Neurodesk: an accessible, flexible and portable data analysis environment for reproducible neuroimaging.

Neuroimaging research requires purpose-built analysis software, which is challenging to install and may produce different results across computing environments. The community-oriented, open-source Neurodesk platform ( https://www.neurodesk.org/ ) harnesses a comprehensive and growing suite of neuroimaging software containers. Neurodesk includes a browser-accessible virtual desktop, command-line interface and computational notebook compatibility, allowing for accessible, flexible, portable and fully reproducible neuroimaging analysis on personal workstations, high-performance computers and the cloud.

Cancer Mutations in SPOP Put a Stop to Its Inter-compartmental Hops.

In this issue of Molecular Cell, Bouchard et al. (2018) identify liquid-liquid phase separation as a mechanism for substrate-triggered localization of SPOP and ubiquitination machinery to different nuclear bodies and describe how cancer mutations disrupt this process.

Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions.

Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.

Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units.

Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or posttranslational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules. Specific interactions between polyubiquitin chains and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Perturbations to either the UBQLN2-binding surface of Ub or the spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model based on polyphasic linkage principles that accurately described the effects of different hubs on UBQLN2 phase separation, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. The extent to which polyubiquitin hubs promote UBQLN2 phase separation is encoded in the spacings between Ub units. This spacing is modulated by chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. The spacing in naturally occurring linear polyubiquitin chains is already optimized to promote phase separation with UBQLN2. We expect our findings to extend to other condensates, emphasizing the importance of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.

Myeloid neoplasm with histiocytosis and spleen tyrosine kinase fusion responds to fostamatinib.

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Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains.

Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 comprises self-associating regions that drive its homotypic liquid-liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11-Ub4 and K48-Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63-Ub4, M1-Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin-binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self-interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin-binding shuttles and adaptors.

Short disordered termini and proline-rich domain are major regulators of UBQLN1/2/4 phase separation.

Highly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2, and UBQLN4 differ in both their specific protein quality control functions and their propensities to localize to stress-induced condensates, cellular aggregates, and aggresomes. We previously showed that UBQLN2 phase separates in vitro, and that the phase separation propensities of UBQLN2 deletion constructs correlate with their ability to form condensates in cells. Here, we demonstrated that full-length UBQLN1, UBQLN2, and UBQLN4 exhibit distinct phase behaviors in vitro. Strikingly, UBQLN4 phase separates at a much lower saturation concentration than UBQLN1. However, neither UBQLN1 nor UBQLN4 phase separates with a strong temperature dependence, unlike UBQLN2. We determined that the temperature-dependent phase behavior of UBQLN2 stems from its unique proline-rich region, which is absent in the other UBQLNs. We found that the short N-terminal disordered regions of UBQLN1, UBQLN2, and UBQLN4 inhibit UBQLN phase separation via electrostatics interactions. Charge variants of the N-terminal regions exhibit altered phase behaviors. Consistent with the sensitivity of UBQLN phase separation to the composition of the N-terminal regions, epitope tags placed on the N-termini of the UBQLNs tune phase separation. Overall, our in vitro results have important implications for studies of UBQLNs in cells, including the identification of phase separation as a potential mechanism to distinguish the cellular roles of UBQLNs and the need to apply caution when using epitope tags to prevent experimental artifacts.

Ion Mobility Mass Spectrometry Unveils Global Protein Conformations in Response to Conditions that Promote and Reverse Liquid-Liquid Phase Separation.

Liquid-liquid phase separation (LLPS) is a process by which biomacromolecules, particularly proteins, condense into a dense phase that resembles liquid droplets. Dysregulation of LLPS is implicated in disease, yet the relationship between protein conformational changes and LLPS remains difficult to discern. This is due to the high flexibility and disordered nature of many proteins that phase separate under physiological conditions and their tendency to oligomerize. Here, we demonstrate that ion mobility mass spectrometry (IM-MS) overcomes these limitations. We used IM-MS to investigate the conformational states of full-length ubiquilin-2 (UBQLN2) protein, LLPS of which is driven by high-salt concentration and reversed by noncovalent interactions with ubiquitin (Ub). IM-MS revealed that UBQLN2 exists as a mixture of monomers and dimers and that increasing salt concentration causes the UBQLN2 dimers to undergo a subtle shift toward extended conformations. UBQLN2 binds to Ub in 2:1 and 2:2 UBQLN2/Ub complexes, which have compact geometries compared to free UBQLN2 dimers. Together, these results suggest that extended conformations of UBQLN2 are correlated with UBQLN2's ability to phase separate. Overall, delineating protein conformations that are implicit in LLPS will greatly increase understanding of the phase separation process, both in normal cell physiology and disease states.

Ubiquitin-Modulated Phase Separation of Shuttle Proteins: Does Condensate Formation Promote Protein Degradation?

Liquid-liquid phase separation (LLPS) has recently emerged as a possible mechanism that enables ubiquitin-binding shuttle proteins to facilitate the degradation of ubiquitinated substrates via distinct protein quality control (PQC) pathways. Shuttle protein LLPS is modulated by multivalent interactions among their various domains as well as heterotypic interactions with polyubiquitin chains. Here, the properties of three different shuttle proteins (hHR23B, p62, and UBQLN2) are closely examined, unifying principles for the molecular determinants of their LLPS are identified, and how LLPS is connected to their functions is discussed. Evidence supporting LLPS of other shuttle proteins is also found. In this review, it is proposed that shuttle protein LLPS leads to spatiotemporal regulation of PQC activities by mediating the recruitment of PQC machinery (including proteasomes or autophagic components) to biomolecular condensates, assembly/disassembly of condensates, selective enrichment of client proteins, and extraction of ubiquitinated proteins from condensates in cells.

Genetic analysis of Vietnamese patients with early-onset Alzheimer's disease.

Purpose of the study: Alzheimer's disease (AD) is the most common type of dementia and its prevalence is rapidly increasing worldwide. Early-onset Alzheimer's disease (EOAD) constitutes of patients with age of onset earlier than 65 year-old and is known to be associated with genetic mutations. In this study, we reported the first genetic analysis of Vietnamese patients with EOAD.Materials and methods: We analyzed targeted sequencing data obtained from a cohort of 51 Vietnamese EOAD patients to identify pathogenic variants in twenty nine well-characterized neurodengerative genes.Results: We identified four missense mutations in APP/PSEN1 genes from six individuals, which accounts for 11.8% of all tested cases. Three of these mutations were previously reported as pathogenic and one mutation in the APP gene was newly identified and might be specific for Vietnamese patients. Our study also found eight individuals carrying homozygous APOE ε4 allele, the main risk factor gene for late-onset AD.Conclusions: Our findings showed that mutation rate in APP/PSEN genes in Vietnamese EOAD patients is consistent with that in other ethnic groups. Although further functional studies are required to validate the pathogenesis of the new mutations, our study demonstrated the necessity of genetic screening for EOAD patients as well as additional genetic data collection in Vietnamese population.

Sealing Treatment of Plasma-Sprayed Cr3C2-NiCr/Al2O3-TiO2 Coating by Aluminum Phosphate Sealant Containing Al2O3 Nanoparticles.

A typical structure of thermal spray coatings consisted of molten particles, semi-molten particles, oxides, pores, and cracks. These factors caused the porosity of sprayed coatings, leading to a significant influence on the coating properties, especially their wear-corrosion resistance. In this study, a post-spray sealing treatment of Cr3C2-NiCr/Al2O3-TiO2 plasma-sprayed coatings was carried out, and then, their corrosion properties were evaluated, before and after the treatment. For the sealing process, aluminum phosphate (APP) containing Al2O3 nanoparticles (~10 nm) was used. The permeability of APP into the sprayed coating was analyzed by SEM-EDS. The treatment efficiency for porosity and corrosion resistance of sprayed coatings was evaluated by electrochemical measurements, such as the potentiodynamic polarization and electrochemical impedance spectroscopy. The wear-corrosion resistance of the coating was examined in 3.5 wt.% NaCl circulation solution containing 0.25% SiO2 particles. The sealing efficiency was evaluated by the percentage of the treated open pores in the coating. The obtained results showed that APP penetrated deeply through the coating and the incorporation of Al2O3 nanoparticles into APP sealant improved the sealing efficiency by 20% of open pores in comparison with the sealant without nano-Al2O3. The effect of the post-treatment on corrosion protection of the sprayed coating has been discussed.

Evaluation of Proliferation and Osteogenic Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Porous Scaffolds.

