Highly Specific Protein Identification by Immunoprecipitation-Mass Spectrometry Using Antifouling Microbeads.

A common method to study protein complexes is immunoprecipitation (IP), followed by mass spectrometry (thus labeled: IP-MS). IP-MS has been shown to be a powerful tool to identify protein-protein interactions. It is, however, often challenging to discriminate true protein interactors from contaminating ones. Here, we describe the preparation of antifouling azide-functionalized polymer-coated beads that can be equipped with an antibody of choice via click chemistry. We show the preparation of generic immunoprecipitation beads that target the green fluorescent protein (GFP) and show how they can be used in IP-MS experiments targeting two different GFP-fusion proteins. Our antifouling beads were able to efficiently identify relevant protein-protein interactions but with a strong reduction in unwanted nonspecific protein binding compared to commercial anti-GFP beads.

Synthesis and characterization of thiocarbonato-linked platinum(IV) complexes.

Herein we show the formation of new oxaliplatin-based platinum(IV) complexes by reaction with DSC-activated thiols via thiocarbonate linkage. Three model complexes based on aliphatic and aromatic thiols, as well as one complex with N-acetylcysteine as biologically active thiol were synthesized. This synthetic strategy affords the expansion of biologically active compounds other than those containing carboxylic, amine or hydroxy groups for coupling to the platinum(IV) center. The complexes were characterized by high-resolution mass spectrometry, NMR spectroscopy (1H, 13C, 195Pt) and elemental analysis. Their biological behavior was evaluated against two ovarian carcinoma cell lines and their cisplatin-resistant analogues. Remarkably, the platinum(IV) samples show modest in vitro cytotoxicity against A2780 cells and comparable effects against A2780cis cells. Two complexes in particular demonstrate improved activity against SKOV3cis cells. The reduction experiment of complex 8, investigated by UHPLC-HRMS, provides evidence of interesting platinum-species formed during reaction with ascorbic acid.

Physical and psychosocial working conditions as predictors of 5-year changes in work ability among 2078 employees in Germany.

OBJECTIVE: To examine 5-year prospective associations between working conditions and work ability among employees in Germany. METHODS: A cohort study (2011/2012-2017), based on a random sample of employees in employments subject to payment of social contributions aged 31-60 years (Study on Mental Health at Work; S-MGA; N = 2,078), included data on physical and quantitative demands, control (influence, possibilities for development, control over working time), relations (role clarity and leadership quality) and work ability (Work Ability Index, WAI; subscale 'subjective work ability and resources'). Data were analysed using linear regression. RESULTS: Physical demands and control were associated with small 5-year changes in work ability (ΔR2 = 1%). Among the subgroup of employees with ≥ 25 sickness days, possibilities for development, control and quality of leadership were associated with changes in work ability (ΔR2 = 8%). CONCLUSIONS: The impact of working conditions on long term changes in work ability seems to be negligible. However, in vulnerable subpopulations experiencing poor health, working conditions may be associated to a larger extent to work ability over this time span.

Workplace bullying and depressive symptoms among employees in Germany: prospective associations regarding severity and the role of the perpetrator.

OBJECTIVES: The aim of this study was to investigate the effect of self-reported workplace bullying on depressive symptoms in a prospective study among a representative sample of employees from Germany. We focused specifically on the role of the perpetrator (co-workers and superiors), which was never done before in a longitudinal design. METHODS: We used data from a nation-wide representative panel study with a 5-year follow-up (N = 2172). Data on bullying exposure were obtained separately for different perpetrators (co-workers and superiors) and degree of severity (severe bullying, i.e., at least weekly). Depressive symptoms were assessed with the Patient Health Questionnaire (PHQ). We used logistic regression analyses to examine the effect of workplace bullying at baseline on depressive symptoms at follow-up. RESULTS: After adjusting for baseline depressive symptoms, severe bullying by co-workers significantly increased the 5-year risk of depressive symptoms (OR = 2.50). Severe bullying by superiors had a nonsignificant effect. CONCLUSIONS: Workplace bullying is a risk factor for depressive symptoms among employees in Germany. The type of perpetrator seems to be an important factor to consider, as indicated by the elevated risk of depressive symptoms when bullying is perpetrated by co-workers.

Simultaneous Silicon Oxide Growth and Electrophoretic Deposition of Graphene Oxide.

