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Alpha-synuclein (α-syn) aggregation and immune activation represent hallmark pathological events in Parkinson's disease (PD). The PD-associated immune response encompasses both brain and peripheral immune cells, although little is known about the immune proteins relevant for such a response. We propose that the upregulation of CD163 observed in blood monocytes and in the responsive microglia in PD patients is a protective mechanism in the disease. To investigate this, we used the PD model based on intrastriatal injections of murine α-syn pre-formed fibrils in CD163 knockout (KO) mice and wild-type littermates. CD163KO females revealed an impaired and differential early immune response to α-syn pathology as revealed by immunohistochemical and transcriptomic analysis. After 6 months, CD163KO females showed an exacerbated immune response and α-syn pathology, which ultimately led to dopaminergic neurodegeneration of greater magnitude. These findings support a sex-dimorphic neuroprotective role for CD163 during α-syn-induced neurodegeneration., (© 2023. The Author(s).)

Objective: Cardiac regeneration in the axolotl has been found to rely on the innate immune system, and especially macrophages have been demonstrated to play a vital role in regulating the regenerative process. In this study we wanted to induce a pro- and anti-inflammatory milieu in the axolotl during heart regeneration to test the resilience of the regenerative response., Results: This was induced via repeated intrapericardial injections of lipopolysaccharide or prednisolone during a 40-day regeneration period in order to challenge the presumably fine-tuned inflammatory response that normally facilitates regeneration. We observed a local and systemic leucocyte response to pro- and anti-inflammatory stimulation, but we found cardiac regeneration to be structurally and functionally unaffected.

Tumor-associated macrophages are among the most abundant non-cancerous cells in the tumor microenvironment and in many cancers macrophage infiltration into the tumor is associated with poor prognosis. Macrophages contribute to tumor development by promoting angiogenesis and immune suppression, and display remarkable phenotypic heterogeneity in the tumor microenvironment. Therapeutic strategies targeting macrophages that currently are in clinical development are mainly focused on a general depletion of tumor-associated macrophages, either by targeting the CSF-1/CSF-1R axis or by inhibiting macrophage recruitment by blocking CCR2/CCL2 signaling. Despite good pre-clinical response rates the treatment strategies focusing on general macrophage targeting have only shown limited clinical success and new approaches that target specific subsets of tumo-associated macrophages are emerging. This chapter will briefly present the functions and heterogeneity of tumor-associated macrophages and provide an overview of the current state of clinical development for pan-targeting strategies as well as discuss new strategies for targeting specific macrophage subsets for future anti-tumor immunotherapies., Competing Interests: Conflict of interest The authors declare no conflict of interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background and Objectives: Most telemedicine solutions are proprietary and disease specific which cause a heterogeneous and silo-oriented system landscape with limited interoperability. Solving the interoperability problem would require a strong focus on data integration and standardization in telemedicine infrastructures. Our objective was to suggest a future-proof architecture, that consisted of small loose-coupled modules to allow flexible integration with new and existing services, and the use of international standards to allow high re-usability of modules, and interoperability in the health IT landscape., Methods: We identified core features of our future-proof architecture as the following (1) To provide extended functionality the system should be designed as a core with modules. Database handling and implementation of security protocols are modules, to improve flexibility compared to other frameworks. (2) To ensure loosely coupled modules the system should implement an inversion of control mechanism. (3) A focus on ease of implementation requires the system should use HL7 FHIR (Fast Interoperable Health Resources) as the primary standard because it is based on web-technologies., Results: We evaluated the feasibility of our architecture by developing an open source implementation of the system called ORDS. ORDS is written in TypeScript, and makes use of the Express Framework and HL7 FHIR DSTU2. The code is distributed on GitHub. All modules have been tested unit wise, but end-to-end testing awaits our first clinical example implementations., Conclusions: Our study showed that highly adaptable and yet interoperable core frameworks for telemedicine can be designed and implemented. Future work includes implementation of a clinical use case and evaluation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: There is no evidence on how motor function is best evaluated in children in a low-risk setting. The method used in the Danish Preventive Child Health Examination Programme (DPCHEP) in general practise has not been validated. The objective of this review was to identify existing motor function tests for 0-2-year-old children that were validated for use in the background population and which are suitable for use in the DPCHEP., Methods: This systematic review was conducted in accordance with the PRISMA guidelines. A systematic literature search was performed in PubMed, Embase, SwedMed, PsycInfo and CINAHL in accordance with the inclusion and exclusion criteria., Results: Five motor function tests were identified. The Alberta Infant Motor Scale (AIMS) exclusively assesses motor function, the Harris Infant Neuromotor Assessment also assesses cognition and the Early Motor Questionnaire (EMQ) additionally assesses perception-action integration skills. The Ages and Stages Questionnaire (ASQ) and The Brigance Infant and Toddler Screen include further aspects of development. All test methods, except for the AIMS, are based on parent involvement., Conclusions: For implementation in the DPCHEP, five motor function tests were potentially adequate. However, the time consumption and extensive use of tools render three of the five tests unsuitable for implementation in the existing programme. The two remaining tests, the ASQ and the EMQ, are parent questionnaires. We suggest that these should be pilot tested with a view to their subsequent implementation in the DPCHEP. It may be considered to present the test elements in a more manageable and systematic way, possibly with illustrations., (Articles published in the DMJ are “open access”. This means that the articles are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits any non-commercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.)

The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.

The Aurora kinases are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumors, and lead to aneuploidy and tumorigenesis. Here we report the discovery of a highly potent and selective small-molecule inhibitor of Aurora kinases, VX-680, that blocks cell-cycle progression and induces apoptosis in a diverse range of human tumor types. This compound causes profound inhibition of tumor growth in a variety of in vivo xenograft models, leading to regression of leukemia, colon and pancreatic tumors at well-tolerated doses. Our data indicate that Aurora kinase inhibition provides a new approach for the treatment of multiple human malignancies.

Angelman syndrome (AS) is a genetic disorder associated with severe developmental delay. The purpose of this study was to investigate cognitive and linguistic development in AS. Piaget's developmental model was used to evaluate the test results. The participants comprised 20 children (14 boys and 6 girls) aged 2-14 years (median age 7.4 years). AS was diagnosed either according to typical clinical criteria or confirmatory genetic testing. Cognitive functioning was evaluated with Griffiths' Mental Development Scale. Language development was also evaluated with Receptive-Expressive Emergent Language Scale 2 (REEL-2). Cognitive functioning, based on results on the Performance Scale, never exceeded Piaget's sensorimotor stage, 0-2 years. The median mental age for language development was 9 months. Expressive verbal vocabulary consisted of less than 2 words (n = 11), 2-3 words (n = 7) and 4-5 words (n = 2). Analyses according to REEL-2 did not indicate a consistent discrepancy between impressive and expressive language.

With the growing concern that environmental chemicals might impair human and animal fertility, it is important to investigate the possible influence of these substances on sexual differentiation and genital development of mammals. Many of these substances are suspected to interfere with endocrine processes, and exposure during critical periods of prenatal development might affect reproductive performance over several generations. Alkylphenols and their metabolites are lipophilic substances exerting apparent estrogenic action in in vitro and in vivo testing systems. With the widespread industrial use of alkylphenols, these are disseminated in the environment with sewage sludge, and domestic animals and humans are likely to be exposed via the food chain. Using the pig as an in vivo model, we studied the effect of intrauterine exposure to tertiary octylphenol (OP) on essential reproductive parameters over 3 generations. Sows were treated daily from D 23 to 85 of pregnancy with either 0, 10 or 1000 micrograms OP/kg body weight. Treatment with OP extended pregnancy length and induced basal cell proliferation in the cervical epithelium of the parental generation. In F1 offspring of sows treated with the low dosage of OP, onset of puberty was accelerated. Furthermore, when F1 gilts and F1 boars originating from sows treated with high dosages of OP were bred, the litter size was reduced. The results of the present study are compared with previous reports on estrogenicity of OP, and the usefulness of in vivo animal or embryo models for the evaluation of possible consequences of human exposure to endocrine disrupting compounds is discussed. Furthermore, possible consequences of exposure to endocrine disrupting compounds for the embryo transfer industry are addressed.

