Societal Impact Statement
Therapeutic protein production in plants is an area of great potential for increasing and improving the production of proteins for the treatment or prevention of disease in ...humans and other animals. There are a number of key benefits of this technique for scientists and society, as well as regulatory challenges that need to be overcome by policymakers. Increased public understanding of the costs and benefits of therapeutic protein production in plants will be instrumental in increasing the acceptance, and thus the medical and veterinary impact, of this approach.
Summary
Therapeutic recombinant proteins are a powerful tool for combating many diseases which have previously been hard to treat. The most utilized expression systems are Chinese Hamster Ovary cells and Escherichia coli, but all available expression systems have strengths and weaknesses regarding development time, cost, protein size, yield, growth conditions, posttranslational modifications and regulatory approval. The plant industry is well established and growing and harvesting crops is easy and affordable using current infrastructure. Growth conditions are generally simple: sunlight, water, and the addition of cheap, available fertilizers. There are multiple options for plant expression systems, including species, genetic constructs and protein targeting, each best suited to a particular type of therapeutic protein production. Transient expression systems provide a mechanism to rapidly transfect plants and produce therapeutic protein in a matter of weeks, rather than the months it can take for many competing expression systems, while proteins targeted to cereal seeds can be harvested, stored and potentially purified much more easily than in competing systems. Current challenges for plant expression systems include a lack of regulatory approval, environmental containment concerns and nonhuman glycosylation, which may limit the scope of the type of therapeutic proteins that can be manufactured in plants. The specific strengths of plant expression systems could facilitate the production of certain therapeutic proteins quickly and cheaply in the near future.
Therapeutic protein production in plants is an area of great potential for increasing and improving the production of proteins for the treatment or prevention of disease in humans and other animals. There are a number of key benefits of this technique for scientists and society, as well as regulatory challenges that need to be overcome by policymakers. Increased public understanding of the costs and benefits of therapeutic protein production in plants will be instrumental in increasing the acceptance, and thus the medical and veterinary impact, of this approach.
Summary Objective Osteoarthritis (OA) is a chronic and slowly progressive disease for which biomarkers may be able to provide a more rapid indication of therapeutic responses to therapy than is ...currently available; this could accelerate and facilitate OA drug discovery and development programs. The goal of this document is to provide a summary and guide to the application of in vitro (biochemical and other soluble) biomarkers in the development of drugs for OA and to outline and stimulate a research agenda that will further this goal. Methods The Biomarkers Working Group representing experts in the field of OA biomarker research from both academia and industry developed this consensus document between 2007 and 2009 at the behest of the Osteoarthritis Research Society International Federal Drug Administration initiative (OARSI FDA initiative). Results This document summarizes definitions and classification systems for biomarkers, the current outcome measures used in OA clinical trials, applications and potential utility of biomarkers for development of OA therapeutics, the current state of qualification of OA-related biomarkers, pathways for biomarker qualification, critical needs to advance the use of biomarkers for drug development, recommendations regarding practices and clinical trials, and a research agenda to advance the science of OA-related biomarkers. Conclusions Although many OA-related biomarkers are currently available they exist in various states of qualification and validation. The biomarkers that are likely to have the earliest beneficial impact on clinical trials fall into two general categories, those that will allow targeting of subjects most likely to either respond and/or progress (prognostic value) within a reasonable and manageable time frame for a clinical study (for instance within 1–2 years for an OA trial), and those that provide early feedback for preclinical decision-making and for trial organizers that a drug is having the desired biochemical effect. As in vitro biomarkers are increasingly investigated in the context of specific drug treatments, advances in the field can be expected that will lead to rapid expansion of the list of available biomarkers with increasing understanding of the molecular processes that they represent.
