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Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head pain, as in disorders such as migraine. Eye squint has emerged as a continuous variable metric that can be measured over time and is effective for predicting pain states in such assays. This paper provides a protocol for the use of DeepLabCut (DLC) to automate and quantify eye squint (Euclidean distance between eyelids) in restrained mice with freely rotating head motions. This protocol enables unbiased quantification of eye squint to be paired with and compared directly against mechanistic measures such as neurophysiology. We provide an assessment of AI training parameters necessary for achieving success as defined by discriminating squint and non-squint periods. We demonstrate an ability to reliably track and differentiate squint in a CGRP-induced migraine-like phenotype at a sub second resolution.

Background: Increased vulnerability to stress is a major risk factor for several mood disorders, including major depressive disorder. Although cellular and molecular mechanisms associated with depressive behaviors following stress have been identified, little is known about the mechanisms that confer the vulnerability that predisposes individuals to future damage from chronic stress., Methods: We used multisite in vivo neurophysiology in freely behaving male and female C57BL/6 mice (n = 12) to measure electrical brain network activity previously identified as indicating a latent stress vulnerability brain state. We combined this neurophysiological approach with single-cell RNA sequencing of the prefrontal cortex to identify distinct transcriptomic differences between groups of mice with inherent high and low stress vulnerability., Results: We identified hundreds of differentially expressed genes (p adjusted < .05) across 5 major cell types in animals with high and low stress vulnerability brain network activity. This unique analysis revealed that GABAergic (gamma-aminobutyric acidergic) neuron gene expression contributed most to the network activity of the stress vulnerability brain state. Upregulation of mitochondrial and metabolic pathways also distinguished high and low vulnerability brain states, especially in inhibitory neurons. Importantly, genes that were differentially regulated with vulnerability network activity significantly overlapped (above chance) with those identified by genome-wide association studies as having single nucleotide polymorphisms significantly associated with depression as well as genes more highly expressed in postmortem prefrontal cortex of patients with major depressive disorder., Conclusions: This is the first study to identify cell types and genes involved in a latent stress vulnerability state in the brain., (Copyright © 2024 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Schizophrenia is an idiopathic psychiatric disorder with a high degree of polygenicity. Evidence from genetics, single-cell transcriptomics, and pharmacological studies suggest an important, but untested, overlap between genes involved in the etiology of schizophrenia and the cellular mechanisms of action of antipsychotics. To directly compare genes with antipsychotic-induced differential expression to genes involved in schizophrenia, we applied single-cell RNA-sequencing to striatal samples from male C57BL/6 J mice chronically exposed to a typical antipsychotic (haloperidol), an atypical antipsychotic (olanzapine), or placebo. We identified differentially expressed genes in three cell populations identified from the single-cell RNA-sequencing (medium spiny neurons [MSNs], microglia, and astrocytes) and applied multiple analysis pipelines to contextualize these findings, including comparison to GWAS results for schizophrenia. In MSNs in particular, differential expression analysis showed that there was a larger share of differentially expressed genes (DEGs) from mice treated with olanzapine compared with haloperidol. DEGs were enriched in loci implicated by genetic studies of schizophrenia, and we highlighted nine genes with convergent evidence. Pathway analyses of gene expression in MSNs highlighted neuron/synapse development, alternative splicing, and mitochondrial function as particularly engaged by antipsychotics. In microglia, we identified pathways involved in microglial activation and inflammation as part of the antipsychotic response. In conclusion, single-cell RNA sequencing may provide important insights into antipsychotic mechanisms of action and links to findings from psychiatric genomic studies., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The architecture whereby activity across many brain regions integrates to encode individual appetitive social behavior remains unknown. Here we measure electrical activity from eight brain regions as mice engage in a social preference assay. We then use machine learning to discover a network that encodes the extent to which individual mice engage another mouse. This network is organized by theta oscillations leading from prelimbic cortex and amygdala that converge on the ventral tegmental area. Network activity is synchronized with cellular firing, and frequency-specific activation of a circuit within this network increases social behavior. Finally, the network generalizes, on a mouse-by-mouse basis, to encode individual differences in social behavior in healthy animals but fails to encode individual behavior in a 'high confidence' genetic model of autism. Thus, our findings reveal the architecture whereby the brain integrates distributed activity across timescales to encode an appetitive brain state underlying individual differences in social behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mentoring is a developmental experience intended to increase the willingness to learn and establish credibility while building positive relationships through networking. In this commentary, we focus on intentional mentoring for underrepresented mentees, including individuals that belong to minority racial, ethnic and gender identity groups in Science, Technology, Engineering, Mathematics and Medicine (STEMM) fields. Intentional mentoring is the superpower action necessary for developing harmony and comprehending the purpose and value of the mentor/mentee relationship. Regardless of a mentor's career stage, we believe the strategies discussed may be used to create a supportive and constructive mentorship environment; thereby improving the retention rates of underrepresented mentees within the scientific community., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

