Mapping brain function in adults and young children during naturalistic viewing with high-density diffuse optical tomography.

Human studies of early brain development have been limited by extant neuroimaging methods. MRI scanners present logistical challenges for imaging young children, while alternative modalities like functional near-infrared spectroscopy have traditionally been limited by image quality due to sparse sampling. In addition, conventional tasks for brain mapping elicit low task engagement, high head motion, and considerable participant attrition in pediatric populations. As a result, typical and atypical developmental trajectories of processes such as language acquisition remain understudied during sensitive periods over the first years of life. We evaluate high-density diffuse optical tomography (HD-DOT) imaging combined with movie stimuli for high resolution optical neuroimaging in awake children ranging from 1 to 7 years of age. We built an HD-DOT system with design features geared towards enhancing both image quality and child comfort. Furthermore, we characterized a library of animated movie clips as a stimulus set for brain mapping and we optimized associated data analysis pipelines. Together, these tools could map cortical responses to movies and contained features such as speech in both adults and awake young children. This study lays the groundwork for future research to investigate response variability in larger pediatric samples and atypical trajectories of early brain development in clinical populations.

Reversibly Reactive Affinity Selection-Mass Spectrometry Enables Identification of Covalent Peptide Binders.

Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.

Long-Circulating Vasoactive 1,18-Octadecanedioic Acid-Terlipressin Conjugate.

Hepatorenal syndrome (HRS) is a life-threatening complication of end-stage liver disease first reported over a century ago, but its management still poses an unmet challenge. A therapeutic agent found to stabilize the condition is a short cyclic peptide, vasopressin analogue, terlipressin (TP). While TP is commonly prescribed for HRS patients in most parts of the world, it was only recently approved for use in the United States. TP exhibits short circulation half-lives and adverse side effects associated with the dose required. Herein, we present a 1,18-octadecanedioic acid (ODDA) conjugate of the cyclic peptide (ODDA-TP), which enables noncovalent binding to serum albumin via native fatty acid binding modes. ODDA-TP is demonstrated to outperform TP alone in studies including in vitro cellular receptor activation, stability in plasma, pharmacokinetics, and performance in vivo in rats. Specifically, ODDA-TP had an elimination half-life 20 times that of TP alone while exhibiting a superior safety profile.

Assessing the Performance of Peptide Force Fields for Modeling the Solution Structural Ensembles of Cyclic Peptides.

Molecular dynamics simulation is a powerful tool for characterizing the solution structural ensembles of cyclic peptides. However, the ability of simulation to recapitulate experimental results and make accurate predictions largely depends on the force fields used. In our work here, we evaluate the performance of seven state-of-the-art force fields in recapitulating the experimental NMR results in water of 12 benchmark cyclic peptides, consisting of 6 cyclic pentapeptides, 4 cyclic hexapeptides, and 2 cyclic heptapeptides. The results show that RSFF2+TIP3P, RSFF2C+TIP3P, and Amber14SB+TIP3P exhibit similar and the best performance, all recapitulating the NMR-derived structure information on 10 cyclic peptides. Amber19SB+OPC successfully recapitulates the NMR-derived structure information on 8 cyclic peptides. In contrast, OPLS-AA/M+TIP4P, Amber03+TIP3P, and Amber14SBonlysc+GB-neck2 could only recapitulate the NMR-derived structure information on 5 cyclic peptides, the majority of which are not well-structured.

Automated Flow Peptide Synthesis Enables Engineering of Proteins with Stabilized Transient Binding Pockets.

Engineering at the amino acid level is key to enhancing the properties of existing proteins in a desired manner. So far, protein engineering has been dominated by genetic approaches, which have been extremely powerful but only allow for minimal variations beyond the canonical amino acids. Chemical peptide synthesis allows the unrestricted incorporation of a vast set of unnatural amino acids with much broader functionalities, including the incorporation of post-translational modifications or labels. Here we demonstrate the potential of chemical synthesis to generate proteins in a specific conformation, which would have been unattainable by recombinant protein expression. We use recently established rapid automated flow peptide synthesis combined with solid-phase late-stage modifications to rapidly generate a set of FK506-binding protein 51 constructs bearing defined intramolecular lactam bridges. This trapped an otherwise rarely populated transient pocket-as confirmed by crystal structures-which led to an up to 39-fold improved binding affinity for conformation-selective ligands and represents a unique system for the development of ligands for this rare conformation. Overall, our results show how rapid automated flow peptide synthesis can be applied to precision protein engineering.

