Dual Stimuli-Responsive Polysaccharide Hydrogels Manufactured by Radiation Technique

Featured Application Stimuli-responsive hydrogels encompassing biobased polymers are anticipated for utilization in various fields, starting from drug delivery systems through temporal actuators and water reservoirs to biodegradable microelements and nutrient delivery depots. This paper describes the results of the radiation-induced crosslinking of polysaccharides modified with hydroxypropyl and carboxymethyl functional groups, hydroxypropylcellulose (HPC) and carboxymethylcellulose (CMC), respectively, without and with poly(ethylene glycol) diacrylate (PEGDA) as a crosslinking agent, to obtain dual stimuli-responsive hydrogels. The gels were characterized in terms of water uptake and gel fraction, parameters that mainly depend on the HPC-CMC compositions, but also on the macromer crosslinker content and the absorbed dose. The swelling of hydrogels is controlled by both the temperature, due to the amphiphilic character of HPC and pH, due to the anionic functional groups of CMC. In spite of a similar degree of substitution in both cellulose derivatives, 1.4 for HPC and 1.2 for CMC, the pH response of hydrogels with an equal content of both polysaccharides is considerably higher-a reduction in swelling of up to 95% with a decrease in the pH to 2 was recorded-than the response to thermal-stimulus-wherein a reduction in swelling of less than 70% with an increasing in temperature to 55 degrees C was found. These biopolymers-based hydrogels of specific, stimuli-responsive swelling properties are anticipated in applications where a combination of two stimuli is essential and biodegradation may be required

Seamless Gene Tagging by Endonuclease-Driven Homologous Recombination

Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions

Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient

Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa

<p>Abstract</p> <p>Background</p> <p>The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment that was obtained with a 1- or 3-step PCR protocol and that carries an antibiotic cassette flanked by a region homologous to the target locus into a strain that expresses the lambda Red recombination system.</p> <p>Results</p> <p>This system has been modified for use in <it>Pseudomonas aeruginosa</it>. Chromosomal DNA deletions of single genes were generated using 3-step PCR products containing flanking regions 400–600 nucleotides (nt) in length that are homologous to the target sequence. A 1-step PCR product with a homologous extension flanking region of only 100 nt was in some cases sufficient to obtain the desired mutant. We further showed that the <it>P. aeruginosa </it>strain PA14 non-redundant transposon library can be used in conjunction with the lambda Red technique to rapidly generate large chromosomal deletions or transfer mutated genes into various PA14 isogenic mutants to create multi-locus knockout mutants.</p> <p>Conclusion</p> <p>The lambda Red-based technique can be used efficiently to generate mutants in <it>P. aeruginosa</it>. The main advantage of this procedure is its rapidity as mutants can be easily obtained in less than a week if the 3-step PCR procedure is used, or in less than three days if the mutation needs to be transferred from one strain to another.</p

MiR-205-driven downregulation of cholesterol biosynthesis through SQLE-inhibition identifies therapeutic vulnerability in aggressive prostate cancer

Prostate cancer (PCa) shows strong dependence on the androgen receptor (AR) pathway. Here, we show that squalene epoxidase (SQLE), an enzyme of the cholesterol biosynthesis pathway, is overexpressed in advanced PCa and its expression correlates with poor survival. SQLE expression is controlled by micro-RNA 205 (miR-205), which is significantly downregulated in advanced PCa. Restoration of miR-205 expression or competitive inhibition of SQLE led to inhibition of de novo cholesterol biosynthesis. Furthermore, SQLE was essential for proliferation of AR-positive PCa cell lines, including abiraterone or enzalutamide resistant derivatives, and blocked transactivation of the AR pathway. Inhibition of SQLE with the FDA approved antifungal drug terbinafine also efficiently blocked orthotopic tumour growth in mice. Finally, terbinafine reduced levels of prostate specific antigen (PSA) in three out of four late-stage PCa patients. These results highlight SQLE as a therapeutic target for the treatment of advanced PCa

An Integrated TCGA Pan-Cancer Clinical Data Resource to Drive High-Quality Survival Outcome Analytics

For a decade, The Cancer Genome Atlas (TCGA) program collected clinicopathologic annotation data along with multi-platform molecular profiles of more than 11,000 human tumors across 33 different cancer types. TCGA clinical data contain key features representing the democratized nature of the data collection process. To ensure proper use of this large clinical dataset associated with genomic features, we developed a standardized dataset named the TCGA Pan-Cancer Clinical Data Resource (TCGA-CDR), which includes four major clinical outcome endpoints. In addition to detailing major challenges and statistical limitations encountered during the effort of integrating the acquired clinical data, we present a summary that includes endpoint usage recommendations for each cancer type. These TCGA-CDR findings appear to be consistent with cancer genomics studies independent of the TCGA effort and provide opportunities for investigating cancer biology using clinical correlates at an unprecedented scale. Analysis of clinicopathologic annotations for over 11,000 cancer patients in the TCGA program leads to the generation of TCGA Clinical Data Resource, which provides recommendations of clinical outcome endpoint usage for 33 cancer types

