Actin waves transport RanGTP to the neurite tip to regulate non-centrosomal microtubules in neurons.

Microtubules (MTs) are the most abundant cytoskeleton in neurons, and control multiple facets of their development. While the MT-organizing center (MTOC) in mitotic cells is typically located at the centrosome, the MTOC in neurons switches to non-centrosomal sites. A handful of cellular components have been shown to promote non-centrosomal MT (ncMT) formation in neurons, yet the regulation mechanism remains unknown. Here, we demonstrate that the small GTPase Ran is a key regulator of ncMTs in neurons. Using an optogenetic tool that enables light-induced local production of RanGTP, we demonstrate that RanGTP promotes ncMT plus-end growth along the neurite. Additionally, we discovered that actin waves drive the anterograde transport of RanGTP. Pharmacological disruption of actin waves abolishes the enrichment of RanGTP and reduces growing ncMT plus-ends at the neurite tip. These observations identify a novel regulation mechanism for ncMTs and pinpoint an indirect connection between the actin and MT cytoskeletons in neurons.

Machine Learning Assisted Handheld Confocal Raman Micro-Spectroscopy for Identification of Clinically Relevant Atopic Eczema Biomarkers.

Atopic dermatitis (AD) is a common chronic inflammatory skin dermatosis condition due to skin barrier dysfunction that causes itchy, red, swollen, and cracked skin. Currently, AD severity clinical scores are subjected to intra- and inter-observer differences. There is a need for an objective scoring method that is sensitive to skin barrier differences. The aim of this study was to evaluate the relevant skin chemical biomarkers in AD patients. We used confocal Raman micro-spectroscopy and advanced machine learning methods as means to classify eczema patients and healthy controls with sufficient sensitivity and specificity. Raman spectra at different skin depths were acquired from subjects' lower volar forearm location using an in-house developed handheld confocal Raman micro-spectroscopy system. The Raman spectra corresponding to the skin surface from all the subjects were further analyzed through partial least squares discriminant analysis, a binary classification model allowing the classification between eczema and healthy subjects with a sensitivity and specificity of 0.94 and 0.85, respectively, using stratified K-fold (K = 10) cross-validation. The variable importance in the projection score from the partial least squares discriminant analysis classification model further elucidated the role of important stratum corneum proteins and lipids in distinguishing two subject groups.

PARG has a robust endo-glycohydrolase activity that releases protein-free poly(ADP-ribose) chains.

Poly(ADP-ribosyl)ation (PARylation) regulates DNA damage response, chromatin structure, and cell-fate. Dynamic regulation of cellular PAR levels is crucial for the maintenance of genomic integrity and excessive cellular PAR activates a PAR-dependent cell death pathway. Thus, PAR serves as a cell-death signal; however, it has been debated how the protein-free PAR is generated. Here, we demonstrate that PAR glycohydrolases (PARGs) from mammals to bacteria have a robust endo-glycohydrolase activity, releasing protein-free PAR chains longer than three ADP-ribose units as early reaction products. Released PAR chains are transient and rapidly degraded to monomeric ADP-ribose, which is consistent with a short half-life of PAR during DNA damage responses. Computational simulations using a tri-ADP-ribose further support that PARG can efficiently bind to internal sites of PAR for the endo-glycosidic cleavage. Our collective results suggest PARG as a key player in producing protein-free PAR during DNA damage signaling and establish bacterial PARG as a useful tool to enrich short PAR chains that emerge as important reagents for biomedical research.

A mechanism for lipid binding to apoE and the role of intrinsically disordered regions coupled to domain-domain interactions.