During electrophoretic deposition of graphene oxide (GO) sheets on silicon substrates, not only deposition but also simultaneous anodic oxidation of the silicon substrate takes place, leading to a three-layered material. Scanning electron microscopy images reveal the presence of GO sheets on the silicon substrate, and this is also confirmed by X-ray photoelectron spectroscopy (XPS), albeit that the carbon portion increases with increasing emission angle, hinting at a thin carbon layer. With increasing applied potential and increasing conductivity of the GO solution, the carbon signal decreases, whereas the overall thickness of the added layer formed on top of the silicon substrate increases. Through XPS spectra in which the Si 2p peaks shifted under those conditions to 103-104 eV, we were able to conclude that significant amounts of oxygen are present, indicative of the formation of an oxide layer. This leads us to conclude that GO can be deposited using electrophoretic deposition, but that at the same time, silicon is oxidized, which may overshadow effects previously assigned to GO deposition.

Systematic Comparison of Zwitterionic and Non-Zwitterionic Antifouling Polymer Brushes on a Bead-Based Platform.

Nonspecific adsorption of biomolecules to solid surfaces, a process called biofouling, is a major concern in many biomedical applications. Great effort has been made in the development of antifouling polymer coatings that are capable of repelling the nonspecific adsorption of proteins, cells, and micro-organisms. In this respect, we herein contribute to understanding the factors that determine which polymer brush results in the best antifouling coating. To this end, we compared five different monomers: two sulfobetaines, a carboxybetaine, a phosphocholine, and a hydroxyl acrylamide. The antifouling coatings were analyzed using our previously described bead-based method with flow cytometry as the read-out system. This method allows for the quick and automated analysis of thousands of beads per second, enabling fast analysis and good statistics. We report the first direct comparison made between a sulfobetaine with opposite charges separated by two and three methylene groups and a carboxybetaine bearing two separating methylene groups. It was concluded that both the distance between opposite charges and the nature of the anionic groups have a distinct effect on the antifouling performance. Phosphocholines and simple hydroxyl acrylamides are not often compared with the betaines. However, here we found that they perform equally well or even better, yielding the following overall antifouling ranking: HPMAA ≥ PCMA-2 ≈ CBMAA-2 > SBMAA-2 > SBMAA-3 ≫ nonmodified beads (HPMAA being the best).

Workplace bullying among employees in Germany: prevalence estimates and the role of the perpetrator.

OBJECTIVES: The aim of this study was to examine the prevalence of workplace bullying in Germany while also taking the perpetrator and severity level (measured by frequency) into account and considering the role of gender, age and socio-economic status. METHODS: We used data from a large representative sample (N = 4143) of employees in Germany subject to social security contributions. Self-reported bullying was assessed for different combinations of perpetrators (co-workers, superiors) and according to severity, i.e., being exposed at all and to severe bullying (at least weekly). RESULTS: Prevalence estimates varied from 2.9% for severe bullying by co-workers to 17.1% for overall bullying (i.e., without distinguishing by perpetrator, less severe bullying also included). Unskilled workers reported more bullying by both perpetrators than academics/managers. We also observed an age trend for severe bullying by superiors (i.e., bossing), with younger employees being more affected from bossing than elder. No gender differences were detected. CONCLUSIONS: The findings indicate that it is crucial to consider type of perpetrator and severity of the behaviors when examining the prevalence of workplace bullying. The way bullying is defined and operationalized strongly contributes to the prevalence estimates. Differences between subgroups and associations or cause-effect relationships should be analyzed with these variations in mind.

Handling properties of a superabsorbent dressing in the management of patients with moderate-to-very high exuding wounds.

OBJECTIVE: Exudate control is an important aspect of wound management in both acute and chronic wounds. Exudate can be an indicator of the wound bed condition, specifically inflammation and infection. This study aimed to evaluate the performance, in terms of usability, handling properties, exudate management, user satisfaction and patient comfort, in daily clinical practice, of a superabsorbent dressing, Vliwasorb Pro (Lohmann & Rauscher), suitable for the management of moderate-to-very high exudate levels. METHOD: The user test was conducted between September 2016 and July 2017, with clinicians from different specialisms in 55 centres across Germany. Both the dressing and user test were supplied by the sponsor. The superabsorbent dressing was used for at least three dressing changes, with frequency dependent on the patient and wound condition. RESULTS: A total of 55 clinicians recruited 171 patients with various wound types. The clinicians rated dressing application as 'easy' for 163 (95.3%) of the patients. The dressing was rated as easy to remove (168 (98.3%) and, according to clinicians, did not soil patients' clothing in 165 (97.1%) of cases. The dressing demonstrated a 'good absorbent capacity', as noted by clinicians in 167 (98.2%) of cases. Clinicians also commented that the dressing reduced foul odour, maceration and improved periwound skin condition. CONCLUSION: The evaluated dressing was easy to use, comfortable and reliable for patients with moderate-to-very high exuding wounds. In view of these results, superabsorbent dressings seem to be interesting for both clinicians and patients. Clinical studies are required to confirm these results.

