Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes.

Increased immune evasion by SARS-CoV-2 variants of concern highlights the need for new therapeutic neutralizing antibodies. Immunization with nanoparticles co-displaying spike receptor-binding domains (RBDs) from eight sarbecoviruses (mosaic-8 RBD-nanoparticles) efficiently elicits cross-reactive polyclonal antibodies against conserved sarbecovirus RBD epitopes. Here, we identified monoclonal antibodies (mAbs) capable of cross-reactive binding and neutralization of animal sarbecoviruses and SARS-CoV-2 variants by screening single mouse B cells secreting IgGs that bind two or more sarbecovirus RBDs. Single-particle cryo-EM structures of antibody-spike complexes, including a Fab-Omicron complex, mapped neutralizing mAbs to conserved class 1/4 RBD epitopes. Structural analyses revealed neutralization mechanisms, potentials for intra-spike trimer cross-linking by IgGs, and induced changes in trimer upon Fab binding. In addition, we identified a mAb-resembling Bebtelovimab, an EUA-approved human class 3 anti-RBD mAb. These results support using mosaic RBD-nanoparticle vaccination to generate and identify therapeutic pan-sarbecovirus and pan-variant mAbs.

A CD38-directed, single-chain T-cell engager targets leukemia stem cells through IFN-γ-induced CD38 expression.

ABSTRACT: Treatment resistance of leukemia stem cells (LSCs) and suppression of the autologous immune system represent major challenges to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), LSCs are frequently enriched in the CD34posCD38neg blast fraction. Here, we report that interferon gamma (IFN-γ) reduces LSCs clonogenic activity and induces CD38 upregulation in both CD38pos and CD38neg LSC-enriched blasts. IFN-γ-induced CD38 upregulation depends on interferon regulatory factor 1 transcriptional activation of the CD38 promoter. To leverage this observation, we created a novel compact, single-chain CD38-CD3 T-cell engager (BN-CD38) designed to promote an effective immunological synapse between CD38pos AML cells and both CD8pos and CD4pos T cells. We demonstrate that BN-CD38 engages autologous CD4pos and CD8pos T cells and CD38pos AML blasts, leading to T-cell activation and expansion and to the elimination of leukemia cells in an autologous setting. Importantly, BN-CD38 engagement induces the release of high levels of IFN-γ, driving the expression of CD38 on CD34posCD38neg LSC-enriched blasts and their subsequent elimination. Critically, although BN-CD38 showed significant in vivo efficacy across multiple disseminated AML cell lines and patient-derived xenograft models, it did not affect normal hematopoietic stem cell clonogenicity and the development of multilineage human immune cells in CD34pos humanized mice. Taken together, this study provides important insights to target and eliminate AML LSCs.

Mass Spectrometric Detection of Formaldehyde-Crosslinked PBMC Proteins in Cell-Free DNA Blood Collection Tubes.

Streck tubes are commonly used to collect blood samples to preserve cell-free circulating DNA. They contain imidazolidinyl urea as a formaldehyde-releasing agent to stabilize cells. We investigated whether the released formaldehyde leads to crosslinking of intracellular proteins. Therefore, we employed a shotgun proteomics experiment on human peripheral blood mononuclear cells (PBMCs) that were isolated from blood collected in Streck tubes, EDTA tubes, EDTA tubes containing formaldehyde, or EDTA tubes containing allantoin. The identified crosslinks were validated in parallel reaction monitoring LC/MS experiments. In total, we identified and validated 45 formaldehyde crosslinks in PBMCs from Streck tubes, which were also found in PBMCs from formaldehyde-treated blood, but not in EDTA- or allantoin-treated samples. Most were derived from cytoskeletal proteins and histones, indicating the ability of Streck tubes to fix cells. In addition, we confirm a previous observation that formaldehyde crosslinking of proteins induces a +24 Da mass shift more frequently than a +12 Da shift. The crosslinking capacity of Streck tubes needs to be considered when selecting blood-collection tubes for mass-spectrometry-based proteomics or metabolomic experiments.

Advancing motion denoising of multiband resting-state functional connectivity fMRI data.

Simultaneous multi-slice (multiband) accelerated functional magnetic resonance imaging (fMRI) provides dramatically improved temporal and spatial resolution for resting-state functional connectivity (RSFC) studies of the human brain in health and disease. However, multiband acceleration also poses unique challenges for denoising of subject motion induced data artifacts, the presence of which is a major confound in RSFC research that substantively diminishes reliability and reproducibility. We comprehensively evaluated existing and novel approaches to volume censoring-based motion denoising in the Human Connectome Project (HCP) dataset. We show that assumptions underlying common metrics for evaluating motion denoising pipelines, especially those based on quality control-functional connectivity (QC-FC) correlations and differences between high- and low-motion participants, are problematic, and appear to be inappropriate in their current widespread use as indicators of comparative pipeline performance and as targets for investigators to use when tuning pipelines for their own datasets. We further develop two new quantitative metrics that are instead agnostic to QC-FC correlations and other measures that rely upon the null assumption that no true relationships exist between trait measures of subject motion and functional connectivity, and demonstrate their use as benchmarks for comparing volume censoring methods. Finally, we develop and validate quantitative methods for determining dataset-specific optimal volume censoring parameters prior to the final analysis of a dataset, and provide straightforward recommendations and code for all investigators to apply this optimized approach to their own RSFC datasets.

Conformational Flexibility of p150Glued(1-191) Subunit of Dynactin Assembled with Microtubules.

Microtubule (MT)-associated proteins perform diverse functions in cells. These functions are dependent on their interactions with MTs. Dynactin, a cofactor of dynein motor, assists the binding of dynein to various organelles and is crucial to the long-distance processivity of dynein-based complexes. The largest subunit of dynactin, the p150Glued, contains an N-terminus segment that is responsible for the MT-binding interactions and long-range processivity of dynactin. We employed solution and magic angle spinning NMR spectroscopy to characterize the structure and dynamics of the p150Glued N-terminal region, both free and in complex with polymerized MTs. This 191-residue region encompasses the cytoskeleton-associated protein glycine-rich domain, the basic domain, and serine/proline-rich (SP-rich) domain. We demonstrate that the basic and SP-rich domains are intrinsically disordered in solution and significantly enhance the binding affinity to MTs as these regions contain the second MT-binding site on the p150Glued subunit. The majority of the basic and SP-rich domains are predicted to be random coil, whereas the segments S111-I116, A124-R132, and K144-T146 in the basic domain contain short α-helical or ß-sheet structures. These three segments possibly encompass the MT-binding site. Surprisingly, the protein retains a high degree of flexibility upon binding to MTs except for the regions that are directly involved in the binding interactions with MTs. This conformational flexibility may be essential for the biological functions of the p150Glued subunit.

A Platform To Enhance Quantitative Single Molecule Localization Microscopy.

Quantitative single molecule localization microscopy (qSMLM) is a powerful approach to study in situ protein organization. However, uncertainty regarding the photophysical properties of fluorescent reporters can bias the interpretation of detected localizations and subsequent quantification. Furthermore, strategies to efficiently detect endogenous proteins are often constrained by label heterogeneity and reporter size. Here, a new surface assay for molecular isolation (SAMI) was developed for qSMLM and used to characterize photophysical properties of fluorescent proteins and dyes. SAMI-qSMLM afforded robust quantification. To efficiently detect endogenous proteins, we used fluorescent ligands that bind to a specific site on engineered antibody fragments. Both the density and nano-organization of membrane-bound epidermal growth factor receptors (EGFR, HER2, and HER3) were determined by a combination of SAMI, antibody engineering, and pair-correlation analysis. In breast cancer cell lines, we detected distinct differences in receptor density and nano-organization upon treatment with therapeutic agents. This new platform can improve molecular quantification and can be developed to study the local protein environment of intact cells.

Relations between cortical thickness, serotonin 1A receptor binding, and structural connectivity: A multimodal imaging study.

