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Abstract: The melanin concentrating hormone (MCH) system is a new target to treat human disorders. Our aim was the preparation of the first PET-tracer for the MCHR1. [11C]SNAP-7941 is a carbon-11 labeled analog of the published MCHR1 antagonist SNAP-7941. The optimum reaction conditions were 2min reaction time, ≤25°C reaction temperature, and 2mg/mL precursor (SNAP-acid) in acetonitrile, using [11C]CH3OTf as methylation agent. [11C]SNAP-7941 was prepared in a reliable and feasible manner with high radiochemical yields (2.9±1.6GBq; 11.5±6.4% EOB, n=15). [Copyright &y& Elsevier]

Emery-Dreifuss muscular dystrophy (EDMD) is mainly inherited in an X-linked (EDMD1), and autosomal dominant (EDMD2) manner, ranging from early onset severe-childhood to slowly progressive adulthood phenotypes. Common features are joint contractures, humero-peroneal muscle weakness with later scapular-pelvic girdle muscles involvement, and cardiopathy. The pathogenic and clinical variability makes the correct diagnostic framing often not initially clear, hence, combining clinical evaluation with serum biomarkers would enable conclusive early diagnosis. Cardiac and skeletal muscles are enriched in a group of specific microRNAs (miRNAs), called myomiRs (miR-1, miR-133a, miR-133b, miR-206), which have a crucial role in myogenic processes and muscle homeostasis as they target several muscle-related genes triggering and sustaining muscle atrophy. Here, we investigated for the first time the involvement and potential clinical use in EDMDs of myomiRs and miR-21, a critical fibrosis promoter. By qRT-PCR, we analysed myomiR and miR-21 expression in EDMD1 patient-derived myoblasts and tenocytes, and in the respective CRISPR-corrected cells. An up-regulation of miR-206, miR-133a, miR-133b and miR-1 specifically in myoblasts after efficient correction of the genotype, and the down-regulation of miR-21 both in corrected tenocytes and myoblasts, were observed. Further, we examined myomiR and miR-21 expression in serum of 21 adult EDMD2 patients, and 21 age-matched healthy controls. We demonstrated a significant serum miR-206 and miR-21 up-regulation in EDMD2 patients compared to controls (p<0.0001). We then extended the miRNA analysis to miR-34a, -34b and -34c, previously associated with Progeria, one of the most severe laminopathies, as senescence/profibrotic markers and found higher miR-34a and -34c levels in EDMD2 patients' sera than controls (p<0.001). Our findings point out myomiRs, miR-21, miR-34a, and -34c as potential diagnostic markers and therapeutic targets for EDMDs. [ABSTRACT FROM AUTHOR]

Lipids play various essential roles in the physiology of animals. They are also highly dependent on cellular metabolism or status. It is therefore crucial to understand to which extent animals can stabilize their lipid composition in the presence of external stressors, such as chemicals that are released into the environment. We developed a MALDI MS imaging workflow for two important aquatic model organisms, the zebrafish (Danio rerio) and water flea (Daphnia magna). Owing to the heterogeneous structure of these organisms, developing a suitable sample preparation workflow is a highly non-trivial but crucial part of this work and needs to be established first. Relevant parameters and practical considerations in order to preserve tissue structure and composition in tissue sections are discussed for each application. All measurements were based on high mass accuracy enabling reliable identification of imaged compounds. In zebrafish we demonstrate that a detailed mapping between histology and simultaneously determined lipid composition is possible at various scales, from extended structures such as the brain or gills down to subcellular structures such as a single axon in the central nervous system. For D. magna we present for the first time a MALDI MSI workflow, that demonstrably maintains tissue integrity during cryosectioning of non-preserved samples, and allows the mapping of lipids in the entire body and the brood chamber inside the carapace. In conclusion, the lipid signatures that we were able to detect with our method provide an ideal basis to analyze changes caused by pollutants in two key aquatic model organisms., (© 2022. The Author(s).)

