Reprogramming epigenetic silencing: artificial transcription factors synergize with chromatin remodeling drugs to reactivate the tumor suppressor mammary serine protease inhibitor

Mammary serine protease inhibitor (maspin) is an important tumor suppressor gene whose expression is associated not only with tumor growth inhibition but also with decreased angiogenesis and metastasis. Maspin expression is down-regulated in metastatic tumors by epigenetic mechanisms, including aberrant promoter hypermethylation. We have constructed artificial transcription factors (ATFs) as novel therapeutic effectors able to bind 18-bp sites in the maspin promoter and reactivate maspin expression in cell lines that harbor an epigenetically silenced promoter. In this article, we have investigated the influence of epigenetic modifications on ATF-mediated regulation of maspin by challenging MDA-MB-231 breast cancer cells, comprising a methylated maspin promoter, with different doses of ATFs and chromatin remodeling drugs: the methyltransferase inhibitor 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor suberoylanilide hydroxamic acid. We found that the ATFs synergized with both inhibitors in reactivating endogenous maspin expression. The strongest synergy was observed with the triple treatment ATF-126 + 5-aza-2′-deoxycytidine + suberoylanilide hydroxamic acid, in which the tumor suppressor was reactivated by 600-fold. Furthermore, this combination inhibited tumor cell proliferation by 95%. Our data suggest that ATFs enhance the efficiency of chromatin remodeling drugs in reactivating silenced tumor suppressors. Our results document the power of a novel therapeutic approach that combines both epigenetic and genetic (sequence-specific ATFs) strategies to reactivate specifically silenced regions of the genome and reprogram cellular phenotypes

Growth of Synechococcus sp. immobilized in chitosan with different times of contact with NaOH

The thickness of the walls of the capsules of chitosan-immobilized Synechococcus cultures was dependent on the time of contact with NaOH and was directly related to culture growth. After an initial lag phase, probably caused by cell damage, the capsules obtained after 80 s in a 0.1 N NaOH solution showed better growth than that of free cell cultures (6.9 and 5.2 divisions in 10 days, respectively)

Single-cell chromosomal imbalances detection by array CGH

Genomic imbalances are a major cause of constitutional and acquired disorders. Therefore, aneuploidy screening has become the cornerstone of preimplantation, prenatal and postnatal genetic diagnosis, as well as a routine aspect of the diagnostic workup of many acquired disorders. Recently, array comparative genomic hybridization (array CGH) has been introduced as a rapid and high-resolution method for the detection of both benign and disease-causing genomic copy-number variations. Until now, array CGH has been performed using a significant quantity of DNA derived from a pool of cells. Here, we present an array CGH method that accurately detects chromosomal imbalances from a single lymphoblast, fibroblast and blastomere within a single day. Trisomy 13, 18, 21 and monosomy X, as well as normal ploidy levels of all other chromosomes, were accurately determined from single fibroblasts. Moreover, we showed that a segmental deletion as small as 34 Mb could be detected. Finally, we demonstrated the possibility to detect aneuploidies in single blastomeres derived from preimplantation embryos. This technique offers new possibilities for genetic analysis of single cells in general and opens the route towards aneuploidy screening and detection of unbalanced translocations in preimplantation embryos in particular

Connector Inversion Probe Technology: A Powerful One-Primer Multiplex DNA Amplification System for Numerous Scientific Applications

We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i.e. “multiplex multiplexing padlocks” (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs

Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field

‘Do you wanna come with me?’: The role of the star image as brand for the commodification of cult in mainstream telefantasy

A key strategy of TV companies in the UK and US since the late 1990s, has been to create distinctive channel brands with flagship TV shows as brands that act to convey the channel’s brand values to audiences. Mainstream quality cult shows, including Doctor Who for the BBC and Game of Thrones for HBO, have conveyed something of the brand of their respective channels in the highly competitive multi-media global TV market. In this essay, I analyse star images as brands (aspects of star image used for economic and promotional purposes), to consider their role as part of the launch and promotion of global cult telefantasy brands through promotional paratexts and their subsequent integration into the TV series narrative. Through the analysis, I argue that key aspects of star brands – particularly authenticity and intertextuality – are a central part of processes of mainstreaming, ‘cult-ification’ and generic balance, necessary to make mainstream cult telefantasy in the contemporary TV landscape. I contend this highlights the increasing importance of cult as a currency in global TV branding and of star brands in shoring up its extensive commodification. In doing so, I add to the understanding of star branding as part of the commodification of cult for mainstream telefantasy, a previously under-studied/theorised aspect of star and television studies

Choice of the initial antiretroviral treatment for HIV-positive individuals in the era of integrase inhibitors

BACKGROUND: We aimed to describe the most frequently prescribed initial antiretroviral therapy (ART) regimens in recent years in HIV-positive persons in the Cohort of the Spanish HIV/AIDS Research Network (CoRIS) and to investigate factors associated with the choice of each regimen. METHODS: We analyzed initial ART regimens prescribed in adults participating in CoRIS from 2014 to 2017. Only regimens prescribed in >5% of patients were considered. We used multivariable multinomial regression to estimate Relative Risk Ratios (RRRs) for the association between sociodemographic and clinical characteristics and the choice of the initial regimen. RESULTS: Among 2874 participants, abacavir(ABC)/lamivudine(3TC)/dolutegavir(DTG) was the most frequently prescribed regimen (32.1%), followed by tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC)/elvitegravir(EVG)/cobicistat(COBI) (14.9%), TDF/FTC/rilpivirine (RPV) (14.0%), tenofovir alafenamide (TAF)/FTC/EVG/COBI (13.7%), TDF/FTC+DTG (10.0%), TDF/FTC+darunavir/ritonavir or darunavir/cobicistat (bDRV) (9.8%) and TDF/FTC+raltegravir (RAL) (5.6%). Compared with ABC/3TC/DTG, starting TDF/FTC/RPV was less likely in patients with CD4100.000 copies/mL. TDF/FTC+DTG was more frequent in those with CD4100.000 copies/mL. TDF/FTC+RAL and TDF/FTC+bDRV were also more frequent among patients with CD4<200 cells//muL and with transmission categories other than men who have sex with men. Compared with ABC/3TC/DTG, the prescription of other initial ART regimens decreased from 2014-2015 to 2016-2017 with the exception of TDF/FTC+DTG. Differences in the choice of the initial ART regimen were observed by hospitals' location. CONCLUSIONS: The choice of initial ART regimens is consistent with Spanish guidelines' recommendations, but is also clearly influenced by physician's perception based on patient's clinical and sociodemographic variables and by the prescribing hospital location