A Global Map of Lipid-Binding Proteins and Their Ligandability in Cells.

Lipids play central roles in physiology and disease, where their structural, metabolic, and signaling functions often arise from interactions with proteins. Here, we describe a set of lipid-based chemical proteomic probes and their global interaction map in mammalian cells. These interactions involve hundreds of proteins from diverse functional classes and frequently occur at sites of drug action. We determine the target profiles for several drugs across the lipid-interaction proteome, revealing that its ligandable content extends far beyond traditionally defined categories of druggable proteins. In further support of this finding, we describe a selective ligand for the lipid-binding protein nucleobindin-1 (NUCB1) and show that this compound perturbs the hydrolytic and oxidative metabolism of endocannabinoids in cells. The described chemical proteomic platform thus provides an integrated path to both discover and pharmacologically characterize a wide range of proteins that participate in lipid pathways in cells.

Stabilization of amyloidogenic immunoglobulin light chains by small molecules.

In Ig light-chain (LC) amyloidosis (AL), the unique antibody LC protein that is secreted by monoclonal plasma cells in each patient misfolds and/or aggregates, a process leading to organ degeneration. As a step toward developing treatments for AL patients with substantial cardiac involvement who have difficulty tolerating existing chemotherapy regimens, we introduce small-molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which can slow or stop the amyloidogenicity cascade at its origin. A protease-coupled fluorescence polarization-based high-throughput screen was employed to identify small molecules that kinetically stabilize LCs. NMR and X-ray crystallographic data demonstrate that at least one structural family of hits bind at the LC-LC dimerization interface within full-length LCs, utilizing variable-domain residues that are highly conserved in most AL patients. Stopping the amyloidogenesis cascade at the beginning is a proven strategy to ameliorate postmitotic tissue degeneration.

Sensory-Motor Mechanisms Increasing Falls Risk in Diabetic Peripheral Neuropathy.

Diabetic peripheral neuropathy (DPN) is associated with peripheral sensory and motor nerve damage that affects up to half of diabetes patients and is an independent risk factor for falls. Clinical implications of DPN-related falls include injury, psychological distress and physical activity curtailment. This review describes how the sensory and motor deficits associated with DPN underpin biomechanical alterations to the pattern of walking (gait), which contribute to balance impairments underpinning falls. Changes to gait with diabetes occur even before the onset of measurable DPN, but changes become much more marked with DPN. Gait impairments with diabetes and DPN include alterations to walking speed, step length, step width and joint ranges of motion. These alterations also impact the rotational forces around joints known as joint moments, which are reduced as part of a natural strategy to lower the muscular demands of gait to compensate for lower strength capacities due to diabetes and DPN. Muscle weakness and atrophy are most striking in patients with DPN, but also present in non-neuropathic diabetes patients, affecting not only distal muscles of the foot and ankle, but also proximal thigh muscles. Insensate feet with DPN cause a delayed neuromuscular response immediately following foot-ground contact during gait and this is a major factor contributing to increased falls risk. Pronounced balance impairments measured in the gait laboratory are only seen in DPN patients and not non-neuropathic diabetes patients. Self-perception of unsteadiness matches gait laboratory measures and can distinguish between patients with and without DPN. Diabetic foot ulcers and their associated risk factors including insensate feet with DPN and offloading devices further increase falls risk. Falls prevention strategies based on sensory and motor mechanisms should target those most at risk of falls with DPN, with further research needed to optimise interventions.

Transgenerational impacts of herbivory and inbreeding on reproductive output in Solanum carolinense.

