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The protein, azurin, has enabled the study of the tryptophan radical. Upon UV excitation of tyrosine-deficient apoazurin and in the presence of a Co(III) electron acceptor, the neutral radical (W48•) is formed. The lifetime of W48• in apoazurin is 41 s, which is shorter than the lifetime of several hours in Zn-substituted azurin. Molecular dynamics simulations revealed enhanced fluctuations of apoazurin which likely destabilize W48•. The photophysics of W48 was investigated to probe the precursor state for ET. The phosphorescence intensity was eliminated in the presence of an electron acceptor while the fluorescence was unchanged; this quenching of the phosphorescence is attributed to ET. The kinetics associated with W48• were examined with a model that incorporates intersystem crossing, ET, deprotonation, and decay of the cation radical. The estimated rate constants for ET (6 × 106 s-1) and deprotonation (3 × 105 s-1) are in agreement with a photoinduced mechanism where W48• is derived from the triplet state. The triplet as the precursor state for ET was supported by photolysis of apoazurin with 280 nm in the absence and presence of triplet-absorbing 405 nm light. Absorption bands from the neutral radical were observed only in the presence of blue light.

PurposeTo determine if treatment with a photobiomodulation (PBM) device results in greater improvement in central subfield thickness (CST) than placebo in eyes with center-involved diabetic macular edema (CI-DME) and good vision.DesignPhase 2 randomized clinical trial.ParticipantsParticipants had CI-DME and visual acuity (VA) 20/25 or better in the study eye and were recruited from 23 clinical sites in the United States.MethodsOne eye of each participant was randomly assigned 1:1 to a 670-nm light-emitting PBM eye patch or an identical device emitting broad-spectrum white light at low power. Treatment was applied for 90 seconds twice daily for 4 months.Main outcome measuresChange in CST on spectral-domain OCT at 4 months.ResultsFrom April 2019 to February 2020, 135 adults were randomly assigned to either PBM (n = 69) or placebo (n = 66); median age was 62 years, 37% were women, and 82% were White. The median device compliance was 92% with PBM and 95% with placebo. OCT CST increased from baseline to 4 months by a mean (SD) of 13 (53) μm in PBM eyes and 15 (57) μm in placebo eyes, with the mean difference (95% confidence interval [CI]) being -2 (-20 to 16) μm (P = 0.84). CI-DME, based on DRCR Retina Network sex- and machine-based thresholds, was present in 61 (90%) PBM eyes and 57 (86%) placebo eyes at 4 months (adjusted odds ratio [95% CI] = 1.30 (0.44-3.83); P = 0.63). VA decreased by a mean (SD) of -0.2 (5.5) letters and -0.6 (4.6) letters in the PBM and placebo groups, respectively (difference [95% CI] = 0.4 (-1.3 to 2.0) letters; P = 0.64). There were 8 adverse events possibly related to the PBM device and 2 adverse events possibly related to the placebo device. None were serious.ConclusionsPBM as given in this study, although safe and well-tolerated, was not found to be effective for the treatment of CI-DME in eyes with good vision.

To defend or burnish our moral reputation, we often tell moral narratives. Moral narratives describe morally relevant actions and explanations of those actions, detailing how people acted and why they did so. When and why does communication about moral events include descriptions of peoples’ actions and explanations? We hypothesize that informational, reputational, and presentational goals of narrators shape whether their communication contains clear actions and explanations. We asked a group of “narrators” to communicate with other people following a moral decision. Another group of “audience” members judged them based on their chosen statement. We find that the informational and reputational goals of narrators can explain what information they decide to reveal. Narrators choose what to say based on inferring the audience’s likely perceptions but underestimate how much audiences in fact expect answers to “what” and “why”. Audiences, however, do not always perceive the lack of expected information as indicative of deceit.

Narratives can communicate moral character by describing one's past actions and motivations. When telling moral narratives, people might twist the truth to appear better than they are, while concealing this goal. We show that audiences evaluate a narrator’s moral character by inferring weights on three goals: providing accurate information, appearing morally good, and projecting an image of informativeness. Participants judged how narrators explained their choice during a “claim” task. Narrators could claim a raise at the risk of causing a co-worker to lose money. They could then lie or tell the truth and be direct or indirect about their motivations (e.g., “I’m not selfish” vs. “It was better for the co-worker”). Our results suggest that to be perceived as morally good, narrators must find a balance: trying too hard to appear generous is costly. We introduce a model for how narratives are constructed through recursive inference of audience perception.