How hydrophobicity, side chains, and salt affect the dimensions of disordered proteins.

Despite the generally accepted role of the hydrophobic effect as the driving force for folding, many intrinsically disordered proteins (IDPs), including those with hydrophobic content typical of foldable proteins, behave nearly as self-avoiding random walks (SARWs) under physiological conditions. Here, we tested how temperature and ionic conditions influence the dimensions of the N-terminal domain of pertactin (PNt), an IDP with an amino acid composition typical of folded proteins. While PNt contracts somewhat with temperature, it nevertheless remains expanded over 10-58°C, with a Flory exponent, ν, >0.50. Both low and high ionic strength also produce contraction in PNt, but this contraction is mitigated by reducing charge segregation. With 46% glycine and low hydrophobicity, the reduced form of snow flea anti-freeze protein (red-sfAFP) is unaffected by temperature and ionic strength and persists as a near-SARW, ν ~ 0.54, arguing that the thermal contraction of PNt is due to stronger interactions between hydrophobic side chains. Additionally, red-sfAFP is a proxy for the polypeptide backbone, which has been thought to collapse in water. Increasing the glycine segregation in red-sfAFP had minimal effect on ν. Water remained a good solvent even with 21 consecutive glycine residues (ν > 0.5), and red-sfAFP variants lacked stable backbone hydrogen bonds according to hydrogen exchange. Similarly, changing glycine segregation has little impact on ν in other glycine-rich proteins. These findings underscore the generality that many disordered states can be expanded and unstructured, and that the hydrophobic effect alone is insufficient to drive significant chain collapse for typical protein sequences.

When simplicity triumphs: niche specialization of gut bacteria exists even for simple fiber structures.

Structurally complex corn bran arabinoxylan (CAX) was used as a model glycan to investigate gut bacteria growth and competition on different AX-based fine structures. Nine hydrolyzate segments of the CAX polymer varying in chemical structure (sugars and linkages), CAX, five less complex non-corn arabinoxylans, and xylose and glucose were ranked from structurally complex to simple. The substrate panel promoted different overall growth and rates of growth of eight Bacteroides xylan-degrading strains. For example, Bacteroides cellulosilyticus DSM 14838 (Bacteroides cellulosilyticus) grew well on an array of complex and simple structures, while Bacteroides ovatus 3-1-23 grew well only on the simple structures. In a competition experiment, B. cellulosilyticus growth was favored over B. ovatus on the complex AX-based structure. On the other hand, on the simple structure, B. ovatus strongly outcompeted B. cellulosilyticus, which was eliminated from the competitive environment by Day 11. This adaptation to fine structure and resulting competition dynamics indicate that dietary fiber chemical structures, whether complex or simple, favor certain gut bacteria. Overall, this work supports a concept that fiber degraders diversify their competitive abilities to access substrates across the spectrum of heterogeneity of fine structural features of dietary fibers.

Novel multiparametric bulk and single EV pipeline for adipose cell-specific biomarker discovery in paired human biospecimens.

Obesity is a global health crisis that contributes to morbidity and mortality worldwide. Obesity's comorbid association with a variety of diseases, from metabolic syndrome to neurodegenerative disease, underscores the critical need to better understand the pathobiology of obesity. Adipose tissue, once seen as an inert storage depot, is now recognized as an active endocrine organ, regulating metabolic and systemic homeostasis. Recent studies spotlight the theranostic utility of extracellular vesicles (EVs) as novel biomarkers and drivers of disease, including obesity-related complications. Adipose-derived EVs (ADEVs) have garnered increased interest for their roles in diverse diseases, however robust isolation and characterization protocols for human, cell-specific EV subsets are limited. Herein, we directly address this technical challenge by establishing a multiparametric analysis framework that leverages bulk and single EV characterization, mRNA phenotyping and proteomics of human ADEVs directly from paired visceral adipose tissue, cultured mature adipocyte conditioned media, and plasma from obese subjects undergoing bariatric surgery. Importantly, rigorous EV phenotyping at the tissue and cell-specific level identified top 'adipose liquid biopsy' candidates that were validated in circulating plasma EVs from the same patient. In summary, our study paves the way toward a tissue and cell-specific, multiparametric framework for studying tissue and circulating adipose EVs in obesity-driven disease.