PREMISE: Plant maternal effects on offspring phenotypes are well documented. However, little is known about how herbivory on maternal plants affects offspring fitness. Furthermore, while inbreeding is known to reduce plant reproductive output, previous studies have not explored whether and how such effects may extend across generations. Here, we addressed the transgenerational consequences of herbivory and maternal plant inbreeding on the reproduction of Solanum carolinense offspring. METHODS: Manduca sexta caterpillars were used to inflict weekly damage on inbred and outbred S. carolinense maternal plants. Cross-pollinations were performed by hand to produce seed from herbivore-damaged outbred plants, herbivore-damaged inbred plants, undamaged outbred plants, and undamaged inbred plants. The resulting seeds were grown in the greenhouse to assess emergence rate and flower production in the absence of herbivores. We also grew offspring in the field to examine reproductive output under natural conditions. RESULTS: We found transgenerational effects of herbivory and maternal plant inbreeding on seedling emergence and reproductive output. Offspring of herbivore-damaged plants had greater emergence, flowered earlier, and produced more flowers and seeds than offspring of undamaged plants. Offspring of outbred maternal plants also had greater seedling emergence and reproductive output than offspring of inbred maternal plants, even though all offspring were outbred. Moreover, the effects of maternal plant inbreeding were more severe when plant offspring were grown in field conditions. CONCLUSIONS: This study demonstrates that both herbivory and inbreeding have fitness consequences that extend across generations even in outbred progeny.

Magnetic resonance enterography for predicting the clinical course of Crohn's disease strictures.

BACKGROUND AND AIMS: Strictures are the most common Crohn's disease complication, but their natural history is unknown. This study aimed to characterize inflammation, predict prognosis, and understand the impact of drug therapy using magnetic resonance enterography (MRE). METHODS: Patients with a stricture diagnosed on MRE over a 5-year period were reviewed for MRE disease extent and inflammation, clinical course, C-reactive protein, response to anti-TNF therapy, endoscopic dilatation, hospitalization, and surgery. RESULTS: 136 patients had 235 strictures (77, one and 59, ≥ 2 strictures). TREATMENT: 46% of patients underwent surgery after a median 6 months; median follow-up for those not requiring surgery was 41 months. Predictors of surgery: Hospitalization because of obstruction predicted subsequent surgery (OR 2.50; 95% CI 1.06-5.90) while anti-TNF therapy commenced at stricture diagnosis was associated with a reduced risk (OR 0.23; 95% CI 0.05-0.99). MRE characteristics associated with surgery were proximal bowel dilatation ≥ 30-mm diameter (OR 2.98; 95% CI 1.36-6.55), stricture bowel wall thickness ≥ 10-mm (OR 2.42; 95% CI 1.11-5.27), and stricture length > 5-cm (OR 2.56; 95% CI 1.21-5.43). 81% of patients with these three adverse MRE features required surgery versus 17% if none were present (P < 0.001). Accuracy for these three MRE variables predicting surgery was high (AUC 0.76). CONCLUSION: Magnetic resonance enterography findings in Crohn's disease strictures are highly predictive of the disease course and the need for future surgery. MRE may also identify who would benefit from treatment intensification. Anti-TNF therapy is associated with reduced risk of surgery and appears to alter the natural history of this complication.

Return to Employment After Stroke in Young Adults: How Important Is the Speed and Energy Cost of Walking?

Background and Purpose- A quarter of individuals who experience a stroke are under the age of 65 years (defined as young adults), and up to 44% will be unable to return to work poststroke, predominantly because of walking difficulties. No research study has comprehensively analyzed walking performance in young adult's poststroke. The primary aim of this study is to investigate how a stroke in young adults affects walking performance (eg, walking speed and metabolic cost) compared with healthy age-matched controls. The secondary aim is to determine the predictive ability of walking performance parameters for return to employment poststroke. Methods- Forty-six individuals (18-40 years: n=6, 41-54 years: n=21, 55-65 years: n=19) who have had a stroke and 15 healthy age-matched able-bodied controls were recruited from 6 hospital sites in Wales, United Kingdom. Type, location, cause of stroke, and demographic factors (eg, employment status) were recorded. Temporal and spatial walking parameters were measured using 3-dimensional gait analysis. Metabolic energy expenditure and metabolic cost of walking were captured during 3 minutes of walking at self-selected speed from measurements of oxygen consumption. Results- Stroke participants walked slower (P<0.004) and less efficiently (P<0.002) than the controls. Only 23% of stroke participants returned to employment poststroke. Walking speed was the strongest predictor (sensitivity, 0.90; specificity, 0.82) for return to work (P=0.004) with a threshold of 0.93 m/s identified: individuals able to walk faster than 0.93 m/s were significantly more likely to return to work poststroke than those who walked slower than this threshold. Conclusions- This study is the first to capture walking performance parameters of young adults who have had a stroke and identifies slower and less efficient walking. Walking speed emerged as the strongest predictor for return to employment. It is recommended that walking speed be used as a simple but sensitive clinical indicator of functional performance to guide rehabilitation and inform readiness for return to work poststroke.

