Overexpression of adaptor protein Ruk/CIN85 in mouse breast adenocarcinoma 4T1 cells induces an increased migration rate and invasion potential

Aim. To study the effect of adaptor protein Ruk/CIN85 overexpression on the dynamics of migration and Matrigel invasion as well as transendothelial migration of murine 4T1 breast adenocarcinoma cells. Methods. Dynamics of 4T1 cells migration/invasion was monitored in real time using the xCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument equipped with a CIM-plate 16. Transendothelial migration (TEM) of 4T1 cells was performed through the layer of primary mouse lung endothelial cells seeded on gelatin-coated 24-well transwell inserts (8-μm pores).The two-tailed Student’s t-test for unequal variances was used for statistical analysis. Results. Ruk/CIN85-overexpression in 4T1 cells are indices a significantly increased motility, Matrigel invasiveness and migration through endothelial cells layer. Conclusions. The Ruk/CIN85 adaptor protein may play a potential role in the control of metastasis in vivo.Мета. Дослідити вплив надекспресії адаптерного протеїну Ruk/CIN85 на динаміку міграції й інвазії через Матригель, а також на ефективність трансендотеліальної міграції клітин аденокарциноми молочної залози миші лінії 4Т1. Методи. Динаміку міграції/інвазії клітин 4Т1 аналізували в режимі реального часу за допомогою приладу XCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument, оснащеного імпедансним планшетом CIM-plate 16. Трансендотеліальну міграцію (ТЕМ) клітин 4Т1 здійснювали через шар первинних ендотеліоцитів легені миші, висіяних на мембрану (розмір пор 8 μм) камери Бойдена. Для статистичного аналізу використовували двовибірковий t-тест Ст’юдента для незалежних вибірок з нерівними дисперсіями. Результати. Встановлено, що надекспресія Ruk/CIN85 у клітинах лінії 4Т1 супроводжується значним зростанням рухливості, здатності до інвазії через Матригель та шар ендотеліальних клітин. Висновки. Отримані результати вказують на потенційну роль адаптерного протеїну Ruk/CIN85 у контролі метастазування in vivo.Цель. Исследовать влияние сверхэкспрессии адаптерного протеина Ruk/CIN85 на динамику миграции и инвазии через Матригель, а также на эффективность трансэндотелиальной миграции клеток аденокарциномы молочной железы мыши линии 4Т1. Методы. Динамику миграции/инвазии клеток 4Т1 анализировали в режиме реального времени с помощью прибора XCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument, оснащенного импедансным планшетом CIM-plate 16. Трансэндотелиальную миграцию (ТЭМ) клеток 4Т1 осуществляли через слой первичных эндотелиоцитов легкого мыши, высеянных на мембрану (размер пор 8 μм) камеры Бойдена. Для статистического анализа использовали двухвыборочный t-тест Стьюдента для независимых выборок с неравными дисперсиями. Результаты. Установлено, что сверхэкспрессия адаптерного протеина Ruk/CIN85 в клетках линии 4Т1 сопровождается значительным ростом подвижности, способности к инвазии через Матригель и слой эндотелиальных клеток. Выводы. Полученные результаты указывают на потенциальную роль адаптерного протеина Ruk/CIN85 в контроле метастазирования in vivo

Diverse Temperate Bacteriophage Carriage in Clostridium difficile 027 Strains

The hypervirulent Clostridium difficile ribotype 027 can be classified into subtypes, but it unknown if these differ in terms of severity of C. difficile infection (CDI). Genomic studies of C. difficile 027 strains have established that they are rich in mobile genetic elements including prophages. This study combined physiological studies, electron microscopy analysis and molecular biology to determine the potential role of temperate bacteriophages in disease and diversity of C. difficile 027.We induced prophages from 91 clinical C. difficile 027 isolates and used transmission electron microscopy and pulsed-field gel electrophoresis to characterise the bacteriophages present. We established a correlation between phage morphology and subtype. Morphologically distinct tailed bacteriophages belonging to Myoviridae and Siphoviridae were identified in 63 and three isolates, respectively. Dual phage carriage was observed in four isolates. In addition, there were inducible phage tail-like particles (PT-LPs) in all isolates. The capacity of two antibiotics mitomycin C and norfloxacin to induce prophages was compared and it was shown that they induced specific prophages from C. difficile isolates. A PCR assay targeting the capsid gene of the myoviruses was designed to examine molecular diversity of C. difficile myoviruses. Phylogenetic analysis of the capsid gene sequences from eight ribotypes showed that all sequences found in the ribotype 027 isolates were identical and distinct from other C. difficile ribotypes and other bacteria species.A diverse set of temperate bacteriophages are associated with C. difficile 027. The observed correlation between phage carriage and the subtypes suggests that temperate bacteriophages contribute to the diversity of C. difficile 027 and may play a role in severity of disease associated with this ribotype. The capsid gene can be used as a tool to identify C. difficile myoviruses present within bacterial genomes

