The HSV-1 Latency-Associated Transcript Functions to Repress Latent Phase Lytic Gene Expression and Suppress Virus Reactivation from Latently Infected Neurons

open access articleHerpes simplex virus 1 (HSV-1) establishes life-long latent infection within sensory neurons, during which viral lytic gene expression is silenced. The only highly expressed viral gene product during latent infection is the latency-associated transcript (LAT), a non-protein coding RNA that has been strongly implicated in the epigenetic regulation of HSV-1 gene expression. We have investigated LAT-mediated control of latent gene expression using chromatin immunoprecipitation analyses and LAT-negative viruses engineered to express firefly luciferase or β-galactosidase from a heterologous lytic promoter. Whilst we were unable to determine a significant effect of LAT expression upon heterochromatin enrichment on latent HSV-1 genomes, we show that reporter gene expression from latent HSV-1 genomes occurs at a greater frequency in the absence of LAT. Furthermore, using luciferase reporter viruses we have observed that HSV-1 gene expression decreases during long-term latent infection, with a most marked effect during LAT-negative virus infection. Finally, using a fluorescent mouse model of infection to isolate and culture single latently infected neurons, we also show that reactivation occurs at a greater frequency from cultures harbouring LAT-negative HSV-1. Together, our data suggest that the HSV-1 LAT RNA represses HSV-1 gene expression in small populations of neurons within the mouse TG, a phenomenon that directly impacts upon the frequency of reactivation and the maintenance of the transcriptionally active latent reservoir

Conversion of CO2 into organic acids by engineered autotrophic yeast

The increase of CO2 emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO2 as a feedstock gains an extensive importance to establish CO2-neutral or CO2-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO2 as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. 13C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO2 as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L−1, while balancing the expression of key genes and modifying screening conditions led to 2 g L−1 itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO2 uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO2, to support sustainability of industrial bioprocesses

The Impact of Parameter Identification Methods on Drug Therapy Control in an Intensive Care Unit

This paper investigates the impact of fast parameter identification methods, which do not require any forward simulations, on model-based glucose control, using retrospective data in the Christchurch Hospital Intensive Care Unit. The integral-based identification method has been previously clinically validated and extensively applied in a number of biomedical applications; and is a crucial element in the presented model-based therapeutics approach. Common non-linear regression and gradient descent approaches are too computationally intense and not suitable for the glucose control applications presented. The main focus in this paper is on better characterizing and understanding the importance of the integral in the formulation and the effect it has on model-based drug therapy control. As a comparison, a potentially more natural derivative formulation which has the same computation speed advantages is investigated, and is shown to go unstable with respect to modelling error which is always present clinically. The integral method remains robust

Design Principles for Fragment Libraries: Maximizing the Value of Learnings from Pharma Fragment-Based Drug Discovery (FBDD) Programs for Use in Academia

Fragment-based drug discovery (FBDD) is well suited for discovering both drug leads and chemical probes of protein function; it can cover broad swaths of chemical space and allows the use of creative chemistry. FBDD is widely implemented for lead discovery in industry but is sometimes. used less systematically in academia. Design principles and implementation approaches for fragment libraries are continually evolving, and the lack of up-to-date guidance may prevent more effective application of FBDD in academia. This Perspective explores many of the theoretical, practical, and strategic considerations that occur within FBDD programs, including the optimal size, complexity, physicochemical profile, and shape profile of fragments in FBDD libraries, as well as compound storage, evaluation; and screening technologies. This:compilation of industry experience in FBDD will hopefully be useful for those pursuing FBDD in academia

Modulators of 14-3-3 Protein-Protein Interactions

Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein-protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as 'undruggable' targets, the last two decades have seen an increasing number of successful examples of PPI modulators resulting in a growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This Perspective focuses on the hub protein 14-3-3, which has several hundred identified protein interaction partners and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide-mimetics and natural products

Structural basis of DNA gyrase inhibition by antibacterial QPT-1, anticancer drug etoposide and moxifloxacin

New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide’s antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a ‘pair of swing-doors’ hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1’s bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested

PhotoAffinity bits : a photoaffinity-based fragment screening platform for efficient identification of protein ligands

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space, however, it remains challenging to develop techniques that are both sufficiently high-throughput and sensitive. We present a fragment screening platform, termed PhABits (PhotoAffinity Bits), which utilises a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. We envision that the PhABits will be widely applicable to novel protein targets, identifying starting points in the development of therapeutic

Round Robin Testing: Exploring Experimental Uncertainties through a Multifacility Comparison of a Hinged Raft Wave Energy Converter

The EU H2020 MaRINET2 project has a goal to improve the quality, robustness and accuracy of physical modelling and associated testing practices for the offshore renewable energy sector. To support this aim, a round robin scale physical modelling test programme was conducted to deploy a common wave energy converter at four wave basins operated by MaRINET2 partners. Test campaigns were conducted at each facility to a common specification and test matrix, providing the unique opportunity for intercomparison between facilities and working practices. A nonproprietary hinged raft, with a nominal scale of 1:25, was tested under a set of 12 irregular sea states. This allowed for an assessment of power output, hinge angles, mooring loads, and six-degree-of-freedom motions. The key outcome to be concluded from the results is that the facilities performed consistently, with the majority of variation linked to differences in sea state calibration. A variation of 5–10% in mean power was typical and was consistent with the variability observed in the measured significant wave heights. The tank depth (which varied from 2–5 m) showed remarkably little influence on the results, although it is noted that these tests used an aerial mooring system with the geometry unaffected by the tank depth. Similar good agreement was seen in the heave, surge, pitch and hinge angle responses. In order to maintain and improve the consistency across laboratories, we make recommendations on characterising and calibrating the tank environment and stress the importance of the device–facility physical interface (the aerial mooring in this case).</jats:p