Bioanalysis of the Bicycle® toxin conjugate BT5528 and released monomethyl auristatin E via liquid chromatography-tandem mass spectrometry.

Background: The Bicycle® toxin conjugate BT5528 is a novel peptide therapeutic conjugated to the cytotoxic agent monomethyl auristatin E (MMAE). A bioanalytical assay was developed to quantify BT5528 and unconjugated MMAE in human plasma. Methodology: BT5528 quantitation used a protein precipitation procedure followed by LC-MS/MS detection. Quantitation of MMAE required a selective offline and online solid-phase extraction with detection via LC-MS/MS. Results: BT5528 was quantified over the assay range of 5-2500 ng/ml and free MMAE was quantified over the assay range of 0.05-50 ng/ml. Conclusion: Bioanalytical methods were used in the bioanalysis of intact BT5528 and released MMAE, in a phase I/IIa clinical trial; to date, over 2000 human patient samples have been analyzed.

Lab Meets World: the Case for Use-Inspired Basic Research in Affective Science.

We join others in envisioning a future for affective science that addresses society's most pressing needs. To move toward this vision, we consider a research paradigm that emerged in other disciplines: use-inspired basic research. This paradigm transcends the traditional basic-applied dichotomy, which pits the basic goal of fundamental scientific understanding against the applied goal of use in solving social problems. In reality, these goals are complementary, and use-inspired basic research advances them simultaneously. Here, we build a case for use-inspired basic research-how it differs from traditional basic science and why affective scientists should engage in it. We first examine how use-inspired basic research challenges problematic assumptions of a strict basic-applied dichotomy. We then discuss how it is consistent with advances in affective science that recognize context specificity as the norm and consider ethical issues of use being a complementary goal. Following this theoretical discussion, we differentiate the implementation of use-inspired basic research from that of traditional basic science. We draw on examples from recent research to illustrate differences: social problems as a starting point, stakeholder and community engagement, and integration of research and service. In conclusion, we invite affective scientists to embrace the "lab meets world" perspective of use-inspired basic research as a promising pathway to real-world impact.

Using Mitra sampling to support first-in-human pharmacokinetic evaluations for PF-07059013.

Aim: A sensitive and selective method for the determination of PF-07059013 in dried blood collected by Mitra™ tips was developed and qualified from 50 to 50,000 ng/ml. Materials & methods: PF-07059013 is isolated from 10 µl of human dried blood by extraction with methanol and analyzed by HPLC-MS/MS. Results & conclusions: In addition to routine validation elements, impact of hematocrit and Mitra tip's lot-to-lot variation on assay accuracy were evaluated. The qualified method was used in one clinical study with excellent performance. Correlation coefficient between blood concentrations obtained from liquid-incurred blood samples and dried-incurred blood samples is 0.95. Clinical Trial Registration: NCT04323124 (ClinicalTrials.gov).

Conjugation of Fluorochromes to Monoclonal Antibodies.

Detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated to a fluorochrome is the most widely used application of flow cytometry. Here, we present protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, we provide a procedure for preparing a PE-Texas Red tandem conjugate dye that can then be used for antibody conjugation. These protocols enable investigators to label antibodies of their choice with multiple fluorochromes and permit more combinations of antibodies for multicolor flow applications. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Labeling an antibody with fluorescein isothiocyanate (FITC) Basic Protocol 2: Labeling an antibody with long-armed biotin Basic Protocol 3: Labeling an antibody with Texas Red-X Basic Protocol 4: Labeling an antibody with a synthetic organic fluor kit Basic Protocol 5: Labeling an antibody with phycobiliproteins Basic Protocol 6: Conjugation of Texas Red to R-phycoerythrin to produce an energy transfer fluorochrome.

Even simple framing effects are rational.

Bermúdez persuasively argues that framing effects are not as irrational as commonly supposed. In focusing on the reasoning of individual decision-makers in complex situations, however, he neglects the crucial role of the social-communicative context for eliciting certain framing effects. We contend that many framing effects are best explained in terms of basic, rational principles of discourse processing and pragmatic reasoning.

Who's the "Real" Victim? How Victim Framing Shapes Attitudes Toward Sexual Assault.