While it is commonly thought that microaggressions are isolated incidents, microaggressions are ingrained throughout the academic research institution (Young, Anderson and Stewart 2015; Lee et al. 2020). Persons Excluded from science because of Ethnicity and Race (PEERs) frequently experience microaggressions from various academicians, including graduate students, postdocs and faculty (Asai 2020; Lee et al. 2020). Here, we elaborate on a rationale for concrete actions to cope with and diminish acts of microaggressions that may otherwise hinder the inclusion of PEERs. We encourage Science, Technology, Engineering and Mathematics (STEM) departments and leadership to affirm PEER scholar identities and promote allyship by infusing sensitivity, responsiveness and anti-bias awareness., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Background: We systematically analyzed the extent of disease in unifocal invasive lobular carcinoma (ILC) using ultrasonography, with the histopathologic findings as the reference standard., Patients and Methods: In the present single-institution retrospective study, 128 cases of ILC were identified during a 5-year period. After exclusions, the analyzed cohort included 66 cases. Ultrasound measurements of the tumor extent along 3 axes were obtained. The tumor size was determined as the largest extent among the 3 axes and the tumor volume by ellipsoidal approximation. Pathology review provided the tumor size and volume. Correlation and regression analyses of tumor size and volume from the ultrasound and pathologic examinations were performed. The tumor stage from the ultrasound and pathologic examinations were used for the concordance analyses., Results: The median and quartiles (Q1, Q3) of tumor size from ultrasonography and pathology were 12.5 mm (Q1, 9 mm; Q3, 19 mm) and 17 mm (Q1, 12 mm; Q3, 25 mm), respectively. The corresponding data for tumor volume were 0.52 cm 3 (Q1, 0.18 cm 3 ; Q3, 1.92 cm 3 ) and 1.04 cm 3 (Q1, 0.45 cm 3 ; Q3, 2.49 cm 3 ). The ultrasound measurements correlated with the pathology-reported tumor size (Spearman ρ = 0.678; P < .0001) and volume (Spearman ρ = 0.699; P < .0001). The ultrasound-measured size and volume differed from the pathology-reported size and volume (P < .0001; Wilcoxon signed ranks test). Concordance between the clinical tumor size stage from ultrasound (cT) and pathology tumor size stage (pT) varied with the pT stage (P = .0003, Fisher's exact test), with the greatest concordance rate of 95.7% (95% confidence limit, 85.2%-99.5%) observed for pT1 tumors., Conclusion: Ultrasonography underestimates the tumor size and volume, with the underestimation increasing for larger tumors. Hence, the concordance rate in tumor size stage between ultrasonography and pathology is tumor size dependent, with the greatest concordance rate observed for pT1 tumors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Brain-wide fluctuations in local field potential oscillations reflect emergent network-level signals that mediate behavior. Cracking the code whereby these oscillations coordinate in time and space (spatiotemporal dynamics) to represent complex behaviors would provide fundamental insights into how the brain signals emotional pathology. Using machine learning, we discover a spatiotemporal dynamic network that predicts the emergence of major depressive disorder (MDD)-related behavioral dysfunction in mice subjected to chronic social defeat stress. Activity patterns in this network originate in prefrontal cortex and ventral striatum, relay through amygdala and ventral tegmental area, and converge in ventral hippocampus. This network is increased by acute threat, and it is also enhanced in three independent models of MDD vulnerability. Finally, we demonstrate that this vulnerability network is biologically distinct from the networks that encode dysfunction after stress. Thus, these findings reveal a convergent mechanism through which MDD vulnerability is mediated in the brain., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The prefrontal cortex plays a critical role in regulating emotional behaviors, and dysfunction of prefrontal cortex-dependent networks has been broadly implicated in mediating stress-induced behavioral disorders including major depressive disorder., Methods: Here we acquired multicircuit in vivo activity from eight cortical and limbic brain regions as mice were subjected to the tail suspension test (TST) and an open field test. We used a linear decoder to determine whether cellular responses across each of the cortical and limbic areas signal movement during the TST and open field test. We then performed repeat behavioral testing to identify which brain areas show cellular adaptations that signal the increase in immobility induced by repeat TST exposure., Results: The increase in immobility observed during repeat TST exposure is linked to a selective functional upregulation of cellular activity in infralimbic cortex and medial dorsal thalamus, and to an increase in the spatiotemporal dynamic interaction between these structures. Inducing this spatiotemporal dynamic using closed-loop optogenetic stimulation is sufficient to increase movement in the TST in stress-naive mice, while stimulating above the carrier frequency of this circuit suppressed movement. This demonstrates that the adaptations in infralimbic cortex-medial dorsal thalamus circuitry observed after stress reflect a compensatory mechanism whereby the brain drives neural systems to counterbalance stress effects., Conclusions: Our findings provide evidence that targeting endogenous spatiotemporal dynamics is a potential therapeutic approach for treating stress-induced behavioral disorders, and that dynamics are a critical axis of manipulation for causal optogenetic studies., (Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Circuits distributed across cortico-limbic brain regions compose the networks that mediate emotional behavior. The prefrontal cortex (PFC) regulates ultraslow (<1 Hz) dynamics across these networks, and PFC dysfunction is implicated in stress-related illnesses including major depressive disorder (MDD). To uncover the mechanism whereby stress-induced changes in PFC circuitry alter emotional networks to yield pathology, we used a multi-disciplinary approach including in vivo recordings in mice and chronic social defeat stress. Our network model, inferred using machine learning, linked stress-induced behavioral pathology to the capacity of PFC to synchronize amygdala and VTA activity. Direct stimulation of PFC-amygdala circuitry with DREADDs normalized PFC-dependent limbic synchrony in stress-susceptible animals and restored normal behavior. In addition to providing insights into MDD mechanisms, our findings demonstrate an interdisciplinary approach that can be used to identify the large-scale network changes that underlie complex emotional pathologies and the specific network nodes that can be used to develop targeted interventions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sezary syndrome is a subtype of cutaneous T cell lymphoma which usually presents as generalized skin disease with erytheroderma. Distal organ involvement is rare and is usually a late finding in the course of the disease. Breast involvement is extremely rare. Herein, we present a case report of a patient whose initial presentation involved an intramammary lymph node prior to the onset of more characteristic skin disease. Sezary syndrome was confirmed by cythopathologic findings., (© 2015 Wiley Periodicals, Inc.)