Ubiquitin-mediated proteolysis regulates the activity of diverse receptor systems. Here, we identify Smurf2, a C2-WW-HECT domain ubiquitin ligase and show that Smurf2 associates constitutively with Smad7. Smurf2 is nuclear, but binding to Smad7 induces export and recruitment to the activated TGF beta receptor, where it causes degradation of receptors and Smad7 via proteasomal and lysosomal pathways. IFN gamma, which stimulates expression of Smad7, induces Smad7-Smurf2 complex formation and increases TGF beta receptor turnover, which is stabilized by blocking Smad7 or Smurf2 expression. Furthermore, Smad7 mutants that interfere with recruitment of Smurf2 to the receptors are compromised in their inhibitory activity. These studies thus define Smad7 as an adaptor in an E3 ubiquitin-ligase complex that targets the TGF beta receptor for degradation.

Mixed-lineage kinase 2 (MLK2) is a cytoplasmic protein kinase expressed at high levels in mammalian brain. The MLK2 structure is composed of a Src homology 3 (SH3) domain, two leucine zippers, a basic motif, a Cdc42/Rac interactive binding motif and a large C-terminal domain rich in proline, serine and threonine residues. To begin to define the role of MLK2 in mammalian brain, we used an MLK2-SH3 domain-glutathione S-transferase fusion protein (GST-MLK2-SH3) to isolate MLK2-binding proteins from rat brain extract. This analysis revealed that the major MLK2-SH3-domain-binding protein in rat brain is the GTPase dynamin. By using two different forms of the dynamin proline-rich domain as affinity ligands, the binding site for MLK2-SH3 was mapped to the C-terminal region of dynamin between residues 832 and 864. In GTPase assays, the addition of MLK2-SH3 stimulated the activity of purified dynamin I by 3-fold over the basal level, whereas the addition of a known dynamin activator, phosphatidylserine (PtdSer), stimulated a 6-fold increase. When MLK2-SH3 was added to the assay together with PtdSer, however, dynamin GTPase activity accelerated by more than 23-fold over basal level. An MLK2 mutant (MLK2-W59A-SH3), with alanine replacing a conserved tryptophan residue in the SH3 domain consensus motif, had no effect on dynamin activity, either alone or in the presence of PtdSer. In the same assay the SH3 domain from the regulatory subunit of phosphatidylinositol 3'-kinase stimulated a similar synergistic acceleration of dynamin GTPase activity in the presence of PtdSer. These results suggest that synergy between phospholipid and SH3 domain binding might be a general mechanism for the regulation of GTP hydrolysis by dynamin.

The mixed lineage kinase 2 (MLK2) protein contains several structurally distinct domains including an src homology (SH) 3 domain, a kinase catalytic domain, two leucine zippers, a basic motif and a cdc42/rac interactive binding motif. These domains have been recognized mainly for their involvement in protein-protein interactions in signal transduction networks. The SH3 domain in particular has been implicated in control of signaling events. To identify proteins that interact with MLK2, the N-terminal 100 amino acids, including the SH3 domain, were expressed as a glutathione S-transferase (GST) fusion protein. This fusion protein (MLK2N) was used as an affinity ligand to isolate binding proteins from lysates of 35S-radiolabeled MDA-MB231 breast carcinoma cells. When the radiolabeled binding proteins were subjected to 2-DE, proteins of Mr 55,000, 31,500 and 34,000 bound consistently to the MLK2N domain fusion protein, but not to the GST control. Two of the binding proteins were isolated from whole cell lysates by preparative 2-DE and subjected to in-gel digestion and capillary or microbore reverse-phase high performance liquid chromatography (RP-HPLC). Resultant peptides were analyzed by peptide mass fingerprinting, N-terminal Edman degradation or tandem mass spectrometry. The 55,000 protein was identified as the cytoskeletal protein, beta-tubulin, and this was verified by immunoblotting of proteins in the MLK2N binding fraction with anti-tubulin antibodies. The 31,500 protein has been identified as prohibitin, a protein that has been implicated in both signal transduction and cell cycle arrest.