Philadelphia chromosome-positive chronic myeloid leukemia (CML) is cytogenetically characterized by the classic translocation t(9;22)(q34;q11), whereas additional non-Philadelphia aberrations (nPhAs) have been studied extensively in adult patients with CML, knowledge on nPhAs in pediatric patients with CML is still sparse. Here, we have determined nPhAs in a cohort of 161 patients younger than 18 years diagnosed with chronic phase CML and consecutively enrolled in the German national CML-PAED-II registry. In 150 cases (93%), an informative cytogenetic analysis had been performed at diagnosis. In total, 21 individuals (13%) showed nPhAs. Of these, 12 (8%) had a variant translocation, 4 (3%) additional chromosomal aberrations (ACAs) and 5 (3%) harbored a complex karyotype. Chromosome 15 was recurrently involved in variant translocations. No significant impact of the cytogenetic subgroup on the time point of cytogenetic response was observed. Patients with a complex karyotype showed an inferior molecular response compared to patients carrying the classic translocation t(9;22)(q34;q11), variant translocations or ACAs. No significant differences in the probability of progression-free survival and overall survival was found between patients with nPhAs and patients with the classic Philadelphia translocation only. Our results highlight the distinct biology of pediatric CML and underline the need for joint international efforts to acquire more data on the disease pathogenesis in this age group.

Acquiring comprehensive knowledge about the uptake of pollutants, impact on tissue integrity and the effects at the molecular level in organisms is of increasing interest due to the environmental exposure to numerous contaminants. The analysis of tissues can be performed by histological examination, which is still time-consuming and restricted to target-specific staining methods. The histological approaches can be complemented with chemical imaging analysis. Chemical imaging of tissue sections is typically performed using a single imaging approach. However, for toxicological testing of environmental pollutants, a multimodal approach combined with improved data acquisition and evaluation is desirable, since it may allow for more rapid tissue characterization and give further information on ecotoxicological effects at the tissue level. Therefore, using the soil model organism Eisenia fetida as a model, we developed a sequential workflow combining Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) for chemical analysis of the same tissue sections. Data analysis of the FTIR spectra via random decision forest (RDF) classification enabled the rapid identification of target tissues (e.g., digestive tissue), which are relevant from an ecotoxicological point of view. MALDI imaging analysis provided specific lipid species which are sensitive to metabolic changes and environmental stressors. Taken together, our approach provides a fast and reproducible workflow for label-free histochemical tissue analyses in E. fetida, which can be applied to other model organisms as well., (© 2021. The Author(s).)

Additional data on blast phase (BP) chronic myeloid leukaemia (CML) in children and adolescents is essential for improving diagnostic and therapeutic approaches of this rare but serious condition. Here, we describe distinct clinical and genetic characteristics of 18 paediatric patients with de novo (n = 10) and secondary (n = 8) BP CML enrolled in the CML-PAED-II trial and registry. Our findings suggest that paediatric patients exhibit a diverse cytogenetic profile compared to adults with BP CML. In addition, patients with de novo BP CML in this cohort presented at a younger age, whereas patients with secondary BP CML more often harboured complex karyotypes., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Bisphenols are important plasticizers currently in use and are released at rates of hundreds of tons each year into the biosphere 1-3 . However, for any bisphenol it is completely unknown if and how it affects the intact adult brain 4-6 , whose powerful homeostatic mechanisms could potentially compensate any effects bisphenols might have on isolated neurons. Here we analyzed the effects of one month of exposition to BPA or BPS on an identified neuron in the vertebrate brain, using intracellular in vivo recordings in the uniquely suited Mauthner neuron in goldfish. Our findings demonstrate an alarming and uncompensated in vivo impact of both BPA and BPS-at environmentally relevant concentrations-on essential communication functions of neurons in mature vertebrate brains and call for the rapid development of alternative plasticizers. The speed and resolution of the assay we present here could thereby be instrumental to accelerate the early testing phase of next-generation plasticizers.

