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Purpose: Patient-reported outcomes have not been sufficiently implemented into the routine care of cancer patients because the existing instruments are often too long and complex or not cancer-specific. The aim of this study is the determination of psychometric properties and item reduction of a newly developed health-related quality of life (HrQoL) questionnaire for use in oncological clinical routines., Methods: This observational study with a repeated measurements design included oncological inpatients and outpatients. A total of 630 patients participated at the first point of measurement and 404 at the second point of measurement. To evaluate the instrument, we conducted hierarchical confirmative factor analyses and for further validation correlated the resulting factors with standardized and validated HrQoL measurements. Test-retest reliability and responsiveness to change were tested., Results: The developed questionnaire "HELP-6" ("Hamburg Inventory for Measuring Quality of Life in Oncological Patients") has a six-factor structure and has moderate-to-good convergent validity ( r = -0.25 --0.68). Test-retest reliability was moderate-to-good ( r =0.56-0.81, p < 0.001). Indications for responsiveness to change were found for three dimensions. The final version of the questionnaire HELP-6 has six dimensions with one item each., Conclusion: With the HELP-6 instrument for measuring HrQoL in cancer patients, we provide a short and practical patient-reported outcome instrument. Though responsiveness to change could not be confirmed for all dimensions in this study, the HELP-6 includes time-efficient completion and evaluation and is informative in relevant HrQoL dimensions of cancer patients. Therefore, the HELP-6 poses an important addition to inpatient and outpatient routine cancer care., Trial Registration: This study was registered at Open Science Framework (https://osf.io/y7xce/), on 9 June 2018., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schrage, Görlach, Betz, Bokemeyer, Kröger, Mueller, Krüll, Schulz and Bleich.)

Purpose: Continuous patient-reported outcomes (PROs) to identify and address patients' needs represent an important addition to current routine care. The aim of this study was to identify and determine important dimensions of health-related quality of life (HrQoL) in routine oncological care., Methods: In a cross-sectional qualitative study, interviews and focus groups were carried out and recorded. The interviewees were asked for their evaluation on HrQoL in general and specifically regarding cancer treatment. The material was transcribed and analyzed using qualitative content analysis based on Mayring. The results were reviewed in an expert discussion., Results: Interviews with patients ( N = 28) and clinicians ( N = 4), as well as five focus groups with clinicians ( N = 18) were conducted. Initially, nine deductive and two inductive categories on HrQoL were built. Four categories ( partnership/sexuality , spirituality/religiousness , health perception , and overall health ) were excluded following the qualitative content analysis because they were hardly or not at all mentioned by participants. Following on from the analysis of the expert discussion, one dimension was added ( dignity ), and two further categories were excluded ( mobility and feeling of security in treatment ). The resulting system consisted of six dimensions: emotional health, physical ailments, autonomy, social functionality, dignity , and resources., Conclusion: The identified dimensions of HrQoL in routine oncological care were found to differ from those used in existing HrQoL measurements for (cancer) patients. Further research is needed to test and evaluate the presented structure in a larger sample of cancer patients to further assess its psychometric properties., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schrage, Görlach, Schulz and Bleich.)

Phytoplasmas are intracellular bacterial plant pathogens that cause devastating diseases in crops and ornamental plants by the secretion of effector proteins. One of these effector proteins, termed SECRETED ASTER YELLOWS WITCHES' BROOM PROTEIN 54 (SAP54), leads to the degradation of a specific subset of floral homeotic proteins of the MIKC-type MADS-domain family via the ubiquitin-proteasome pathway. In consequence, the developing flowers show the homeotic transformation of floral organs into vegetative leaf-like structures. The molecular mechanism of SAP54 action involves binding to the keratin-like domain of MIKC-type proteins and to some RAD23 proteins, which translocate ubiquitylated substrates to the proteasome. The structural requirements and specificity of SAP54 function are poorly understood, however. Here, we report, based on biophysical and molecular biological analyses, that SAP54 folds into an α-helical structure. Insertion of helix-breaking mutations disrupts correct folding of SAP54 and compromises SAP54 binding to its target proteins and, concomitantly, its ability to evoke disease phenotypes in vivo. Interestingly, dynamic light scattering data together with electrophoretic mobility shift assays suggest that SAP54 preferentially binds to multimeric complexes of MIKC-type proteins rather than to dimers or monomers of these proteins. Together with data from literature, this finding suggests that MIKC-type proteins and SAP54 constitute multimeric α-helical coiled coils. Our investigations clarify the structure-function relationship of an important phytoplasma effector protein and may thus ultimately help to develop treatments against some devastating plant diseases.

Background: Cancer patients often suffer from the physical and psychological burden of their disease and its treatment. This is frequently insufficiently identified and addressed in clinical practice. In the context of improving patient-centered care in oncological patients, patient-reported outcomes (PROs) represent an important addition to current routine care. So far, available PRO questionnaires for cancer patients are unsuitable for routine procedures due to their length and complexity., Objective: This study aimed to develop and psychometrically test a short questionnaire to measure health-related quality of life (HrQoL) in cancer patients for use in routine care., Methods: This observational study consists of two parts: (1) a qualitative study to develop a short questionnaire measuring HrQoL and (2) a quantitative study to psychometrically test this questionnaire in five oncological departments of a comprehensive cancer center. In part 1 of the study, semistructured interviews with 28 cancer patients, as well as five focus groups with 22 clinicians and nurses, were conducted to identify clinically relevant dimensions of HrQoL. The identified dimensions were complemented with related dimensions from empirical studies and reviewed via expert discussion. Based on this, a short instrument was developed. In part 2 of the study, the developed questionnaire was tested in cancer in- and outpatients at five participating oncological clinics using additional standardized questionnaires assessing HrQoL and other important PROs. The questionnaire was presented to more than 770 patients twice during treatment., Results: The project started in May 2017 with recruitment for study phase I beginning in December 2017. Recruitment for study phases I and II ended in April 2018 and February 2019, respectively. After study phase II and psychometrical analyses, the newly developed questionnaire measuring the HrQoL of all cancer entities in routine care was finalized., Conclusions: With five to six dimensions and one item per dimension, the developed questionnaire is short enough to not disrupt routine procedures during treatment and is profound enough to inform clinicians about the patient's HrQoL impairments and status., Trial Registration: Open Science Framework Registries 10.17605/OSF.IO/Y7XCE; https://osf.io/y7xce/., International Registered Report Identifier (irrid): RR1-10.2196/17854., (©Theresa Schrage, Mirja Görlach, Christian Stephan Betz, Carsten Bokemeyer, Nicolaus Kröger, Volkmar Mueller, Cordula Petersen, Andreas Krüll, Holger Schulz, Christiane Bleich. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 29.07.2020.)

Oxidative modification of cysteine residues has been shown to regulate the activity of several protein-tyrosine kinases. We explored the possibility that Fms-like tyrosine kinase 3 (FLT3), a hematopoietic receptor-tyrosine kinase, is subject to this type of regulation. An underlying rationale was that the FLT3 gene is frequently mutated in Acute Myeloid Leukemia patients, and resulting oncogenic variants of FLT3 with 'internal tandem duplications (FLT3ITD)' drive production of reactive oxygen in leukemic cells. FLT3 was moderately activated by treatment of intact cells with hydrogen peroxide. Conversely, FLT3ITD signaling was attenuated by cell treatments with agents inhibiting formation of reactive oxygen species. FLT3 and FLT3ITD incorporated DCP-Bio1, a reagent specifically reacting with sulfenic acid residues. Mutation of FLT3ITD cysteines 695 and 790 reduced DCP-Bio1 incorporation, suggesting that these sites are subject to oxidative modification. Functional characterization of individual FLT3ITD cysteine-to-serine mutants of all 8 cytoplasmic cysteines revealed phenotypes in kinase activity, signal transduction and cell transformation. Replacement of cysteines 681, 694, 695, 807, 925, and 945 attenuated signaling and blocked FLT3ITD-mediated cell transformation, whereas mutation of cysteine 790 enhanced activity of both FLT3ITD and wild-type FLT3. These effects were not related to altered FLT3ITD dimerization, but likely caused by changed intramolecular interactions. The findings identify the functional relevance of all cytoplasmic FLT3ITD cysteines, and indicate the potential for redox regulation of this clinically important oncoprotein., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Standard evolutionary theories of aging postulate that reduced extrinsic mortality leads to evolution of longevity. Clownfishes of the genus Amphiprion live in a symbiotic relationship with sea anemones that provide protection from predators. We performed a survey and identified at least two species with a lifespan of over 20 years. Given their small size and ease of captive reproduction, clownfish lend themselves as experimental models of exceptional longevity. To identify genetic correlates of exceptional longevity, we sequenced the transcriptomes of Amphiprion percula and A. clarkii and performed a scan for positively-selected genes (PSGs)., Results: The PSGs that we identified in the last common clownfish ancestor were compared with PSGs detected in long-lived mole rats and short-lived killifishes revealing convergent evolution in processes such as mitochondrial biogenesis. Among individual genes, the Mitochondrial Transcription Termination Factor 1 (MTERF1), was positively-selected in all three clades, whereas the Glutathione S-Transferase Kappa 1 (GSTK1) was under positive selection in two independent clades. For the latter, homology modelling strongly suggested that positive selection targeted enzymatically important residues., Conclusions: These results indicate that specific pathways were recruited in independent lineages evolving an exceptionally extended or shortened lifespan and point to mito-nuclear balance as a key factor.