Entrepreneurs’ achieved success: developing a multi-faceted measure

Firm performance is typically measured via objective financial indicators. However, researchers increasingly acknowledge that entrepreneurs do not measure their success solely in financial terms but that a range of often subjective indicators matter to them. This article contributes to the debate on entrepreneurial performance by studying how entrepreneurs assess their achieved success. ‘Entrepreneurs’ achieved success’ was conceptualized as a multi-faceted construct that includes entrepreneurs’ self-reported achievement of firm performance, workplace relationships, personal fulfilment, community impact, and personal financial rewards. It was measured via the Subjective Entrepreneurial Success–Achievement Scale (SES-AS). Over the course of three studies (N = 390) the factorial structure of ‘entrepreneurs’ achieved success’ was established and largely replicated in two cultures. Based on a nomological network, we documented relationships among ‘entrepreneurs’ achieved success’, quasi-objective indicators of firm performance, and entrepreneurs’ financial satisfaction, creativity, and health. Based on our research, we propose a new conceptual framework to study performance in the context of entrepreneurship. This framework acknowledges both the success criteria that entrepreneurs wish to achieve and those that they actually achieve, and extends our understanding of firm performance

Sec12 Binds to Sec16 at Transitional ER Sites

COPII vesicles bud from an ER domain known as the transitional ER (tER). Assembly of the COPII coat is initiated by the transmembrane guanine nucleotide exchange factor Sec12. In the budding yeast Pichia pastoris, Sec12 is concentrated at tER sites. Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner. We now show that this binding partner is Sec16, a peripheral membrane protein that functions in ER export and tER organization. One line of evidence is that overexpression of Sec12 delocalizes Sec12 to the general ER, but simultaneous overexpression of Sec16 retains overexpressed Sec12 at tER sites. Additionally, when P. pastoris Sec12 is expressed in S. cerevisiae, the exogenous Sec12 localizes to the general ER, but when P. pastoris Sec16 is expressed in the same cells, the exogenous Sec12 is recruited to tER sites. In both of these experimental systems, the ability of Sec16 to recruit Sec12 to tER sites is abolished by deleting a C-terminal fragment of Sec16. Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12. Similarly, we demonstrate that human Sec12 is concentrated at tER sites, likely due to association with a C-terminal fragment of Sec16A. These results suggest that a Sec12–Sec16 interaction has a conserved role in ER export

MicroRNA-375 plays a dual role in prostate carcinogenesis

Background: Prostate cancer (PCa), a highly incident and heterogeneous malignancy, mostly affects men from developed countries. Increased knowledge of the biological mechanisms underlying PCa onset and progression are critical for improved clinical management. MicroRNAs (miRNAs) deregulation is common in human cancers, and understanding how it impacts in PCa is of major importance. MiRNAs are mostly downregulated in cancer, although some are overexpressed, playing a critical role in tumor initiation and progression. We aimed to identify miRNAs overexpressed in PCa and subsequently determine its impact in tumorigenesis. Results: MicroRNA expression profiling in primary PCa and morphological normal prostate (MNPT) tissues identified 17 miRNAs significantly overexpressed in PCa. Expression of three miRNAs, not previously associated with PCa, was subsequently assessed in large independent sets of primary tumors, in which miR-182 and miR-375 were validated, but not miR-32. Significantly higher expression levels of miR-375 were depicted in patients with higher Gleason score and more advanced pathological stage, aswellaswithregionallymph nodesmetastases. Forced expression of miR-375 in PC-3 cells, which display the lowest miR-375 levels among PCa cell lines, increased apoptosis and reduced invasion ability and cell viability. Intriguingly, in 22Rv1 cells, which displayed the highest miR-375 expression, knockdown experiments also attenuated the malignant phenotype. Gene ontology analysis implicated miR-375 in several key pathways deregulated in PCa, including cell cycle and cell differentiation. Moreover, CCND2 was identified as putative miR-375 target in PCa, confirmed by luciferase assay. Conclusions: A dual role for miR-375 in prostate cancer progression is suggested, highlighting the importance of cellular context on microRNA targeting.Research Center of Portuguese Oncology Institute - Porto (CI-IPOP 4–2012) and by the Federal funds through Programa Operacional Temático Factores de Competitividade (COMPETE) with co-participation from the European Community Fund (FEDER) and by the National funds through Fundação para a Ciência e Tecnología (FCT) under the projects EXPL/BIM-ONC/0556/2012. FQV and JRC were or are supported by FCT-Fundação para a Ciência e a Tecnologia grants (SFRH/BD/70564/2010 and SFRH/BD/71293/2010, respectively)

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts