HECT E3 Ubiquitin Ligase Itch Functions as a Novel Nega

The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases

Characterization of the denaturation and renaturation of human plasma vitronectin II. Investigation into the mechanism of formation of multimers

Unfolding and refolding of plasma vitronectin appear irreversible under near physiological conditions, with rearrangements of disulfides and self-association to a multimeric form observed as prominent structural alterations which accompany denaturation. A mechanism for the folding reactions of vitronectin has been proposed (Zhuang, P., Blackburn, M. NPeterson, C. B. (1996) J. Biol. Chem. 270, 14323-14332) in which vitronectin acquires a partially folded intermediate structure which is highly prone to oligomerize into a multimeric form. Strongly oxidizing conditions adopted for refolding from urea were effective at preventing disulfide rearrangement which disrupts distal disulfides near the C terminus of the protein. Prohibiting disulfide rearrangement under these conditions, however, was not sufficient to achieve reversibility in folding. In contrast, variations in the ionic strength of the refolding medium affect the partitioning of species so that refolded monomers are obtained at high ionic strength, and self-association is precluded. The effects of ionic strength on the partially folded intermediate in the vitronectin folding pathway appear to favor intramolecular hydrophobic collapse to form a stable hydrophobic core for the monomer versus intermolecular hydrophobic interactions which stabilize multimeric vitronectin. Although both ionic and hydrophobic interactions presumably contribute to subunit interfaces within the multimer, the basic heparin-binding region near the C terminus of the protein does not provide binding interactions which are important for self-association of vitronectin

Trauma Immediately Preceding REM-Behavior Disorder:A Valuable Prognostic Marker?

Background: The definition of rapid eye movement (REM) sleep behavior disorder (RBD) has varied over the years. Rapid eye movement sleep behavior disorder can be considered isolated or idiopathic or can occur in the context of other disorders, including trauma-associated sleep disorder (TSD) and overlap parasomnia. However, whether trauma in RBD carries any prognostic specificity is currently unknown. Study Objectives: To test the hypothesis that RBD secondary to trauma is less likely to result in the development of neurodegeneration compared to idiopathic RBD (iRBD) without trauma in the general population. Methods: A retrospective cohort study of 122 consecutive RBD patients (103 males) at two tertiary sleep clinics in Europe between 2005 and 2020 was studied. Patients were diagnosed as having iRBD by video polysomnography (vPSG) and had a semi-structured interview at presentation, including specifically eliciting any history of trauma. Patients with secondary RBD to recognized causes were excluded from the study. Patients with iRBD were categorized into three groups according to reported trauma history: (1) No history of trauma, (2) traumatic experience at least 12 months prior to RBD symptom onset, and (3) traumatic experience within 12 months of RBD symptom onset. Idiopathic RBD duration was defined as the interval between estimated onset of RBD symptoms and last hospital visit or death. Follow-up duration was defined as the interval between iRBD diagnosis and last hospital visit or death. Results: In a follow-up period of up to 18 years, no patient who experienced trauma within 12 months preceding their iRBD diagnosis received a diagnosis of a neurodegenerative disorder (n = 35), whereas 38% of patients without trauma within the 12 months of symptom onset developed a neurodegenerative illness. These patients were also significantly more likely to have a family history of α-synucleinopathy or tauopathy. Conclusions: The development of RBD within 12 months of experiencing a traumatic life event, indistinguishable clinically from iRBD, did not lead to phenoconversion to a neurodegenerative disorder even after 18 years (mean follow up 6 years). We suggest that a sub-type of RBD be established and classified as secondary RBD due to trauma. Additionally, we advocate that a thorough psychological and trauma history be undertaken in all patients presenting with dream enactment behaviors (DEB)

Enhanced Approaches for Identifying Amadori Products: Application to Peanut Allergens

The dry roasting of peanuts is suggested to influence allergic sensitization as a result of the formation of advanced glycation end products (AGEs) on peanut proteins. Identifying AGEs is technically challenging. The AGEs of a peanut allergen were probed with nano-scale liquid chromatography−electrospray ionization−mass spectrometry (nanoLC−ESI−MS) and tandem mass spectrometry (MS/MS) analyses. Amadori product ions matched to expected peptides and yielded fragments that included a loss of three waters and HCHO. As a result of the paucity of b and y ions in the MS/MS spectrum, standard search algorithms do not perform well. Reactions with isotopically labeled sugars confirmed that the peptides contained Amadori products. An algorithm was developed on the basis of information content (Shannon entropy) and the loss of water and HCHO. Results with test data show that the algorithm finds the correct spectra with high precision, reducing the time needed to manually inspect data. Computational and technical improvements allowed for better identification of the chemical differences between modified and unmodified proteins