Relative to the apolipoprotein E (apoE) E3 allele of the APOE gene, apoE4 strongly increases the risk for the development of late-onset Alzheimer's disease. However, apoE4 differs from apoE3 by only a single amino acid at position 112, which is arginine in apoE4 and cysteine in apoE3. It remains unclear why apoE3 and apoE4 are functionally different. Described here is a proposal for understanding the functional differences between these two isoforms with respect to lipid binding. A mechanism is proposed that is based on the full-length monomeric structure of the protein, on hydrogen-deuterium exchange mass spectrometry data, and on the role of intrinsically disordered regions to control protein motions. It is proposed that lipid binds between the N-terminal and C-terminal domains and that separation of the two domains, along with the presence of intrinsically disordered regions, controls this process. The mechanism explains why apoE3 differs from apoE4 with respect to different lipid-binding specificities, why lipid increases the binding of apoE to its receptor, and why specific residues are conserved.

TRIM9 controls growth cone responses to netrin through DCC and UNC5C.

The guidance cue netrin-1 promotes both growth cone attraction and growth cone repulsion. How netrin-1 elicits these diverse axonal responses, beyond engaging the attractive receptor DCC and repulsive receptors of the UNC5 family, remains elusive. Here we demonstrate that murine netrin-1 induces biphasic axonal responses in cortical neurons: attraction at lower concentrations and repulsion at higher concentrations using both a microfluidic-based netrin-1 gradient and bath application of netrin-1. TRIM9 is a brain-enriched E3 ubiquitin ligase previously shown to bind and cluster the attractive receptor DCC at the plasma membrane and regulate netrin-dependent attractive responses. However, whether TRIM9 also regulated repulsive responses to netrin-1 remained to be seen. In this study, we show that TRIM9 localizes and interacts with both the attractive netrin receptor DCC and the repulsive netrin receptor, UNC5C, and that deletion of murine Trim9 alters both attractive and repulsive responses to murine netrin-1. TRIM9 was required for netrin-1-dependent changes in surface levels of DCC and total levels of UNC5C in the growth cone during morphogenesis. We demonstrate that DCC at the membrane regulates growth cone area and show that TRIM9 negatively regulates FAK activity in the absence of netrin-1. We investigate membrane dynamics of the UNC5C receptor using pH-mScarlet fused to the extracellular domain of UNC5C. Minutes after netrin addition, levels of UNC5C at the plasma membrane drop in a TRIM9-independent fashion, however TRIM9 regulated the mobility of UNC5C in the plasma membrane in the absence of netrin-1. Together this work demonstrates that TRIM9 interacts with and regulates both DCC and UNC5C during attractive and repulsive axonal responses to netrin-1.

A genetic atlas for the butterflies of continental Canada and United States.

Multi-locus genetic data for phylogeographic studies is generally limited in geographic and taxonomic scope as most studies only examine a few related species. The strong adoption of DNA barcoding has generated large datasets of mtDNA COI sequences. This work examines the butterfly fauna of Canada and United States based on 13,236 COI barcode records derived from 619 species. It compiles i) geographic maps depicting the spatial distribution of haplotypes, ii) haplotype networks (minimum spanning trees), and iii) standard indices of genetic diversity such as nucleotide diversity (π), haplotype richness (H), and a measure of spatial genetic structure (GST). High intraspecific genetic diversity and marked spatial structure were observed in the northwestern and southern North America, as well as in proximity to mountain chains. While species generally displayed concordance between genetic diversity and spatial structure, some revealed incongruence between these two metrics. Interestingly, most species falling in this category shared their barcode sequences with one at least other species. Aside from revealing large-scale phylogeographic patterns and shedding light on the processes underlying these patterns, this work also exposed cases of potential synonymy and hybridization.

BOLD v4: A Centralized Bioinformatics Platform for DNA-Based Biodiversity Data.

BOLD, the Barcode of Life Data System, supports the acquisition, storage, validation, analysis, and publication of DNA barcodes, activities requiring the integration of molecular, morphological, and distributional data. Its pivotal role in curating the reference library of DNA barcodes, coupled with its data management and analysis capabilities, makes it a central resource for biodiversity science. It enables rapid, accurate identification of specimens and also reveals patterns of genetic diversity and evolutionary relationships among taxa.Launched in 2005, BOLD has become an increasingly powerful tool for advancing the understanding of planetary biodiversity. It currently hosts 17 million specimen records and 14 million barcodes that provide coverage for more than a million species from every continent and ocean. The platform has the long-term goal of providing a consistent, accurate system for identifying all species of eukaryotes.BOLD's integrated analytical tools, full data lifecycle support, and secure collaboration framework distinguish it from other biodiversity platforms. BOLD v4 brought enhanced data management and analysis capabilities as well as novel functionality for data dissemination and publication. Its next version will include features to strengthen its utility to the research community, governments, industry, and society-at-large.

