficient in their ability to induce or respond to IFN showed a 100-150-fold increase in infectious virus production, indicating that the primary role of bile acids was not the inactivation of the innate immune response. Furthermore, the use of IFN-deficient cell lines enabled more efficient recovery of PSaV from cDNA constructs. Overall, the highly efficient cell culture and reverse genetics system established here for PSaV highlighted the key role of the innate immune response in the restriction of PSaV infection and should greatly facilitate further molecular studies on sapovirus host-cell interactions.This research was supported by funding from the Wellcome Trust (Ref: WT097997MA), Biotechnology and Biological Sciences Research Council (Ref: BB/I012303/1) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2014R1A2A2A01004292). IG is a Wellcome senior fellow.This is the final version of the article. It first appeared from the Society for General Microbiology via http://dx.doi.org/10.1099/vir.0.071365-

A lab-on-a-disc platform enables serial monitoring of individual CTCs associated with tumor progression during EGFR-targeted therapy for patients with NSCLC

Rationale: Unlike traditional biopsy, liquid biopsy, which is a largely non-invasive diagnostic and monitoring tool, can be performed more frequently to better track tumors and mutations over time and to validate the efficiency of a cancer treatment. Circulating tumor cells (CTCs) are considered promising liquid biopsy biomarkers; however, their use in clinical settings is limited by high costs and a low throughput of standard platforms for CTC enumeration and analysis. In this study, we used a label-free, high-throughput method for CTC isolation directly from whole blood of patients using a standalone, clinical setting-friendly platform. Methods: A CTC-based liquid biopsy approach was used to examine the efficacy of therapy and emergent drug resistance via longitudinal monitoring of CTC counts, DNA mutations, and single-cell-level gene expression in a prospective cohort of 40 patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer. Results: The change ratio of the CTC counts was associated with tumor response, detected by CT scan, while the baseline CTC counts did not show association with progression-free survival or overall survival. We achieved a 100% concordance rate for the detection of EGFR mutation, including emergence of T790M, between tumor tissue and CTCs. More importantly, our data revealed the importance of the analysis of the epithelial/mesenchymal signature of individual pretreatment CTCs to predict drug responsiveness in patients. Conclusion: The fluid-assisted separation technology disc platform enables serial monitoring of CTC counts, DNA mutations, as well as unbiased molecular characterization of individual CTCs associated with tumor progression during targeted therapy

A microfluidic chip for screening individual cancer cells via eavesdropping on autophagyinducing crosstalk in the stroma niche

Autophagy is a cellular homeostatic mechanism where proteins and organelles are digested and recycled to provide an alternative source of building blocks and energy to cells. The role of autophagy in cancer microenvironment is still poorly understood. Here, we present a microfluidic system allowing monitoring of the crosstalk between single cells. We used this system to study how tumor cells induced autophagy in the stromal niche. Firstly, we could confirm that transforming growth factor beta 1 (TGF beta 1) secreted from breast tumor cells is a paracrine mediator of tumor-stroma interaction leading to the activation of autophagy in the stroma component fibroblasts. Through proof of concept experiments using TGF beta 1 as a model factor, we could demonstrate real time monitoring of autophagy induction in fibroblasts by single tumor cells. Retrieval of individual tumor cells from the microfluidic system and their subsequent genomic analysis was possible, allowing us to determine the nature of the factor mediating tumor-stroma interactions. Therefore, our microfluidic platform might be used as a promising tool for quantitative investigation of tumor-stroma interactions, especially for and high-throughput screening of paracrine factors that are secreted from heterogeneous tumor cell populations

Characteristics of Hearing Loss Among Older Adults in the Korean Genome and Epidemiology Study: A Community-Based Longitudinal Cohort Study With an 8-Year Follow-up

Objectives. This study investigated the 8-year incidence and progression of hearing loss (HL) and its types and examined the risk factors for changes in HL. Methods. This longitudinal cohort study analyzed data from the Korean Genome and Epidemiology Study (KoGES), an ongoing, prospective, community-based cohort study that has been conducted since 2001. Altogether, 1,890 residents of urban areas in Korea aged 45–75 years at time 1 (baseline) were included in the study. Pure-tone audiometry (PTA) testing was performed twice, at time 1 (2008–2009) and time 2 (2015–2018, follow-up), 8 years apart. HL grades were defined as seven mutually exclusive categories following the revised World Health Organization classification. Incidence was defined as PTA >20 dB HL in the better ear at time 2 among those without HL at time 1. Progression was defined as the progressive deterioration of HL among those with HL at time 1. The three types of HL constituted sensorineural (SNHL), conductive, and mixed HL. Results. At time 1, 36.40% of patients were diagnosed with HL, which increased to 51.64% at time 2. The 8-year incidence of HL was 27.20%, and progressive deterioration of HL occurred in 23.11% of those with HL. SNHL was the most common type of HL, and its prevalence markedly increased at time 2. Multivariate analysis demonstrated that the incidence of HL was significantly associated with increasing age, male sex (odds ratio [OR], 1.73; 95% confidence interval [CI], 1.07–2.81), and diabetes mellitus (OR, 1.43; 95% CI, 1.04–1.96). Alcohol consumption was a risk factor for HL deterioration among those with HL at time 1. Conclusion. The prevalence and deterioration of HL were extremely high among older adults, and age was the strongest risk factor for these changes. Therefore, timely screening and intervention are necessary to prevent HL and delay its deterioration among older adults