The genetics of lifespan determination is poorly understood. Most research has been done on short-lived animals and it is unclear if these insights can be transferred to long-lived mammals like humans. Some African mole-rats (Bathyergidae) have life expectancies that are multiple times higher than similar sized and phylogenetically closely related rodents. To gain new insights into genetic mechanisms determining mammalian lifespans, we obtained genomic and transcriptomic data from 17 rodent species and scanned eleven evolutionary branches associated with the evolution of enhanced longevity for positively selected genes (PSGs). Indicating relevance for aging, the set of 250 identified PSGs showed in liver of long-lived naked mole-rats and short-lived rats an expression pattern that fits the antagonistic pleiotropy theory of aging. Moreover, we found the PSGs to be enriched for genes known to be related to aging. Among these enrichments were "cellular respiration" and "metal ion homeostasis", as well as functional terms associated with processes regulated by the mTOR pathway: translation, autophagy and inflammation. Remarkably, among PSGs are RHEB, a regulator of mTOR, and IGF1, both central components of aging-relevant pathways, as well as genes yet unknown to be aging-associated but representing convincing functional candidates, e.g. RHEBL1, AMHR2, PSMG1 and AGER. Exemplary protein homology modeling suggests functional consequences for amino acid changes under positive selection. Therefore, we conclude that our results provide a meaningful resource for follow-up studies to mechanistically link identified genes and amino acids under positive selection to aging and lifespan determination.

Twenty amino acid substitutions in the thymidine kinase (TK) of clinical herpes simplex virus type 1 strains were assessed for conferring acyclovir (ACV) resistance. Site-directed mutagenesis, cell-free protein synthesis and protein expression in Escherichia coli were performed to obtain recombinant TK proteins, which were authenticated by Western blotting. A modified enzyme-linked immunosorbent assay (ELISA) was carried out to determine the phosphorylation activity of the mutants towards 5-bromo-2'-deoxyuridine (BrdU). The activity against ACV and deoxythymidine (dT) was analyzed by high performance liquid chromatography/ultraviolet spectroscopy (HPLC/UV) following incubation of recombinant TK with ACV and dT. Using ELISA, seven substitutions (G61E, A93V, M121K, R163G, P173del, V238F, G264V) showing negative activity could be classified likely as resistance-related, eleven (Q15K, R20C, R32H, E43A, E43D, R89H, A156V, P269S, G271V, S276N, I326V) with high activity as natural polymorphisms, and two (N244H and N376stop) with low phosphorylation activity. Since the N244H protein did not show any activity towards ACV, but activity towards dT using HPLC/UV, it was classified as TK with altered substrate specificity. In conclusion, the ELISA determining activity towards BrdU is suitable for the characterization of substitutions regarding their significance for resistance. Ambiguous results can be re-assessed by HPLC/UV, which classifies TK with altered substrate specificity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Small regulatory RNAs (sRNAs) are the most prominent post-transcriptional regulators in all kingdoms of life. A few of them, e.g. SR1 from Bacillus subtilis, are dual-function sRNAs. SR1 acts as a base-pairing sRNA in arginine catabolism and as an mRNA encoding the small peptide SR1P in RNA degradation. Both functions of SR1 are highly conserved among 23 species of Bacillales. Here, we investigate the interaction between SR1P and GapA by a combination of in vivo and in vitro methods. De novo prediction of the structure of SR1P yielded five models, one of which was consistent with experimental circular dichroism spectroscopy data of a purified, synthetic peptide. Based on this model structure and a comparison between the 23 SR1P homologues, a series of SR1P mutants was constructed and analysed by Northern blotting and co-elution experiments. The known crystal structure of Geobacillus stearothermophilus GapA was used to model SR1P onto this structure. The hypothetical SR1P binding pocket, composed of two α-helices at both termini of GapA, was investigated by constructing and assaying a number of GapA mutants in the presence and absence of wild-type or mutated SR1P. Almost all residues of SR1P located in the two highly conserved motifs are implicated in the interaction with GapA. A critical lysine residue (K332) in the C-terminal α-helix 14 of GapA corroborated the predicted binding pocket.

Motivation: Core service units have become an organisational hallmark in many research institutions world wide. Such service cores provide complex state-of-the-art technologies and expertise to the research community. Typically, a user delivers material or raw data to a core. The core defines work packages for ensuing analysis and returns results back to the user. This core activity can be quite complex and time consuming and usually does not communicate itself to the outside. Naturally, the user is highly interested to follow the progress of a project once handed over to the core unit. This generates a time-intensive direct communication activity back and forth. A more effective, convenient and less disruptive way to track the status of a given project by the researcher, but also by core managers, appears highly desirable. Hence, we developed a lightweight and readily implementable web application that allows efficient progress tracking of core unit projects., Results: The web application TraqBio allows for the convenient tracking of projects. Following project set-up by the core, the user receives an e-mail containing links for tracking the project status. Examples are provided for three common core units, namely genomics, proteomics, and bioinformatics units. TraqBio is a secure lightweight web application that can be either used in a standalone setup or incorporated into an existing web server infrastructure. Being accessible not only from classical desktop computers but also from mobile devices such as smartphones and tablets, TraqBio offers easy integration into every day work., Competing Interests: The authors have declared that no competing interests exist.

The RF pulse scheme RN[N-CA HEHAHA]NH, which provides a convenient approach to the acquisition of different multidimensional chemical shift correlation NMR spectra leading to backbone resonance assignments, including those of the proline residues of intrinsically disordered proteins (IDPs), is experimentally demonstrated. Depending on the type of correlation data required, the method involves the generation of in-phase ((15) N)(x) magnetisation via different magnetisation transfer pathways such as H→N→CO→N, HA→CA→CO→N, H→N→CA→N and H→CA→N, the subsequent application of (15) N-(13) C(α) heteronuclear Hartmann-Hahn mixing over a period of ≈100 ms, chemical-shift labelling of relevant nuclei before and after the heteronuclear mixing step and amide proton detection in the acquisition dimension. It makes use of the favourable relaxation properties of IDPs and the presence of (1) JCαN and (2) JCαN couplings to achieve efficient correlation of the backbone resonances of each amino acid residue "i" with the backbone amide resonances of residues "i-1" and "i+1". It can be implemented in a straightforward way through simple modifications of the RF pulse schemes commonly employed in protein NMR studies. The efficacy of the approach is demonstrated using a uniformly ((15) N,(13) C) labelled sample of α-synuclein. The different possibilities for obtaining the amino-acid-type information, simultaneously with the connectivity data between the backbone resonances of sequentially neighbouring residues, have also been outlined., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To assess the basis of the different half-lives of long-acting human granulocyte colony-stimulating factor (G-CSF) drugs, the effect of neutrophil elastase on lipegfilgrastim and pegfilgrastim was investigated. Sensitivity to human neutrophil elastase (HNE) was evaluated by incubating the drugs with HNE followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Drugs were also incubated with isolated human neutrophils followed by Western blot analysis. Lipegfilgrastim was more resistant to degradation with HNE or neutrophils than pegfilgrastim and appeared more intact on SDS-PAGE gels and Western blots. Lipegfilgrastim retained more functional activity than pegfilgrastim after incubation with HNE (67% vs ∼ 9%, respectively) or neutrophils (80% vs ∼ 4%, respectively) as assessed in an NFS-60 cell-based [(3) H]-thymidine incorporation assay. The binding and affinity of untreated lipegfilgrastim and pegfilgrastim for G-CSF receptors were evaluated using an NFS-60 competitive G-CSF receptor-binding assay and surface plasmon resonance. Untreated drugs were also evaluated in the functional NFS-60 thymidine incorporation assay. G-CSF receptor binding, receptor affinity, and functional activity were comparable between untreated drugs. The results showed a greater resistance to neutrophil elastase degradation and concomitant retention of functional activity of lipegfilgrastim compared with pegfilgrastim, which potentially explains the clinical observations of a longer half-life of lipegfilgrastim versus pegfilgrastim., (© 2015, The American College of Clinical Pharmacology.)