Serotonin 1A (5-HT1A ) receptors play a direct role in neuronal development, cell proliferation, and dendritic branching. We hypothesized that variability in 5-HT1A binding can affect cortical thickness, and may account for a subtype of major depressive disorder (MDD) in which both are altered. To evaluate this, we measured cortical thickness from structural magnetic resonance imaging (MRI) and 5-HT1A binding by positron emission tomography (PET) in an exploratory study. To examine a range of 5-HT1A binding and cortical thickness values, we recruited 25 healthy controls and 19 patients with MDD. We hypothesized increased 5-HT1A binding in the raphe nucleus (RN) would be negatively associated with cortical thickness due to reduced serotonergic transmission. Contrary to our hypothesis, raphe 5-HT1A binding was positively correlated with cortical thickness in right posterior cingulate cortex (PCC), a region implicated in the default mode network. Cortical thickness was also positively correlated with 5-HT1A in each cortical region. We further hypothesized that the strength of 5-HT1A -cortical thickness correlation depends on the number of axons between the raphe nucleus and each region. To explore this we related 5-HT1A -cortical thickness correlation coefficients to the number of tracts connecting that region and the raphe, as measured by diffusion tensor imaging (DTI) in an independent sample. The 5-HT1A -cortical thickness association correlated significantly with the number of tracts to each region, supporting our hypothesis. We posit a defect in the raphe may affect the PCC within the default mode network in MDD through serotonergic fibers, resulting in increased ruminative processing.

Template-Catalyzed, Disulfide Conjugation of Monoclonal Antibodies Using a Natural Amino Acid Tag.

The high specificity and favorable pharmacological properties of monoclonal antibodies (mAbs) have prompted significant interest in re-engineering this class of molecules to add novel functionalities for enhanced therapeutic and diagnostic potential. Here, we used the high affinity, meditope-Fab interaction to template and drive the rapid, efficient, and stable site-specific formation of a disulfide bond. We demonstrate that this template-catalyzed strategy provides a consistent and reproducible means to conjugate fluorescent dyes, cytotoxins, or "click" chemistry handles to meditope-enabled mAbs (memAbs) and memFabs. More importantly, we demonstrate this covalent functionalization is achievable using natural amino acids only, opening up the opportunity to genetically encode cysteine meditope "tags" to biologics. As proof of principle, genetically encoded, cysteine meditope tags were added to the N- and/or C-termini of fluorescent proteins, nanobodies, and affibodies, each expressed in bacteria, purified to homogeneity, and efficiently conjugated to different memAbs and meFabs. We further show that multiple T-cell and Her2-targeting bispecific molecules using this strategy potently activate T-cell signaling pathways in vitro. Finally, the resulting products are highly stable as evidenced by serum stability assays (>14 d at 37 °C) and in vivo imaging of tumor xenographs. Collectively, the platform offers the opportunity to build and exchange an array of functional moieties, including protein biologics, among any cysteine memAb or Fab to rapidly create, test, and optimize stable, multifunctional biologics.

Disruption of TWIST1-RELA binding by mutation and competitive inhibition to validate the TWIST1 WR domain as a therapeutic target.

BACKGROUND: Most cancer deaths result from tumor cells that have metastasized beyond their tissue of origin, or have developed drug resistance. Across many cancer types, patients with advanced stage disease would benefit from a novel therapy preventing or reversing these changes. To this end, we have investigated the unique WR domain of the transcription factor TWIST1, which has been shown to play a role in driving metastasis and drug resistance. METHODS: In this study, we identified evolutionarily well-conserved residues within the TWIST1 WR domain and used alanine substitution to determine their role in WR domain-mediated protein binding. Co-immunoprecipitation was used to assay binding affinity between TWIST1 and the NFκB subunit p65 (RELA). Biological activity of this complex was assayed using a dual luciferase assay system in which firefly luciferase was driven by the interleukin-8 (IL-8) promoter, which is upregulated by the TWIST1-RELA complex. Finally, in order to inhibit the TWIST1-RELA interaction, we created a fusion protein comprising GFP and the WR domain. Cell fractionation and proteasome inhibition experiments were utilized to elucidate the mechanism of action of the GFP-WR fusion. RESULTS: We found that the central residues of the WR domain (W190, R191, E193) were important for TWIST1 binding to RELA, and for increased activation of the IL-8 promoter. We also found that the C-terminal 245 residues of RELA are important for TWIST1 binding and IL-8 promoter activation. Finally, we found the GFP-WR fusion protein antagonized TWIST1-RELA binding and downstream signaling. Co-expression of GFP-WR with TWIST1 and RELA led to proteasomal degradation of TWIST1, which could be inhibited by MG132 treatment. CONCLUSIONS: These data provide evidence that mutation or inhibition of the WR domain reduces TWIST1 activity, and may represent a potential therapeutic modality.