Cytoskeleton: Septin wreaths regulate actin in neuritogenesis.

The shape of a neuron changes dramatically during development. New work reports a novel septin cytoskeleton network that is important in establishing proper neuronal morphology.

Erinacine S from Hericium erinaceus mycelium promotes neuronal regeneration by inducing neurosteroids accumulation.

Erinacines derived from Hericium erinaceus have been shown to possess various health benefits including neuroprotective effect against neurodegenerative diseases, yet the underlying mechanism remains unknown. Here we found that erinacine S enhances neurite outgrowth in a cell autonomous fashion. It promotes post-injury axon regeneration of PNS neurons and enhances regeneration on inhibitory substrates of CNS neurons. Using RNA-seq and bioinformatic analyses, erinacine S was found to cause the accumulation of neurosteroids in neurons. ELISA and neurosteroidogenesis inhibitor assays were performed to validate this effect. This research uncovers a previously unknown effect of erinacine S on raising the level of neurosteroids.

IRAK4 degrader in hidradenitis suppurativa and atopic dermatitis: a phase 1 trial.

Toll-like receptor-driven and interleukin-1 (IL-1) receptor-driven inflammation mediated by IL-1 receptor-associated kinase 4 (IRAK4) is involved in the pathophysiology of hidradenitis suppurativa (HS) and atopic dermatitis (AD). KT-474 (SAR444656), an IRAK4 degrader, was studied in a randomized, double-blind, placebo-controlled phase 1 trial where the primary objective was safety and tolerability. Secondary objectives included pharmacokinetics, pharmacodynamics and clinical activity in patients with moderate to severe HS and in patients with moderate to severe AD. KT-474 was administered as a single dose and then daily for 14 d in 105 healthy volunteers (HVs), followed by dosing for 28 d in an open-label cohort of 21 patients. Degradation of IRAK4 was observed in HV blood, with mean reductions after a single dose of ≥93% at 600-1,600 mg and after 14 daily doses of ≥95% at 50-200 mg. In patients, similar IRAK4 degradation was achieved in blood, and IRAK4 was normalized in skin lesions where it was overexpressed relative to HVs. Reduction of disease-relevant inflammatory biomarkers was demonstrated in the blood and skin of patients with HS and patients with AD and was associated with improvement in skin lesions and symptoms. There were no drug-related infections. These results, from what, to our knowledge, is the first published clinical trial using a heterobifunctional degrader, provide initial proof of concept for KT-474 in HS and AD to be further confirmed in larger trials. ClinicalTrials.gov identifier: NCT04772885 .

Modulation of soleus H reflex by spinal DC stimulation in humans.

Transcranial direct current stimulation (tDCS) of the human motor cortex induces changes in excitability within cortical and spinal circuits that occur during and after the stimulation. Recently, transcutaneous spinal direct current stimulation (tsDCS) has been shown to modulate spinal conduction properties, as assessed by somatosensory-evoked potentials, and transynaptic properties of the spinal neurons, as tested by postactivation depression of the H reflex or by the RIII nociceptive component of the flexion reflex in the lower limb. To further explore tsDCS-induced plastic changes in spinal excitability, we examined, in a double-blind crossover randomized study, the stimulus-response curves of the soleus H reflex before, during, at current offset and 15 min after anodal, cathodal, and sham tsDCS delivered at the Th11 level (2.5 mA, 15 min, 0.071 mA/cm(2), 0.064 C/cm(2)) in 17 healthy subjects. Anodal tsDCS induced a progressive leftward shift of the recruitment curve of the soleus H reflex during the stimulation; the effects persisted for at least 15 min after current offset. In contrast, both cathodal and sham tsDCS had no significant effects. This exploratory study provides further evidence for the use of tsDCS as an expedient, noninvasive tool to induce long-lasting plastic changes in spinal circuitry. Increased spinal excitability after anodal tsDCS may have potential for spinal neuromodulation in patients with central nervous system lesions.

