Intracellular characterization of Gag-GFP VLP production upon PEImediated transient transfection of HEK 293 cells

Transient Gene Expression is a fast, flexible, and cost-effective approach to produce high-quality products that circumvents the time and cost required for the generation of stably transfected cell lines. However, the levels of recombinant protein produced by TGE, tend to be significantly lower than those of stable cell lines. Despite the continued interest in transient gene expression approaches, little is known about the transfection process at intracellular level, particularly for complex products such as VLPs. The kinetics of PEI-mediated transient transfection was studied with the aim of characterizing and understanding the complete process leading to VLP generation, and identifying important events to drive process improvement. For this purpose, DNA/PEI polyplexes were tracked using Cy3 DNA staining and the production of Gag-GFP VLPs was monitored by flow cytometry, confocal microscopy, and fluorometry. Flow cytometry and confocal microscopy assays show that using a standard transfection protocol DNA:PEI polyplexes interact with the cell membrane from time point zero. A linear increase in transfection efficiency is observed until 60 minutes of contact between cells and polyplexes. No change in transfection efficiency (percentage of GFP positive cells) or VLP production levels is obtained when additional contact time is allowed, reaching a maximum transfection efficiency of 60% and VLP production of 10x109 VLPs/mL harvested at 48 hours post transfection (hpt). After 1.5 hpt, polyplexes are detected in the cytoplasm of transfected cells and reach the nucleus around 4 hpt. Of note, all cells show the presence of DNA/PEI complex in the cytoplasm after transfection but only a fraction of cells express the fluorescent Gag protein. By flow cytometry analysis of isolated nuclei, it was determined that polyplexes are only present in 60% of the nuclei at 6 hpt (concomitant with the GFP expressing cells), suggesting that the entrance of polyplexes to the nucleus is one of the limiting steps of the transfection process. After 10 hpt, GFP fluorescence is detected homogenously inside the cells, but generalized budding of VLPs is not observed until 48 hpt. As mentioned before, a unique population of cells Cy3+ (with a polyplex inside) appears from the very beginning of the transfection. A new population of cells that do not contain any polyplex inside (Cy3-) and do not express the protein (GFP-) appears at 24 hpt suggesting plasmid loss after this time point. The VLP production kinetics was also studied, observing that fluorescence in the supernatant is always 40% less than total fluorencense (supernatant plus pellet). Maximum VLP levels in the cell culture supernatant, while keeping cell culture viability still high, are observed at 72 hpt, which was determined to be the optimal harvest time. Three bottlenecks in VLP production could be identified in this work: polyplexes entry into the nucleus, plasmid loss during the production phase and VLP buddin

Physical properties of binary mixtures : ionic liquids + alcohols

Information on the interactions between ionic liquids and molecular solvents are essential for the understanding of the function of ionic liquids in related procedures, and excess properties are sensitive probe for studying these interactions. In this paper, physical properties such as densities and speeds of sound of the binary systems containing 1–propyl–4 methylpyridiniumbis(trifluoromethylsulfonil)imide and 1propyl–4–methylpyridiniumbis(trifluoromethylsulfonil)imide, ethanol and propanol, over the whole composition range were measured at T=(298.15, 308.15, 318.15) K and at atmospheric. These data were used to calculate the corresponding derived properties such as excess molar volumes and excess molar isentropic compressions, which were fitted with the Redlich-Kister equation. For the studied systems, the excess molar volume values show a sinusoidal behaviour with a minimum at high concentrations of alcohol due to changes from negative to positive values when the alcohol´s alkyl chain length increases. The excess molar isentropic compression values are negative over the whole composition range and presenting also a minimum at high concentrations of alcohol. It is possible observed that an increase of the temperature causes that the negative excess molar volume and excess molar isentropic compression values for the alcoholic mixtures deviate more from ideality.Papers presented to the 12th International Conference on Heat Transfer, Fluid Mechanics and Thermodynamics, Costa de Sol, Spain on 11-13 July 2016

Microbial Activity in Subterranean Ecosystems: Recent Advances

Of the several critical challenges present in environmental microbiology today, one is the assessment of the contribution of microorganisms in the carbon cycle in the Earth-climate system. Karstic subterranean ecosystems have been overlooked until recently. Covering up to 25% of the land surface and acting as a rapid CH4 sink and alternately as a CO2 source or sink, karstic subterranean ecosystems play a decisive role in the carbon cycle in terms of their contribution to the global balance of greenhouse gases. Recent data indicate that microbiota must play a significant ecological role in the biogeochemical processes that control the composition of the subterranean atmosphere, as well as in the availability of nutrients for the ecosystem. Nevertheless, there are still essential gaps in our knowledge concerning the budgets of greenhouse gases at the ecosystem scale and the possible feedback mechanisms between environmental-microclimatic conditions and the rates and type of activity of microbial communities in subterranean ecosystems. Another challenge is searching for bioactive compounds (antibiotics) used for treating human diseases. At present, there is a global health emergency and a strong need for novel biomolecules. In recent decades, great research efforts have been made to extract antibiotics from marine organisms. More recently, caves have been receiving considerable attention in search of novel antibiotics. Cave methanotrophic and heterotrophic bacteria are producers of bioactive compounds and may be potential sources of metabolites with antibacterial, antifungal or anticancer activities of interest in pharmacological and medical research, as well as enzymes with a further biotechnological use. Here we also show that bacteria isolated from mines, a still unexplored niche for scientists in search of novel compounds, can be a source of novel secondary metabolites.Financial support was obtained through project 0483_PROBIOMA_5_E, co-financed by the European Regional Development Fund within the framework of the Interreg V-A Spain-Portugal program (POCTEP) 2014–2020. This work was also supported by the Spanish Ministry of Economy and Competitiveness through projects CGL2016-75590-P and PID2019-110603RB-I00, AEI/FEDER, UE