Psychological stress contributes to the onset and exacerbation of nearly all neuropsychiatric disorders. Individual differences in stress-regulatory circuits can therefore dramatically affect vulnerability to these illnesses. Here we identify neural circuit mechanisms underlying individual differences in vulnerability to stress using a murine model of chronic social defeat stress. In chronically stressed mice, we find that the degree of prefrontal cortex (PFC) control of amygdala activity predicts stress susceptibility in individual mice. Critically, we also find that individual differences in PFC activation (that is, reactivity) during exposure to an aggressor mouse predict the emergence stress-induced behavioural deficits in stress-naïve mice. Finally, we show that naturally occurring differences in PFC reactivity directly correspond to the intrinsic firing rate of PFC neurons. This demonstrates that naturally occurring differences in PFC function underlie individual differences in vulnerability to stress, raising the hypothesis that PFC modulation may prevent stress-induced psychiatric disorders.

The disruption of neurotransmitter and neurotrophic factor signaling in the central nervous system (CNS) is implicated as the root cause of neuropsychiatric disorders, including schizophrenia, epilepsy, chronic pain, and depression. Therefore, identifying the underlying molecular mechanisms by which neurotransmitter and neurotrophic factor signaling regulates neuronal survival or growth may facilitate identification of more effective therapies for these disorders. Previously, our lab found that the heterotrimeric G protein, Gz, mediates crosstalk between G protein-coupled receptors and neurotrophin signaling in the neural cell line PC12. These data, combined with Gαz expression profiles--predominantly in neuronal cells with higher expression levels corresponding to developmental times of target tissue innervation--suggested that Gαz may play an important role in neurotrophin signaling and neuronal development. Here, we provide evidence in cortical neurons, both manipulated ex vivo and those cultured from Gz knockout mice, that Gαz is localized to axonal growth cones and plays a significant role in the development of axons of cortical neurons in the CNS. Our findings indicate that Gαz inhibits BDNF-stimulated axon growth in cortical neurons, establishing an endogenous role for Gαz in regulating neurotrophin signaling in the CNS., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Protein S-acylation is a major posttranslational modification whereby a cysteine thiol is converted to a thioester. A prototype is S-palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modification is a well-recognized determinant of protein function and localization, current techniques to study cellular S-acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S-nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S-acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S-acylation. Acyl-RAC should therefore find general applicability in studies of both global and individual protein S-acylation in mammalian cells.