Computationally driven discovery of SARS-CoV-2 Mpro inhibitors: from design to experimental validation.

We report a fast-track computationally driven discovery of new SARS-CoV-2 main protease (Mpro) inhibitors whose potency ranges from mM for the initial non-covalent ligands to sub-µM for the final covalent compound (IC50 = 830 ± 50 nM). The project extensively relied on high-resolution all-atom molecular dynamics simulations and absolute binding free energy calculations performed using the polarizable AMOEBA force field. The study is complemented by extensive adaptive sampling simulations that are used to rationalize the different ligand binding poses through the explicit reconstruction of the ligand-protein conformation space. Machine learning predictions are also performed to predict selected compound properties. While simulations extensively use high performance computing to strongly reduce the time-to-solution, they were systematically coupled to nuclear magnetic resonance experiments to drive synthesis and for in vitro characterization of compounds. Such a study highlights the power of in silico strategies that rely on structure-based approaches for drug design and allows the protein conformational multiplicity problem to be addressed. The proposed fluorinated tetrahydroquinolines open routes for further optimization of Mpro inhibitors towards low nM affinities.

A molecular-based identification resource for the arthropods of Finland.

To associate specimens identified by molecular characters to other biological knowledge, we need reference sequences annotated by Linnaean taxonomy. In this study, we (1) report the creation of a comprehensive reference library of DNA barcodes for the arthropods of an entire country (Finland), (2) publish this library, and (3) deliver a new identification tool for insects and spiders, as based on this resource. The reference library contains mtDNA COI barcodes for 11,275 (43%) of 26,437 arthropod species known from Finland, including 10,811 (45%) of 23,956 insect species. To quantify the improvement in identification accuracy enabled by the current reference library, we ran 1000 Finnish insect and spider species through the Barcode of Life Data system (BOLD) identification engine. Of these, 91% were correctly assigned to a unique species when compared to the new reference library alone, 85% were correctly identified when compared to BOLD with the new material included, and 75% with the new material excluded. To capitalize on this resource, we used the new reference material to train a probabilistic taxonomic assignment tool, FinPROTAX, scoring high success. For the full-length barcode region, the accuracy of taxonomic assignments at the level of classes, orders, families, subfamilies, tribes, genera, and species reached 99.9%, 99.9%, 99.8%, 99.7%, 99.4%, 96.8%, and 88.5%, respectively. The FinBOL arthropod reference library and FinPROTAX are available through the Finnish Biodiversity Information Facility (www.laji.fi) at https://laji.fi/en/theme/protax. Overall, the FinBOL investment represents a massive capacity-transfer from the taxonomic community of Finland to all sectors of society.

The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis.

TRIM9 and TRIM67 are neuronally enriched E3 ubiquitin ligases essential for appropriate morphogenesis of cortical and hippocampal neurons and fidelitous responses to the axon guidance cue netrin-1. Deletion of murine Trim9 or Trim67 results in neuroanatomical defects and striking behavioral deficits, particularly in spatial learning and memory. TRIM9 and TRIM67 interact with cytoskeletal and exocytic proteins, but the full interactome is not known. Here we performed the unbiased proximity-dependent biotin identification (BioID) approach to define TRIM9 and TRIM67 protein-protein proximity network in developing cortical neurons and identified putative neuronal TRIM interaction partners. Candidates included cytoskeletal regulators, cytosolic protein transporters, exocytosis and endocytosis regulators, and proteins necessary for synaptic regulation. A subset of high-priority candidates was validated, including Myo16, Coro1A, MAP1B, ExoC1, GRIP1, PRG-1, and KIF1A. For a subset of validated candidates, we utilized total internal reflection fluorescence microscopy to demonstrate dynamic colocalization with TRIM proteins at the axonal periphery, including at the tips of filopodia. Further analysis demonstrated that the RNA interference-based knockdown of the unconventional myosin Myo16 in cortical neurons altered growth cone filopodia density and axonal branching patterns in a TRIM9- and netrin-1-dependent manner. Future analysis of other validated candidates will likely identify novel proteins and mechanisms by which TRIM9 and TRIM67 regulate neuronal form and function. [Media: see text].

