Medium-throughput processing of whole mount in situ hybridisation experiments into gene expression domains

This is the final version of the article. Available from the publisher via the DOI in this record.Understanding the function and evolution of developmental regulatory networks requires the characterisation and quantification of spatio-temporal gene expression patterns across a range of systems and species. However, most high-throughput methods to measure the dynamics of gene expression do not preserve the detailed spatial information needed in this context. For this reason, quantification methods based on image bioinformatics have become increasingly important over the past few years. Most available approaches in this field either focus on the detailed and accurate quantification of a small set of gene expression patterns, or attempt high-throughput analysis of spatial expression through binary pattern extraction and large-scale analysis of the resulting datasets. Here we present a robust, "medium-throughput" pipeline to process in situ hybridisation patterns from embryos of different species of flies. It bridges the gap between high-resolution, and high-throughput image processing methods, enabling us to quantify graded expression patterns along the antero-posterior axis of the embryo in an efficient and straightforward manner. Our method is based on a robust enzymatic (colorimetric) in situ hybridisation protocol and rapid data acquisition through wide-field microscopy. Data processing consists of image segmentation, profile extraction, and determination of expression domain boundary positions using a spline approximation. It results in sets of measured boundaries sorted by gene and developmental time point, which are analysed in terms of expression variability or spatio-temporal dynamics. Our method yields integrated time series of spatial gene expression, which can be used to reverse-engineer developmental gene regulatory networks across species. It is easily adaptable to other processes and species, enabling the in silico reconstitution of gene regulatory networks in a wide range of developmental contexts.The laboratory of Johannes Jaeger and this study in particular was funded by the MEC-EMBL agreement for the EMBL/CRG Research Unit in Systems Biology, by grant 153 (MOPDEV) of the ERANet: ComplexityNET program, by SGR grant 406 from the Catalan funding agency AGAUR, by grant BFU2009-10184 from the Spanish Ministry of Science, and by European Commission grant FP7-KBBE-2011-5/289434 (BioPreDyn)

Efficient reverse-engineering of a developmental gene regulatory network

This is the final version of the article. Available from the publisher via the DOI in this record.Understanding the complex regulatory networks underlying development and evolution of multi-cellular organisms is a major problem in biology. Computational models can be used as tools to extract the regulatory structure and dynamics of such networks from gene expression data. This approach is called reverse engineering. It has been successfully applied to many gene networks in various biological systems. However, to reconstitute the structure and non-linear dynamics of a developmental gene network in its spatial context remains a considerable challenge. Here, we address this challenge using a case study: the gap gene network involved in segment determination during early development of Drosophila melanogaster. A major problem for reverse-engineering pattern-forming networks is the significant amount of time and effort required to acquire and quantify spatial gene expression data. We have developed a simplified data processing pipeline that considerably increases the throughput of the method, but results in data of reduced accuracy compared to those previously used for gap gene network inference. We demonstrate that we can infer the correct network structure using our reduced data set, and investigate minimal data requirements for successful reverse engineering. Our results show that timing and position of expression domain boundaries are the crucial features for determining regulatory network structure from data, while it is less important to precisely measure expression levels. Based on this, we define minimal data requirements for gap gene network inference. Our results demonstrate the feasibility of reverse-engineering with much reduced experimental effort. This enables more widespread use of the method in different developmental contexts and organisms. Such systematic application of data-driven models to real-world networks has enormous potential. Only the quantitative investigation of a large number of developmental gene regulatory networks will allow us to discover whether there are rules or regularities governing development and evolution of complex multi-cellular organisms.Funding: The laboratory of Johannes Jaeger and this study in particular was funded by the MEC-EMBL agreement for the EMBL/CRG Research Unit in Systems Biology, by Grant 153 (MOPDEV) of the ERANet: ComplexityNET program, by SGR Grant 406 from the Catalan funding agency AGAUR, by grant BFU2009-10184 from the Spanish Ministry of Science, and by European Commission grant FP7-KBBE-2011-5/289434 (BioPreDyn). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Block of death-receptor apoptosis protects mouse cytomegalovirus from macrophages and is a determinant of virulence in immunodeficient hosts.

