Client depletion assay comparison of paclitaxel to Hsp90 inhibitors

Many researchers have claimed they have found a successful inhibitor of Hsp90; however, we suspect they are only successful when the inhibitor is used in large quantities. On top of being one of the easiest ways to show an effective inhibitor, researchers are claiming they have found an inhibitor when only meeting as few as two hallmarks. Due to the researchers' inhibitors only meeting two of the criteria, we are trying to show whether these limited expectations are a valid way in identifying Hsp90 inhibitors. We think the client depletion assay test is not specific to Hsp90 inhibitors but instead is a generalized cell response to cell death. At this point in our research, we know a lot about client depletion assays and Hsp90s; however, we do not know if the claims these researchers are making are valid claims because of the amount of Hsp90 inhibitor they are using.Howard Hughes Medical Institute Science Education ProgramBiochemistry and Molecular Biolog

Studies of intercellular interactions using synthetic Notch receptors

Prawidłowe funkcjonowanie organizmu wielokomórkowego jest zależne od licznych interakcji pomiędzy poszczególnymi komórkami. Szczególnie istotne dla komórek są odziaływania z ich specyficznym mikrośrodowiskiem, tzw. niszą. Obecnie precyzyjne badanie oddziaływań komórkowych, zwłaszcza między populacjami rzadkich komórek a ich niszą w warunkach in vivo jest mocno ograniczone. W niniejszej pracy proponujemy i sprawdzamy czy można w tym celu zastosować syntetyczne receptory Notch. Celem pracy było zbadanie skuteczności oraz specyficzności systemu SynNotch w warunkach in vitro, tak aby w przyszłość można było go zastosować do badań interakcji komórkowych w warunkach in vivo. W celu stworzenia systemu SynNotch wykorzystano plazmidy: UAS-BFP-PGK-mCherry, eGFP-ligand oraz Lag17-SynNotch-Gal4VP64, które wykorzystano do wyprodukowania wektorów lentiwirusowych. Wyprodukowane wektory zostały zagęszczone dzięki wirowaniu przy wysokim współczynniku wirowania g. Otrzymane miana wektorów lentiwirusowych wyniosły rzędu 109 wirusów/ml. Wektory lentiwirusowe zostały następnie użyte do transdukcji komórek HEK 293T, HMEC-1, CT- 26, w celu ekspresji systemu SynNotch w komórkach. Zastosowany system składał się z receptora SynNotch, który rozpoznaje białko GFP i indukuje ekspresję białka fluorescencyjnego. Komórki, w których ekspresji miał ulegać system SynNotch transdukowano początkowo wektorem UAS- BFP-PGK-mCherry, zawierającym kasetę reporterową. Następnie transdukowane komórki odsortowano, a otrzymywaną stabilnie zmodyfikowaną linię komórkową transdukowano kolejno wektorem z receptorem SynNotch, Lag17-SynNotch Gal4VP64. Komórki, w których ekspresji miał ulegać ligand dla syntetycznego receptora SynNotch, transdukowano wektorem eGFP ligand. Finalnie, otrzymane linie komórkowe odsortowano, tak aby zawierały jedynie stabilnie stransdukowane komórki. Następnie zmodyfikowane linie komórkowe zostały wykorzystane do przeprowadzenia właściwych eksperymentów. W eksperymentach hodowano komórki w kokulturze, z różnym stosunkiem komórek z ligandem do komórek z receptorem SynNotch. Do oceny działania systemu SynNotch wykorzystano techniki: mikroskopii fluorescencyjnej, mikroskopii konfokalnej oraz cytometrii przepływowej.Podsumowując przeprowadzone doświadczenia potwierdzają możliwość zastosowania syntetycznych receptorów Notch do identyfikacji i izolacji oddziałujących ze sobą komórek.The proper functioning of the multicellular organism depends on interactions occurring between cells. Especially important are interactions between cells and their specific microenvironment, referred to as niche. Currently, studing the interactions between individual cells, especially between rare population of cells and their niches in vivo is technically limtited. Here we propose and test the synthetic Notch receptors as a tool for this purpose. The aim of this study was to investigate the efficacy and specificity of the SynNotch platform in vitro. SynNotch platform could be used in the future to study cellular interaction in vivo.To create the SynNotch platform we used plasmids: UAS-BFP-PGK-mCherry, eGFP ligand and Lag17 SynNotch Gal4VP64. These plasmids were used to produce lentiviral vectors. The production of vectors include step of their concentration by ultra-centrifugation. The titer of the obtained lentiviral vectors was of the order 109 viruses/ml. The constructed lentiviral vectors were then used to transduce HEK 293T, HMEC-1 and CT-26 cells to express the SynNotch platform in the cells. The system we used consists of the receptor the recognized extracellular GFP and induces the expression of fluorescent marker. The cells expressing the SynNotch platform were transduced initially with the UAS-BFP-PGK-mCherry vector (reporter cassete). Then the cells were sorted and the modified cell line was subsequently transduced with the Lag17-SynNotch-Gal4VP64 vector with receptor. The cells with ligand were transduced with the eGFP ligand vector. At the end, the modified cell lines were sorted to contained only stably transduced cells.Then the modified cell lines were used to the appropriate experiments. In the experiments, we co-culutred cells with GFP-ligand and cells with the SynNotch receptor at different ratios The following techniques were used to measure the activity of the SynNotch platform: fluorescence microscopy, confocal microscopy and flow cytometry.In summary, the experiments based on synthetic Notch receptors give positive results on the activity and specificity of SynNotch receptors