View-dependent streamlines for 3D vector fields.

This paper introduces a new streamline placement and selection algorithm for 3D vector fields. Instead of considering the problem as a simple feature search in data space, we base our work on the observation that most streamline fields generate a lot of self-occlusion which prevents proper visualization. In order to avoid this issue, we approach the problem in a view-dependent fashion and dynamically determine a set of streamlines which contributes to data understanding without cluttering the view. Since our technique couples flow characteristic criteria and view-dependent streamline selection we are able achieve the best of both worlds: relevant flow description and intelligible, uncluttered pictures. We detail an efficient GPU implementation of our algorithm, show comprehensive visual results on multiple datasets and compare our method with existing flow depiction techniques. Our results show that our technique greatly improves the readability of streamline visualizations on different datasets without requiring user intervention.

Fast pulsatile blood flow measurement in deep tissue through a multimode detection fiber.

SIGNIFICANCE: Noninvasive in vivo fast pulsatile blood flow measurement in deep tissue is important because the blood flow waveform is correlated with physiological parameters, such as blood pressure and elasticity of blood vessels. Compromised blood flow may cause diseases, such as stroke, foot ulcer, and myocardial ischemia. There is great clinical demand for a portable and cost-effective device for noninvasive pulsatile blood flow measurement. AIM: A diffuse-optics-based method, diffuse speckle pulsatile flowmetry (DSPF), was developed for fast measurement (∼300 Hz) of deep tissue blood flow noninvasively. To validate its performance, both a phantom experiment and in vivo demonstration were conducted. APPROACH: Over the past two decades, single-mode fibers have been used as detection fibers in most diffuse-optics-based deep tissue blood flow measurement modalities. We used a multimode (MM) detection fiber with a core size of 200 µm for diffused speckle pattern detection. A background intensity correction algorithm was implemented for speckle contrast calculation. The MM detection fiber helped to achieve a level of deep tissue blood flow measurement similar to that of conventional modalities, such as diffuse correlation spectroscopy and diffuse speckle contrast analysis, but it increases the measurement rate of blood flow to 300 Hz. RESULTS: The design and implementation of the DSPF system were introduced. The theory of the background intensity correction for the diffused speckle pattern detected by the MM fiber was explained. A flow phantom was built for validation of the performance of the DSPF system. An in vivo cuff-induced occlusion experiment was performed to demonstrate the capability of the proposed DSPF system. CONCLUSIONS: An MM detection fiber can help to achieve fast (∼300 Hz) pulsatile blood flow measurement in the proposed DSPF method. The cost-effective device and the fiber-based flexible probe increase the usability of the DSPF system significantly.

Handheld confocal Raman spectroscopy (CRS) for objective assessment of skin barrier function and stratification of severity in atopic dermatitis (AD) patients.

BACKGROUND: We developed the first-of-its-kind handheld confocal Raman spectroscopy (CRS) system to quantify the concentration of natural moisturizing factors in the skin. OBJECTIVE: To evaluate the feasibility of our handheld CRS system and propose a novel quantitative index to measure skin barrier function. METHODS: This prospective study included 30 atopic dermatitis (AD) patients and 14 healthy volunteers. All AD participants were assessed using the Scoring Atopic Dermatitis (SCORAD) severity instrument, a vapometer for trans-epidermal water loss and a moisture meter for skin surface moisture. A handheld CRS operating at 785 nm laser was used to measure the biochemical constituents of the skin up to a depth of ∼100 µm. We trained a linear kernel-based support vector machine (SVM) model for eczema classification based on the water, ceramide and urocanic acid content. A novel Eczema Biochemical Index (EBI) was then formulated using the skin constituents measured from the AD participants to stage disease severity. RESULTS: The SVM model used to classify healthy participants and AD patients obtained high cross-validated area under the curve of 0.857 and accuracy of 0.841, with high sensitivity and specificity values of 0.857 and 0.833 respectively. EBI can be used to stratify AD patients of varying severity, based on the biochemical constituents in the skin. CONCLUSION: As compared to the standard CRS system, the handheld CRS offers higher portability and provides Raman measurements at various body regions with similar sensitivity. This suggests that a handheld CRS device could be a valuable point-of-care resource in both research and clinical use.