The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC(-/-)), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system

Cytotoxic polyfunctionality maturation of cytomegalovirus-pp65-specific CD4 + and CD8 + T-cell responses in older adults positively correlates with response size

Cytomegalovirus (CMV) infection is one of the most common persistent viral infections in humans worldwide and is epidemiologically associated with many adverse health consequences during aging. Previous studies yielded conflicting results regarding whether large, CMV-specific T-cell expansions maintain their function during human aging. In the current study, we examined the in vitro CMV-pp65-reactive T-cell response by comprehensively studying five effector functions (i.e., interleukin-2, tumor necrosis factor-α, interferon-γ, perforin, and CD107a expression) in 76 seropositive individuals aged 70 years or older. Two data-driven, polyfunctionality panels (IL-2-associated and cytotoxicity-associated) derived from effector function co-expression patterns were used to analyze the results. We found that, CMV-pp65-reactive CD8 + and CD4 + T cells contained similar polyfunctional subsets, and the level of polyfunctionality was related to the size of antigen-specific response. In both CD8 + and CD4 + cells, polyfunctional cells with high cytotoxic potential accounted for a larger proportion of the total response as the total response size increased. Notably, a higher serum CMV-IgG level was positively associated with a larger T-cell response size and a higher level of cytotoxic polyfunctionality. These findings indicate that CMV-pp65-specific CD4 + and CD8 + T cell undergo simultaneous cytotoxic polyfunctionality maturation during aging

KECAP BARANG DINA RUBRIK “KATUMBIRI” MAJALAH MANGLÉ : Ulikan Struktur jeung Semantik

Ieu panalungtikan dikasangtukangan ku ayana wangun kecap barang jembar nu teu saluyu jeung tiori katatabasaan Sunda, ayana pola anyar kecap barang jembar, sarta ayana kecap barang jémbar nu ngabogaan wangun, fungsi, jeung harti gramatikal anu béda. Dumasar kana kasang tukang masalah, ieu panalungtikan miboga tujuan pikeun ngadéskripsikeun sarta mikanyaho wangun, fungsi gramatikal, jeung harti gramatikal kecap barang anu aya dina Rubrik “Katumbiri” Majalah Manglé. Dina ieu panalungtikan digunakeun pamarekan kualitatif kalayan métode déskriptif. Ari sumber data anu digunakeun nyaéta Rubrik “Katumbiri” Majalah Manglé bulan Séptémber-Oktober No. 2698 - No. 2702. Hasil kecap barang dina ieu panalungtikan kapanggih aya 323 kecap barang jembar. Disawang tina wangunna, éta kecap barang jembar nyoko kana kecap barang rundayan (139 kecap), kecap barang rajékan (25 kecap), kecap barang kantétan (36 kecap), jeung kecap barang wancahan (123 kecap). Disawang tina fungsi grammatikalna, fungsi gramatikal infléktif (209 kecap) jeung fungsi gramatikal dérivatif (114 kecap). Sarta disawang tina harti gramatikalna kapanggih aya 22 rupa harti, nyaéta harti ‘nuduhkeun (anafora)’ (67 kecap), anu harti ‘nu dibéré D’, harti ‘nu ngalakukeun D’, harti ‘alat paranti ngaD’, harti ‘nu miboga sipat D’, harti ‘nu dianggap D’, harti ‘nu aya dina D’, harti ‘nu patali jeung D’ (29 kecap), harti ‘anu di-D’ (opat kecap), harti ‘nu dipi-D’ (dua kecap), harti ‘nu jadi D’ (genep kecap), harti ‘nu di-D-keun’ (opat kecap), harti ‘hasil D’ (dua kecap), harti ‘tempat’ (tujuh kecap), harti ‘palaku D’ (dua kecap), harti ‘jalma nu D’ (dua kecap), harti ‘jama’ (27 kecap), harti ‘nu nunjukeun D loba’ (dua kecap), harti ‘jinis’ (4 kecap), harti ‘sabada’ (dua kecap), harti ‘ahli/tukang’ (dua kecap), jeung harti ‘jalma nu jadi D’ (dua kecap). Kecap Galeuh: kecap barang jembar, wangun, fungsi gramatikal, harti gramatikal ------- Penelitian ini dilatarbelakangi karena adanya bentuk kata benda turunan yang tidak sesuai dengan kaidah bahasa Sunda, adanya pola kata benda turunan yang baru, dan adanya kata benda turunan yang mempnyai bentuk, fungsi, dan makna gramatikal yang berbeda. Berdasarkan latar belakang masalah, penelitian ini bertujuan untuk mendeskripsikan dan mengetahui bentuk, fungsi gramatikal, serta makna gramatikal dalam Rubrik “Katumbiri” Majalah Manglé. Dalam penelitian ini digunakan pendekatan deskriptif dengan metode kualitatif. Sedangkan sumber data yang digunakan yaitu Rubrik “Katumbiri” Majalah Manglé bulan September-Oktober No. 2698 - No. 2702. Hasil Kata Benda dalam penelitian ini terdapat 323 kata benda turunan. Dilihat dari segi bentuk terdapat kata benda afiksasi (139 kata), kata benda Reduplikasi (25 kata), kata benda komposisi (36 kata), dan kata benda singkatan (123 kata). Dari fungsi kegramatikalan terdapat: fungsi gramatikal infléktif (209 kata) dan fungsi gramatikal dérivatif (114 kata). Sarta dilihat dari segi makna gramatikalnya, terdapat 22 jenis makna, yaitu makna ‘menunjukan (anafora)’ (67 kata), makna ‘yang diberi D’, makna ‘yang melakukan D’, makna ‘alat untuk men-D’, makna ‘yang mempunyai sifat D’, makna ‘yang dianggap D’, makna ‘yang ada dalam D’, makna ‘yang berhubungan dengan D’ (29 kata), makna ‘yang di-D’ (empat kata), makna ‘yang sangat di-D’ (dua kata), makna ‘yang menjadi D’ (enam kata), makna ‘yang di-D-kan’ (empat kata), harti ‘hasil’ (dua kecap), makna ‘tempat’ (tujuh kata), harti ‘pelaku D’ (dua kecap), makna ‘orang yang D’ (dua kecap), makna ‘jamak’ (27 kata), makna ‘yang menunjukan D banyak’ (dua kata), makna ‘jenis’ (empat kata), makna ‘sesudah’ (dua kata), makna ‘ahli’ (dua kata), dan makna ‘orang yang menjadi D’ (dua kata). Kata kunci: kata beda turunan, wangun, fungsi gramatikal, makna gramatikal The backgrounds of this research are the inappropriate of derivarive nouns with Sundanese language structure, the new form of derivative noun patterns, and the derivative nouns that have different gramatical from, function and meaning. Based on the background, this studi aim to describe an to understand the gramatical from, grammatical function, and gramatical meaning in Katumbiri Rubric from Mangle’s magazine. This study used a descriptive approach with qualitative method. Data used in this researce are Katumbiri Rubric between September-October No. 2698 - No. 2702. The results of noun that have been identified are 232 derivate nouns. In refer to grammatical from there are affixed nound (139 words), Reduplication nouns (25 words), composition nouns (36 words), and abbreviated nouns (123 words). In refer to grammatical functions there are in inflrctive grammatical functions (209 words) and derivative grammatical functions (114 words). In refer to grammatical meaning there are 22 types of meaning, which are the meaning of ‘showing (anafhora) (67 words), the meaning of ‘given D’, ‘the meaning of ‘the D-doing’, the meaning of ‘D-instrument’, the meaning of ‘that has D-characteristic’, the meaning of ‘that considered as D’, the meaning of ‘what inside D’, the meaning of ‘related to D’ (29 words), the meaning of ‘D-ing’ (two words), the meaning of ‘thas very D’ (six words), the meaning of ‘result’ (two words), the meaning of ‘setting (seven words)’, the meaning of ‘D character’(two words), the meaning of ‘D person’ (two words), the meaning of ‘plural’ (27 words), the meaning of ‘that shows a lot of D’ (two words), the meaning of ‘kind’ (four words), the meaning of ‘after’ (two words), the meaning of ‘expert’ (two words), and the meaning of ‘person who’ve become D’ (two words). Keywords: derivative nouns, awakening, grammatical function,grammatical meanin