Solvation and hydration are key factors for determining the stability and folding of proteins, as well as the formation of amyloid fibrils and related polypeptide aggregates. Using attenuated total reflectance Fourier-transform infrared and solid-state NMR spectroscopy, we find that the Aβ peptide experiences a remarkable conformational switch from β to α secondary structure upon solvent removal by lyophilization of oligomers. This transition is, contrary to Aβ fibrils, independent of concentration of organic co-solvents or co-solutes and is reversible upon re-addition of the solvent. Our data illuminate a previously unnoted secondary structural plasticity of the Aβ peptide in amyloid oligomers that could bear relevance for Aβ's interactions with cellular structures of low polarity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in the laboratory. Its rapid growth, early sexual maturation, fast aging, and arrested embryonic development (diapause) make it an attractive model organism in biomedical research. Here, we report a draft sequence of its genome that allowed us to uncover an intra-species Y chromosome polymorphism representing-in real time-different stages of sex chromosome formation that display features of early mammalian XY evolution "in action." Our data suggest that gdf6Y, encoding a TGF-β family growth factor, is the master sex-determining gene in N. furzeri. Moreover, we observed genomic clustering of aging-related genes, identified genes under positive selection, and revealed significant similarities of gene expression profiles between diapause and aging, particularly for genes controlling cell cycle and translation. The annotated genome sequence is provided as an online resource (http://www.nothobranchius.info/NFINgb)., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A simple triple resonance NMR experiment that leads to the correlation of the backbone amide resonances of each amino acid residue 'i' with that of residues 'i-1' and 'i+1' in ((13)C, (15)N) labelled intrinsically disordered proteins (IDPs) is presented. The experimental scheme, {HN-NCA heteronuclear TOCSY-NH}, exploits the favourable relaxation properties of IDPs and the presence of (1) J CαN and (2) J CαN couplings to transfer the (15)N x magnetisation from amino acid residue 'i' to adjacent residues via the application of a band-selective (15)N-(13)C(α) heteronuclear cross-polarisation sequence of ~100 ms duration. Employing non-uniform sampling in the indirect dimensions, the efficacy of the approach has been demonstrated by the acquisition of 3D HNN chemical shift correlation spectra of α-synuclein. The experimental performance of the RF pulse sequence has been compared with that of the conventional INEPT-based HN(CA)NH pulse scheme. As the availability of data from both the HCCNH and HNN experiments will make it possible to use the information extracted from one experiment to simplify the analysis of the data of the other and lead to a robust approach for unambiguous backbone and side-chain resonance assignments, a time-saving strategy for the simultaneous collection of HCCNH and HNN data is also described.

The generation of efficient RN n (ν)s,(ν)k symmetry-based low-power RF pulse schemes for simultaneous (15)N-(13)CA and (15)N-(13)CO dipolar recoupling is demonstrated. The method involves mixing schemes employing phase and amplitude-modulated dual band-selective 180° pulses as basic "R" element and tailoring of the RF field-modulation profile of the 180° pulses so as to obtain efficient magnetisation transfer characteristics over the resonance offset range of the nuclei involved. Mixing schemes leading to simultaneous (15)N-(13)CA and (15)N-(13)CO dipolar recoupling would permit the one-shot acquisition of different chemical shift correlation spectra that are typically utilized for protein backbone resonance assignments and thereby save data acquisition time. At representative MAS frequencies the efficacies of the mixing schemes presented here have been experimentally demonstrated via the simultaneous acquisition of {3D CONH and 3D CANH}, {3D CONH and 3D CO(CA)NH} and {3D CONH, 3D CANH, 3D CO(CA)NH and 3D CA(CO)NH} spectra generated via the magnetisation transfer pathways (1)H → (13)CO → (15)N → (1)H (CONH), (1)H → (13)CA → (15)N → (1)H (CANH) and (1)H → (13)CO → (13)CA → (15)N → (1)H (CO(CA)NH) and (1)H → (13)CA → (13)CO → (15)N → (1)H (CA(CO)NH).