Monochromatic aberrations of the human eye in a large population

From both a fundamental and a clinical point of view, it is necessary to know the distribution of the eye's aberrations in the normal population and to be able to describe them as efficiently as possible. We used a modified Hartmann-Shack wave-front sensor to measure the monochromatic wave aberration of both eyes for 109 normal human subjects across a 5.7-mm pupil. We analyzed the distribution of the eye's aberrations in the population and found that most Zernike modes are relatively uncorrelated with each other across the population. A principal components analysis was applied to our wave-aberration measurements with the resulting principal components providing only a slightly more compact description of the population data than Zernike modes. This indicates that Zernike modes are efficient basis functions for describing the eye's wave aberration. Even though there appears to be a random variation in the eye's aberrations from subject to subject, many aberrations in the left eye were found to be significantly correlated with their counterparts in the right eye

Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons

Artificial Inclusion Environments—Replicating Industry in the Laboratory

The authors present a series of complementary test methods which were developed and used to investigate reactions between high aluminium steel and silica rich inclusions. Non-metallic inclusions (NMIs) cause many defects in the final steel product, therefore the ability to track their size, morphology and composition and correlate this with fundamental reaction kinetics provides important knowledge to support the production of clean quality steel products. Novel steel grades such as TRIP, TWIP and low-density steels have high aluminium contents; aluminium is a readily oxidisable species presenting the potential for instability and excessive reaction with commonly used mould powders that contain silica. A novel combination of techniques including HT-CLSM (High-Temperature Confocal Laser Scanning Microscope), XCT (X-ray computed tomography) and SEM/EDS (scanning electron microscopy/electron dispersive spectroscopy) have been used to study the interaction of entrained mould powder inclusions with steel at high temperatures simulating industrial conditions. This report presents a discussion on the development of techniques and samples to achieve representative and repeatable results that can provide information on the complex chemical and physical interaction phenomena with confidence. Each experimental technique had its own learning points and consequent results. Outcomes presented include possible confirmation of the chemical reaction rate controlling step being aluminium mass transfer; heterogeneous local environmental conditions including fluidity and chemical composition; and occurrence of spontaneous emulsification where the mould powder inclusion breaks apart into a cloud of smaller fragments

Benchmarking Relatedness Inference Methods with Genome-Wide Data from Thousands of Relatives

Inferring relatedness from genomic data is an essential component of genetic association studies, population genetics, forensics, and genealogy. While numerous methods exist for inferring relatedness, thorough evaluation of these approaches in real data has been lacking. Here, we report an assessment of 12 state-of-the-art pairwise relatedness inference methods using a data set with 2485 individuals contained in several large pedigrees that span up to six generations. We find that all methods have high accuracy (92–99%) when detecting first- and second-degree relationships, but their accuracy dwindles to \u3c43% for seventh-degree relationships. However, most identical by descent (IBD) segment-based methods inferred seventh-degree relatives correct to within one relatedness degree for \u3e76% of relative pairs. Overall, the most accurate methods are Estimation of Recent Shared Ancestry (ERSA) and approaches that compute total IBD sharing using the output from GERMLINE and Refined IBD to infer relatedness. Combining information from the most accurate methods provides little accuracy improvement, indicating that novel approaches, such as new methods that leverage relatedness signals from multiple samples, are needed to achieve a sizeable jump in performance

Retired A Stars and Their Companions II: Jovian planets orbiting kappa Coronae Borealis and HD167042

We report precise Doppler measurements of two evolved stars, kappa CrB (HD142091) and HD 167042, obtained at Lick Observatory as part of our search for planets orbiting intermediate-mass subgiants. Periodic variations in the radial velocities of both stars reveal the presence of substellar orbital companions. These two stars are notably massive with stellar masses of 1.80 Msun and 1.64 Msun, indicating that they are former A-type dwarfs that have evolved off of the main sequence and are now K-type subgiants. The planet orbiting kappa CrB has a minimum mass Msini = 1.8 Mjup, eccentricity e = 0.146 and a 1208 day period, corresponding to a semimajor axis of 2.7 AU. The planet around HD167042 has a minimum mass Msini = 1.7 Mjup and a 412.6 day orbit, corresponding to a semimajor axis of 1.3 AU. The eccentricity of HD167042b is consistent with circular (e = 0.027+/-0.04), adding to the rare class of known exoplanets in long-period, circular orbits similar to the Solar System gas giants. Like all of the planets previously discovered around evolved A stars, kappa CrBb and HD167042b orbit beyond 0.8 AU.Comment: 8 pages, 3 figures, 4 tables, ApJ Accepte

A practical and general amidation method from isocyanates enabled by flow technology

The addition of carbon nucleophiles to isocyanates represents a conceptually flexible and efficient approach to the preparation of amides. This general synthetic strategy has, however, been relatively underutilized, due to narrow substrate tolerance, and the requirement for less favourable reaction conditions. Herein, we disclose a high‐yielding, mass efficient, and scalable method with appreciable functional group tolerance for the formation of amides by reaction of Grignard reagents with isocyanates. Through the application of flow chemistry, and the use of sub‐stoichiometric amounts of CuBr2, this process has been developed to encompass a broad range of substrates, including reactants found to be incompatible with previously published protocols