INTRODUCTION: Human umbilical cord-derived mesenchymal stem cells (UCMSCs) are multiple potential stem cells that can differentiate into various kinds of functional cells, including adipocytes, osteoblasts, and chondroblasts. Thus, UCMSCs have recently been used in both stem cell therapy and tissue engineering applications to produce various functional tissues. This study aimed to evaluate the proliferation and differentiation of UCMSCs on porous scaffolds. METHODS: UCMSCs were established in a previous study and kept in liquid nitrogen. They were thawed and expanded in vitro to yield enough cells for further experiments. The cells were characterized as having MSC phenotype. They were seeded onto culture medium-treated porous scaffolds or on non-treated porous scaffolds at different densities of UCMSCs (105, 2.1 × 105, and 5 × 105 cells/0.005 g scaffold). The existence of UCMSCs on the scaffold was evaluated by nucleic staining using Hoechst 33342 dye, while cell proliferation on the scaffold was determined by MTT assay. Osteogenic differentiation was evaluated by changes in cellular morphology, accumulation of extracellular calcium, and expression of osteoblast-specific genes (including runx2, osteopontin (OPN), and osteocalcin (OCN)). RESULTS: The data showed that UCMSCs could attach, proliferate, and differentiate on both treated and non-treated scaffolds but were better on the treated scaffold. At a cell density of 105 cells/0.005 g scaffold, the adherent and proliferative abilities of UCMSCs were higher than that of the other densities after 14 days of culture (p < 0.05). Adherent UCMSCs on the scaffold could be induced into osteoblasts in the osteogenic medium after 21 days of induction. These cells accumulated calcium in the extracellular matrix that was positive with Alizarin Red staining. They also expressed some genes related to osteoblasts, including runx2, OPN, and OCN. CONCLUSION: UCMSCs could adhere, proliferate, and differentiate into osteoblasts on porous scaffolds. Therefore, porous scaffolds (such as Variotis) may be suitable scaffolds for producing bone tissue in combination with UCMSCs.

In Vitro Production of Cartilage Tissue from Rabbit Bone Marrow-Derived Mesenchymal Stem Cells and Polycaprolactone Scaffold.

In vitro production of tissues or tissue engineering is a promising approach to produce artificial tissues for regenerative medicine. There are at least three important components of tissue engineering, including stem cells, scaffolds and growth factors. This study aimed to produce cartilage tissues in vitro from culture and chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMMSCs), induced by chondrogenesis medium, on biodegradable polycaprolactone (PCL) scaffolds. BMMSCs were isolated from rabbit bone marrow according to the standard protocol. The adherence, proliferation and differentiation of BMMSCs on scaffolds were investigated using two scaffold systems: PCL scaffolds and collagen-coated PCL (PCL/col) scaffolds. The results showed that BMMSCs could attach and grow on both PCL and PCL/col scaffolds. However, the adhesion efficacy of BMMSCs on the PCL/col scaffolds was significantly better than on PCL scaffolds. Under induced conditions, BMMSCs on PLC/col scaffolds showed increased aggrecan accumulation and upregulated expression of chondrogenesis-associated genes (e.g. collagen type II, collagen type I, aggrecan and collagen type X) after 3, 7, 21 and 28 days of induction. These in vitro cartilage tissues could form mature chondrocyte-like cells after they were grafted into rabbits. The results suggest that use of BMMSCs in combination with polycaprolactone scaffolds and chondrogenesis medium can be a way to form in vitro cartilage tissue.

A microfluidic enrichment platform with a recombinase polymerase amplification sensor for pathogen diagnosis.

Rapid and sensitive detection of low amounts of pathogen in large samples is needed for early diagnosis and treatment of patients and surveillance of pathogen. In this study, we report a microfluidic platform for detection of low pathogen levels in a large sample volume that couples an Magainin 1 based microfluidic platform for pathogen enrichment and a recombinase polymerase amplification (RPA) sensor for simultaneous pathogenic DNA amplification and detection in a label-free and real-time manner. Magainin 1 is used as a pathogen enrichment agent with a herringbone microfluidic chip. Using this enrichment platform, the detection limit was found to be 20 times more sensitive in 10 ml urine with Salmonella and 10 times more sensitive in 10 ml urine with Brucella than that of real-time PCR without the enrichment process. Furthermore, the combination system of the enrichment platform and an RPA sensor that based on an isothermal DNA amplification method with rapidity and sensitivity for detection can detect a pathogen at down to 50 CFU in 10 ml urine for Salmonella and 102 CFU in 10 ml urine for Brucella within 60 min. This system will be useful as it has the potential for better diagnosis of pathogens by increasing the capture efficiency of the pathogen in large samples, subsequently enhancing the detection limit of pathogenic DNA.

Allogeneic Adipose-Derived Mesenchymal Stem Cell Transplantation Enhances the Expression of Angiogenic Factors in a Mouse Acute Hindlimb Ischemic Model.