Calmodulin (CaM) is a small (148 amino acid), ubiquitously expressed eukaryotic protein essential for Ca(2+) regulation and signaling. This highly acidic polypeptide (pI<4) has two homologous domains (N and C), each consisting of two EF-hand Ca(2+)-binding sites. Despite significant homology, the domains have intrinsic differences in their Ca(2+)-binding properties and separable roles in regulating physiological targets such as kinases and ion channels. In mammalian full-length CaM, sites III and IV in the C-domain bind Ca(2+) cooperatively with ~10-fold higher affinity than sites I and II in the N-domain. However, the difference is only twofold when CaM is severed at residue 75, indicating that anticooperative interactions occur in full-length CaM. The Ca(2+)-binding properties of sites I and II are regulated by several factors including the interplay of interdomain linker residues far from the binding sites. Our prior thermodynamic studies showed that these residues inhibit thermal denaturation and decrease calcium affinity. Based on high-resolution structures and NMR spectra, there appear to be interactions between charged residues in the sequence 75-80 and those near the amino terminus of CaM. To explore electrostatic contributions to interdomain interactions in CaM, KCl was used to perturb the Ca(2+)-binding affinity, thermal stability, and hydrodynamic size of a nested set of recombinant mammalian CaM (rCaM) fragments terminating at residues 75, 80, 85, or 90. Potassium chloride is known to decrease Ca(2+)-binding affinity of full-length CaM. It may act directly by competition with acidic side chains that chelate Ca(2+) in the binding sites, and indirectly elsewhere in the molecule by changing tertiary constraints and conformation. In all proteins studied, KCl decreased Ca(2+)-affinity, decreased Stokes radius, and increased thermal stability, but not monotonically. Crystallographic structures of Ca(2+)-saturated rCaM(1-75) (3B32.pdb) and rCaM(1-90) (3IFK.pdb) were determined, offering cautionary notes about the effect of packing interactions on flexible linkers. This chapter describes an array of methods for characterizing system-specific thermodynamic properties that in concert govern structure and function., (Copyright © 2009 Elsevier Inc. All rights reserved.)

A hydrophobic core is a widely accepted determinant of protein stability. However, regulatory proteins undergoing ligand-induced conformational switching may expose interior residues to solvent and cannot afford to be extremely rigid. Optimizing the energetic balance between stability and binding is challenging. The addition of five interdomain residues to rat and Paramecium calmodulin N-domain fragments (residues 1-75) increased their thermostability by 9 degrees C and lowered their calcium affinity by a factor of 4. This demonstrates that the flexible linker regulates functional properties as well as tethering the neighboring domains and that protein stability may be increased markedly by minor modifications of the C-terminus. The sensitivity of this domain to few and conservative variations in helices A and D (D2E, S17A, T70S and M71L) is demonstrated by the rat CaM fragments having lower stability and higher calcium affinity than fragments of the same length derived from Paramecium CaM.

A comparison of the Q3 and Hybrid III 3-year-old crash test dummies is presented in this paper. The performance of the dummies were compared in sixty biofidelity tests, seventy-seven static out-of-position airbag tests and sixty-three calibration tests. Various time histories and other data pertaining to accelerations, deflections, forces and moments are compared. In addition, the ease of positioning, handling, and the durability of the dummies in various out-of-position test configurations was assessed. Both the Q3 and Hybrid III 3-year-old dummies were calibrated to their respective specifications. The Hybrid III 3-year-old met its calibration requirements, while the Q3 did not always meet its own calibration requirements. The calibration specifications of the Q3 dummy need to be re-examined and possibly refined. The biofidelity of the Q3 and Hybrid III 3-year-old dummies were evaluated in both frontal and lateral test modes. Each dummy was evaluated against its own and the other's specified requirements, when possible. In the frontal test mode, the Hybrid III 3-year-old acceptably met all of its requirements. The Q3 dummy did not meet all of its own frontal biofidelity requirements. Based on these results, the Hybrid III 3-year-old is more biofidelic for primarily frontal loading conditions. With respect to the lateral biofidelity specifications, neither the Hybrid III 3-year-old nor the Q3 dummy met the requirements for the thorax and pelvis tests performed. Both dummies met the head drop requirements. Neither dummy is recommended for lateral loading conditions. For lateral testing where only the head is impacted, the Hybrid III 3-year-old could be used. In general, the responses of both dummies were repeatable in both the frontal and lateral biofidelity tests performed. The Hybrid III 3-year-old and the Q3 dummies were evaluated in static out-of-position airbag tests with three different side airbag systems (two seat-mounted and one door-mounted system), and one frontal passenger airbag system. Throughout this testing, the Q3 resultant head accelerations exhibited an excessive amount of high-frequency noise causing this dummy to be unacceptable for static out-of-position airbag testing. No significant issues were found with the Hybrid III 3-yearold. It was also determined that the Q3 dummy was more difficult to position repeatedly than the Hybrid III 3-yearold. This was due to the dummy's construction and its lack of rigid landmarks.