People accused of sexual assault are often described as the "real" victim by their defenders, but the impact of "victim framing" on public opinion is unknown. We investigated this issue across four experiments (N = 2,614). Online U.S. adult participants read a report about an alleged sexual assault that framed the female accuser as the victim (of assault), framed the male alleged perpetrator as the victim (of false accusations), or was neutral about victimhood (baseline). Relative to those in the baseline condition, participants in the assault- and allegation-victim conditions generally expressed more support for the victim-framed protagonist and less support for the other protagonist. The consistency of these effects varied with how often the victim frame was instantiated and whether the report described a fictionalized or real-world case. Across all contexts, however, participants who identified the victim-related language as influencing their evaluations exhibited strong framing effects. This suggests that social-pragmatic reasoning is a key mechanism by which victim framing shapes moral judgments.

STAT1 Gain-of-Function Mutations Cause High Total STAT1 Levels With Normal Dephosphorylation.

Signal transducer and activator of transcription (STAT1)1 gain of function (GOF) pathogenic variants have been associated with increased levels of phosphorylated STAT1 and STAT1-dependent cellular responses. Delayed dephosphorylation was proposed as the underlying mechanism leading to the characteristically raised pSTAT1 levels. We examined the levels of STAT1 protein and message as well as rates of STAT1 phosphorylation, dephosphorylation, and degradation associated with STAT1 GOF pathogenic variants. Fresh peripheral blood mononuclear cells (PBMC) from 14 STAT1 GOF patients carrying 10 different pathogenic variants in the coiled-coil, DNA binding, and SH2 domains and healthy donors were used to study STAT1 levels and phosphorylation (pSTAT1) following IFNγ and IFNα stimulation. STAT1 protein levels were measured by flow cytometry and immunoblot. STAT1 mRNA levels were measured using quantitative reverse transcription PCR. STAT1 protein degradation was studied using cycloheximide. Patient IFNγ and IFNα induced peak pSTAT1 was higher than in healthy controls. The velocity of pSTAT1 dephosphorylation after treatment of IFNγ stimulated CD14+ monocytes with the Janus Kinase (JAK)-inhibitor ruxolitinib was significantly faster in patient cells. STAT1 protein levels in patient CD14+ monocytes and CD3+ T cells were higher than in healthy donors. There was a strong and positive correlation between CD14+ STAT1 protein levels and peak pSTAT1 levels. Patient fresh PBMC STAT1 mRNA levels were increased at rest and after 16 h of incubation. STAT1 protein degradation was similar in patient and healthy volunteer cells. Patient IFNγ receptors 1 and 2 and JAK2 levels were normal. One patient in our cohort was treated with the oral JAK inhibitor ruxolitinib. Treatment was associated with normalization of both STAT1 protein and peak pSTAT1 levels. After JAK inhibitor treatment was stopped the patient's CD14+ monocyte STAT1 protein and peak phosphorylation levels increased proportionally. These findings suggest that patients with STAT1 GOF mutations have higher levels of total STAT1 protein, leading to high levels of pSTAT1 after stimulation, despite rapid STAT1 dephosphorylation and normal degradation.

Is Emotional Magnitude Spatialized? A Further Investigation.

Accumulating evidence suggests that different magnitudes (e.g., number, size, and duration) are spatialized in the mind according to a common left-right metric, consistent with a generalized system for representing magnitude. A previous study conducted by two of us (Holmes & Lourenco, ) provided evidence that this metric extends to the processing of emotional magnitude, or the intensity of emotion expressed in faces. Recently, however, Pitt and Casasanto () showed that the earlier effects may have been driven by a left-right mapping of mouth size rather than emotional magnitude, and they found no evidence for an emotional magnitude mapping when using words as stimuli. Here, we report two new experiments that further examine these conclusions. In Experiment 1, using face stimuli with mouths occluded, we replicate the original finding: Less emotional faces were associated with the left and more emotional faces with the right. However, we also find that people can reliably infer the sizes of the occluded mouths, and that these inferred mouth sizes can explain the observed left-right mapping. In Experiment 2, we show that comparative judgments of emotional words yield a left-right mapping of emotional magnitude not attributable to stimulus confounds. Based on these findings, we concur with Pitt and Casasanto that faces pose challenges for isolating the forces driving spatialization, but we suggest that emotional magnitude, when assessed using unconfounded stimuli in a sufficiently sensitive task, may indeed be spatialized as originally proposed. Suggestions for further research on the spatialization of emotional magnitude are discussed.