In animal husbandry, livestock industry and research facilities, anaesthetic agents are frequently used to moderate stressful intervention. For mammals and birds, procedures have been established to fine-tune anaesthesia according to the intervention. In ectothermic vertebrates, however, and despite changes in legislation and growing evidence on their cognitive abilities, the presently available information is insufficient to make similarly informed decisions. Here we suggest a straightforward way for rapidly filling this gap. By recording from a command neuron in the brain of fish whose crucial role requires it to integrate and process information from all sensory systems and to relay it to motor output pathways, the specific effects of candidate anaesthesia on central processing of sensory information can directly and efficiently be probed. Our approach allows a rapid and reliable way of deciding if and at which concentration a given anaesthetic affects the central nervous system and sensory processing. We employ our method to four anaesthetics commonly used in fish and demonstrate that our method quickly and with small numbers of animals provides the critical data for informed decisions on anaesthetic use.

Since high MAO-B levels are present in early stages of AD, the MAO-B system can be designated as an appropriate and prospective tracer target of molecular imaging biomarkers for the detection of early AD. According to the preceding investigations of Mishra et al. the aim of this work was the development of a compound library of selective and reversible MAO-B inhibitors by performing bioisosteric modifications of the core structure of 3-(anthracen-9-yl)-5-phenyl-4,5-dihydro-1H-pyrazoles. In conclusion, 13 new pyrazoline based derivatives have been prepared, which will serve as precursor substances for future radiolabeling as well as reference compounds for the investigation of increased MAO-B levels in AD., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A new series of substituted tri-/tetraazabenzo[3,2-a]fluorene-5,6-diones and their corresponding oxime derivatives have been synthesized and spectroscopically characterized. The antiproliferative activities of all compounds were evaluated on at least three different cell lines., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The MCH receptor has been revealed as a target of great interest in positron emission tomography imaging. The receptor's eponymous substrate melanin-concentrating hormone (MCH) is a cyclic peptide hormone, which is located predominantly in the hypothalamus with a major influence on energy and weight regulation as well as water balance and memory. Therefore, it is thought to play an important role in the pathophysiology of adiposity, which is nowadays a big issue worldwide. Based on the selective and high-affinity MCH receptor 1 antagonist SNAP-7941, a series of novel SNAP derivatives has been developed to provide different precursors and reference compounds for the radiosyntheses of the novel PET radiotracers [(11)C]SNAP-7941 and [(18)F]FE@SNAP. Positron emission tomography promotes a better understanding of physiologic parameters on a molecular level, thus giving a deeper insight into MCHR1 related processes as adiposity.

The melanin-concentrating hormone (MCH) system is a new target for the treatment of human disorders. Since the knowledge of the MCH system's involvement in a variety of pathologies (obesity, diabetes, and deregulation of metabolic feedback mechanism) is based on in vitro or preclinical studies, a suitable positron emission tomography (PET) tracer needs to be developed. We herein present the preparation and first preclinical evaluation of [(18)F]FE@SNAP - a new PET tracer for MCH receptor-1 (MCHR1). The synthesis was performed using a microfluidic device. Preclinical evaluation included binding affinity, plasma stability, plasma free fraction, stability against the cytochrome P-450 (CYP450) system using liver microsomes, stability against carboxyl-esterase, and methods to assess the penetration of the blood-brain barrier (BBB) such as logD analysis and immobilized artificial membrane (IAM) chromatography. Levels at 374 ± 202 MBq [(18)F]FE@SNAP were obtained after purification. The obtained K d value of [(18)F]FE@SNAP was 2.9 nM. [(18)F]FE@SNAP evinced high stability against carboxylesterase, CYP450 enzymes, and in human plasma. LogD (3.83) and IAM chromatography results (Pm=0.51) were in the same range as for known BBB-penetrating compounds. The synthesis of [(18)F]FE@SNAP was reliable and successful. Due to high binding affinity and stability, [(18)F]FE@SNAP is a promising tracer for MCHR1.