A cytomegalovirus-based vaccine provides long-lasting protection against lethal Ebola virus challenge after a single dose

This is the author accepted manuscript. The final version is available fromElsevier via the DOI in this record.Ebola virus (Zaire ebolavirus; EBOV) is a highly lethal hemorrhagic disease virus that most recently was responsible for two independent 2014 outbreaks in multiple countries in Western Africa, and the Democratic Republic of the Congo, respectively. Herein, we show that a cytomegalovirus (CMV)-based vaccine provides durable protective immunity from Ebola virus following a single vaccine dose. This study has implications for human vaccination against ebolaviruses, as well as for development of a 'disseminating' vaccine to target these viruses in wild African great apes.We thank Dr U. Koszinowski (Max von Pettenkofer-Institute, Ludwig-Maximilians-University, Germany) for providing the pSMfr3 MCMV BAC, and Dr D. Court (NCI-Frederick, MD) for providing the lambda-based recombination system used to construct the original MCMV/ZEBOV-NPCTL construct. We appreciate K. Marshall (VGTI, OR) and J. Bailey (NIAID, MT) for their organization and coordination of animals used in the study. We also thank the members of Rocky Mountain Veterinary Branch (DIR, NIAID, NIH) for assistance with animal care. Finally, we thank Drs H. Ebihara (DIR, NIAID, NIH), A. Marzi (DIR, NIAID, NIH), P. Barry (University of California at Davis, CA), M. Cranfield (Mountain Gorilla Veterinary Project, Baltimore, MD) for insightful discussions. This study was supported by R21 (AI088442) and the Intramural Research Program of the NIAID, NIH; and University of Plymouth, School of Biomedical and Healthcare Sciences internal funding

Accelerated in vivo proliferation of memory phenotype CD4+ T-cells in human HIV-1 infection irrespective of viral chemokine co-receptor tropism.