Enhancing Organic Semiconductor-Surface Plasmon Polariton Coupling with Molecular Orientation.

Due to strong electric field enhancements, surface plasmon polaritons (SPPs) are capable of drastically increasing light-molecule coupling in organic optoelectronic devices. The electric field enhancement, however, is anisotropic, offering maximal functional benefits if molecules are oriented perpendicular to the interface. To provide a clear demonstration of this orientation dependence, we study SPP dispersion and SPP-mediated photoluminescence at a model Au/small-molecule interface where identical molecules can be deposited with two very different molecular backbone orientations depending on processing conditions. First, we demonstrate that thin films of p-SIDT(FBTTh2)2 can be deposited with either all "in-plane" (parallel to substrate) or a 50/50 mix of in-plane/"out-of-plane" (perpendicular to substrate) optical transition dipoles by the absence or presence, respectively, of diiodooctane during spin-coating. In contrast to typical orientation control observed in organic thin films, for this particular molecule, this corresponds to films with conjugated backbones purely in-plane, or with a 50/50 mix of in-plane/out-of-plane backbones. Then, using momentum-resolved reflectometry and momentum-resolved photoluminescence, we study and quantify changes in SPP dispersion and photoluminescence intensity arising solely from changes in molecular orientation. We demonstrate increased SPP momentum and a 2-fold enhancement in photoluminescence for systems with out-of-plane oriented transition dipoles. These results agree well with theory and have direct implications for the design and analysis of organic optoelectronic devices.

Identification of Neutrophil Exocytosis Inhibitors (Nexinhibs), Small Molecule Inhibitors of Neutrophil Exocytosis and Inflammation: DRUGGABILITY OF THE SMALL GTPase Rab27a.

Neutrophils constitute the first line of cellular defense in response to bacterial and fungal infections and rely on granular proteins to kill microorganisms, but uncontrolled secretion of neutrophil cargos is injurious to the host and should be closely regulated. Thus, increased plasma levels of neutrophil secretory proteins, including myeloperoxidase and elastase, are associated with tissue damage and are hallmarks of systemic inflammation. Here, we describe a novel high-throughput screening approach to identify small molecule inhibitors of the interaction between the small GTPase Rab27a and its effector JFC1, two central regulators of neutrophil exocytosis. Using this assay, we have identified small molecule inhibitors of Rab27a-JFC1 binding that were also active in cell-based neutrophil-specific exocytosis assays, demonstrating the druggability of Rab GTPases and their effectors. These compounds, named Nexinhibs (neutrophil exocytosis inhibitors), inhibit exocytosis of azurophilic granules in human neutrophils without affecting other important innate immune responses, including phagocytosis and neutrophil extracellular trap production. Furthermore, the compounds are reversible and potent inhibitors of the extracellular production of superoxide anion by preventing the up-regulation of the granule membrane-associated subunit of the NADPH oxidase at the plasma membrane. Nexinhibs also inhibit the up-regulation of activation signature molecules, including the adhesion molecules CD11b and CD66b. Importantly, by using a mouse model of endotoxin-induced systemic inflammation, we show that these inhibitors have significant activity in vivo manifested by decreased plasma levels of neutrophil secretory proteins and significantly decreased tissue infiltration by inflammatory neutrophils. Altogether, our data present the first neutrophil exocytosis-specific inhibitor with in vivo anti-inflammatory activity, supporting its potential use as an inhibitor of systemic inflammation.

Concentrations of Adalimumab and Infliximab in Mothers and Newborns, and Effects on Infection.