Importância dos Saca-Rabos (Herpestes Ichneumon) como Reservatório de Mycobacterium avium subsp. paratuberculosis. Deteção por Técnicas Tradicionais e Moleculares

Poster apresentado nas IV Jornadas de Genética, realizadas na UTAD, Vila Real, nos dias 1,2 e 3 de Março de 2012.Os saca-rabos (Herpestes ichneumon) também conhecidos por mangustos, são carnívoros diurnos selvagens que juntamente com a geneta (Genetta genetta) representam os exemplares da família Viverridae em Portugal. É uma espécie cinegética de caça menor que se alimenta de coelhos, roedores, aves, cobras, insectos e ovos. Neste estudo colheram-se amostras de 8 animais mortos por atropelamento e em ações de controlo de predadores, durante os anos de 2010 e 2011, nos concelhos de Idanha-a-Nova e Penamacor do distrito de Castelo Branco. As amostras colhidas foram fígado, pulmão, baço, intestino, rim, gânglio mesentérico, retrofaríngeo, mediastínico, amígdalas e fezes. As amostras foram submetidas à técnica de PCR e a cultura microbiológica em meios específicos. Em três saca-rabos (37,5%) detectou-se Mycobacterium avium subsp. paratuberculosis (Map) através da técnica de biologia molecular. Dois eram machos e um era fêmea. Map foi confirmado também em cultura nos dois machos. Sete saca-rabos (87,5%) apresentaram bactérias álcool-ácido resistentes compatíveis com Map em esfregaços de diferentes tecidos, quando corados pelo método de Ziehl-Neelsen. Estes resultados preliminares confirmam os saca-rabos como reservatório de Map no nosso país. Atualmente, estão a ser desenvolvidos mais estudos para a avaliação dos saca-rabos na dinâmica da infeção de Map em mamíferos selvagens

The Benefits and Risks of Prophylactic Central Neck Dissection for Papillary Thyroid Carcinoma: Prospective Cohort Study

Objectives. This study evaluated the benefits of performing prophylactic central neck dissection (CND) with total thyroidectomy (TT) in management of papillary thyroid carcinoma (PTC) patients who were clinically node-negative at presentation. Methods. A total of 257 patients with stage T1 or T2 PTC and without preoperative evidence of lymph node involvement (N0) were enrolled in this prospective study. The patients were randomly assigned to two groups: (1) a total thyroidectomy (TT) group (n=104) or (2) a TT plus CND group (n=153). The two groups were compared for their perioperative data, complication rates, disease recurrence rates, and clinical outcomes. Results. The two groups of patients were similar in age, sex ratio, follow-up duration, and tumor size (P=0.227, 0.359, 0.214, and 0.878, resp.). The two groups showed similar rates of disease recurrence (3.9% in the TT group versus 3.3% in the TT plus CND group); however, complications occurred more frequently in the TT plus CND group; especially transient hypocalcemia (P=0.043). Conclusions. Patients treated with TT plus CND had a higher rate of complications with similar recurrence rate. We believe that CND may not be routinely recommended when treating patients with PTC

Full-length genomic analysis of korean porcine sapelovirus strains.

Porcine sapelovirus (PSV), a species of the genus Sapelovirus within the family Picornaviridae, is associated with diarrhea, pneumonia, severe neurological disorders, and reproductive failure in pigs. However, the structural features of the complete PSV genome remain largely unknown. To analyze the structural features of PSV genomes, the full-length nucleotide sequences of three Korean PSV strains were determined and analyzed using bioinformatic techniques in comparison with other known PSV strains. The Korean PSV genomes ranged from 7,542 to 7,566 nucleotides excluding the 3' poly(A) tail, and showed the typical picornavirus genome organization; 5'untranslated region (UTR)-L-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3'UTR. Three distinct cis-active RNA elements, the internal ribosome entry site (IRES) in the 5'UTR, a cis-replication element (CRE) in the 2C coding region and 3'UTR were identified and their structures were predicted. Interestingly, the structural features of the CRE and 3'UTR were different between PSV strains. The availability of these first complete genome sequences for PSV strains will facilitate future investigations of the molecular pathogenesis and evolutionary characteristics of PSV