Cell migration and molecular mechanisms during healing of damaged vascular or muscle tissues are emerging fields of interest worldwide. The study herein focuses on evaluating the role of allogenic adipose-derived mesenchymal stem cells (ADMSCs) in restoring damaged tissues. Using a hindlimb ischemic mouse model, ADMSC-mediated induction of cell migration and gene expression related to myocyte regeneration and angiogenesis were evaluated. ADMSCs were labeled with GFP (ADMSC-GFP). The proximal end of the femoral blood vessel of mice (over 6 months of age) are ligated at two positions then cut between the two ties. Hindlimb ischemic mice were randomly divided into two groups: Group I (n = 30) which was injected with PBS (100 µL) and Group II (n = 30) which was transplanted with ADMSC-GFP (106 cells/100 µL PBS) at the rectus femoris muscle. The migration of ADMSC-GFP in hindlimb was analyzed by UV-Vis system. The expression of genes related to angiogenesis and muscle tissue repair was quantified by real-time RT-PCR. The results showed that ADMSCs existed in the grafted hindlimb for 7 days. Grafted cells migrated to other damaged areas such as thigh and heel. In both groups the ischemic hindlimb showed an increased expression of several angiogenic genes, including Flt-1, Flk-1, and Ang-2. In particular, the expression of Ang-2 and myogenic-related gene MyoD was significantly increased in the ADMSC-treated group compared to the PBS-treated (control) group; the expression increased at day 28 compared to day 3. The other factors, such as VE-Cadherin, HGF, CD31, Myf5, and TGF-ß, were also more highly expressed in the ADMSC-treated group than in the control group. Thus, grafted ADMSCs were able to migrate to other areas in the injured hindlimb, persist for approximately 7 days, and have a significantly positive impact on stimulating expression of myogenic- and angiogenesis-related genes.

Highly polarized C-terminal transition state of the leucine-rich repeat domain of PP32 is governed by local stability.

The leucine-rich repeat domain of PP32 is composed of five ß-strand-containing repeats anchored by terminal caps. These repeats differ in sequence but are similar in structure, providing a means to connect topology, sequence, and folding pathway selection. Through kinetic studies of PP32, we find folding to be rate-limited by the formation of an on-pathway intermediate. Destabilizing core substitutions reveal a transition state ensemble that is highly polarized toward the C-terminal repeat and cap. To determine if this nucleus for folding corresponds to the most stable region of PP32, we monitored amide hydrogen exchange by NMR spectroscopy. Indeed, we find the highest protection to be biased toward the C terminus. Sequence manipulations that destabilize the C terminus spread out the transition state toward the middle of the protein. Consistent with results for helical ankyrin repeat proteins, these results suggest that local stabilities determine folding pathways.

High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape.

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific 1H-15N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms.

Vapor inhalation exposure to soman in conscious untreated rats: preliminary assessment of neurotoxicity.

Neurological toxicity and brain injury following vapor inhalation exposure to the chemical warfare nerve agent (CWNA) soman (GD) were examined in untreated non-anesthetized rats. In this study, male Sprague-Dawley rats (300-350 g) were exposed to 600 mg × min/m(3) of soman or vehicle in a customized head-out inhalation system for 7 min. Convulsant animals were observed for clinical signs and various regions of the brain (dorsolateral thalamus, basolateral amygdala, piriform cortex, and lateral cortex) were collected for pathological observations 24 h post-exposure. Signs of CWNA-induced cholinergic crises including salivation, lacrimation, increased urination and defecation, and tremors were observed in all soman-exposed animals. Soman-exposed animals at 24 h post-exposure lost 11% of their body weight in comparison to 2% in vehicle-exposed animals. Whole blood acetylcholinesterase (AChE) activity was significantly inhibited in all soman-exposed groups in comparison to controls. Brain injury was confirmed by the neurological assessment of hematoxylin-eosin (H&E) staining and microscopy in the piriform cortex, dorsolateral thalamus, basolateral amygdala, and lateral cortex. Severe damage including prominent lesions, edematous, congested, and/or hemorrhagic tissues was observed in the piriform cortex, dorsolateral thalamus, and lateral cortex in soman-exposed animals 24 h post-exposure, while only minimal damage was observed in the basolateral amygdala. These results indicate that inhalation exposure to soman vapor causes neurological toxicity and brain injury in untreated unanesthetized rats. This study demonstrates the ability of the described soman vapor inhalation exposure model to cause neurological damage 24 h post-exposure in rats.

Pathogenic mutations in UBQLN2 exhibit diverse aggregation propensity and neurotoxicity.

The ubiquitin-adaptor protein UBQLN2 promotes degradation of several aggregate-prone proteins implicated in neurodegenerative diseases. Missense UBQLN2 mutations also cause X-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previously we demonstrated that the liquid-like properties of UBQLN2 molecular assemblies are altered by a specific pathogenic mutation, P506T, and that the propensity of UBQLN2 to aggregate correlated with neurotoxicity. Here, we systematically assess the effects of multiple, spatially distinct ALS/FTD-linked missense mutations on UBQLN2 aggregation propensity, neurotoxicity, phase separation, and autophagic flux. In contrast to what we observed for the P506T mutation, no other tested pathogenic mutant exhibited a clear correlation between aggregation propensity and neurotoxicity. These results emphasize the unique nature of pathogenic UBQLN2 mutations and argue against a generalizable link between aggregation propensity and neurodegeneration in UBQLN2-linked ALS/FTD.