Computational studies on fatty acid synthesis: from mechanisms to drug design

The first committed steps of the Fatty Acid synthesis pathway involves the de/carboxylation reactions of biotin. By understanding this step, potential novel antimicrobial agents could be discovered. The current tools of drug discovery can only help the research in finding and modifying potential hits. Finding a lead candidate from these programs are often equated to finding a needle in a haystack, which is due to the many assumptions used in molecular docking. The fundamental reaction kinetics can not be described by these techniques and a detailed study of the decarboxylation reaction is investigated using ab initio molecular dynamics. In this particular study, Car-Parrinello molecular dynamics is used and how the biotin model is protonated was found to play an important role in its reaction barrier. Although stable in low acidic solutions, a crucial nitrogen protonation is shown to have the lowest free energy barrier which could play a pivotal role in the enzymatic mechanism. The molecular docking knowledge of potential ligand inhibitors via a low level modeling technique connected to high level quantum mechanical reaction modeling provides a synergistic route in the search for inhibitors

A bivariate first order autoregressive time series model in exponential variables (BEAR (1))

A simple time series model for bivariate exponential variables having first-order auto-regressive structure is presented. The linear random coefficient difference equation model is an adaptation of the New Exponential Autoregressive model (NEAR (2)). The process is Markovian in the bivariate sense and has correlation structure analogous to that of the Gaussian AR(1) bivariate time series model. The model exhibits a full range of positive correlations and cross-correlations. With some modification in either the innovation or the random coefficients, the model admits some negative values for the cross- correlations. The marginal processes are shown to have correlation structure of ARMA (2,1) modelsPrepared for: Naval Postgraduate School Monterey, CAhttp://archive.org/details/bivariatefirstor00dewaNAN

Subsurface investigation of the Nellie Bly Formation in Creek and Okfuskee Counties, Oklahoma

Geolog

Heparanase 2, mutated in urofacial syndrome, mediates peripheral neural development in Xenopus

Urofacial syndrome (UFS; previously Ochoa syndrome) is an autosomal recessive disease characterized by incomplete bladder emptying during micturition. This is associated with a dyssynergia in which the urethral walls contract at the same time as the detrusor smooth muscle in the body of the bladder. UFS is also characterized by an abnormal facial expression upon smiling, and bilateral weakness in the distribution of the facial nerve has been reported. Biallelic mutations in HPSE2 occur in UFS. This gene encodes heparanase 2, a protein which inhibits the activity of heparanase. Here, we demonstrate, for the first time, an in vivo developmental role for heparanase 2. We identified the Xenopus orthologue of heparanase 2 and showed that the protein is localized to the embryonic ventrolateral neural tube where motor neurons arise. Morpholino-induced loss of heparanase 2 caused embryonic skeletal muscle paralysis, and morphant motor neurons had aberrant morphology including less linear paths and less compactly-bundled axons than normal. Biochemical analyses demonstrated that loss of heparanase 2 led to upregulation of fibroblast growth factor 2/phosphorylated extracellular signal-related kinase signalling and to alterations in levels of transcripts encoding neural- and muscle-associated molecules. Thus, a key role of heparanase 2 is to buffer growth factor signalling in motor neuron development. These results shed light on the pathogenic mechanisms underpinning the clinical features of UFS and support the contention that congenital peripheral neuropathy is a key feature of this disorder

The Generation of an Engineered Interleukin-10 Protein With Improved Stability and Biological Function

Interleukin-10 (IL-10) is an immunoregulatory cytokine that plays a pivotal role in modulating inflammation. IL-10 has inhibitory effects on proinflammatory cytokine production and function in vitro and in vivo; as such, IL-10 is viewed as a potential treatment for various inflammatory diseases. However, a significant drawback of using IL-10 in clinical application is the fact that the biologically active form of IL-10 is an unstable homodimer, which has a short half-life and is easily degraded in vivo. Consequently, IL-10 therapy using recombinant native IL-10 has had only limited success in the treatment of human disease. To improve the therapeutic potential of IL-10, we have generated a novel form of IL-10, which consists of two IL-10 monomer subunits linked in a head to tail fashion by a flexible linker. We show that the linker length per se did not affect the expression and biological activity of the stable IL-10 molecule, which was more active than natural IL-10, both in vitro and in vivo. We confirmed that the new form of IL-10 had a much-improved temperature- and pH-dependent biological stability compared to natural IL-10. The IL-10 dimer protein binds to the IL-10 receptor similarly to the natural IL-10 protein, as shown by antibody blocking and through the genetic modifications of one monomer in the IL-10 dimer specifically at the IL-10 receptor binding site. Finally, we showed that stable IL-10 is more effective at suppressing LPS-induced-inflammation in vivo compared to the natural IL-10. In conclusion, we have developed a new stable dimer version of the IL-10 protein with improved stability and efficacy to suppress inflammation. We propose that this novel stable IL-10 dimer could serve as the basis for the development of targeted anti-inflammatory drugs

Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens

From Springer Nature via Jisc Publications RouterHistory: received 2021-04-28, rev-recd 2021-06-02, accepted 2021-06-08, registration 2021-06-11, pub-electronic 2021-06-28, online 2021-06-28, pub-print 2021-07Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/P004237/1Abstract: Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of ‘difficult-to-express’ bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd.’s Clostridium difficile vaccine programme. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. Further, proteolytic cleavage of Ant2-3 was observed. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. However, screening of different buffer/additives showed that the addition of 1–15 mM dithiothreitol alone decreased the formation of insoluble aggregates and improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3, and solubility-based prediction tools were employed to determine the role of hydrophobicity and charge on protein production. The results showed that a large non-polar region (containing hydrophobic amino acids) was detected on the surface of Ant2 structures, whereas positively charged regions (containing lysine and arginine amino acids) were observed for Ant3, both of which were associated with poor protein solubility. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression and stability of single and potentially multi-domain (or fusion) antigens in bacterial expression systems

Technology Development Roadmap: A Technology Development Roadmap for a Future Gravitational Wave Mission

Humankind will detect the first gravitational wave (GW) signals from the Universe in the current decade using ground-based detectors. But the richest trove of astrophysical information lies at lower frequencies in the spectrum only accessible from space. Signals are expected from merging massive black holes throughout cosmic history, from compact stellar remnants orbiting central galactic engines from thousands of close contact binary systems in the Milky Way, and possibly from exotic sources, some not yet imagined. These signals carry essential information not available from electromagnetic observations, and which can be extracted with extraordinary accuracy. For 20 years, NASA, the European Space Agency (ESA), and an international research community have put considerable effort into developing concepts and technologies for a GW mission. Both the 2000 and 2010 decadal surveys endorsed the science and mission concept of the Laser Interferometer Space Antenna (LISA). A partnership of the two agencies defined and analyzed the concept for a decade. The agencies partnered on LISA Pathfinder (LPF), and ESA-led technology demonstration mission, now preparing for a 2015 launch. Extensive technology development has been carried out on the ground. Currently, the evolved Laser Interferometer Space Antenna (eLISA) concept, a LISA-like concept with only two measurement arms, is competing for ESA's L2 opportunity. NASA's Astrophysics Division seeks to be a junior partner if eLISA is selected. If eLISA is not selected, then a LISA-like mission will be a strong contender in the 2020 decadal survey. This Technology Development Roadmap (TDR) builds on the LISA concept development, the LPF technology development, and the U.S. and European ground-based technology development. The eLISA architecture and the architecture of the Mid-sized Space-based Gravitational-wave Observatory (SGO Mid)-a competitive design with three measurement arms from the recent design study for a NASA-led mission after 2020-both use the same technologies. Further, NASA participation in an ESA-led mission would likely augment the eLISA architecture with a third arm to become the SGO Mid architecture. For these reasons, this TDR for a future GW mission applies to both designs and both programmatic paths forward. It is adaptable to the different timelines and roles for an ESA-led or a NASA-led mission, and it is adaptable to available resources. Based on a mature understanding of the interaction between technology and risk, the authors of this TDR have chosen a set of objectives that are more expansive than is usual. The objectives for this roadmap are: (1) reduce technical and development risks and costs; (2) understand and, where possible, relieve system requirements and consequences; (3) increase technical insight into critical technologies; and (4) validate the design at the subsystem level. The emphasis on these objectives, particularly the latter two, is driven by outstanding programmatic decisions, namely whether a future GW mission is ESA-led or NASA-led, and availability of resources. The relative emphasis is best understood in the context of prioritization

A Review of the Global Literature on Dental Therapists: In the Context of the Movement to Add Dental Therapists to the Oral Health Workforce in the United States (Executive Summary)

This executive summary documents evidence that dental therapists can effectively expand access to dental care, especially for children, and is technically competent, safe and effective