An executive-functions-based reading training enhances sensory-motor systems integration during reading fluency in children with dyslexia.

The Simple View of Reading model suggests that intact language processing and word decoding lead to proficient reading comprehension, with recent studies pointing at executive functions as an important component contributing to reading proficiency. Here, we aimed to determine the underlying mechanism(s) for these changes. Participants include 120 8- to 12-year-old children (n = 55 with dyslexia, n = 65 typical readers) trained on an executive functions-based reading program, including pre/postfunctional MRI and behavioral data collection. Across groups, improved word reading was related to stronger functional connections within executive functions and sensory networks. In children with dyslexia, faster and more accurate word reading was related to stronger functional connections within and between sensory networks. These results suggest greater synchronization of brain systems after the intervention, consistent with the "neural noise" hypothesis in children with dyslexia and support the consideration of including executive functions as part of the Simple View of Reading model.

Development of an α-Klotho Recognizing High-Affinity Peptide Probe from In-Solution Enrichment.

The kidney, parathyroid gland, and choroid plexus express the aging-related transmembrane protein α-Klotho, a coreceptor of the fibroblast growth factor 23 (FGF23) receptor complex. Reduced α-Klotho levels are correlated with chronic kidney disease and other age-related diseases, wherein they are released from membranes into circulation. Klotho's potential physiological action as a hormone is of current scientific interest. Part of the challenges associated with advancing these studies, however, has been the long-standing difficulty in detecting soluble α-Klotho in biofluids. Here, we describe the discovery of peptides that recognize α-Klotho with high affinity and selectivity by applying in-solution size-exclusion-based affinity selection-mass spectrometry (AS-MS). After two rounds of AS-MS and subsequent N-terminal modifications, the peptides improved their binding affinity to α-Klotho by approximately 2300-fold compared to the reported starting peptide Pep-10, previously designed based on the C-terminal region of FGF23. The lead peptide binders were shown to enrich α-Klotho from cell lysates and to label α-Klotho in kidney cells. Our results further support the utility of in-solution, label-free AS-MS protocols to discover peptide-based binders to target proteins of interest with high affinity and selectivity, resulting in functional probes for biological studies.

Development of a synthetic relaxin-3/INSL5 chimeric peptide ligand for NanoBiT complementation binding assays.

INSL5 and relaxin-3 are relaxin family peptides with important roles in gut and brain function, respectively. They mediate their actions through the class A GPCRs RXFP4 and RXFP3. RXFP4 has been proposed to be a therapeutic target for colon motility disorders whereas RXFP3 targeting could be effective for neurological conditions such as anxiety. Validation of these targets has been limited by the lack of specific ligands and the availability of robust ligand-binding assays for their development. In this study, we have utilized NanoBiT complementation to develop a SmBiT-conjugated tracer for use with LgBiT-fused RXFP3 and RXFP4. The low affinity between LgBiT:SmBiT should result in a low non-specific luminescence signal and enable the quantification of binding without the tedious separation of non-bound ligands. We used solid-phase peptide synthesis to produce a SmBiT-labelled RXFP3/4 agonist, R3/I5, where SmBiT was conjugated to the B-chain N-terminus via a PEG12 linker. Both SmBiT-R3/I5 and R3/I5 were synthesized and purified in high purity and yield. Stable HEK293T cell lines expressing LgBiT-RXFP3 and LgBiT-RXFP4 were produced and demonstrated normal signaling in response to the synthetic R3/I5 peptide. Binding was first characterized in whole-cell binding kinetic assays validating that the SmBiT-R3/I5 bound to both cell lines with nanomolar affinity with minimal non-specific binding without bound and free SmBiT-R3/I5 separation. We then optimized membrane binding assays, demonstrating easy and robust analysis of both saturation and competition binding from frozen membranes. These assays therefore provide an appropriate rigorous binding assay for the high-throughput analysis of RXFP3 and RXFP4 ligands.