Genome size evolution at the speciation level: The cryptic species complex Brachionus plicatilis (Rotifera)

<p>Abstract</p> <p>Background</p> <p>Studies on genome size variation in animals are rarely done at lower taxonomic levels, e.g., slightly above/below the species level. Yet, such variation might provide important clues on the tempo and mode of genome size evolution. In this study we used the flow-cytometry method to study the evolution of genome size in the rotifer <it>Brachionus plicatilis</it>, a cryptic species complex consisting of at least 14 closely related species.</p> <p>Results</p> <p>We found an unexpectedly high variation in this species complex, with genome sizes ranging approximately seven-fold (haploid '1C' genome sizes: 0.056-0.416 pg). Most of this variation (67%) could be ascribed to the major clades of the species complex, i.e. clades that are well separated according to most species definitions. However, we also found substantial variation (32%) at lower taxonomic levels - within and among genealogical species - and, interestingly, among species pairs that are not completely reproductively isolated. In one genealogical species, called <it>B</it>. 'Austria', we found greatly enlarged genome sizes that could roughly be approximated as multiples of the genomes of its closest relatives, which suggests that whole-genome duplications have occurred early during separation of this lineage. Overall, genome size was significantly correlated to egg size and body size, even though the latter became non-significant after controlling for phylogenetic non-independence.</p> <p>Conclusions</p> <p>Our study suggests that substantial genome size variation can build up early during speciation, potentially even among isolated populations. An alternative, but not mutually exclusive interpretation might be that reproductive isolation tends to build up unusually slow in this species complex.</p

A Genome-Wide Survey of Switchgrass Genome Structure and Organization

The perennial grass, switchgrass (Panicum virgatum L.), is a promising bioenergy crop and the target of whole genome sequencing. We constructed two bacterial artificial chromosome (BAC) libraries from the AP13 clone of switchgrass to gain insight into the genome structure and organization, initiate functional and comparative genomic studies, and assist with genome assembly. Together representing 16 haploid genome equivalents of switchgrass, each library comprises 101,376 clones with average insert sizes of 144 (HindIII-generated) and 110 kb (BstYI-generated). A total of 330,297 high quality BAC-end sequences (BES) were generated, accounting for 263.2 Mbp (16.4%) of the switchgrass genome. Analysis of the BES identified 279,099 known repetitive elements, >50,000 SSRs, and 2,528 novel repeat elements, named switchgrass repetitive elements (SREs). Comparative mapping of 47 full-length BAC sequences and 330K BES revealed high levels of synteny with the grass genomes sorghum, rice, maize, and Brachypodium. Our data indicate that the sorghum genome has retained larger microsyntenous regions with switchgrass besides high gene order conservation with rice. The resources generated in this effort will be useful for a broad range of applications

The genome-wide dynamics of purging during selfing in maize

Self-fertilization (also known as selfing) is an important reproductive strategy in plants and a widely applied tool for plant genetics and plant breeding. Selfing can lead to inbreeding depression by uncovering recessive deleterious variants, unless these variants are purged by selection. Here we investigated the dynamics of purging in a set of eleven maize lines that were selfed for six generations. We show that heterozygous, putatively deleterious single nucleotide polymorphisms are preferentially lost from the genome during selfing. Deleterious single nucleotide polymorphisms were lost more rapidly in regions of high recombination, presumably because recombination increases the efficacy of selection by uncoupling linked variants. Overall, heterozygosity decreased more slowly than expected, by an estimated 35% to 40% per generation instead of the expected 50%, perhaps reflecting pervasive associative overdominance. Finally, three lines exhibited marked decreases in genome size due to the purging of transposable elements. Genome loss was more likely to occur for lineages that began with larger genomes with more transposable elements and chromosomal knobs. These three lines purged an average of 398 Mb from their genomes, an amount equivalent to three Arabidopsis thaliana genomes per lineage, in only a few generations

Oncogene advance online publication

The p53 gene is often mutated during cancer development. Frequency and functional consequences of these mutations vary in different tumor types. We analysed conformation and temperature dependency of 23 partially inactivating temperature-dependent (td) p53 mutants derived from various human tumors in yeast. We found considerable differences in transactivation capabilities and discriminative character of various p53 mutants. No correlations in transactivation rates and conformations of the td p53 proteins were detected. Amifostine-induced p53 reactivation occurred only in 13 of 23 td mutants, and this effect was temperature dependent and responsive element specific. The most of the p53 mutations (10/13) reactivated by amifostine were located in the part of the p53 gene coding for hydrophobic b-sandwich structure of the DNA-binding domain