Dystonia 16 (DYT16) is caused by mutations in PACT, the protein activator of interferon-induced double-stranded RNA-activated protein kinase (PKR). PKR regulates the integrated stress response (ISR) ...via phosphorylation of the translation initiation factor eIF2α. This post-translational modification attenuates general protein synthesis while concomitantly triggering enhanced translation of a few specific transcripts leading either to recovery and homeostasis or cellular apoptosis depending on the intensity and duration of stress signals. PKR plays a regulatory role in determining the cellular response to viral infections, oxidative stress, endoplasmic reticulum (ER) stress, and growth factor deprivation. In the absence of stress, both PACT and PKR are bound by their inhibitor transactivation RNA-binding protein (TRBP) thereby keeping PKR inactive. Under conditions of cellular stress these inhibitory interactions dissociate facilitating PACT-PACT interactions critical for PKR activation. While both PACT-TRBP and PKR-TRBP interactions are pro-survival, PACT-PACT and PACT-PKR interactions are pro-apoptotic. In this study we evaluate if five DYT16 substitution mutations alter PKR activation and ISR. Our results indicate that the mutant DYT16 proteins show stronger PACT-PACT interactions and enhanced PKR activation. In DYT16 patient derived lymphoblasts the enhanced PACT-PKR interactions and heightened PKR activation leads to a dysregulation of ISR and increased apoptosis. More importantly, this enhanced sensitivity to ER stress can be rescued by luteolin, which disrupts PACT-PKR interactions. Our results not only demonstrate the impact of DYT16 mutations on regulation of ISR and DYT16 etiology but indicate that therapeutic interventions could be possible after a further evaluation of such strategies.
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•Inherited PACT mutations cause neuromuscular movement disorder dystonia 16 (DYT16).•DYT16 PACT mutants activate protein kinase PKR with enhanced efficiency.•Enhanced activation of PKR in DYT16 promotes apoptosis.•DYT16 patient cells exhibit heightened sensitivity to stress signals.•Our work elucidates DYT16 pathomechanism and offers plausible therapeutic strategies.
Background and purpose: The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed ...extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5‐lipoxygenase (LOX) enzymes. The anti‐inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated.
Experimental approach: LPS‐stimulated (1 µg·mL−1) peritoneal rat macrophages were co‐incubated with different concentrations of flavocoxid (32–128 µg·mL−1) or RPMI medium for different incubation times. Inducible COX‐2, 5‐LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB‐α (IκB‐α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF‐κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor‐α (TNF‐α) gene and protein expression were measured by real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated.
Key results: LPS stimulation induced a pro‐inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL−1) significantly inhibited COX‐2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5‐LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX‐1 expression. PGE2 and LTB4 levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB‐α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF‐κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the enhanced TNF‐α mRNA levels (LPS = 8 ± 0.9; flavocoxid = 1.9 ± 0.8 n‐fold/β‐actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS‐stimulated macrophages.
Conclusion and implications: Flavocoxid might be useful as a potential anti‐inflammatory agent, acting at the level of gene and protein expression.
BACKGROUND AND PURPOSE
Inflammation plays a key role in the development of benign prostatic hyperplasia (BPH). Eicosanoids derived from the COX and 5‐lipoxygenase (5‐LOX) pathways are elevated in the ...enlarging prostate. Flavocoxid is a novel flavonoid–based ‘dual inhibitor’ of the COX and 5‐LOX enzymes. This study evaluated the effects of flavocoxid in experimental BPH.
EXPERIMENTAL APPROACH
Rats were treated daily with testosterone propionate (3 mg·kg−1 s.c.) or its vehicle for 14 days to induce BPH. Animals receiving testosterone were randomized to receive vehicle (1 mL·kg−1, i.p.) or flavocoxid (20 mg·kg−1, i.p.) for 14 days. Histological changes, eicosanoid content and mRNA and protein levels for apoptosis‐related proteins and growth factors were assayed in prostate tissue. The effects of flavocoxid were also tested on human prostate carcinoma PC3 cells.
KEY RESULTS
Flavocoxid reduced prostate weight and hyperplasia, blunted inducible expression of COX‐2 and 5‐LOX as well as the increased production of PGE2 and leukotriene B4 (LTB4), enhanced pro‐apoptotic Bax and caspase‐9 and decreased the anti‐apoptotic Bcl‐2 mRNA. Flavocoxid also reduced EGF and VEGF expression. In PC3 cells, flavocoxid stimulated apoptosis and inhibited growth factor expression. Flavocoxid‐mediated induction of apoptosis was inhibited by the pan‐caspase inhibitor, Z‐VAD‐FMK, in PC3 cells, suggesting an essential role of caspases in flavocoxid‐mediated apoptosis during prostatic growth.
