PROTEOFORMER: deep proteome coverage through ribosome profiling and MS integration

An increasing amount of studies integrate mRNA sequencing data into MS-based proteomics to complement the translation product search space. However, several factors, including extensive regulation of mRNA translation and the need for three- or six-frame-translation, impede the use of mRNA-seq data for the construction of a protein sequence search database. With that in mind, we developed the PROTEOFORMER tool that automatically processes data of the recently developed ribosome profiling method (sequencing of ribosome-protected mRNA fragments), resulting in genome-wide visualization of ribosome occupancy. Our tool also includes a translation initiation site calling algorithm allowing the delineation of the open reading frames (ORFs) of all translation products. A complete protein synthesis-based sequence database can thus be compiled for mass spectrometry-based identification. This approach increases the overall protein identification rates with 3% and 11% (improved and new identifications) for human and mouse, respectively, and enables proteome-wide detection of 5'-extended proteoforms, upstream ORF translation and near-cognate translation start sites. The PROTEOFORMER tool is available as a stand-alone pipeline and has been implemented in the galaxy framework for ease of use

A comprehensive overview of genomic imprinting in breast and its deregulation in cancer

Genomic imprinting plays an important role in growth and development. Loss of imprinting (LOI) has been found in cancer, yet systematic studies are impeded by data-analytical challenges. We developed a methodology to detect monoallelically expressed loci without requiring genotyping data, and applied it on The Cancer Genome Atlas (TCGA, discovery) and Genotype-Tissue expression project (GTEx, validation) breast tissue RNA-seq data. Here, we report the identification of 30 putatively imprinted genes in breast. In breast cancer (TCGA), HM13 is featured by LOI and expression upregulation, which is linked to DNA demethylation. Other imprinted genes typically demonstrate lower expression in cancer, often associated with copy number variation and aberrant DNA methylation. Downregulation in cancer frequently leads to higher relative expression of the (imperfectly) silenced allele, yet this is not considered canonical LOI given the lack of (absolute) re-expression. In summary, our novel methodology highlights the massive deregulation of imprinting in breast cancer

Clinically relevant aberrant Filip1l DNA methylation detected in a murine model of cutaneous squamous cell carcinoma

Background: Cutaneous squamous cell carcinomas (cSCC) are among the most common and highly mutated human malignancies. Understanding the impact of DNA methylation in cSCC may provide avenues for new therapeutic strategies. Methods: We used reduced-representation bisulfite sequencing for DNA methylation analysis of murine cSCC. Differential methylation was assessed at the CpG level using limma. Next, we compared with human cSCC Infinium HumanMethylation BeadArray data. Genes were considered to be of major relevance when they featured at least one significantly differentially methylated CpGs (RRBS) / probes (Infinium) with at least a 30% difference between tumour vs. control in both a murine gene and its human orthologue. The human EPIC Infinium data were used to distinguish two cSCC subtypes, stem-cell-like and keratinocyte-like tumours. Findings: We found increased average methylation in mouse cSCC (by 12.8%, p = 0.0011) as well as in stem-cell like (by 3.1%, p=0.002), but not keratinocyte-like (0.2%, p = 0.98), human cSCC. Comparison of differentially methylated genes revealed striking similarities between human and mouse cSCC. Locus specific methylation changes in mouse cSCC often occurred in regions of potential regulatory function, including enhancers and promoters. A key differentially methylated region was located in a potential enhancer of the tumour suppressor gene Filip1l and its expression was reduced in mouse tumours. Moreover, the FILIP1L, locus showed hypermethylation in human cSCC and lower expression in human cSCC cell lines. Interpretation: Deregulation of DNA methylation is an important feature of murine and human cSCC that likely contributes to silencing of tumour suppressor genes, as shown for Filip1l. 2021 The Author(s). Published by Elsevier B.V

In-situ Plasma Studies using a Direct Current Microplasma in a Scanning Electron Microscope

Microplasmas can be used for a wide range of technological applications and to improve our understanding of fundamental physics. Scanning electron microscopy, on the other hand, provides insights into the sample morphology and chemistry of materials from the mm-down to the nm-scale. Combining both would provide direct insight into plasma-sample interactions in real-time and at high spatial resolution. Up till now, very few attempts in this direction have been made, and significant challenges remain. This work presents a stable direct current glow discharge microplasma setup built inside a scanning electron microscope. The experimental setup is capable of real-time in-situ imaging of the sample evolution during plasma operation and it demonstrates localized sputtering and sample oxidation. Further, the experimental parameters such as varying gas mixtures, electrode polarity, and field strength are explored and experimental $V$-$I$ curves under various conditions are provided. These results demonstrate the capabilities of this setup in potential investigations of plasma physics, plasma-surface interactions, and materials science and its practical applications. The presented setup shows the potential to have several technological applications, e.g., to locally modify the sample surface (e.g., local oxidation and ion implantation for nanotechnology applications) on the $\mu$m-scale.Comment: LG, DC, and RDM contributed equally to this work. The videos mentioned in the manuscript can be found in the Zenodo repository linked in the pape