An efficient approach to NMR assignments in intrinsically disordered proteins is presented, making use of the good dispersion of cross peaks observed in [(15) N,(13) C']- and [(13) C',(1) H(N) ]-correlation spectra. The method involves the simultaneous collection of {3D (H)NCO(CAN)H and 3D (HACA)CON(CA)HA} spectra for backbone assignments via sequential H(N) and H(α) correlations and {3D (H)NCO(CACS)HS and 3D (HS)CS(CA)CO(N)H} spectra for side-chain (1) H and (13) C assignments, employing sequential (1) H data acquisitions with direct detection of both the amide and aliphatic protons. The efficacy of the approach for obtaining resonance assignments with complete backbone and side-chain chemical shifts is demonstrated experimentally for the 61-residue [(13) C,(15) N]-labelled peptide of a voltage-gated potassium channel protein of the Kv1.4 channel subunit. The general applicability of the approach for the characterisation of moderately sized globular proteins is also demonstrated., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical 'wings' of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The synthesis of poly(2-oxazoline)-based block copolymers consisting of a cationic and a hydrophilic segment is described. The self-assembly of these macromolecules in organic solvents results in the formation of micelles and vesicles, respectively, depending on the solvent used. To transfer the systems into water, cross-linking using glutaraldehyde was applied, followed by the consumption of excessive aldehyde functions by either diethylamine or 6-aminofluorescein (6AF). The cross-linked assemblies were analyzed regarding their size and shape by electron microscopy and light scattering methods, as well as for their chemical composition by solid state NMR spectroscopy. 6AF associated samples were examined with respect to their absorption and fluorescence behavior in aqueous environment, revealing an alkaline microenvironment within the presented nanostructures. The toxicity of the systems against mouse fibroblast cell line L929 was examined by the XTT assay and was found to be insignificant for concentrations of up to 2.5 mg mL -1 . Flow cytometry and fluorescence microscopy analysis revealed an efficient concentration and time dependent cellular uptake of the nanogels.

Amyloid oligomers are nonfibrillar polypeptide aggregates linked to diseases, such as Alzheimer's and Parkinson's. Here we show that these aggregates possess a compact, quasi-crystalline architecture that presents significant nanoscale regularity. The amyloid oligomers are dynamic assemblies and are able to release their individual subunits. The small oligomeric size and spheroid shape confer diffusible characteristics, electrophoretic mobility, and the ability to enter hydrated gel matrices or cells. We finally showed that the amyloid oligomers can be labeled with both fluorescence agents and iron oxide nanoparticles and can target macrophage cells. Oligomer amyloids may provide a new biological nanomaterial for improved targeting, drug release, and medical imaging.

Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

High-fidelity replication guarantees the stable inheritance of genetic information stored in the DNA of living organisms. The minichromosome maintenance (MCM) complex functions as replicative DNA-unwinding helicase and has been identified as one key player in the replication process of archea and eukarya. Despite the availability of considerable structural information on archeal MCMs, such information was missing for their C-terminal domain. In order to obtain more detailed structural information, we assigned the NMR chemical shifts for backbone and side chain nuclei for the MCM C-terminal winged helix domains of the archeal species Methanothermobacter thermautrophicus and Sulfolobus solfataricus.

A procedure for the simultaneous acquisition of {HNCOCANH & HCCCONH} chemical shift correlation spectra employing sequential [Formula: see text] data acquisition for moderately sized proteins is presented. The suitability of the approach for obtaining sequential resonance assignments, including complete [Formula: see text] and [Formula: see text] chemical shift information, is demonstrated experimentally for a [Formula: see text] and [Formula: see text] labelled sample of the C-terminal winged helix (WH) domain of the minichromosome maintenance (MCM) complex of Sulfolobus solfataricus. The chemical shift information obtained was used to calculate the global fold of this winged helix domain via CS-Rosetta. This demonstrates that our procedure provides a reliable and straight-forward protocol for a quick global fold determination of moderately-sized proteins.