Two-dimensional polyacrylamide gel electrophoresis (2-DE) in combination with mass spectrometry is an extremely powerful tool for characterizing complex mixtures of proteins. In many cases, the success of this approach relies upon the ability to recover peptides at high concentrations and free of interfering artifacts from in-gel and/or on-membrane enzymatic digests. In previous studies, we demonstrated that capillary or microcolumn (< 350 microm ID) reversed-phase high performance liquid chromatography (RP-HPLC) is a powerful microseparation technique for proteins and peptides (Moritz, R. L. and Simpson, R. J., J. Chromatogr. 1992, 599, 119-130). Here we evaluate various capillary column RP-HPLC/mass spectrometric approaches for identifying and characterizing 2-DE resolved proteins. For these studies, stable and efficient 0.20 mm and 0.32 mm internal diameter (ID) fused-silica columns with hydrophilic polyvinylidene difluoride (PVDF) frits were fabricated and slurry packed with 7 microm spherical, 300 A pore size, C8 bonded phase silica particles. We show that capillary column chromatography is a rapid and efficient desalting/concentrating (ON/OFF) technique for sample cleanup prior to protein identification by peptide-mass fingerprinting using matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry. While marginally more peptide mass information can be obtained by stepped elution of the peptide mixture with increasing concentrations of organic solvent, best results were obtained by fractionation of the peptide mixture using a linear 60 min gradient. One salient feature of this study was the observation that, in contrast to the stepped elution and gradient approaches, the ionization of peptide T1 (m/z 2402.2 SGETEDTFIADLVV(PeCys)TGQIK) was almost completely suppressed using the ON/OFF approach. Maximal amino acid sequence coverage, a necessary prerequisite for complete characterization of a protein, was accomplished using a capillary column (0.2 mm ID) directly coupled with an electrospray ionization (ESI) ion-trap tandem mass spectrometer. For example, from an in situ tryptic digest of alpha-enolase isolated by 2-DE from the human breast carcinoma cell line MDA-MB231, 71% of the amino acid sequence was obtained. In addition to identifying two possible N-terminal acetylated alpha-enolase variants, Asn153Asp and Ile152Asp/Asn153Ile, the tandem mass spectrometric data revealed the presence of a number of process-induced modifications of alpha-enolase such as methionine oxidation and cysteine amidoethylation.

Previously, we reported a two-dimensional gel map and database with molecular weight/isoelectric point (Mr/pI) loci for 22 proteins expressed in the breast carcinoma cell line, MDA-MB231 (Rasmussen et al., Electrophoresis 1997, 18, 588-598). Here we update this database with Mr/pI loci for a further nine cytoplasmic proteins and three Triton X-114 solubilised membrane proteins from MDA-MB231 cells. In addition, a novel protein, previously represented only in expressed sequence tag (EST) databases, has been identified as a Triton X-114 soluble protein and assigned an Mr/pI locus. During the course of isolating proteins from the Triton X-114 fraction, we compared recoveries of proteins in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels after isoelectric focusing (IEF) using either immobilised pH gradients or carrier ampholytes. In these experiments, a significantly higher proportion of membrane proteins were visible in SDS-polyacrylamide gels after the use of carrier ampholytes for the first dimension. We also report our mass spectrometric-based procedure for identifying two-dimensional electrophoresis (2-DE) gel-resolved proteins, combining in-gel enzymatic digestion, 0.2 mm internal diameter (ID) capillary column reversed-phase high-performance liquid chromatography (RP-HPLC) peptide mapping and electrospray ionisation--ion trap--mass spectrometry.