BACKGROUND & AIMS: Little is known about in utero exposure to and postnatal clearance of anti-tumor necrosis factor (anti-TNF) agents in neonates. We investigated the concentrations of adalimumab and infliximab in umbilical cord blood of newborns and rates of clearance after birth, and how these correlated with drug concentrations in mothers at birth and risk of infection during the first year of life. METHODS: We performed a prospective study of 80 pregnant women with inflammatory bowel diseases at tertiary hospitals in Denmark, Australia, and New Zealand from March 2012 through November 2014: 36 received adalimumab and 44 received infliximab; 39 received concomitant thiopurines during pregnancy. Data were collected from medical records on disease activity and treatment before, during, and after pregnancy. Concentrations of anti-TNF agents were measured in blood samples from women at delivery and in umbilical cords, and in infants for every 3 months until the drug was no longer detected. RESULTS: The time from last exposure to anti-TNF agent during pregnancy correlated inversely with the concentration of the drugs in the umbilical cord (adalimumab: r = -0.64, P = .0003; infliximab: r = -0.77, P < .0001) and in mothers at time of birth (adalimumab, r = -0.80; infliximab, r = -0.80; P < .0001 for both). The median ratio of infant:mother drug concentration at birth was 1.21 for adalimumab (95% confidence interval [CI], 0.94-1.49) and 1.97 for infliximab (95% CI, 1.50-2.43). The mean time to drug clearance in infants was 4.0 months for adalimumab (95% CI, 2.9-5.0) and 7.3 months for infliximab (95% CI, 6.2-8.3; P < .0001). Drugs were not detected in infants after 12 months of age. Bacterial infections developed in 4 infants (5%) and viral infections developed in 16 (20%), all with benign courses. The relative risk for infection was 2.7 in infants whose mothers received the combination of an anti-TNF agent and thiopurine, compared with anti-TNF monotherapy (95% CI, 1.09-6.78; P = .02). CONCLUSIONS: In a prospective study of infants born to mothers who received anti-TNF agents during pregnancy, we detected the drugs until 12 months of age. There was an inverse correlation between the time from last exposure during pregnancy and drug concentration in the umbilical cord. Infliximab was cleared more slowly than adalimumab from the infants. The combination of an anti-TNF agent and thiopurine therapy during pregnancy increased the relative risk for infant infections almost 3-fold compared with anti-TNF monotherapy. Live vaccines therefore should be avoided for up to 1 year unless drug clearance is documented, and pregnant women should be educated on the risks of anti-TNF use.

Serologic antibodies in relation to outcome in postoperative Crohn's disease.

BACKGROUND AND AIM: Disease recurs frequently after Crohn's disease resection. The role of serological antimicrobial antibodies in predicting recurrence or as a marker of recurrence has not been well defined. METHODS: A total of 169 patients (523 samples) were prospectively studied, with testing peri-operatively, and 6, 12 and 18 months postoperatively. Colonoscopy was performed at 18 months postoperatively. Serologic antibody presence (perinuclear anti-neutrophil cytoplasmic antibody [pANCA], anti-Saccharomyces cerevisiae antibodies [ASCA] IgA/IgG, anti-OmpC, anti-CBir1, anti-A4-Fla2, anti-Fla-X) and titer were tested. Quartile sum score (range 6-24), logistic regression analysis, and correlation with phenotype, smoking status, and endoscopic outcome were assessed. RESULTS: Patients with ≥ 2 previous resections were more likely to be anti-OmpC positive (94% vs 55%, ≥ 2 vs < 2, P = 0.001). Recurrence at 18 months was associated with anti-Fla-X positivity at baseline (49% vs 29%; positive vs negative, P = 0.033) and 12 months (52% vs 31%, P = 0.04). Patients positive (n = 28) for all four antibacterial antibodies (anti-CBir1, anti-OmpC, anti-A4-Fla2, and anti-Fla-X) at baseline were more likely to experience recurrence at 18 months than patients negative (n = 32) for all four antibodies (82% vs 18%, P = 0.034; odds ratio 6.4, 95% confidence interval 1.16-34.9). The baseline quartile sum score for all six antimicrobial antibodies was higher in patients with severe recurrence (Rutgeert's i3-i4) at 18 months, adjusted for clinical risk factors (odds ratio 1.16, 95% confidence interval 1.01-1.34, P = 0.039). Smoking affected antibody status. CONCLUSIONS: Anti-Fla-X and presence of all anti-bacterial antibodies identifies patients at higher risk of early postoperative Crohn's disease recurrence. Serologic screening pre-operatively may help identify patients at increased risk of recurrence.