Internal dynamics of dynactin CAP-Gly is regulated by microtubules and plus end tracking protein EB1.

CAP-Gly domain of dynactin, a microtubule-associated activator of dynein motor, participates in multiple cellular processes, and its point mutations are associated with neurodegenerative diseases. Recently, we have demonstrated that conformational plasticity is an intrinsic property of CAP-Gly. To understand its origin, we addressed internal dynamics of CAP-Gly assembled on polymeric microtubules, bound to end-binding protein EB1 and free, by magic angle spinning NMR and molecular dynamics simulations. The analysis of residue-specific dynamics of CAP-Gly on time scales spanning nano- through milliseconds reveals its unusually high mobility, both free and assembled on polymeric microtubules. On the contrary, CAP-Gly bound to EB1 is significantly more rigid. Molecular dynamics simulations indicate that these motions are strongly temperature-dependent, and loop regions are surprisingly mobile. These findings establish the connection between conformational plasticity and internal dynamics in CAP-Gly, which is essential for the biological functions of CAP-Gly and its ability to bind to polymeric microtubules and multiple binding partners. In this work, we establish an approach, for the first time, to probe atomic resolution dynamic profiles of a microtubule-associated protein assembled on polymeric microtubules. More broadly, the methodology established here can be applied for atomic resolution analysis of dynamics in other microtubule-associated protein assemblies, including but not limited to dynactin, dynein, and kinesin motors assembled on microtubules.

RWD Domain as an E2 (Ubc9)-Interaction Module.

An RWD domain is a well conserved domain found through bioinformatic analysis of the human proteome sequence; however, its function has been unknown. Ubiquitin-like modifications require the catalysis of three enzymes generally known as E1, E2, and E3. We solved the crystal structure of the E2 for the small ubiquitin-like modifiers (SUMO) in complex with an RWD domain and confirmed the structure using solution NMR analysis. The binding surface of RWD on Ubc9 is located near the N terminus of Ubc9 that is known to be involved in noncovalent binding of the proteins in the conjugation machinery, including a domain of E1, SUMO, and an E3 ligase. NMR data indicate that the RWD domain does not bind to SUMO and E1. The interaction between RWD and Ubc9 has a Kd of 32 ± 4 µM. Consistent with the structure and binding affinity and in contrast to a previous report, the RWD domain and RWDD3 have minimal effects on global SUMOylation. The structural and biochemical information presented here forms the basis for further investigation of the functions of RWD-containing proteins.

L1CAM is an independent predictor of poor survival in endometrial cancer - An analysis of The Cancer Genome Atlas (TCGA).

BACKGROUND: L1-cell adhesion molecule (L1CAM) was previously reported to carry a poor prognosis in Stage I, low-risk endometrial cancer (EC). We evaluated the role of L1CAM among all stages and histologies in ECs in The Cancer Genome Atlas (TCGA). METHODS: Clinical information and RNA-Seq expression data were derived from TCGA uterine cancer cohort. Associations between L1CAM expression and clinical factors were tested with linear and logistic regression. Differences in survival between "high" and "low" expression groups (defined by median expression) of L1CAM were compared using Cox regression analysis, with p-values calculated via log-rank test. Kaplan-Meier curves were tested with the log-rank test. RESULTS: Patient characteristics of 545 primary tumors with RNA-Seq gene expression data were analyzed. Median age was 64years (range 31-90). Stage I, II, III, and IV comprised 62%, 10%, 23%, and 5%, respectively. 75% were endometrioid; 21% serous. Grade 1, 2, and 3 comprised 18%, 22%, and 60%, respectively. Median follow-up was 23.0months. High L1CAM expression was associated with advanced stage (OR 3.2), high grade (OR=6.8), serous histology (OR=16.3), positive cytology (OR=3.5), positive pelvic (OR=21.8) and para-aortic lymph nodes (OR=10.3) (all p≤0.001). High L1CAM was associated with a median overall survival (OS) of 107months, versus not reached for low L1-expressing ECs (HR=3.46, CI 1.97-6.07, p<0.001). On multivariate analysis, L1CAM expression remained an independent prognostic variable in predicting OS in EC. CONCLUSIONS: L1CAM expression is an independent predictor of poor survival in endometrial cancer, and is associated with advanced stage, high-risk endometrial cancer.

Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies.

Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximab (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Finally, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent "linker" for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.