The effect of TGF-β on migration and metastasis markers of murine breast cancer E0771 in vitro

TGF-β jest cytokiną wpływającą na migrację, proliferację, różnicowanie i apoptozę wielu typów komórek. Bierze on również udział w indukcji przejścia epitelialno – mezenchymalnego, przez co staje się potencjalnym czynnikiem wpływającym na progresję nowotworu. TGF-β prowadzi do wzrostu ekspresji N-kadheryn, wimentyny oraz czynników transkrypcyjnych EMT takich jak Snail.W celu zbadania wpływu TGF-β na komórki linii E0771 traktowano je TGF-β w stężeniu 1ng/ml przez cztery tygodnie. Wykonywano testy proliferacji, aktywności migracyjnej komórek oraz barwienia markerów przerzutowania: Snail-1, wimentyna, N-kadheryna oraz Cx43.Analiza proliferacji nie wykazała istotnych różnic pomiędzy komórkami kontrolnymi a traktowanymi TGF-β. Zauważono jednak promujący wpływ badanej cytokiny na ruchliwość komórek. Po traktowaniu komórki poruszały się szybciej i przebywały dłuższą drogę w porównaniu do komórek hodowanych w standardowych warunkach. Również ekspresja markerów przerzutowania takich jak Snail-1, wimentyna czy N-kadheryna uległa wzrostowi po potraktowaniu komórek TGF-β. Nie wykazano jednak istotnych różnic w ilości koneksyny 43. Na podstawie przeprowadzonych doświadczeń można wnioskować, że TGF-β pełni kluczową rolę w indukcji przejścia epitelialno – mezenchymalnego w komórkach E0771 i może przyczyniać się do nabywania przez komórki nowotworowe fenotypu inwazyjnego.TGF-β is a cytokine that affects migration, proliferation, differentiation and apoptosis in many cell types. TGF-β plays also an important role in the induction of the epitelial - mesenchymal transition (EMT), and therefore can affect the progression of a cancer. TGF-β causes an enhancement in expression of N-cadherin, wimentin and EMT transcription factors, such as Snail.To investigate effect of TGF-β on E0771 cells, cells were treated with TGF-β at a concentration of 1 ng/ml for 4 weeks. After this period proliferation and migration tests were done. Additionally, staining for metastasis markers, including Snail-1, vimentin, N-cadherin and Cx43, was performed.Proliferation test have not shown any significant differences between control and treated cells. However, TGF-β enhanced cell motility. Stimulated cells migrated faster and further compared to cells cultured in standard conditions. Also level of the metastasis markers such as Snail-1, vimentin, or N-cadherin increased after treatment with TGF-β. Any significant changes in expression of connexin 43 was not observed.This study has shown that TGF-β plays a key role in the induction of epitellial-mesenchymal transition in E0771 cells and may contribute to enhancement of invasiveness of cancer cells

Oncolytic Virus Therapy Alters the Secretome of Targeted Glioblastoma Cells

Oncolytic virus (OV) therapy, which is being tested in clinical trials for glioblastoma, targets cancer cells, while triggering immune cells. Yet OV sensitivity varies from patient to patient. As OV therapy is regarded as an anti-tumor vaccine, by making OV-infected cancer cells secrete immunogenic proteins, linking these proteins to transcriptome would provide a measuring tool to predict their sensitivity. A set of six patient-derived glioblastoma cells treated ex-vivo with herpes simplex virus type 1 (HSV1) modeled a clinical setting of OV infection. The cellular transcriptome and secreted proteome (separated into extracellular vesicles (EV) and EV-depleted fractions) were analyzed by gene microarray and mass-spectroscopy, respectively. Data validation and in silico analysis measured and correlated the secretome content with the response to infection and patient survival. Glioblastoma cells reacted to the OV infection in a seemingly dissimilar fashion, but their transcriptomes changed in the same direction. Therefore, the upregulation of transcripts encoding for secreted proteins implies a common thread in the response of cancer cells to infection. Indeed, the OV-driven secretome is linked to the immune response. While these proteins have distinct membership in either EV or EV-depleted fractions, it is their co-secretion that augments the immune response and associates with favorable patient outcomes

A large-scale whole genome sequence-based analysis discovered novel genetic variants influencing bone mineral density: Results from the GEFOS and UK10K Consortia

International audienc

Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture

The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is mainly unknown. Bone mineral density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 × 10(-14)), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 × 10(-11); ncases = 98,742 and ncontrols = 409,511). Using an En1(cre/flox) mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 × 10(-11)). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.</p