Bitopic Sphingosine 1-Phosphate Receptor 3 (S1P3) Antagonist Rescue from Complete Heart Block: Pharmacological and Genetic Evidence for Direct S1P3 Regulation of Mouse Cardiac Conduction.

The molecular pharmacology of the G protein-coupled receptors for sphingosine 1-phosphate (S1P) provides important insight into established and new therapeutic targets. A new, potent bitopic S1P3 antagonist, SPM-354, with in vivo activity, has been used, together with S1P3-knockin and S1P3-knockout mice to define the spatial and functional properties of S1P3 in regulating cardiac conduction. We show that S1P3 is a key direct regulator of cardiac rhythm both in vivo and in isolated perfused hearts. 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol in vivo and S1P in isolated hearts induced a spectrum of cardiac effects, ranging from sinus bradycardia to complete heart block, as measured by a surface electrocardiogram in anesthetized mice and in volume-conducted Langendorff preparations. The agonist effects on complete heart block are absent in S1P3-knockout mice and are reversed in wild-type mice with SPM-354, as characterized and described here. Homologous knockin of S1P3-mCherry is fully functional pharmacologically and is strongly expressed by immunohistochemistry confocal microscopy in Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4)-positive atrioventricular node and His-Purkinje fibers, with relative less expression in the HCN4-positive sinoatrial node. In Langendorff studies, at constant pressure, SPM-354 restored sinus rhythm in S1P-induced complete heart block and fully reversed S1P-mediated bradycardia. S1P3 distribution and function in the mouse ventricular cardiac conduction system suggest a direct mechanism for heart block risk that should be further studied in humans. A richer understanding of receptor and ligand usage in the pacemaker cells of the cardiac system is likely to be useful in understanding ventricular conduction in health, disease, and pharmacology.

Chemical Proteomic Profiling of Human Methyltransferases.

Methylation is a fundamental mechanism used in Nature to modify the structure and function of biomolecules, including proteins, DNA, RNA, and metabolites. Methyl groups are predominantly installed into biomolecules by a large and diverse class of S-adenosyl methionine (SAM)-dependent methyltransferases (MTs), of which there are ∼200 known or putative members in the human proteome. Deregulated MT activity contributes to numerous diseases, including cancer, and several MT inhibitors are in clinical development. Nonetheless, a large fraction of the human MT family remains poorly characterized, underscoring the need for new technologies to characterize MTs and their inhibitors in native biological systems. Here, we describe a suite of S-adenosyl homocysteine (SAH) photoreactive probes and their application in chemical proteomic experiments to profile and enrich a large number of MTs (>50) from human cancer cell lysates with remarkable specificity over other classes of proteins. We further demonstrate that the SAH probes can enrich MT-associated proteins and be used to screen for and assess the selectivity of MT inhibitors, leading to the discovery of a covalent inhibitor of nicotinamide N-methyltransferase (NNMT), an enzyme implicated in cancer and metabolic disorders. The chemical proteomics probes and methods for their utilization reported herein should prove of value for the functional characterization of MTs, MT complexes, and MT inhibitors in mammalian biology and disease.

Crohn's disease management after intestinal resection: a randomised trial.