A series of indazolo[4,3-gh]isoquinolinones derivatives have been synthesized to decrease cardiotoxic side effects in comparison to Mitoxantrone. The antiproliferative effects of different side chains were investigated and tested on at least four different cell lines of cervix, ovarian, CNS, NSCLC (non-small-cell lung cancer) and colon carcinoma. In addition to antiproliferative activities, influence on cell cycle and intercalation behavior have been tested., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

IL-1 is a key inflammatory and immune mediator in many diseases, including dry-eye disease, and its inhibition is clinically efficacious in rheumatoid arthritis and cryopyrin-associated periodic syndromes. To treat ocular surface disease with a topical biotherapeutic, the uniqueness of the site necessitates consideration of the agent's size, target location, binding kinetics, and thermal stability. Here we chimerized two IL-1 receptor ligands, IL-1β and IL-1Ra, to create an optimized receptor antagonist, EBI-005, for topical ocular administration. EBI-005 binds its target, IL-1R1, 85-fold more tightly than IL-1Ra, and this increase translates to an ∼100-fold increase in potency in vivo. EBI-005 preserves the affinity bias of IL-1Ra for IL-1R1 over the decoy receptor (IL-1R2), and, surprisingly, is also more thermally stable than either parental molecule. This rationally designed antagonist represents a unique approach to therapeutic design that can potentially be exploited for other β-trefoil family proteins in the IL-1 and FGF families.

Changes in the expression of the melanin concentrating hormone receptor 1 (MCHR1) are involved in a variety of pathologies, especially obesity and anxiety disorders. To monitor these pathologies in-vivo positron emission tomography (PET) is a suitable method. After the successful radiosynthesis of [(11)C]SNAP-7941-the first PET-Tracer for the MCHR1, we aimed to synthesize its [(18)F]fluoroethylated analogue: [(18)F]FE@SNAP. Therefore, microfluidic and vessel-based approaches were tested. [(18)F]fluoroethylation was conducted via various [(18)F]fluoroalkylated synthons and direct [(18)F]fluorination. Only the direct [(18)F]fluorination of a tosylated precursor using a flow-through microreactor was successful, affording [(18)F]FE@SNAP in 44.3 ± 2.6%., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A series of 6-azanaphthoquinone pyrrolo-annelated derivatives carrying different basic side chains have been synthesized. The antiproliferative activities of all compounds were evaluated on at least four different cell lines with Mitoxantrone as reference compound. Cytotoxic effects and DNA intercalation behavior were investigated., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Advances in single-use technologies can enable greater speed, flexibility, and a smaller footprint for multi-product production facilities, such as at a contract manufacturer. Recent efforts in the area of cell line and media optimization have resulted in bioreactor productivities that exceed 8 g/L in fed-batch processes or 25 g/L in high-density cell culture processes. In combination with the development of single-use stirred tank bioreactors with larger working volumes, these intensified upstream processes can now be fit into a single-use manufacturing setting. Contrary to these upstream advances, downstream single-use technologies have been slower to follow, mostly limited by low capacity, high cost, and poor scalability. In this study we describe a downstream process based solely on single-use technologies that meets the challenges posed by expression of a mAb (IgG(1)) in a high-density suspension culture of PER.C6 cells. The cell culture harvest was clarified by enhanced cell settling (ECS) and depth filtration. Precipitation was used for crude purification of the mAb. A high capacity chromatographic membrane was then used in bind/elute mode, followed by two membranes in flow-through (FT) mode for polishing. A proof of concept of the entire disposable process was completed for two different scales of the purification train.

A series of azanaphthoquinone pyrrolo-annelated derivatives attached to basic side chains have been synthesized. The antiproliferative activities of all compounds were evaluated on at least four different cell lines. The effects on cell cycle and intercalation were investigated., (2010 Elsevier Ltd. All rights reserved.)

Herpesviridae comprises over 120 viruses infecting a wide range of vertebrates including humans and livestock. Herpesvirus infections typically produce dermal lesions or immune cell destruction, but can also lead to oncogenesis, especially with KSHV (Kaposi's sarcoma-associated herpesvirus). All herpesviruses are nuclear replicating viruses that subvert cellular processes such as nucleocytoplasmic transport for their advantage. For virus replication to take over the cell and produce lytic infection requires that virus gene expression outpace that of the host cell. KSHV ORF57 (open reading frame 57) appears to play a major role in this by (i) serving as a nuclear export receptor to carry intronless viral mRNAs out of the nucleus and (ii) inhibiting expression of intron-containing host mRNAs. As most of the virally encoded mRNAs are intronless compared with host cell mRNAs, these two mechanisms are critical to overcoming host gene expression.