Efficient and Tunable Three-Dimensional Functionalization of Fully Zwitterionic Antifouling Surface Coatings.

To enhance the sensitivity and selectivity of surface-based (bio)sensors, it is of crucial importance to diminish background signals that arise from the nonspecific binding of biomolecules, so-called biofouling. Zwitterionic polymer brushes have been shown to be excellent antifouling materials. However, for sensing purposes, antifouling does not suffice but needs to be combined with the possibility to efficiently modify the brush with recognition units. So far this has been achieved only at the expense of either antifouling properties or binding capacity. Herein we present a conceptually new approach by integrating both characteristics into a single tailor-made monomer: a novel sulfobetaine-based zwitterionic monomer equipped with a clickable azide moiety. Copolymerization of this monomer with a well-established standard sulfobetaine monomer results in highly antifouling surface coatings with a large yet tunable number of clickable groups present throughout the entire brush. Subsequent functionalization of the azido brushes via widely used strain-promoted alkyne azide click reactions yields fully zwitterionic 3D-functionalized coatings with a recognition unit of choice that can be tailored for any specific application. Here we show a proof of principle with biotin-functionalized brushes on Si3N4 that combine excellent antifouling properties with specific avidin binding from a protein mixture. The signal-to-noise ratio is significantly improved over that of traditional chain-end modification of sulfobetaine polymer brushes, even if the azide content is lowered to 1%. This therefore offers a viable approach to the development of biosensors with greatly enhanced performance on any surface.

Local Light-Induced Modification of the Inside of Microfluidic Glass Chips.

The ability to locally functionalize the surface of glass allows for myriad biomedical and chemical applications. This would be the case if the surface functionalization can be induced using light with wavelengths for which standard glass is almost transparent. To this aim, we present the first example of a photochemical modification of hydrogen-terminated glass (H-glass) with terminal alkenes. Both flat glass surfaces and the inside of glass microchannels were modified with a well-defined, covalently attached organic monolayer using a range of wavelengths, including sub-band-gap 302 nm ultraviolet light. A detailed characterization thereof was conducted by measurements of the static water contact angle, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and scanning Auger microscopy (SAM). Germanium attenuated total reflection Fourier transform infrared (GATR-FTIR) indicates that the mechanism of the surface modification proceeds via an anti-Markovnikov substitution. Reacting H-glass with 10-trifluoro-acetamide-1-decene (TFAAD) followed by basic hydrolysis affords the corresponding primary amine-terminated monolayer, enabling additional functionalization of the substrate. Furthermore, we show the successful formation of a photopatterned amine layer by the specific attachment of fluorescent nanoparticles in very discrete regions. Finally, a microchannel was photochemically patterned with a functional linker allowing for surface-directed liquid flow. These results demonstrate that H-glass can be modified with a functional tailor-made organic monolayer, has highly tunable wetting properties, and displays significant potential for further applications.

Ancestral AlaX editing enzymes for control of genetic code fidelity are not tRNA-specific.

Accurate protein synthesis requires the hydrolytic editing of tRNAs incorrectly aminoacylated by aminoacyl-tRNA synthetases (ARSs). Recognition of cognate tRNAs by ARS is less error-prone than amino acid recognition, and, consequently, editing domains are generally believed to act only on the tRNAs cognate to their related ARSs. For example, the AlaX family of editing domains, including the editing domain of alanyl-tRNA synthetase and the related free-standing trans-editing AlaX enzymes, are thought to specifically act on tRNA(Ala), whereas the editing domains of threonyl-tRNA synthetases are specific for tRNA(Thr). Here we show that, contrary to this belief, AlaX-S, the smallest of the extant AlaX enzymes, deacylates Ser-tRNA(Thr) in addition to Ser-tRNA(Ala) and that a single residue is important to determine this behavior. Our data indicate that promiscuous forms of AlaX are ancestral to tRNA-specific AlaXs. We propose that former AlaX domains were used to maintain translational fidelity in earlier stages of genetic code evolution when mis-serylation of several tRNAs was possible.

pH response and molecular recognition in a low molecular weight peptide hydrogel.