Developing insulin-like peptide 5-based antagonists for the G protein-coupled receptor, RXFP4.

Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (∼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (∼3 nM) with reduced partial agonist activity, enhanced antagonist potency (∼10 nM) and maximum agonist inhibition (∼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.

Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons.

Antigen processing is critical for therapeutic vaccines to generate epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often limits the quantity of epitopes released. We address this challenge using machine learning to ascribe a proteasomal degradation score to epitope sequences. Epitopes with varying scores were translocated into cells using nontoxic anthrax proteins. Epitopes with a low score show pronounced immunogenicity due to antigen processing, but epitopes with a high score show limited immunogenicity. This work sheds light on the sequence-activity relationships between proteasomal degradation and epitope immunogenicity. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by these vaccines in clinical settings.

An organometallic swap strategy for bottlebrush polymer-protein conjugate synthesis.

Polymer-protein bioconjugation offers a powerful strategy to alter the physical properties of proteins, and various synthetic polymer compositions and architectures have been investigated for this purpose. Nevertheless, conjugation of molecular bottlebrush polymers (BPs) to proteins remains an unsolved challenge due to the large size of BPs and a general lack of methods to transform the chain ends of BPs into functional groups suitable for bioconjugation. Here, we present a strategy to address this challenge in the context of BPs prepared by "graft-through" ring-opening metathesis polymerization (ROMP), one of the most powerful methods for BP synthesis. Quenching ROMP of PEGylated norbornene macromonomers with an activated enyne terminator facilitates the transformation of the BP Ru alkylidene chain ends into Pd oxidative addition complexes (OACs) for facile bioconjugation. This strategy is shown to be effective for the synthesis of two BP-protein conjugates (albumin and ERG), setting the stage for a new class of BP-protein conjugates for future therapeutic and imaging applications.

Peptide Self-Assembly into Amyloid Fibrils: Unbiased All-Atom Simulations.

Protein self-assembly plays an important role in biological systems, accounting for the formation of mesoscopic structures that can be highly symmetric as in the capsid of viruses or disordered as in molecular condensates or exhibit a one-dimensional fibrillar morphology as in amyloid fibrils. Deposits of the latter in tissues of individuals with degenerative diseases like Alzheimer's and Parkinson's has motivated extensive efforts to understand the sequence of molecular events accounting for their formation. These studies aim to identify on-pathway intermediates that may be the targets for therapeutic intervention. This detailed knowledge of fibril formation remains obscure, in part due to challenges with experimental analyses of these processes. However, important progress is being achieved for short amyloid peptides due to advances in our ability to perform completely unbiased all-atom simulations of the self-assembly process. This perspective discusses recent developments, their implications, and the hurdles that still need to be overcome to further advance the field.

Activation of Multiple G Protein Pathways to Characterize the Five Dopamine Receptor Subtypes Using Bioluminescence Technology.

G protein-coupled receptors show preference for G protein subtypes but can recruit multiple G proteins with various downstream signaling cascades. This functional selection can guide drug design. Dopamine receptors are both stimulatory (D1-like) and inhibitory (D2-like) with diffuse expression across the central nervous system. Functional selectivity of G protein subunits may help with dopamine receptor targeting and their downstream effects. Three bioluminescence-based assays were used to characterize G protein coupling and function with the five dopamine receptors. Most proximal to ligand binding was the miniG protein assay with split luciferase technology used to measure recruitment. For endogenous and selective ligands, the G-CASE bioluminescence resonance energy transfer (BRET) assay measured G protein activation and receptor selectivity. Downstream, the BRET-based CAMYEN assay quantified cyclic adenosine monophosphate (cAMP) changes. Several dopamine receptor agonists and antagonists were characterized for their G protein recruitment and cAMP effects. G protein selectivity with dopamine revealed potential Gq coupling at all five receptors, as well as the ability to activate subtypes with the "opposite" effects to canonical signaling. D1-like receptor agonist (+)-SKF-81297 and D2-like receptor agonist pramipexole showed selectivity at all receptors toward Gs or Gi/o/z activation, respectively. The five dopamine receptors show a wide range of potentials for G protein coupling and activation, reflected in their downstream cAMP signaling. Targeting these interactions can be achieved through drug design. This opens the door to pharmacological treatment with more selectivity options for inducing the correct physiological events.