BACKGROUND: Most patients with Crohn's disease need an intestinal resection, but a majority will subsequently experience disease recurrence and require further surgery. This study aimed to identify the optimal strategy to prevent postoperative disease recurrence. METHODS: In this randomised trial, consecutive patients from 17 centres in Australia and New Zealand undergoing intestinal resection of all macroscopic Crohn's disease, with an endoscopically accessible anastomosis, received 3 months of metronidazole therapy. Patients at high risk of recurrence also received a thiopurine, or adalimumab if they were intolerant to thiopurines. Patients were randomly assigned to parallel groups: colonoscopy at 6 months (active care) or no colonoscopy (standard care). We used computer-generated block randomisation to allocate patients in each centre to active or standard care in a 2:1 ratio. For endoscopic recurrence (Rutgeerts score ≥i2) at 6 months, patients stepped-up to thiopurine, fortnightly adalimumab with thiopurine, or weekly adalimumab. The primary endpoint was endoscopic recurrence at 18 months. Patients and treating physicians were aware of the patient's study group and treatment, but central reading of the endoscopic findings was undertaken blind to the study group and treatment. Analysis included all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT00989560. FINDINGS: Between Oct 13, 2009, and Sept 28, 2011, 174 (83% high risk across both active and standard care groups) patients were enrolled and received at least one dose of study drug. Of 122 patients in the active care group, 47 (39%) stepped-up treatment. At 18 months, endoscopic recurrence occurred in 60 (49%) patients in the active care group and 35 (67%) patients in the standard care group (p=0.03). Complete mucosal normality was maintained in 27 (22%) of 122 patients in the active care group versus four (8%) in the standard care group (p=0.03). In the active care arm, of those with 6 months recurrence who stepped up treatment, 18 (38%) of 47 patients were in remission 12 months later; conversely, of those in remission at 6 months who did not change therapy recurrence occurred in 31 (41%) of 75 patients 12 months later. Smoking (odds ratio [OR] 2.4, 95% CI 1.2-4.8, p=0.02) and the presence of two or more clinical risk factors including smoking (OR 2.8, 95% CI 1.01-7.7, p=0.05) increased the risk of endoscopic recurrence. The incidence and type of adverse and severe adverse events did not differ significantly between patients in the active care and standard care groups (100 [82%] of 122 vs 45 [87%] of 52; p=0.51) and (33 [27%] of 122 vs 18 [35%] of 52; p=0.36), respectively. INTERPRETATION: Treatment according to clinical risk of recurrence, with early colonoscopy and treatment step-up for recurrence, is better than conventional drug therapy alone for prevention of postoperative Crohn's disease recurrence. Selective immune suppression, adjusted for early recurrence, rather than routine use, leads to disease control in most patients. Clinical risk factors predict recurrence, but patients at low risk also need monitoring. Early remission does not preclude the need for ongoing monitoring. FUNDING: AbbVie, Gutsy Group, Gandel Philanthropy, Angior Foundation, Crohn's Colitis Australia, and the National Health and Medical Research Council.

Measurement of fecal calprotectin improves monitoring and detection of recurrence of Crohn's disease after surgery.

BACKGROUND & AIMS: Crohn's disease (CD) usually recurs after intestinal resection; postoperative endoscopic monitoring and tailored treatment can reduce the chance of recurrence. We investigated whether monitoring levels of fecal calprotectin (FC) can substitute for endoscopic analysis of the mucosa. METHODS: We analyzed data collected from 135 participants in a prospective, randomized, controlled trial, performed at 17 hospitals in Australia and 1 hospital in New Zealand, that assessed the ability of endoscopic evaluations and step-up treatment to prevent CD recurrence after surgery. Levels of FC, serum levels of C-reactive protein (CRP), and Crohn's disease activity index (CDAI) scores were measured before surgery and then at 6, 12, and 18 months after resection of all macroscopic Crohn's disease. Ileocolonoscopies were performed at 6 months after surgery in 90 patients and at 18 months after surgery in all patients. RESULTS: Levels of FC were measured in 319 samples from 135 patients. The median FC level decreased from 1347 µg/g before surgery to 166 µg/g at 6 months after surgery, but was higher in patients with disease recurrence (based on endoscopic analysis; Rutgeerts score, ≥i2) than in patients in remission (275 vs 72 µg/g, respectively; P < .001). Combined 6- and 18-month levels of FC correlated with the presence (r = 0.42; P < .001) and severity (r = 0.44; P < .001) of CD recurrence, but the CRP level and CDAI score did not. Levels of FC greater than 100 µg/g indicated endoscopic recurrence with 89% sensitivity and 58% specificity, and a negative predictive value (NPV) of 91%; this means that colonoscopy could have been avoided in 47% of patients. Six months after surgery, FC levels less than 51 µg/g in patients in endoscopic remission predicted maintenance of remission (NPV, 79%). In patients with endoscopic recurrence at 6 months who stepped-up treatment, FC levels decreased from 324 µg/g at 6 months to 180 µg/g at 12 months and 109 µg/g at 18 months. CONCLUSIONS: In this analysis of data from a prospective clinical trial, FC measurement has sufficient sensitivity and NPV values to monitor for CD recurrence after intestinal resection. Its predictive value might be used to identify patients most likely to relapse. After treatment for recurrence, the FC level can be used to monitor response to treatment. It predicts which patients will have disease recurrence with greater accuracy than CRP level or CDAI score.