Structural characterization of the α-N-acetylglucosaminidase, a key enzyme in the pathogenesis of Sanfilippo syndrome B.

Mucopolysaccharidosis III B (MPS III-B) is a rare lysosomal storage disorder caused by deficiencies in Alpha-N-acetylglucosaminidase (NAGLU) for which there is currently no cure, and present treatment is largely supportive. Understanding the structure of NAGLU may allow for identification of novel therapeutic targets for MPS III-B. Here we describe the first crystal structure of human NAGLU, determined to a resolution of 2.3 Å. The crystal structure reveals a novel homotrimeric configuration, maintained primarily by hydrophobic and electrostatic interactions via domain II of three contiguous domains from the N- to C-terminus. The active site cleft is located between domains II and III. Catalytic glutamate residues, E316 and E446, are located at the top of the (α/ß)8 barrel structure in domain II. We utilized the three-dimensional structure of NAGLU to map several MPS III-B mutations, and hypothesize their functional consequences. Revealing atomic level structural information about this critical lysosomal enzyme paves the way for the design of novel therapeutics to target the underlying causes of MPS III-B.

Novel Impactor and Microsphere-Based Assay Used to Measure Containment of Aerosols Generated in a Flow Cytometer Cell Sorter.

Today's state-of-the-art cell sorting flow cytometers are equipped with aerosol containment systems designed to evacuate aerosols from the sort chamber during a sort. This biosafety device is especially important when the sort operator is sorting infectious or potentially infections samples. Hence, it is critical to evaluate the performance for this system in normal operation and in "failure" mode to determine the efficacy of containment. In the past decade, the most popular published method for evaluating containment has been the Glo-Germ bead procedure. These highly fluorescent and multisize particles can easily be detected on a microscope slide and enumerated using a fluorescent microscope. Collecting particles on this slide is accomplished using an Aerotech impactor. This sampler collects potentially escaping aerosols from the sort chamber before enumerating any particles. Although the Glo-Germ procedure has been adopted by many labs, there are several drawbacks with the procedure that have limited its adoption by cell sorter laboratories: The Aerotech impactor is a reusable device that requires rigorous cleaning between measurements. The surface area of the collection slide is large and difficult to scan on a fluorescence microscope. These beads produce a wide variation in sizes resulting in inconsistency in flow rates. Here, we describe a novel and replacement method utilizing a Cyclex-d impactor and Dragon Green beads. This method was compared for sensitivity of detection of escaped aerosols with a published method for aerosol detection which utilizes a UV-APS aerodynamic particle sizer and a UV-excitable dye. One of the advantages of the Cyclex-d system is the narrow-defined field of collection as compared to the standard Glo-Germ bead procedure, this means a smaller sampling area is used in the Cyclex-d impactor as compared to the AeroTech impactor. In addition, the sensitivity of detection was found to be better using the Cyclex-d collection device as compared to the standard Glo-Germ bead procedure. © 2018 International Society for Advancement of Cytometry.

Vascular Endothelial Growth Factor Enhances Compensatory Lung Growth in Piglets.

BACKGROUND: Vascular endothelial growth factor has been found to accelerate compensatory lung growth after left pneumonectomy in mice. The aim of this study was to determine the natural history and the effects of vascular endothelial growth factor on compensatory lung growth in a large animal model. METHODS: To determine the natural history of compensatory lung growth, female Yorkshire piglets underwent a left pneumonectomy on days of life 10-11. Tissue harvest and volume measurement of the right lung were performed at baseline (n = 5) and on postoperative days 7 (n = 5), 14 (n = 4), and 21 (n = 5). For pharmacokinetic studies, vascular endothelial growth factor was infused via a central venous catheter, with plasma vascular endothelial growth factor levels measured at various time points. To test the effect of vascular endothelial growth factor on compensatory lung growth, 26 female Yorkshire piglets underwent a left pneumonectomy followed by daily infusion of vascular endothelial growth factor at 200 µg/kg or isovolumetric 0.9% NaCl (saline control). Lungs were harvested on postoperative day 7 for volume measurement and morphometric analyses. RESULTS: Compared with baseline, right lung volume after left pneumonectomy increased by factors of 2.1 ± 0.6, 3.3 ± 0.6, and 3.6 ± 0.4 on postoperative days 7, 14, and 21, respectively. The half-life of VEGF ranged from 89 to 144 minutes. Lesser doses of vascular endothelial growth factor resulted in better tolerance, volume of distribution, and clearance. Compared with the control group, piglets treated with vascular endothelial growth factor had greater lung volume (P < 0.0001), alveolar volume (P = 0.001), septal surface area (P = 0.007) and total alveolar count (P = 0.01). CONCLUSION: Vascular endothelial growth factor enhanced alveolar growth in neonatal piglets after unilateral pneumonectomy.