Assessment of National CLAS Standards in Rural and Urban Local Health Departments in Kentucky.

CONTEXT: Findings from the Centers for Disease Control and Prevention suggest that addressing persistent health disparities based on race and ethnicity must become a national priority. The field of cultural and linguistic competency has gained national attention by improving access to and quality of health care, patient-provider communication, health outcomes, and health equity for minority groups and other vulnerable or special needs populations. OBJECTIVES: (1) To measure how local health departments (LHDs) in Kentucky comply with the National Standards for Culturally and Linguistically Appropriate Services (CLAS); and (2) to provide policy recommendation based on the findings. This study is the first to assess a statewide public health system under the lens of CLAS. DESIGN: Analysis of cross-sectional survey. SETTING: Electronic surveys administered to LHD administrators in Kentucky. PARTICIPANTS: Public health directors, nurse leaders, and program managers. MAIN OUTCOME MEASURE: Levels of compliance with various CLAS standards were examined for rural and urban counties using a novel scoring method. RESULTS: A total of 159 LHD administrators received the survey. Response rate was 67% (106 participants). Rural and urban LHDs achieved moderate compliance on domains of plans and policies, quality monitoring and improvement for needs assessment, management information systems for clients, and staff training and development. Both geographic groups exhibited lesser compliance on domains of organizational governance, culturally inclusive health care environment for educational materials, and diversity of personnel recruitment. CONCLUSION: County and district LHDs in Kentucky have implemented activities and policies that comply with CLAS standards at levels that vary by domain. Areas requiring particular attention include organizational governance, culturally inclusive health care environment for educational materials, and diversity of personnel. Improvements in these areas may help LHDs better meet the needs of vulnerable populations, racial and ethnic minorities, and special needs groups. CLAS practices may allow organizations to adhere to national public health accreditation standards.

Optogenetic versus Electrical Stimulation of Human Cardiomyocytes: Modeling Insights.

Optogenetics provides an alternative to electrical stimulation to manipulate membrane voltage, and trigger or modify action potentials (APs) in excitable cells. We compare biophysically and energetically the cellular responses to direct electrical current injection versus optical stimulation mediated by genetically expressed light-sensitive ion channels, e.g., Channelrhodopsin-2 (ChR2). Using a computational model of ChR2(H134R mutant), we show that both stimulation modalities produce similar-in-morphology APs in human cardiomyocytes, and that electrical and optical excitability vary with cell type in a similar fashion. However, whereas the strength-duration curves for electrical excitation in ventricular and atrial cardiomyocytes closely follow the theoretical exponential relationship for an equivalent RC circuit, the respective optical strength-duration curves significantly deviate, exhibiting higher nonlinearity. We trace the origin of this deviation to the waveform of the excitatory current-a nonrectangular self-terminating inward current produced in optical stimulation due to ChR2 kinetics and voltage-dependent rectification. Using a unifying charge measure to compare energy needed for electrical and optical stimulation, we reveal that direct electrical current injection (rectangular pulse) is more efficient at short pulses, whereas voltage-mediated negative feedback leads to self-termination of ChR2 current and renders optical stimulation more efficient for long low-intensity pulses. This applies to cardiomyocytes but not to neuronal cells (with much shorter APs). Furthermore, we demonstrate the cell-specific use of ChR2 current as a unique modulator of intrinsic activity, allowing for optical control of AP duration in atrial and, to a lesser degree, in ventricular myocytes. For self-oscillatory cells, such as Purkinje, constant light at extremely low irradiance can be used for fine control of oscillatory frequency, whereas constant electrical stimulation is not feasible due to electrochemical limitations. Our analysis offers insights for designing future new energy-efficient stimulation strategies in heart or brain.

Photocleavable dimerizer for the rapid reversal of molecular trap antagonists.

Herein, we report the development of a photocleavable analog of AP20187, a cell-permeable molecule used to dimerize FK506-binding protein (FKBP) fusion proteins and initiate biological signaling cascades and gene expression or disrupt protein-protein interactions. We demonstrate that this reagent permits the unique ability to rapidly and specifically antagonize a molecular interaction in vitro and follow a biological process due to this acute antagonism (e.g. endosome dispersion) and to release the trap upon photocleavage to follow the cell's return to homeostasis. In addition, this photocleavable AP20187 analog can be used in other systems where the dimerization of FKBP has been used to initiate signaling pathways, offering the ability to correlate the duration of a signaling event and a cellular response.