Impressions of others’ moral character are key to our social lives, but we rarely directly witness immoral acts, and rather rely on the stories we hear. This creates significant opportunities for narrators to distort their stories in an effort to appear more moral. How does the detection of such persuasion goals affect readers' impressions of the authors' character? Participants read autobiographical stories written by other participants about morally questionable actions they did – written once with no goal, and then again with the goal of appearing morally good or bad. Readers were very accurate in detecting the authors’ goals, but these were nonetheless effective in modulating character impressions. Critically, this effect vanished when readers thought the author didn't care about communicating information accurately. This suggests that audiences sometimes fail to discount narrators' goals when evaluating their character, but only when goals don't come at the cost of communicating information accurately.

For three decades, the LPL-specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.

Cryo-electron microscopy is an essential tool for high-resolution structural studies of biological systems. This method relies on the use of phase contrast imaging at high defocus to improve information transfer at low spatial frequencies at the expense of higher spatial frequencies. Here we demonstrate that electron ptychography can recover the phase of the specimen with continuous information transfer across a wide range of the spatial frequency spectrum, with improved transfer at lower spatial frequencies, and as such is more efficient for phase recovery than conventional phase contrast imaging. We further show that the method can be used to study frozen-hydrated specimens of rotavirus double-layered particles and HIV-1 virus-like particles under low-dose conditions (5.7 e/Å2) and heterogeneous objects in an Adenovirus-infected cell over large fields of view (1.14 × 1.14 μm), thus making it suitable for studies of many biologically important structures.

α-Linolenic acid (ALA) is an essential fatty acid and the precursor for long-chain n-3 PUFA. However, biosynthesis of n-3 PUFA is limited in a Western diet likely due to an overabundance of n-6 PUFA. We hypothesized that dietary reduction of n-6/n-3 PUFA ratio is sufficient to promote the biosynthesis of long-chain n-3 PUFA, leading to an attenuation of high fat (HF) diet-induced obesity and inflammation. C57BL/6 J mice were fed a HF diet from ALA-enriched butter (n3Bu, n-6/n-3=1) in comparison with isocaloric HF diets from either conventional butter lacking both ALA and LA (Bu, n-6/n-3=6), or margarine containing a similar amount of ALA and abundant LA (Ma, n-6/n-3=6). Targeted lipidomic analyses revealed that n3Bu feeding promoted the bioconversion of long-chain n-3 PUFA and their oxygenated metabolites (oxylipins) derived from ALA and EPA. The n3Bu supplementation attenuated hepatic TG accumulation and adipose tissue inflammation, resulting in improved insulin sensitivity. Decreased inflammation by n3Bu feeding was attributed to the suppression of NF-κB activation and M1 macrophage polarization. Collectively, our work suggests that dietary reduction of the n-6/n-3 PUFA ratio, as well as total n-3 PUFA consumed, is a crucial determinant that facilitates n-3 PUFA biosynthesis and subsequent lipidomic modifications, thereby conferring metabolic benefits against obesity-induced inflammation and insulin resistance.

Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely diffusing NDs. In this article, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism, and ultraviolet resonance Raman spectroscopy to confirm the spontaneous folding of outer membrane protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The circular dichroism spectra of OmpA in NDs and SUVs were similar and indicated β-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by ultraviolet resonance Raman spectroscopy, which revealed the intense 785 cm-1 structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was two- to threefold slower in NDs compared to in SUVs, and this decreased rate can tentatively be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments and offer benefits of optical clarity, sample homogeneity, control of ND:protein ratios, and greater stability.

A key characteristic of color perception is that it both categorical and continuous. This is reflected in graded color ad-jective use. This red fruit is redder than the other red fruit sounds more natural than this red fruit is redder than the bluefruit (Kennedy & McNally, 2010). We examined the contribution of first-person sensory experience to color gradabilityunderstanding by working with congenitally blind adults. Blind (n=20) and sighted (n=15) adults rated the naturalness ofstatements describing two objects of the same color (two red mugs), dissimilar colors (red mug, blue mug) or similar col-ors (red mug, purple mug). Both groups judged redder as most natural for two red objects, least for objects with differentcolors (red/blue) and intermediate for objects with similar colors (red/purple). Color similarity had a larger effect for thesighted group. Understanding color gradability does not require first-person perception.