Compound Words Reflect Cross-Culturally Shared Bodily Metaphors.

Parts of the body are often embedded in the structure of compound words, such as heartbreak and brainchild. We explored the relationships between the semantics of compounds and their constituent body parts, asking whether these relationships are largely arbitrary or instead reflect deeper metaphorical mappings shared across languages and cultures. In three studies, we found that U.S. English speakers associated the English translation equivalents of Chinese compounds with their constituent body parts at rates well above chance, even for compounds with highly abstract meanings and even when accounting for the semantic relatedness of the compounds and body parts. English speakers in India and Chinese speakers in Hong Kong showed similar intuitions about these associations. Our results suggest that the structure of compound words can provide insight into cross-culturally shared ways of connecting meaning to the body.

Revisiting the role of language in spatial cognition: Categorical perception of spatial relations in English and Korean speakers.

The spatial relation of support has been regarded as universally privileged in nonlinguistic cognition and immune to the influence of language. English, but not Korean, obligatorily distinguishes support from nonsupport via basic spatial terms. Despite this linguistic difference, previous research suggests that English and Korean speakers show comparable nonlinguistic sensitivity to the support/nonsupport distinction. Here, using a paradigm previously found to elicit cross-language differences in color discrimination, we provide evidence for a difference in sensitivity to support/nonsupport between native English speakers and native Korean speakers who were late English learners and tested in a context that privileged Korean. Whereas the former group showed categorical perception (CP) when discriminating spatial scenes capturing the support/nonsupport distinction, the latter did not. An additional group of native Korean speakers-relatively early English learners tested in an English-salient context-patterned with the native English speakers in showing CP for support/nonsupport. These findings suggest that obligatory marking of support/nonsupport in one's native language can affect nonlinguistic sensitivity to this distinction, contra earlier findings, but that such sensitivity may also depend on aspects of language background and the immediate linguistic context.

Categorical Perception Beyond the Basic Level: The Case of Warm and Cool Colors.

Categories can affect our perception of the world, rendering between-category differences more salient than within-category ones. Across many studies, such categorical perception (CP) has been observed for the basic-level categories of one's native language. Other research points to categorical distinctions beyond the basic level, but it does not demonstrate CP for such distinctions. Here we provide such a demonstration. Specifically, we show CP in English speakers for the non-basic distinction between "warm" and "cool" colors, claimed to represent the earliest stage of color lexicon evolution. Notably, the advantage for discriminating colors that straddle the warm-cool boundary was restricted to the right visual field-the same behavioral signature previously observed for basic-level categories. This pattern held in a replication experiment with increased power. Our findings show that categorical distinctions beyond the basic-level repertoire of one's native language are psychologically salient and may be spontaneously accessed during normal perceptual processing.

Right idea, wrong magnitude system.

Leibovich et al. claim that number representations are non-existent early in life and that the associations between number and continuous magnitudes reside in stimulus confounds. We challenge both claims - positing, instead, that number is represented independently of continuous magnitudes already in infancy, but is nonetheless more deeply connected to other magnitudes through adulthood than acknowledged by the "sense of magnitude" theory.

Registered Replication Report: Strack, Martin, & Stepper (1988).