In this article we report the preparation and characterization of a peptide-based hydrogel, which possesses characteristic rheological properties, is pH responsive and can be functionalized at its thiol function. The tripeptide N-(fluorenyl-9-methoxycarbonyl)-L-Cys(acetamidomethyl)-L-His-L-Cys-OH 1 forms stable supramolecular aggregates in water leading to hydrogels above 1.5 wt%. Rheological analysis of the hydrogel revealed visco-elastic and shear thinning properties of samples containing 1.5 wt% of peptide 1. The hydrogel reversibly responds to pH changes. Below and above pH 6, electrostatic repulsion of the peptide results in a weakening of the three-dimensional gel network. Based on atomic force microscopy, small angle X-ray scattering and molecular dynamics simulations, it is proposed that the peptide assembles into nanostructures that tend to entangle at higher concentrations in water. The development of functional materials based on the peptide assemblies was possible via thiol-ene-click chemistry of the free thiol function at the C-terminal cysteine unit. As a proof of concept, the functionalization with adamantyl units to give 1-Ad was shown by molecular recognition of ß-cyclodextrin vesicles. These vesicles were used as supramolecular cross-linkers of the assemblies of peptide 1 mixed with peptide 1-Ad leading to gel networks at a reduced peptide concentration.

Measuring slips and lapses when they occur - ambulatory assessment in application to cognitive failures.

Cognitive failures are lapses in attention, cognition, and actions that everybody experiences in everyday life. Self-reports are mainly used for assessment but those instruments are memory-biased and more related to personality aspects than to actual behavior. Ambulatory assessment is already used for capturing emotions or addictive behavior, but not yet for cognitive failures. The newly developed Questionnaire for Cognitive Failures in Everyday Life (KFA) was applied via mobile phones (electronic KFA) wherein an acoustic signal asked participants (N=91, 60-76 years) 4 times daily to answer 13 questions for 1 week. The new instrument showed satisfying reliability and was compared with a self-report method (Cognitive Failures Questionnaire; Broadbent, Cooper, Fitzgerald, and Parkes, 1982) in terms of correlations with cognitive abilities (working memory capacity, short-term memory, switching ability, and reasoning), personality traits, and demographical aspects. Although further validation is needed, first results are promising and eKFA enriches cognitive failures research.

A helical RGD motif promoting cell adhesion: crystal structures of the Helicobacter pylori type IV secretion system pilus protein CagL.

RGD tripeptide motifs frequently mediate ligand binding to integrins. The type IV secretion system (T4SS) protein CagL of the gastric pathogen Helicobacter pylori also contains an RGD motif. CagL decorates the T4SS pilus and may function as an adhesin for host cells. Whether CagL binds integrins via its RGD motif is under debate. Here, we present crystal structures of CagL revealing an elongated four-helix bundle that appears evolutionarily unrelated to the proposed VirB5 orthologs. The RGD motif is surface-exposed but located within a long α helix. This is unprecedented as previously characterized integrin-binding RGD motifs are located within extended or flexible loops. Yet, adhesion of gastric epithelial cells to CagL was strictly RGD-dependent. Comparison of seven crystallographically independent molecules reveals substantial structural flexibility. Intramolecular disulfide bonds engineered to reduce CagL flexibility resulted in more stable protein, but unable to support cell adhesion. CagL may thus partly unfold during receptor binding.

Aminopropylindenes derived from Grundmann's ketone as a novel chemotype of oxidosqualene cyclase inhibitors.

A series of aminopropylindenes, designed as mimics of a cationic high energy intermediate in the oxidosqualene cyclase(1) (OSC)-mediated cyclization of 2,3-oxidosqualen to lanosterol was prepared from Grundmann's ketone. Screening on OSCs from five different organisms revealed interesting activities and selectivities of some of the compounds. A N,N-dimethylaminopropyl derivative showed promising inhibition of Trypanosoma cruzi OSC in combination with low cytotoxicity, and showed significant reduction of cholesterol biosynthesis in a human cell line.