Mirror-image ligand discovery enabled by single-shot fast-flow synthesis of D-proteins.

Widespread adoption of mirror-image biological systems presents difficulties in accessing the requisite D-protein substrates. In particular, mirror-image phage display has the potential for high-throughput generation of biologically stable macrocyclic D-peptide binders with potentially unique recognition modes but is hindered by the individualized optimization required for D-protein chemical synthesis. We demonstrate a general mirror-image phage display pipeline that utilizes automated flow peptide synthesis to prepare D-proteins in a single run. With this approach, we prepare and characterize 12 D-proteins - almost one third of all reported D-proteins to date. With access to mirror-image protein targets, we describe the successful discovery of six macrocyclic D-peptide binders: three to the oncoprotein MDM2, and three to the E3 ubiquitin ligase CHIP. Reliable production of mirror-image proteins can unlock the full potential of D-peptide drug discovery and streamline the study of mirror-image biology more broadly.

Non-destructive environmental safety assessment of threatened and endangered plants in weed biological control.

Assessing the risk of nontarget attack (NTA) for federally listed threatened and endangered (T&E) plant species confamilial to invasive plants targeted for classical biological control, is one of the most important objectives of pre-release environmental safety assessments in the United States. However, evaluating potential NTA on T&E species is often complicated by restrictive agency requirements for obtaining propagules, or the ability to propagate plants and rear agents to the appropriate phenostages synchronously for testing, or both. Here, we assessed whether plant cues associated with a host recognition can be used for testing the attractiveness of four T&E and one rare single population plant species non-destructively for a candidate biocontrol agent. We used the seed-feeding weevil, Mogulones borraginis, a candidate for the biological control of the invasive plant, Cynoglossum officinale (Boraginaceae) as the study system. We collected olfactory and visual cues in the form of flowering sprigs from T&E plant species confamilial to the invasive plant in a non-destructive manner and used them to measure behavioral responses and searching time of weevils. Female weevils preferred C. officinale to all tested plant species in dual-choice bioassays using either olfactory or visual cues in a modified y-tube device. Furthermore, female weevils were repelled by the combined olfactory and visual cues from all tested T&E plant species in a dual-choice test against controls (e.g., purified air in an empty arm), indicating that it would be extremely unlikely for the weevil to attack any of these species upon release in the United States. Principal component analysis based on 61 volatile organic compounds effectively separated the five confamilial plant species and C. officinale, corroborating the results of behavioral bioassays. We conclude that studies on pre-alighting host selection behavior and the underlying physiological mechanisms of how organisms select host plants they exploit can aid in environmental safety testing of weed biological control agents.

Child-adolescent emergency psychiatry: addressing false positive admissions.

Current literature emphasizes the necessary and increasing role of the emergency department (ED) psychologist. This perspective paper will illustrate that the recent focus on an ED psychologist is necessary, but insufficient. Equally important, is an understanding of when a patient in a potential crisis does not require an ED admission, but rather an assessment that is made prior to the patient going to the ED. The essential role of an outpatient crisis team is vital in differentiating when an ED admission is indicated for a psychiatric crisis (true positive) and when an ED admission is not indicated for a psychiatric crisis (false positive). Evaluating crises prior to ED admissions accomplishes two critical healthcare objectives in a parallel process: 1) accurately assessing the proper level of care needed when a patient reports they are experiencing acute psychiatric symptoms (which may or may not necessitate emergency department level of care, and 2) reducing burden on an already over-extended ED when emergency care is not indicated. Our findings are uniquely drawn from a highly diverse youth patient population in Northern California, United States.