Purpose: The application of diamond knives has steadily increased in ophthalmic surgery. However, the geometry of the blade, its thickness and the sharpness of the cutting edge are limited by the abrasive diamond polishing process, e. g. the crystalline morphology of the bulk material and the grinding powder used. A new generation of diamond blades is presented herewith allowing free choice of blade shape and thickness and possessing excellent sharpness due to a new polishing process., Methods: The new production method is based on a high-quality CVD (chemical vapour deposition) diamond film of some tenths of microns thickness, deposited on a silicon wafer as microchip technology. The mechanical properties of this synthetic diamond film are almost equal to those of a natural diamond and the surface of this film is mirror-like after deposition without requiring post-polishing. The shape of the blade can be freely defined and is transferred into the diamond film by a plasma polishing process adopted from microsystem technology., Results: The new production method results in highly reproducible diamond blades. Concave blades and round shapes can now be realised without the restrictions of the conventional production process. The force-free fabrication method even allows realisation of miniaturized blades (e. g. width < 0.125 mm, thickness < 50 microm) far beyond the possibilities of conventional diamond blade production. Plasma polishing by means of gas atoms results in extreme sharpness with the cutting edge radius in the range of approx. 3 nm., Conclusions: Utilising microsystem technology we were able to manufacture reproducible artificial diamond blades. The new process offers for the first time surgeons a possibility of designing blades with a geometry close to their personal needs. Furthermore, the potential of facet-free ergonomically shaped diamond blades may stimulate further improvements towards novel surgical techniques.

Tobacco pollen tubes were used as a standard in vitro system to investigate cell growth aberrations caused by some of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme chemicals and other toxic compounds. Changes in cytoskeletal pattern were observed in the tube cells by using tubulin immunofluorescence and rhodamin-phalloidin fluorescence for the localisation of microtubules and actin filaments, respectively. Four different types of cell malformation were found: screw-like growth, isodiametric tip swelling, hook formation, and pollen grain enlargement. We suggest that these malformations resulted from an interference by the chemicals with the cytosolic calcium gradient which controls tip growth and the orientation of the pollen tube. The results may contribute to a general understanding of toxicity-based cell malformations.

The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. The lamina is proposed to be an important determinant of nuclear structure, but there has been little direct testing of this idea. To investigate lamina functions, we have characterized a novel lamin B1 mutant lacking the middle approximately 4/5 of its alpha-helical rod domain. Though retaining only 10 heptads of the rod, this mutant assembles into intermediate filament-like structures in vitro. When expressed in cultured cells, it concentrates in patches at the nuclear envelope. Concurrently, endogenous lamins shift from a uniform to a patchy distribution and lose their complete colocalization, and nuclei become highly lobulated. In vitro binding studies suggest that the internal rod region is important for heterotypic associations of lamin B1, which in turn are required for proper organization of the lamina. Accompanying the changes in lamina structure induced by expression of the mutant, nuclear pore complexes and integral membrane proteins of the inner membrane cluster, principally at the patches of endogenous lamins. Considered together, these data indicate that lamins play a major role in organizing other proteins in the nuclear envelope and in determining nuclear shape.

We present here a detailed analysis of a rat polypeptide termed Nup50 (formerly NPAP60) that was previously found to be associated with the nuclear pore complex (F. Fan et al., Genomics 40:444-453, 1997). We have found that Nup50 (and/or a related 70-kDa polypeptide) is present in numerous rat cells and tissues. By immunofluorescence microscopy, Nup50 was found to be highly concentrated at the nuclear envelope of rat liver nuclei, whereas in cultured NRK cells it also is abundant in intranuclear regions. On the basis of immunogold electron microscopy of both rat liver nuclear envelopes and NRK cells, we determined that Nup50 is specifically localized in the nucleoplasmic fibrils of the pore complex. Microinjection of anti-Nup50 antibodies into the nucleus of NRK cells resulted in strong inhibition of nuclear export of a protein containing a leucine-rich nuclear export sequence, whereas nuclear import of a protein containing a classical nuclear localization sequence was unaffected. Correspondingly, CRM1, the export receptor for leucine-rich export sequences, directly bound to a fragment of Nup50 in vitro, whereas several other import and export receptors did not significantly interact with this fragment. Taken together, our data indicate that Nup50 has a direct role in nuclear protein export and probably serves as a binding site on the nuclear side of the pore complex for export receptor-cargo complexes.