Correction to: Usefulness of Genetic Testing in Hypertrophic Cardiomyopathy: an Analysis Using Real-World Data

post-print123 K

Pest categorisation of Venturia nashicola

The Panelon Plant Health performed a pest categorisation of Venturianashicola, the causal agent of Asian pear scab, for the European Union (EU). The pathogen is a well-defined, distinguishable fungal species affecting Pyruspyrifolia var. culta, P.ussuriensis and P.bretschneideri in Asian countries. P.communis (European pear) is not a host of V.nashicola, but the host status of other Pyrus species is unclear. V.nashicola is not known to occur in the EU. It is listed in Annex IIAI of Directive 2000/29/EC. The pathogen could potentially enter the EU on host plants for planting and fruit originated in infested countries. There are no climatic factors limiting the potential establishment and spread of the pathogen in the EU, as its epidemiology is similar to those of Venturiainaequalis (apple scab) and Venturiapyrina (European pear scab), which are well-established in the EU. The hosts are present in the EU, but no data were found on their abundance and distribution. In the infested areas, V.nashicola causes premature leaf and fruit drop and fruit distortion resulting in considerable yield/quality losses. The introduction of the pathogen into the EU could cause yield/quality losses and environmental consequences because of the additional fungicide sprays for disease control. Cultural practices and chemical measures applied in the infested areas reduce the inoculum sources but they cannot eliminate the pathogen. Phytosanitary measures are available to mitigate the risk of introduction and spread of the pathogen in the EU. All criteria assessed by EFSA for consideration as a potential Union quarantine pest are met. As V.nashicola is not known to occur in the EU, this criterion assessed by EFSA to consider it as a Union regulated non-quarantine pest is not met

Data fusion framework for planetary and orbital robotics applications

In space robotics, a wide range of sensor data fusion methods are required to accomplish challenging objectives for exploration, science and commercial purposes. This includes navigation for planetary and guidance for orbital robotics, scientific prospecting, and on-orbit servicing. In Fuse provides a comprehensive data fusion framework or toolset to fuse and interpret sensor data from multiple sensors. This project represents an optimal approach to develop software for robotics: a standardized and comprehensive development environment for industrial applications, with particular focus on space applications where components can be connected, tested offline, evaluated and deployed in any preferred robotic framework, including those devised for space or terrestrial applications. This paper discusses the results of verification and validation of data fusion methods for robots deployed in orbital and planetary scenarios using data sets collected in simulation and outdoor analogue campaigns

Diagnosis and management in Rubinstein-Taybi syndrome:first international consensus statement

Rubinstein-Taybi syndrome (RTS) is an archetypical genetic syndrome that is characterised by intellectual disability, well-defined facial features, distal limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in either of two genes (CREBBP, EP300) which encode for the proteins CBP and p300, which both have a function in transcription regulation and histone acetylation. As a group of international experts and national support groups dedicated to the syndrome, we realised that marked heterogeneity currently exists in clinical and molecular diagnostic approaches and care practices in various parts of the world. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria for types of RTS (RTS1: CREBBP; RTS2: EP300), molecular investigations, long-term management of various particular physical and behavioural issues and care planning. The recommendations as presented here will need to be evaluated for improvements to allow for continued optimisation of diagnostics and care.</p

Diagnosis and management in Rubinstein-Taybi syndrome:first international consensus statement

Rubinstein-Taybi syndrome (RTS) is an archetypical genetic syndrome that is characterised by intellectual disability, well-defined facial features, distal limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in either of two genes (CREBBP, EP300) which encode for the proteins CBP and p300, which both have a function in transcription regulation and histone acetylation. As a group of international experts and national support groups dedicated to the syndrome, we realised that marked heterogeneity currently exists in clinical and molecular diagnostic approaches and care practices in various parts of the world. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria for types of RTS (RTS1: CREBBP; RTS2: EP300), molecular investigations, long-term management of various particular physical and behavioural issues and care planning. The recommendations as presented here will need to be evaluated for improvements to allow for continued optimisation of diagnostics and care.</p

Understanding the genetic complexity of puberty timing across the allele frequency spectrum.

Pubertal timing varies considerably and is associated with later health outcomes. We performed multi-ancestry genetic analyses on ~800,000 women, identifying 1,080 signals for age at menarche. Collectively, these explained 11% of trait variance in an independent sample. Women at the top and bottom 1% of polygenic risk exhibited ~11 and ~14-fold higher risks of delayed and precocious puberty, respectively. We identified several genes harboring rare loss-of-function variants in ~200,000 women, including variants in ZNF483, which abolished the impact of polygenic risk. Variant-to-gene mapping approaches and mouse gonadotropin-releasing hormone neuron RNA sequencing implicated 665 genes, including an uncharacterized G-protein-coupled receptor, GPR83, which amplified the signaling of MC3R, a key nutritional sensor. Shared signals with menopause timing at genes involved in DNA damage response suggest that the ovarian reserve might signal centrally to trigger puberty. We also highlight body size-dependent and independent mechanisms that potentially link reproductive timing to later life disease