CD4(+) T-cell loss is the hallmark of HIV-1 infection. CD4 counts fall more rapidly in advanced disease when CCR5-tropic viral strains tend to be replaced by X4-tropic viruses. We hypothesized: (i) that the early dominance of CCR5-tropic viruses results from faster turnover rates of CCR5(+) cells, and (ii) that X4-tropic strains exert greater pathogenicity by preferentially increasing turnover rates within the CXCR4(+) compartment. To test these hypotheses we measured in vivo turnover rates of CD4(+) T-cell subpopulations sorted by chemokine receptor expression, using in vivo deuterium-glucose labeling. Deuterium enrichment was modeled to derive in vivo proliferation (p) and disappearance (d*) rates which were related to viral tropism data. 13 healthy controls and 13 treatment-naive HIV-1-infected subjects (CD4 143-569 cells/ul) participated. CCR5-expression defined a CD4(+) subpopulation of predominantly CD45R0(+) memory cells with accelerated in vivo proliferation (p = 2.50 vs 1.60%/d, CCR5(+) vs CCR5(-); healthy controls; P<0.01). Conversely, CXCR4 expression defined CD4(+) T-cells (predominantly CD45RA(+) naive cells) with low turnover rates. The dominant effect of HIV infection was accelerated turnover of CCR5(+)CD45R0(+)CD4(+) memory T-cells (p = 5.16 vs 2.50%/d, HIV vs controls; P<0.05), naïve cells being relatively unaffected. Similar patterns were observed whether the dominant circulating HIV-1 strain was R5-tropic (n = 9) or X4-tropic (n = 4). Although numbers were small, X4-tropic viruses did not appear to specifically drive turnover of CXCR4-expressing cells (p = 0.54 vs 0.72 vs 0.44%/d in control, R5-tropic, and X4-tropic groups respectively). Our data are most consistent with models in which CD4(+) T-cell loss is primarily driven by non-specific immune activation

The Role of Arabic Language Teachers in Developing Mahārat Al-Kalām in Class VII Students of MTs. Ira Tembung

The purpose of this study was to analyze the role of Arabic Language teacher and the language skills of MTs Ira Tembung students. This research method uses a qualitative descriptive method with primary data sources in the form of interviews with MTs Ira Tembung's Arabic teacher. The results of this study indicate that the role of the class VII Arabic teacher at MTs Ira Tembung as an educator is able to become a figure, role model, and identification for students and their environment. When the teacher teaches patiently and diligently, his efforts will be seen by the students. They will make their teacher as a role model in the future. The Teacher As Trainer, the education and learning process requires skill training, both intellectual and ability, so that it requires the teacher&nbsp; to act as a trainer. Therefore, the class VII Arabic teacher at MTs Ira Tembung provides training to train students who are not yet able and involve students in many activities and competitions. The development of students' Arabic language skills in Class VII Students of MTs Ira Tembung uses the method of repeating words or sentences (Taqrîr). This initial method is very suitable for low grade students, namely class VII because they are an early stage class who do not understand what Arabic is, so they only need to hear the teacher pronounce new vocabulary and then be followed by class VII students of MTs Ira Tembung and not only the ability to hear the ability to speak (Mahārat Al-Kalām) is the most important ability in language learning because the ability to speak is a basic skill in learning a foreign language. The ability to speak is part of an active and productive language skill. In seeking to develop Arabic speaking skills&nbsp; by practicing pronouncing and distinguishing certain sounds. To practice pronouncing and distinguishing certain sounds students will learn directly through conversation (hiwār). Practice reciting daily conversations using Arabic during lessons in cla

Pembudidayaan Tanaman Apotek Hidup dengan Memanfaatkan Lahan Kosong di Kantor Desa Blang Krueng, Kecamatan Baitussalam, Kabupaten Aceh Besar

A living pharmacy is the use of part of a plot of land to be planted with medicinal plants and daily necessities. The cultivation of living pharmacy plants using empty land in the yard is a positive activity. There are various types of living pharmacy plants that can be cultivated and have various benefits such as being an alternative to herbal medicine, moving land that looks arid, and becoming a spice in everyday cooking. The types of living pharmacy plants include turmeric, ginger, galangal, kencur, ginger, and lemongrass. Based on the usefulness of a living pharmacy for the community, the planting of living pharmacy plants is carried out on the empty land in the Blang Krueng village office yard as an effort to provide an overview of the easy planting process and increase people's insight about the benefits of a living pharmacy. &nbsp; Keywords: Living Pharmacy, Plants, Vacant Land. &nbsp