[PSI+] is a genetic element in yeast for which a heritable change in phenotype appears to be caused by a heritable change in the conformational state of the Sup35 protein. The inheritance of [PSI+] and the physical state of Sup35 in vivo depend on the protein chaperone Hsp104 (heat shock protein 104). Although these observations provide a strong genetic argument in support of the "protein-only" or "prion" hypothesis for [PSI+], there is, as yet, no direct evidence of an interaction between the two proteins. We report that when purified Sup35 and Hsp104 are mixed, the circular dichroism (CD) spectrum differs from that predicted by the addition of the proteins' individual spectra, and the ATPase activity of Hsp104 is inhibited. Similar results are obtained with two other amyloidogenic substrates, mammalian PrP and beta-amyloid 1-42 peptide, but not with several control proteins. With a group of peptides that span the PrP protein sequence, those that produced the largest changes in CD spectra also caused the strongest inhibition of ATPase activity in Hsp104. Our observations suggest that (i) previously described genetic interactions between Hsp104 and [PSI+] are caused by direct interaction between Hsp104 and Sup35; (ii) Sup35 and PrP, the determinants of the yeast and mammalian prions, respectively, share structural features that lead to a specific interaction with Hsp104; and (iii) these interactions couple a change in structure to the ATPase activity of Hsp104.

The [PSI+] factor of S. cerevisiae represents a new form of inheritance: cytosolic transmission of an altered phenotype is apparently based upon inheritance of an altered protein structure rather than an altered nucleic acid. The molecular basis of its propagation is unknown. We report that purified Sup35 and subdomains that induce [PSI+] elements in vivo form highly ordered fibers in vitro. Fibers bind Congo red and are rich in beta sheet, characteristics of amyloids found in certain human diseases, including the prion diseases. Some fibers have distinct structures and these, once initiated, are self-perpetuating. Preformed fibers greatly accelerate fiber formation by unpolymerized protein. These data support a "protein-only" seeded polymerization model for the inheritance of [PSI+].

The HSP100/Clp proteins are a newly discovered family with a great diversity of functions, such as increased tolerance to high temperatures, promotion of proteolysis of specific cellular substrates and regulation of transcription. HSP100/Clp proteins are also synthesized in a variety of specific patterns and, in eukaryotes, are localized to different subcellular compartments. Recent data suggest that a common ability to disassemble higher-order protein structures and aggregates unifies the molecular functions of this diverse family.

Human herpesvirus 6 (HHV-6) is a newly identified lymphotropic herpesvirus. We have analyzed viral and host DNA replication in peripheral blood lymphocytes infected in the absence of drugs or infected in the presence of phosphonoacetic acid (PAA) or acyclovir (ACV). The results revealed the following: (i) Infection with HHV-6 resulted in the shutoff of host DNA replication. (ii) PAA at concentrations of 100 and 300 micrograms/ml significantly reduced virus replication. The drug inhibited viral DNA replication, whereas host cell DNA replication was not affected. This strongly suggests that HHV-6 encodes a PAA sensitive viral DNA polymerase. (iii) ACV at 20 microM did not interfere with virus production and virus spread. ACV at 100 microM only partly interfered with virus replication, whereas at 400 microM the block was more complete. Viral DNA replication was not affected by ACV at 20 microM. However, approximately 60 and 85% inhibition in viral DNA replication was observed in the presence of 100 and 400 microM of ACV. (iv) Assays for viral thymidine kinase (TK) revealed no significant increase in TK activity, whereas increased TK activity was noted following infection of the same peripheral blood lymphocytes with herpes simplex virus. Thus, either HHV-6 does not encode a tk enzyme which can phosphorylate ACV or the inefficient block may reflect lower sensitivity of the HHV-6 DNA polymerase to the drug.