According to the facial feedback hypothesis, people's affective responses can be influenced by their own facial expression (e.g., smiling, pouting), even when their expression did not result from their emotional experiences. For example, Strack, Martin, and Stepper (1988) instructed participants to rate the funniness of cartoons using a pen that they held in their mouth. In line with the facial feedback hypothesis, when participants held the pen with their teeth (inducing a "smile"), they rated the cartoons as funnier than when they held the pen with their lips (inducing a "pout"). This seminal study of the facial feedback hypothesis has not been replicated directly. This Registered Replication Report describes the results of 17 independent direct replications of Study 1 from Strack et al. (1988), all of which followed the same vetted protocol. A meta-analysis of these studies examined the difference in funniness ratings between the "smile" and "pout" conditions. The original Strack et al. (1988) study reported a rating difference of 0.82 units on a 10-point Likert scale. Our meta-analysis revealed a rating difference of 0.03 units with a 95% confidence interval ranging from -0.11 to 0.16.

Gamble on gaze: Eye movements reflect the numerical value of blackjack hands.

We used a novel task-a blackjack game that naturally involves mental summation of numerical values-to investigate the role of attention in the mental number line (MNL) and to provide insight into the ecological validity of this representational format. By analyzing the spatial position of participants' spontaneous, task-irrelevant eye movements, we avoided some of the limitations of previous research on the MNL, in which the findings could be attributed to task-specific factors such as the use of overt spatial cues. In two experiments, we found that eye movements along the horizontal axis reflected the overall numerical value of participants' hands, with smaller-value hands eliciting fixations toward the left of the screen and larger-value hands eliciting fixations toward the right. This pattern held even when controlling for the number of cards in the hand and the value of the card most recently dealt-suggesting that the effects were driven by mental summation of values, not merely by the processing of serial order or individual numbers. Vertical eye movements, in contrast, reflected hand value less reliably. In showing that spontaneous eye movements along the horizontal axis track the magnitude of internally computed sums in an ecologically relevant task, our findings provide evidence for a dynamic MNL that supports magnitude-driven shifts of attention and that may be recruited during everyday forms of numerical reasoning.

Equal access for all? Access to medical information for European psychiatric trainees.

Access to medical information is important as lifelong scientific learning is in close relation with a better career satisfaction in psychiatry. This survey aimed to investigate how medical information sources are being used among members of the European Federation of Psychiatric Trainees. Eighty-three psychiatric trainees completed our questionnaire. A significant variation was found, and information availability levels were associated with training duration and average income. The most available sources were books and websites, but the most preferred ones were scientific journals. Our findings suggest that further steps should be taken to provide an equal access to medical information across Europe.

PSD-95 regulates CRFR1 localization, trafficking and ß-arrestin2 recruitment.

Corticotropin-releasing factor (CRF) is a neuropeptide commonly associated with the hypothalamic-pituitary adrenal axis stress response. Upon release, CRF activates two G protein-coupled receptors (GPCRs): CRF receptor 1 (CRFR1) and CRF receptor 2 (CRFR2). Although both receptors contribute to mood regulation, CRFR1 antagonists have demonstrated anxiolytic and antidepressant-like properties that may be exploited in the generation of new pharmacological interventions for mental illnesses. Previous studies have demonstrated CRFR1 capable of heterologously sensitizing serotonin 2A receptor (5-HT2AR) signaling: another GPCR implicated in psychiatric disease. Interestingly, this phenomenon was dependent on Postsynaptic density 95 (PSD-95)/Disc Large/Zona Occludens (PDZ) interactions on the distal carboxyl termini of both receptors. In the current study, we demonstrate that endogenous PSD-95 can be co-immunoprecipitated with CRFR1 from cortical brain homogenate, and this interaction appears to be primarily via the PDZ-binding motif. Additionally, PSD-95 colocalizes with CRFR1 within the dendritic projections of cultured mouse neurons in a PDZ-binding motif-dependent manner. In HEK 293 cells, PSD-95 overexpression inhibited CRFR1 endocytosis, whereas PSD-95 shRNA knockdown enhanced CRFR1 endocytosis. Although PSD-95 does not appear to play a significant role in CRF-mediated cAMP or ERK1/2 signaling, PSD-95 was demonstrated to suppress ß-arrestin2 recruitment: providing a potential mechanism for PSD-95's inhibition of endocytosis. In revisiting previously documented heterologous sensitization, PSD-95 shRNA knockdown did not prevent CRFR1-mediated enhancement of 5-HT2AR signaling. In conclusion, we have identified and characterized a novel functional relationship between CRFR1 and PSD-95 that may have implications in the design of new treatment strategies for mental illness.