Astrocytes in paper chips and their interaction with hybrid vesicles

© 2022 The Authors. Advanced Biology published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.The role of astrocytes in brain function has received increased attention lately due to their critical role in brain development and function under physiological and pathophysiological conditions. However, the biological evaluation of soft material nanoparticles in astrocytes remains unexplored. Here, the interaction of crosslinked hybrid vesicles (HVs) and either C8-D1A astrocytes or primary astrocytes cultured in polystyrene tissue culture or floatable paper-based chips is investigated. The amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) (P1) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine lipids are used for the assembly of HVs with crosslinked membranes. The assemblies show no short-term toxicity towards the C8-D1A astrocytes and the primary astrocytes, and both cell types internalize the HVs when cultured in 2D cell culture. Further, it is demonstrated that both the C8-D1A astrocytes and the primary astrocytes could mature in paper-based chips with preserved calcium signaling and glial fibrillary acidic protein expression. Last, it is confirmed that both types of astrocytes could internalize the HVs when cultured in paper-based chips. These findings lay out a fundamental understanding of the interaction between soft material nanoparticles and astrocytes, even when primary astrocytes are cultured in paper-based chips offering a 3D environment.This project was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. 818890), the Lundbeck Foundation (R346-2020-1617), and the Carlsberg Foundation (CF 20–0418). The HRM Queen Margrethe II's travel grant (C.A.M.) is acknowledged for support. Tinfo:eu-repo/semantics/publishedVersio

Occurrence of nematoda and protozoa in cats with tutors in the city of Porto Alegre, RS, Brazil

Este estudo avalia a ocorrência de parasitos gastrintestinais e pulmonares em 266 gatos domésticos através de exames de fezes realizados no ano de 2018. Os gatos eram domiciliados, adultos de diversas raças, com tutores residentes na cidade de Porto Alegre, Rio Grande do Sul, Brasil. As amostras foram processadas pelos métodos de Baermann, Willis-Mollay, Lutz e Faust. A ocorrência de gatos parasitados foi de 21% (56/266). Foram identificados os gêneros parasitários: Giardia (26,8%), Toxocara (26,8%), Cystoisospora (17,8%), Ancylostoma (5,3%), Entamoeba (3,6%), Dipylidium (1,8%), Toxocara/Cystoisospora (1,8%), Toxocara/Dipylidium (1,8%) e larvas L1 de Aelurostrongylus abstrusus (14,3%). A ocorrência de infecções parasitárias foi significativa para parasitos zoonóticos.This study evaluates the occurrence of gastrointestinal and pulmonary parasites in 266 domestic cats through stool examinations performed in 2018. The cats were adults of several breeds, with tutors and residents in the city of Porto Alegre, Rio Grande do Sul, Brazil. The samples were processed by the methods of Baermann, Willis-Mollay, Lutz and Faust. The occurrence of parasitized cats was 21% (56/266). The parasitic genera were identified: Giardia (26,8%), Toxocara (26,8%), Cystoisospora (17,8%), Ancylostoma (5,3%), Entamoeba (3,6%), Dipylidium (1,8%), Toxocara/Cystoisospora (1,8%), Toxocara/Dipylidium (1,8%) and L1 larvae of Aelurostrongylus abstrusus (14,3%). The occurrence of parasitic infections was significant for zoonotic parasites

Editorial

Informações sobre o conteúdo da Urdimento v.1, n.28, 2017

Hepatitis C viral evolution in genotype 1 treatment-naïve and treatment-experienced patients receiving telaprevir-based therapy in clinical trials