A systems thinking approach to analysis of the Patient Protection and Affordable Care Act.

The public health community is challenged with understanding the many complexities presented by systems thinking and its applications in systems modeling. The model presented encompasses multiple variables needed (eg, model building) for the construction of a conceptual system model of the Patient Protection and Affordable Care Act (ACA). The model tracks the ACA from inception, through passage, March 2010, to its current state. Justification for the need to reform the current health care system rests, in part, on the heels of social justice. Proponents of the ACA have long argued that health reform was needed by the millions of uninsured person who suffered from health disparities, took little advantage of health prevention advice, and faced issues of access to providers as well as insurers. In addition the ACA seeks to address our uncontrollable spending on health care delivery. This article highlights the ACA from a systems perspective. The conceptual model presented encompasses both health reform variables (eg, health care provisions, key legislative components, system environment) and system variables (eg, inputs, outputs, feedback, and throughput) needed to understand current health care reform efforts from a systems perspective. The model presented shows how the interrelationships and interconnections of elements of a system come together to achieve its purpose or goal.

Computational optogenetics: empirically-derived voltage- and light-sensitive channelrhodopsin-2 model.

Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silico prediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools.

A tale of two n-backs: Diverging associations of dorsolateral prefrontal cortex activation with n-back task performance.

Background: In studying the neural correlates of working memory (WM) ability via functional magnetic resonance imaging (fMRI) in health and disease, it is relatively uncommon for investigators to report associations between brain activation and measures of task performance. Additionally, how the choice of WM task impacts observed activation-performance relationships is poorly understood. We sought to illustrate the impact of WM task on brain-behavior correlations using two large, publicly available datasets. Methods: We conducted between-participants analyses of task-based fMRI data from two publicly available datasets: the Human Connectome Project (HCP; n = 866) and the Queensland Twin Imaging (QTIM) Study (n = 459). Participants performed two distinct variations of the n-back WM task with different stimuli, timings, and response paradigms. Associations between brain activation ([2-back - 0-back] contrast) and task performance (2-back % correct) were investigated separately in each dataset, as well as across datasets, within the dorsolateral prefrontal cortex (dlPFC), medial prefrontal cortex, and whole cortex. Results: Global patterns of activation to task were similar in both datasets. However, opposite associations between activation and task performance were observed in bilateral pre-supplementary motor area and left middle frontal gyrus. Within the dlPFC, HCP participants exhibited a significantly greater activation-performance relationship in bilateral middle frontal gyrus relative to QTIM Study participants. Conclusions: The observation of diverging activation-performance relationships between two large datasets performing variations of the n-back task serves as a critical reminder for investigators to exercise caution when selecting WM tasks and interpreting neural activation in response to a WM task.

Characterization and Mitigation of a Simultaneous Multi-Slice fMRI Artifact: Multiband Artifact Regression in Simultaneous Slices.

Simultaneous multi-slice (multiband) acceleration in fMRI has become widespread, but may be affected by novel forms of signal artifact. Here, we demonstrate a previously unreported artifact manifesting as a shared signal between simultaneously acquired slices in all resting-state and task-based multiband fMRI datasets we investigated, including publicly available consortium data from the Human Connectome Project (HCP) and Adolescent Brain Cognitive Development (ABCD) Study. We propose Multiband Artifact Regression in Simultaneous Slices (MARSS), a regression-based detection and correction technique that successfully mitigates this shared signal in unprocessed data. We demonstrate that the signal isolated by MARSS correction is likely non-neural, appearing stronger in neurovasculature than grey matter. Additionally, we evaluate MARSS both against and in tandem with sICA+FIX denoising, which is implemented in HCP resting-state data, to show that MARSS mitigates residual artifact signal that is not modeled by sICA+FIX. MARSS correction leads to study-wide increases in signal-to-noise ratio, decreases in cortical coefficient of variation, and mitigation of systematic artefactual spatial patterns in participant-level task betas. Finally, MARSS correction has substantive effects on second-level t-statistics in analyses of task-evoked activation. We recommend that investigators apply MARSS to multiband fMRI datasets with moderate or higher acceleration factors, in combination with established denoising methods.