Model-blind characterization of thin-film optical constants with momentum-resolved reflectometry.

Determining optical constants of thin material films is important for characterizing their electronic excitations and for the design of optoelectronic devices. Spectroscopic ellipsometry techniques have emerged as the predominant approach for measuring thin-film optical constants. However, ellipsometry methods suffer from complications associated with highly model-dependent, multi-parameter spectral fitting procedures. Here, we present a model-blind, momentum-resolved reflectometry technique that yields accurate and precise optical constants, with quantifiable error estimates, even for film thicknesses less than 50 nm. These capabilities are demonstrated by interrogating an optical absorption resonance in films of the polymer P(NDI2OD-T2). We show that this approach produces exceptional agreement with UV-Vis-NIR absorption measurements, while simultaneously avoiding the need to construct complicated multi-oscillator spectral models. Finally, we use this procedure to resolve subtle differences in the out-of-plane optical properties of different film morphologies that were previously obscured in ellipsometry measurements.

Sphingosine 1-phosphate receptor 1 (S1P(1)) upregulation and amelioration of experimental autoimmune encephalomyelitis by an S1P(1) antagonist.

Sphingosine 1-phosphate receptor 1 (S1P(1)) is a G protein-coupled receptor that is critical for proper lymphocyte development and recirculation. Agonists to S1P(1) are currently in use clinically for the treatment of multiple sclerosis, and these drugs may act on both S1P(1) expressed on lymphocytes and S1P(1) expressed within the central nervous system. Agonists to S1P(1) and deficiency in S1P(1) both cause lymphocyte sequestration in the lymph nodes. In the present study, we show that S1P(1) antagonism induces lymphocyte sequestration in the lymph nodes similar to that observed with S1P(1) agonists while upregulating S1P(1) on lymphocytes and endothelial cells. Additionally, we show that S1P(1) antagonism reverses experimental autoimmune encephalomyelitis in mice without acting on S1P(1) expressed within the central nervous system, demonstrating that lymphocyte sequestration via S1P(1) antagonism is sufficient to alleviate autoimmune pathology.

Small-molecule procaspase activators identified using fluorescence polarization.

Wake up, protein! Small molecules that directly activate proteins are rare and their discovery opens new avenues for the development of drugs and chemical tools to probe the functions and mechanisms of protein targets. To address the one-sided dichotomy between enzyme inhibition and activation, we describe a series of procaspase activators as chemical tools in the study of caspase biology.

Morphology-dependent light trapping in thin-film organic solar cells.

The active layer materials used in organic photovoltaic (OPV) cells often self-assemble into highly ordered morphologies, resulting in significant optical anisotropies. However, the impact of these anisotropies on light trapping in nanophotonic OPV architectures has not been considered. In this paper, we show that optical anisotropies in a canonical OPV material, P3HT, strongly affect absorption enhancements in ultra-thin textured OPV cells. In particular we show that plasmonic and gap-mode solar cell architectures redistribute electromagnetic energy into the out-of-plane field component, independent of the active layer orientation. Using analytical and numerical calculations, we demonstrate how the absorption in these solar cell designs can be significantly increased by reorienting polymer domains such that strongly absorbing axes align with the direction of maximum field enhancement.

Selective inhibitors and tailored activity probes for lipoprotein-associated phospholipase A(2).

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) or PLA(2)G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA(2) has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA(2) have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA(2) inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA(2) and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA(2) in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA(2) function in biological systems.

A sphingosine 1-phosphate receptor 2 selective allosteric agonist.

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.