RF pulse schemes for the simultaneous acquisition of heteronuclear multi-dimensional chemical shift correlation spectra, such as {HA(CA)NH & HA(CACO)NH}, {HA(CA)NH & H(N)CAHA} and {H(N)CAHA & H(CC)NH}, that are commonly employed in the study of moderately-sized protein molecules, have been implemented using dual sequential (1)H acquisitions in the direct dimension. Such an approach is not only beneficial in terms of the reduction of experimental time as compared to data collection via two separate experiments but also facilitates the unambiguous sequential linking of the backbone amino acid residues. The potential of sequential (1)H data acquisition procedure in the study of RNA is also demonstrated here.

Two different NMR pulse schemes involving sequential (1)H data acquisition are presented for achieving protein backbone sequential resonance assignments: (i) acquisition of 3D {HCCNH and HNCACONH} and (ii) collection of 3D {HNCOCANH and HNCACONH} chemical shift correlation spectra using uniformly (13)C,(15)N labelled proteins. The sequential acquisition of these spectra reduces the overall experimental time by a factor of ≈2 as compared to individual acquisitions. The suitability of this approach is experimentally demonstrated for the C-terminal winged helix (WH) domain of the minichromosome maintenance (MCM) complex of Sulfolobus solfataricus., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The use of granulocyte colony stimulating factor (G-CSF) biosimilars for peripheral blood hematopoietic stem cell (PBSC) mobilization has stimulated an ongoing debate regarding their efficacy and safety. However, the use of biosimilar G-CSF was approved by the European Medicines Agency (EMA) for all the registered indications of the originator G-CSF (Neupogen (®) ) including mobilization of stem cells. Here, we performed a comprehensive review of published reports on the use of biosimilar G-CSF covering patients with hematological malignancies as well as healthy donors that underwent stem cell mobilization at multiple centers using site-specific non-randomized regimens with a biosimilar G-CSF in the autologous and allogeneic setting. A total of 904 patients mostly with hematological malignancies as well as healthy donors underwent successful autologous or allogeneic stem cell mobilization, respectively, using a biosimilar G-CSF (520 with Ratiograstim®/Tevagrastim, 384 with Zarzio®). The indication for stem cell mobilization in hematology patients included 326 patients with multiple myeloma, 273 with Non-Hodgkin's lymphoma (NHL), 79 with Hodgkin's lymphoma (HL), and other disease. 156 sibling or volunteer unrelated donors were mobilized using biosimilar G-CSF. Mobilization resulted in good mobilization of CD34+ stem cells with side effects similar to originator G-CSF. Post transplantation engraftment did not significantly differ from results previously documented with the originator G-CSF. The side effects experienced by the patients or donors mobilized by biosimilar G-CSF were minimal and were comparable to those of originator G-CSF. In summary, the efficacy of biosimilar G-CSFs in terms of PBSC yield as well as their toxicity profile are equivalent to historical data with the reference G-CSF.

NMR-based structure determination of a protein requires the assignment of resonances as indispensable first step. Even though heteronuclear through-bond correlation methods are available for that purpose, challenging situations arise in cases where the protein in question only yields samples of limited concentration and/or stability. Here we present a strategy based upon specific individual unlabeling of all 20 standard amino acids to complement standard NMR experiments and to achieve unambiguous backbone assignments for the fast precipitating 23 kDa catalytic domain of human aprataxin of which only incomplete standard NMR data sets could be obtained. Together with the validation of this approach utilizing the protein GB1 as a model, a comprehensive insight into metabolic interconversion ("scrambling") of NH and CO groups in a standard Escherichia coli expression host is provided.

Motivation: Circular dichroism (CD) spectroscopy is one of the most versatile tools to study protein folding and to validate the proper fold of purified proteins. Here, we aim to provide a readily accessible, user-friendly and platform-independent tool capable of analysing multiple CD datasets of virtually any format and returning results as high-quality graphical output to the user., Results: CAPITO (CD Anaylsis and Plotting Tool) is a novel web server-based tool for analysing and plotting CD data. It allows reliable estimation of secondary structure content utilizing different approaches. CAPITO accepts multiple CD datasets and, hence, is well suited for a wide application range such as the analysis of temperature or pH-dependent (un)folding and the comparison of mutants., Availability: http://capito.nmr.fli-leibniz.de., Contact: cwiede@fli-leibniz.de or mago@fli-leibniz.de, Supplementary Information: Supplementary data are available at Bioinformatics online.

The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein in vivo. Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.