Visualizing Alzheimer's Disease Mouse Brain with Multispectral Optoacoustic Tomography using a Fluorescent probe, CDnir7.

Alzheimer's disease (AD) is now clinically considered as a chronic inflammation-based neurodegenerative disease. The CDnir7 probe was previously developed as an optical imaging probe to target macrophages in order to image mouse inflammation using in vivo optical imaging modalities such as In Vivo imaging system (IVIS) and fluorescent molecular tomography (FMT). Here, we demonstrate the application of CDnir7 in AD mouse brain imaging via multispectral optoacoustic tomography (MSOT). Longitudinal MSOT imaging of CDnir7 showed higher CDnir7 localization in AD mouse cerebral cortex compared to that of normal mice. MSOT signals of CDnir7 localization in mouse brain were verified by ex vivo near-infrared (NIR) imaging and immunohistochemistry. Histological evaluation showed strong CDnir7 staining in AD cerebral cortex, hippocampus, basal ganglia and thalamus area. Based on the supporting evidence, CDnir7 has great potential as a molecular imaging probe for AD brain imaging.

A reference library for Canadian invertebrates with 1.5 million barcodes, voucher specimens, and DNA samples.

The reliable taxonomic identification of organisms through DNA sequence data requires a well parameterized library of curated reference sequences. However, it is estimated that just 15% of described animal species are represented in public sequence repositories. To begin to address this deficiency, we provide DNA barcodes for 1,500,003 animal specimens collected from 23 terrestrial and aquatic ecozones at sites across Canada, a nation that comprises 7% of the planet's land surface. In total, 14 phyla, 43 classes, 163 orders, 1123 families, 6186 genera, and 64,264 Barcode Index Numbers (BINs; a proxy for species) are represented. Species-level taxonomy was available for 38% of the specimens, but higher proportions were assigned to a genus (69.5%) and a family (99.9%). Voucher specimens and DNA extracts are archived at the Centre for Biodiversity Genomics where they are available for further research. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, and the Global Genome Biodiversity Network Data Portal.

Laminectomy and fusion after spinal cord injury: national inpatient complications and outcomes.

There is little information about national in-hospital complication rates, adverse outcomes, and mortality after spinal fusion for spinal cord injury (SCI). The National Inpatient Sample (NIS) was utilized to identify 31,381 admissions of acute spinal cord injured patients who underwent spinal decompression with laminectomy and/or fusion (lam/fusion) in the United States from 1993 to 2002. Multivariate analysis was performed to analyze the effects of patient and hospital characteristics on outcomes such as mortality, complications, and discharge disposition, which were then stratified by age, level, and type of injury. The overall mortality was 3.0%, with a complication rate of 26.3% and mean length of stay (LOS) of 17 days. Pulmonary complications (14.4%) and postoperative hemorrhages or hematomas (3.8%) were the most common complications reported. One postoperative complication doubled the length of stay, increased the mortality rate by fivefold and added over $50,000 to hospital charges. Age and comorbidities were the main significant predictors of mortality on multivariate analysis. Patients aged >85 or 65-84 had a 44- and 14-fold greater risk of dying compared with patients in the 18-44 age group respectively. Patients with >3 comorbidities also had an increased risk of mortality (odds ratio [OR] = 1.8). Alcohol abuse was the most common medical comorbidity (present in 12% of patients treated). This study represents the first major national estimate of in-hospital mortality and complication rates after nonoperative and operative treatment for SCI.