CONCLUSION AND IMPLICATIONS
Our results show that a ‘dual inhibitor’ of the COX and 5‐LOX enzymes, such as flavocoxid, might represent a rational approach to reduce BPH through modulation of eicosanoid production and a caspase‐induced apoptotic mechanism.
Abstract
Using the Radial Velocity Experiment (RAVE) survey, we recently brought to light a gradient in the mean galactocentric radial velocity of stars in the extended solar neighbourhood. This ...gradient likely originates from non-axisymmetric perturbations of the potential, among which a perturbation by spiral arms is a possible explanation. Here, we apply the traditional density wave theory and analytically model the radial component of the two-dimensional velocity field. Provided that the radial velocity gradient is caused by relatively long-lived spiral arms that can affect stars substantially above the plane, this analytic model provides new independent estimates for the parameters of the Milky Way spiral structure. Our analysis favours a two-armed perturbation with the Sun close to the inner ultra-harmonic 4:1 resonance, with a pattern speed and a small amplitude per cent of the background potential (14 per cent of the background density). This model can serve as a basis for numerical simulations in three dimensions, additionally including a possible influence of the Galactic bar and/or other non-axisymmetric modes.
The β₂-adrenergic receptor (β₂AR) has been a model system for understanding regulatory mechanisms of G-protein–coupled receptor (GPCR) actions and plays a significant role in cardiovascular and ...pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β₂AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 (4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide, exhibiting a unique chemotype and low micromolar affinity for the β₂AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β₂AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β₂AR. In cell-signaling studies, 15 inhibits cAMP production through the β₂AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β₂AR. This study presents an allosteric small-molecule ligand for the β₂AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.
Introduction of oil and gas extraction wastewaters (OGWs) to surface water leads to elevated halide levels from geogenic bromide and iodide, as well as enhanced formation of brominated and iodinated ...disinfection byproducts (DBPs) when treated. OGWs contain high levels of chemical additives used to optimize extraction activities, such as surfactants, which have the potential to serve as organic DBP precursors in OGW-impacted water sources. We report the first identification of olefin sulfonate surfactant-derived DBPs from laboratory-disinfected gas extraction wastewater. Over 300 sulfur-containing DBPs, with 43 unique molecular formulas, were found by high-resolution mass spectrometry, following bench-scale chlor(am)ination. DBPs consisted of mostly brominated species, including bromohydrin sulfonates, dihalo-bromosulfonates, and bromosultone sulfonates, with chlorinated/iodinated analogues formed to a lesser extent. Disinfection of a commercial C12-olefin sulfonate surfactant mixture revealed dodecene sulfonate as a likely precursor for most detected DBPs; disulfur-containing DBPs, like bromosultone sulfonate and bromohydrin disulfonate, originated from olefin disulfonate species, present as side-products of olefin sulfonate production. Disinfection of wastewaters increased mammalian cytotoxicity several orders of magnitude, with chloraminated water being more toxic. This finding is important to OGW-impacted source waters because drinking water plants with high-bromide source waters may switch to chloramination to meet DBP regulations.
•Pedestrian speed from vehicle speed and damage, anthropometry and head strike.•Vehicle speed from vehicle damage, pedestrian speed, anthropometry and head strike.•Accident reconstruction pedestrian ...computer positioning tool for gait and speed.
Pedestrian accident reconstruction is necessary to establish cause of death, i.e. establishing vehicle collision speed as well as circumstances leading to the pedestrian being impacted and determining culpability of those involved for subsequent court enquiry. Understanding the complexity of the pedestrian attitude during an accident investigation is necessary to ascertain the causes leading to the tragedy. A generic new method, named Pedestrian Crossing Speed Calculator (PCSC), based on vector algebra, is proposed to compute the pedestrian crossing speed at the moment of impact. PCSC uses vehicle damage and pedestrian anthropometric dimensions to establish a combination of head projection angles against the windscreen; this angle is then compared against the combined velocities angle created from the vehicle and the pedestrian crossing speed at the time of impact. This method has been verified using one accident fatality case in which the exact vehicle and pedestrian crossing speeds were known from Police forensic video analysis. PCSC was then applied on two other accident scenarios and correctly corroborated with the witness statements regarding the pedestrians crossing behaviours. The implications of PCSC could be significant once fully validated against further future accident data, as this method is reversible, allowing the computation of vehicle impact velocity from pedestrian crossing speed as well as verifying witness accounts.
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