Background: In patients with genotype 1 chronic hepatitis C infection, telaprevir (TVR) in combination with peginterferon and ribavirin (PR) significantly increased sustained virologic response (SVR) rates compared with PR alone. However, genotypic changes could be observed in TVR-treated patients who did not achieve an SVR. Methods: Population sequence analysis of the NS3•4A region was performed in patients who did not achieve SVR with TVR-based treatment. Results: Resistant variants were observed after treatment with a telaprevir-based regimen in 12% of treatment-naïve patients (ADVANCE; T12PR arm), 6% of prior relapsers, 24% of prior partial responders, and 51% of prior null responder patients (REALIZE, T12PR48 arms). NS3 protease variants V36M, R155K, and V36M+R155K emerged frequently in patients with genotype 1a and V36A, T54A, and A156S/T in patients with genotype 1b. Lower-level resistance to telaprevir was conferred by V36A/M, T54A/S, R155K/T, and A156S variants; and higher-level resistance to telaprevir was conferred by A156T and V36M+R155K variants. Virologic failure during telaprevir treatment was more common in patients with genotype 1a and in prior PR nonresponder patients and was associated with higher-level telaprevir-resistant variants. Relapse was usually associated with wild-type or lower-level resistant variants. After treatment, viral populations were wild-type with a median time of 10 months for genotype 1a and 3 weeks for genotype 1b patients. Conclusions: A consistent, subtype-dependent resistance profile was observed in patients who did not achieve an SVR with telaprevir-based treatment. The primary role of TVR is to inhibit wild-type virus and variants with lower-levels of resistance to telaprevir. The complementary role of PR is to clear any remaining telaprevir-resistant variants, especially higher-level telaprevir-resistant variants. Resistant variants are detectable in most patients who fail to achieve SVR, but their levels decline over time after treatment

PARASITOS EM RÉPTEIS DO NÚCLEO DE CONSERVAÇÃO E REABILITAÇÃO DE ANIMAIS SILVESTRES DE UMA UNIVERSIDADE PÚBLICA-BRASIL

The medical clinic for reptiles has grown in recent years, whether for attending free-living animals in rehabilitation centers or for pets. The knowledge of the parasitic fauna of the Reptilia Class is important for the identification of pathogenic agents and effective therapy. Fecal samples were collected from 35 animals of the Orders Crocodylia (N=3), Squamata (N=3) and Testudines (N=29) attended from 2013 to 2018 at the Center for Conservation and Rehabilitation of Wild Animals (PRESERVAS) of the Universidade Federal do Rio Grande do Sul (UFRGS). Fecal samples were processed by the methods of Willis, Lutz and Sheather at the Laboratory of Helminthology at UFRGS. The percentage of positive samples was 17.14% (6/35): three Trachemys dorbogni (Duméril & Bibron 1835) presenting Serpinema spp., Physaloptera spp. and Strongyloidea, respectively; two Caiman latirostris (Daudin, 1802) with Strongyloides spp. and Augusticaecum spp., respectively, and Iguana iguana (Linnaeus, 1758) with Eimeria spp. This study expands the list of parasites in reptiles from the state of Rio Grande do Sul.La clínica médica para reptiles ha crecido en los últimos años, ya sea para atender animales de vida libre en centros de rehabilitación o para mascotas. El conocimiento de la fauna parasitaria de la clase Reptilia es importante para la identificación de agentes patógenos y una terapia eficaz. Se recolectaron muestras fecales de 35 animales de los Órdenes Crocodylia (N = 3), Squamata (N = 3) y Testudines (N = 29) atendidos de 2013 a 2018 en el Centro de Conservación y Rehabilitación de Animales Silvestres (PRESERVAS) de la Universidade Federal do Rio Grande do Sul (UFRGS). Las muestras fecales fueron procesadas por los métodos de Willis, Lutz y Sheather en el Laboratorio de Helmintología de la UFRGS. El porcentaje de muestras positivas fue del 17,14% (6/35): tres Trachemys dorbogni (Duméril & Bibron 1835) presentaron Serpinema spp., Physaloptera spp. y Strongyloidea, respectivamente; dos Caiman latirostris (Daudin, 1802) con Strongyloides spp. y Augusticaecum spp., respectivamente, e Iguana iguana (Linnaeus, 1758) con Eimeria spp. Este estudio amplía la lista de parásitos en reptiles del estado de Rio Grande do Sul.A clínica médica de répteis tem crescido nos últimos anos, seja pelo atendimento de animais de vida livre em centros de reabilitação ou pela aquisição como pets. O conhecimento da fauna parasitária da Classe Reptilia é importante para identificação de agentes patogênicos e terapêutica efetiva. Foram coletadas amostras fecais de 35 animais das Ordens Crocodylia (N= 3), Squamata (N=3) e Testudines (N=29) atendidos de 2013 à 2018 no Núcleo de Conservação e Reabilitação de Animais Silvestres (PRESERVAS) da Universidade Federal do Rio Grande do Sul (UFRGS). Amostras fecais foram processadas pelos métodos de Willis, Lutz e Sheather no Laboratório de Helmintologia da UFRGS. A porcentagem de amostras positivas foi de 17, 14 % (6/35): três Trachemys dorbogni (Duméril & Bibron 1835) apresentando Serpinema spp., Physaloptera spp. e Strongyloidea, respectivamente; dois Caiman latirostris (Daudin, 1802) com Strongyloides spp. e Augusticaecum spp., respectivamente é uma Iguana iguana (Linnaeus, 1758) com Eimeria spp. Este estudo amplia a lista de parasitos em répteis do estado do Rio Grande do Sul