Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through ...incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.
The therapeutic action of drugs is predicated on their physical engagement with cellular targets. Here we describe a broadly applicable method using bioluminescence resonance energy transfer (BRET) ...to reveal the binding characteristics of a drug with selected targets within intact cells. Cell-permeable fluorescent tracers are used in a competitive binding format to quantify drug engagement with the target proteins fused to Nanoluc luciferase. The approach enabled us to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. Our analysis was directed particularly to the clinically approved prodrug FK228 (Istodax/Romidepsin) because of its unique and largely unexplained mechanism of sustained intracellular action. Analysis of the binding kinetics by BRET revealed remarkably long intracellular residence times for FK228 at HDAC1, explaining the protracted intracellular behaviour of this prodrug. Our results demonstrate a novel application of BRET for assessing target engagement within the complex milieu of the intracellular environment.
Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate ...bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4-mediator complex subunit 1 foci with an EC
of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design-simultaneous targeting of two separate domains with a drug-like small molecule-providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.
Congenital hyperinsulinism is characterised by the inappropriate release of insulin during hypoglycaemia. This potentially life-threatening disorder can occur in isolation, or present as a feature of ...syndromic disease. Establishing the underlying aetiology of the hyperinsulinism is critical for guiding medical management of this condition especially in children with diazoxide-unresponsive hyperinsulinism where the underlying genetics determines whether focal or diffuse pancreatic disease is present. Disease-causing single nucleotide variants affecting over 30 genes are known to cause persistent hyperinsulinism with mutations in the KATP channel genes (
ABCC8
and
KCNJ11
) most commonly identified in children with severe persistent disease. Defects in methylation, changes in chromosome number, and large deletions and duplications disrupting multiple genes are also well described in congenital hyperinsulinism, further highlighting the genetic heterogeneity of this condition. Next-generation sequencing has revolutionised the approach to genetic testing for congenital hyperinsulinism with targeted gene panels, exome, and genome sequencing being highly sensitive methods for the analysis of multiple disease genes in a single reaction. It should though be recognised that limitations remain with next-generation sequencing with no single application able to detect all reported forms of genetic variation. This is an important consideration for hyperinsulinism genetic testing as comprehensive screening may require multiple investigations.
The substance P/neurokinin 1 receptor pathway is critical in chronic pruritus; anecdotal evidence suggests that antagonism of this pathway can reduce chronic itch.
To assess the safety and efficacy ...of the substance P/neurokinin 1 receptor antagonist serlopitant in treating chronic pruritus.
Eligible patients with severe chronic pruritus who were refractory to antihistamines or topical steroids were randomized to serlopitant, 0.25, 1, or 5 mg, or to placebo, administered once daily for 6 weeks as monotherapy or with midpotency steroids and emollients. The primary efficacy end point was percentage change in visual analog scale pruritus score from baseline.
Serlopitant treatment resulted in a dose-dependent decrease in pruritus. The mean percentage decreases from baseline visual analog scale pruritus scores were statistically significantly larger with the 1- and 5-mg doses of serlopitant (P = .022 and P = .013, respectively) than with placebo at week 6. No significant safety or tolerability differences were detected among the groups.
The sample size was insufficient for subgroup analyses of the efficacy of serlopitant for chronic pruritus on the basis of underlying conditions.
Serlopitant, 1 mg and 5 mg daily, was associated with a statistically significant reduction in chronic pruritus and was well tolerated (NCT01951274).
Finding new causes of monogenic diabetes helps understand glycaemic regulation in humans. To find novel genetic causes of maturity-onset diabetes of the young (MODY), we sequenced MODY cases with ...unknown aetiology and compared variant frequencies to large public databases. From 36 European patients, we identify two probands with novel RFX6 heterozygous nonsense variants. RFX6 protein truncating variants are enriched in the MODY discovery cohort compared to the European control population within ExAC (odds ratio = 131, P = 1 × 10
). We find similar results in non-Finnish European (n = 348, odds ratio = 43, P = 5 × 10
) and Finnish (n = 80, odds ratio = 22, P = 1 × 10
) replication cohorts. RFX6 heterozygotes have reduced penetrance of diabetes compared to common HNF1A and HNF4A-MODY mutations (27, 70 and 55% at 25 years of age, respectively). The hyperglycaemia results from beta-cell dysfunction and is associated with lower fasting and stimulated gastric inhibitory polypeptide (GIP) levels. Our study demonstrates that heterozygous RFX6 protein truncating variants are associated with MODY with reduced penetrance.Maturity-onset diabetes of the young (MODY) is the most common subtype of familial diabetes. Here, Patel et al. use targeted DNA sequencing of MODY patients and large-scale publically available data to show that RFX6 heterozygous protein truncating variants cause reduced penetrance MODY.