Why do we describe blueberries as blue as opposed to white (their inside color)? People might label object colors entirelyaccording to what they see most frequently. We hypothesized instead that labeling takes into account typical viewingconditions (outside/daytime) and object causal history (colors relationship to function; Cohen, 2004). We further predictedthat these intuitions develop independently of visual experience. Sighted (n=15) and congenitally blind (n=20) participantschose one of two color labels for novel objects, described as having different colors (or textures) on the inside/outsideor during daylight/nighttime. On some day/night trials, objects had nighttime-intended functions. Sighted and blindindividuals alike chose observer-centric outside and day colors by default, but switched to nighttime colors when objectshad nighttime functions. First-person visual experience is not required for color-labeling to take into account observercharacteristics and object causal history.

Locke argued that persons born blind do not possess true knowledge about color. While prior studies find some knowledgeof color among blind individuals, questions remain about the depth of this knowledge. Do blind individuals merely learninferentially shallow verbal associations (e.g., bananayellow)? We hypothesized instead that blind individuals are morelikely to acquire causally-relevant color information. Blind (n=20) and sighted adults (n=20) reported colors of naturalkinds (e.g. banana) and artifacts (e.g. car) and judged the likelihood that two instances of a type have the same color.Relative to the sighted, blind participants were less likely to know specific object colors (e.g. banana-yellow), but madeidentical inferences about color consistency (more consistent colors for natural kinds). Inferences were similar acrossgroups even for novel objects. Further, blind individuals gave detailed and coherent causal explanations of color origins.Inferentially rich knowledge of sensory categories can develop without first-person experience.

Ultraviolet resonance Raman (UVRR) spectroscopy is a vibrational technique that reveals structures and dynamics of biological macromolecules without the use of extrinsic labels. By tuning the Raman excitation wavelength to the deep UV region (e.g., 228 nm), Raman signal from tryptophan and tyrosine residues are selectively enhanced, allowing for the study of these functionally relevant amino acids in lipid and aqueous environments. In this chapter, we present methods on the UVRR data acquisition and analysis of the tryptophan vibrational modes of a model β-barrel membrane protein, OmpA, in folded and unfolded conformations.

Abstract Background Research has documented that housing conditions can negatively impact the health of residents. Asthma has many known indoor environmental triggers including dust, pests, smoke and mold, as evidenced by the 25 million people in the U.S. population who have asthma. The paper describes a follow-up study involving elder adults with asthma who participated in a multifaceted home educational and environmental intervention shown to produce significant health benefits. On average the time between the end of the prior intervention study and the follow-up was 2.3 years. The objective of this study was to evaluate whether improvements in environmental conditions and health outcomes resulting from the original Older Adult Study (OAS, multifaceted educational and environmental interventions) would be maintained or decline over time for these low income seniors with asthma. Methods Health assessment included data on respiratory health outcomes included the Saint George’s Respiratory Questionnaire (SGRQ) and Asthma Control Test from the original Older Adult Study (OAS) and this follow-up Older Adult Study (OAFS) along with health care utilization data. Environmental assessments included evaluation of asthma trigger activities (ATAs) and exposures before and after the original healthy homes intervention (questionnaire, home survey) and at this follow-up. Assessments were conducted in English, Khmer and Spanish. Results At assessment in the Older Adult Follow-up Study (OAFS), the older adults maintained some of the health improvements gained during the OAS when compared to the OAS pre-intervention baseline. However, health outcomes declined from the OAS final assessment to the OAFS (only the SGRQ Impact scores were significantly different). Conclusion These findings suggest that further study with a larger population is needed to determine if the significant health outcome improvements from multifaceted home educational and environmental interventions (OAS) could be more strongly maintained by providing additional follow-up “booster” interventions to this older adult population with asthma.

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER-Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. Since Hsp70, the major inducible hsp, has been detected on surface of stressed cells, we propose that membrane interaction is the first step in the export process. The question that emerges is how does this charged cytosolic protein interact with lipid membranes? Prior studies have shown that Hsp70 formed ion conductance pathways within artificial lipid bilayers. These early observations have been extended herewith using a liposome insertion assay. We showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high-molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments and the region embedded into the lipid membrane was mapped toward the C-terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted into a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.

BackgroundFluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.MethodologyHere, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.ConclusionsOur results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library.