High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations

Effects of endosperm vitreousness and kernel processing of corn silage on fiber and starch metabolism

Three corn hybrids with different endosperm types (flinty, intermediate, floury) were chopped (nonprocessed: 0.95 cm or processed: 1.91 cm chop at a 1-mm roller clearance) for silage. Kernel processing eliminated whole kernels. Processing reduced the medium particle fraction (0.95 to 1.91 cm) resulting in an increased small particle fraction (\u3c0.95 cm). Floury compared with flinty silage resulted in a greater small particle fraction. Processing numerically increased starch content of small particles, and NDF and ADF content of medium particles. Flinty compared with floury silage increased in vitro and in situ NDF digestibility of small particles. Ruminal digestion kinetics of processed versus nonprocessed, and flinty versus floury silage were compared using a standard and a new macro in situ procedure. The standard procedure showed a reduction in NDF digestibility for floury compared with flinty silage. The macro procedure resulted in an increased DM digestibility for processed compared with nonprocessed silage, and an increased DM digestibility and decreased NDF digestibility for floury compared with flinty silage. Eight cannulated, multiparous (BW = 649 kg) and four primiparous (BW = 638 kg) Holstein cows were used to evaluate effects of kernel processing and endosperm type of corn silage on digestibility and milk production. A replicated 4 x 4 Latin square with a 2 x 2 factorial arrangement of treatments was used. Processing improved daily milk production (9.9%), 4% FCM (8.3%), efficiency of 4% FCM produced (12%), and starch digestibility (8.9%), while reducing (14.7%) NDF digestibility. Floury endosperm corn silage improved efficiency (5.1%) of 4% FCM production, and DM (7.5%), OM (8.3) and starch (6.4%) digestibilities. Milk production and feed efficiency can be improved by kernel processing corn silage. Furthermore, selecting floury verses flinty endosperm hybrids can increase milk production efficiency. Changes in physicochemical characteristics of particles result in improved ruminal and total tract digestibilities with processed and floury endosperm silages. Therefore, when formulating lactating dairy cows diets, endosperm type and processing method need to be considered

Application of Healthcare ‘Big Data’ in CNS Drug Research:The Example of the Neurological and mental health Global Epidemiology Network (NeuroGEN)

Neurological and psychiatric (mental health) disorders have a large impact on health burden globally. Cognitive disorders (including dementia) and stroke are leading causes of disability. Mental health disorders, including depression, contribute up to one-third of total years lived with disability. The Neurological and mental health Global Epidemiology Network (NeuroGEN) is an international multi-database network that harnesses administrative and electronic medical records from Australia, Asia, Europe and North America. Using these databases NeuroGEN will investigate medication use and health outcomes in neurological and mental health disorders. A key objective of NeuroGEN is to facilitate high-quality observational studies to address evidence-practice gaps where randomized controlled trials do not provide sufficient information on medication benefits and risks that is specific to vulnerable population groups. International multi-database research facilitates comparisons across geographical areas and jurisdictions, increases statistical power to investigate small subpopulations or rare outcomes, permits early post-approval assessment of safety and effectiveness, and increases generalisability of results. Through bringing together international researchers in pharmacoepidemiology, NeuroGEN has the potential to be paradigm-changing for observational research to inform evidence-based prescribing. The first focus of NeuroGEN will be to address evidence-gaps in the treatment of chronic comorbidities in people with dementia

Phosphorus Cycling in a Freshwater Estuary Impacted by Cyanobacterial Blooms

The availability of reactive phosphorus (P) may promote cyanobacterial blooms, a worldwide increasing phenomenon. Cyanobacteria may also regulate benthic P cycling through labile organic input to sediments, favouring reduced conditions and P release, ultimately acting as self-sustainment mechanism for the phytoplankton blooms. To analyse P–cyanobacteria feedbacks and compare external versus internal loads, we investigated P cycling in the Curonian Lagoon, a freshwater estuary with recurrent summer blooms. At two sites representing the dominant sediment types, we characterised P pools and mobility, via combined pore water analysis, calculation of diffusive exchanges and flux measurements via sediment core incubations. Annual P budgets were also calculated, to analyse the whole lagoon role as net sink or source. Muddy sediments, representing nearly 50 % of the lagoon surface, displayed higher P content if compared with sandy sediments, and most of this pool was reactive. The muddy site had consequently higher pore water dissolved inorganic phosphorus (DIP) concentrations maintaining high diffusive gradients. However, measured fluxes suggested that both sediment types were mostly P sinks except for a large DIP regeneration (nearly 30 μmol m−2 h−1) recorded at the muddy site during an intense cyanobacteria bloom. Such internal regeneration had the same order of magnitude as the annual external P load and may offset the net annual DIP sink role of the estuary. It may also prolong the duration of the bloom. Our results suggest that positive feedbacks can regulate N-fixing cyanobacteria blooms and internal P recycling, through either diffusive fluxes or sediment settling and resuspension