One long-recognized consequence of the tension between popular sovereignty and democratic values like liberty and equality is public opinion backlash, which occurs when individuals recoil in response ...to some salient event. For decades, scholars have suggested that opinion backlash impedes policy gains by marginalized groups. Public opinion research, however, suggests that widespread attitude change that backlash proponents theorize is likely to be rare. Examining backlash against gays and lesbians using a series of online and natural experiments about marriage equality, and large-sample survey data, we find no evidence of opinion backlash among the general public, by members of groups predisposed to dislike gays and lesbians, or from those with psychological traits that may predispose them to lash back. The important implication is that groups pursuing rights should not be dissuaded by threats of backlash that will set their movement back in the court of public opinion.
Across much of the eastern United States, oak forests are undergoing mesophication as shade‐tolerant competitors become more abundant and suppress oak regeneration. Given the historical role of ...anthropogenic surface fires in promoting oak dominance, prescribed fire has become important in efforts to reverse mesophication and sustain oaks. In 2000 we established the Ohio Hills Fire and Fire Surrogate (FFS) study to examine whether repeated prescribed fire (Fire), mechanical partial harvest (Mech), and their combined application (Mech + Fire) reduced the dominance of subcanopy mesophytic competitors, increased the abundance of large oak–hickory advance regeneration, created a more diverse and productive ground‐layer flora, and produced fuel beds more conducive to prescribed fire, reducing the risk of high‐severity wildfire. Here we report on the ~20‐year effects of treatments on vegetation and fuels and examine the support for interactive effects across a topographic‐moisture and energy gradient. In general, we found that Fire and Mech + Fire treatments tended to reverse mesophication while the Mech‐only treatment did not. The moderate and occasionally high‐intensity fires resulted in effects that were ultimately very similar between the two fire treatments but were modulated by topography with increasing fire severity on drier sites. In particular, we found support for an interaction effect between treatment and topography on forest structure and tree regeneration responses. Fire generally reduced mesophytic tree density in the midstory and sapling strata across all site conditions, while leading to substantial gains in the abundance of large oak–hickory advance regeneration on dry and intermediate landscape positions. Fire also promoted ground‐layer diversity and created compositionally distinct communities across all site conditions, primarily through the increased richness of native perennial herbs. However, the fire had limited effects on fine surface fuel loading and increased the loading of large woody fuels, potentially increasing the risk of high‐severity wildfire during drought conditions. We conclude that two decades of repeated fires, with and without mechanical density reduction, significantly shifted the trajectory of mesophication across most of the landscape, particularly on dry and intermediate sites, highlighting the capacity of a periodic fire regime to sustain eastern oak forests and promote plant diversity but modulated by topography.
Economic impacts of invasive species worldwide are substantial. Management strategies have been incorporated in population models to assess the effectiveness of management for reducing density, with ...the implicit assumption that economic impact of the invasive species will also decline. The optimal management effort, however, is that which minimizes the sum of both the management and impact costs. The relationship between population density and economic impact (what we call the "density—impact curve") is rarely examined in a management context and could take several nonlinear forms. Here we determine the effects of population dynamics and density—impact curves of different shapes on optimal management effort and discover cases where management is either highly effective or a waste of resources. When an inaccurate density—impact curve is used, the increase in total costs due to over- or underinvestment in management can be large. We calculate the increase in total costs incurred if the density—impact curve is incorrect and find that the greater the maximum impact caused by an invasive species, the more important it is not only to reduce its density, but also to know the shape of the density—impact relationship accurately. Lack of information regarding the relationship between density and economic impact causes the most acute problems for invaders that cause high impact at low density, where management typically will be too little, too late. For species that are only problematic at high density, ignorance of the density—impact curve can lead to overinvestment in management with little reduction in impact.
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