Capillary column (< or = 320-micron ID) liquid chromatography is an essential tool for the separation and concentration of low-picomole amounts of proteins and peptides for mass-spectrometric based structural analysis. We describe a detailed procedure for the fabrication of stable and efficient 50- to 180-micron ID polyimide fused-silica columns. Columns were packed by conventional slurry packing with reversed-phase silica-based supports followed by column bed consolidation with acetonitrile and sonication. PVDF membrane or internal fused-silica particles were employed for column end-frit construction. The ability of these columns to withstand high back pressures (300-400 bar) enabled their use for rapid chromatography (> 3400 cm/hr; i.e., approximately 40 microliters/min for 200-micron ID columns) and the loading of large sample volumes (up to 500 microliters). The accurate low flow rates (0.4-4.0 microliters/min) and precise gradient formation necessary to operate these columns were achieved by a simple modification of conventional HPLC systems [Moritz et al. (1992), J. Chromatogr. 599, 119-130]. Column performance was evaluated for ability to resolve low-fmol amounts of all components of a mixture of PTH-amino acids and to separate peptides for on-line LC/MS analysis of peptide mixtures derived from in situ digestion of 2-DE resolved protein spots.

MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al. (Eur. J. Biochem. 1993, 213, 701-710), comprises several distinct structural domains including an src homology-3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif. Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction. To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N-terminal 100 amino acids of MLK2 (MLK2N), including the SH3 domain (residues 23-77), fused to glutathione S-transferase. This fusion protein was expressed in Escherichia coli, purified using gluthathione-Sepharose affinity chromatography and employed in an affinity approach to isolate MLK2-SH3 domain binding proteins from lysates of 35S-labelled MDA-MB231 human breast tumour cells. Electrophoretic analysis of bound proteins revealed that two low-abundance proteins with a molecular weights (Mr) of approximately 31,500 and approximately 34,000, bound consistently to the MLK2N protein. To establish accurately the Mt / isoelectric point (pI) loci of these MLK2-SH3 domain binding proteins, a number of abundant proteins in a two-dimensional electrophoresis (2-DE) master gel were identified to serve as triangulation marker points. Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in-gel proteolysis and fractionation by rapid (approximately 12 min) microbore column (2.1 mm ID) reversed-phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide-mass fingerprinting and electrospray (ESI)-mass spectrometry (MS)-MS utilizing capillary (0.2-0.3 mm ID) column chromatography, or (iv) immunoblot analysis. Using this information, a preliminary 2-DE protein database for the human breast carcinoma cell line MDA-MB231, comprising 21 identified proteins, has been constructed and can be accessed via the World Wide Web (URL address: http:(/)/ www.ludwig.edu.au/www/jpsl/jpslhome.htm l).

In spite of adequate dietary regimen, many patients with galactosemia have developmental abnormalities. We studied 8 patients with galactosemia, aged 9 months to 19 years who had all been treated with a galactose-free diet from an early stage. Neurological functioning, general developmental, and language and speech development were assessed in all cases. The results show that even medically well treated children and young adults with galactosemia are at risk to develop disabilities, including mental retardation, speech and language disabilities. Verbal dyspraxia was diagnosed in 3 of 6 patients, who had acquired verbal language, all with IQ 70 or below. This may indicate that verbal dyspraxia is just one symptom among others in patients with galactosemia and mental retardation.

Fatty acid synthesis from acetate in extracts of Saccharomyces cerevisiae strain LK2G12 was shown to be stimulated by alpha-glycerophosphate and citrate, and by a number of compounds related to them. Magnesium was shown to stimulate fatty acid synthesis from acetyl-coenzyme A but not from malonyl-coenzyme A, thus indicating the site of stimulation of fatty acid synthesis to be the acetyl-coenzyme A step.

The mechanism proposed for the activation of animal acetyl-coenzyme A (CoA) carboxylase by alpha-glycerophosphate, namely, the removal of inhibitory palmityl-CoA via glyceride synthesis, is not the only possible one in the yeast system because extracts exhibiting marked stimulation of acetyl-CoA carboxylase activity by alpha-glyerophosphate show a lack of acyl-CoA compounds.