Impact of Peptide Sequences on Their Structure and Function: Mimicking of Virus-Like Nanoparticles for Nucleic Acid Delivery.

We rationally designed a series of amphiphilic hepta-peptides enriched with a chemically conjugated guanidiniocarbonylpyrrole (GCP) unit at the lysine side chain. All peptides are composed of polar (GCP) and non-polar (cyclohexyl alanine) residues but differ in their sequence periodicity, resulting in different secondary as well as supramolecular structures. CD spectra revealed the assembly of ß-sheet-, α-helical and random structures for peptides 1, 2 and 3, respectively. Consequently, this enabled the formation of distinct supramolecular assemblies such as fibres, nanorod-like or spherical aggregates. Notably, all three cationic peptides are equipped with the anion-binding GCP unit and thus possess a nucleic acid-binding centre. However, only the helical (2) and the unstructured (3) peptide were able to assemble into small virus-like DNA-polyplexes and effectively deliver DNA into cells. Notably, as both peptides (2 and 3) were also capable of siRNA-delivery, they could be utilized to downregulate expression of the caner-relevant protein Survivin.

Preparedness as a key factor for human biomonitoring programs after chemical incidents.

BACKGROUND: Human biomonitoring (HBM) has been repeatedly recommended for and applied to post-incident chemical exposure assessment. The applicability of HBM and the validity of its results, however, closely depend on the existence and quality of preparatory measures such as information and instruction materials, sampling procedures, transport and storage facilities, and on the selection of appropriate biomarkers, sampling time, transport and storage conditions. OBJECTIVE: To establish a standardized HBM program for emergency responders of a large chemical production site, considering the aforementioned aspects. METHODS: An HBM program based on a comprehensive questionnaire, information and training of emergency responders, and availability of sampling material was established. The quantitative determination of metabolites of hazardous substances was carried out based on quality-controlled analytical methods. RESULTS: The use of HBM after emergency operations was significantly increased immediately after the implementation of the program. Only in single cases, however, established HBM assessment values were exceeded. After one major incident, an increased exposure to benzene exceeding the internal action value was observed after firefighting and safeguarding. SIGNIFICANCE: The experience with several minor and one major incident at a chemical production site suggests that the implementation of easily accessible and applicable routines is one paramount prerequisite for the success of HBM programs after chemical incidents.

Specific inhibition of the Survivin-CRM1 interaction by peptide-modified molecular tweezers.

Survivin's dual function as apoptosis inhibitor and regulator of cell proliferation is mediated via its interaction with the export receptor CRM1. This protein-protein interaction represents an attractive target in cancer research and therapy. Here, we report a sophisticated strategy addressing Survivin's nuclear export signal (NES), the binding site of CRM1, with advanced supramolecular tweezers for lysine and arginine. These were covalently connected to small peptides resembling the natural, self-complementary dimer interface which largely overlaps with the NES. Several biochemical methods demonstrated sequence-selective NES recognition and interference with the critical receptor interaction. These data were strongly supported by molecular dynamics simulations and multiscale computational studies. Rational design of lysine tweezers equipped with a peptidic recognition element thus allowed to address a previously unapproachable protein surface area. As an experimental proof-of-principle for specific transport signal interference, this concept should be transferable to any protein epitope with a flanking well-accessible lysine.

Quantitative determination of urinary metabolites of geraniol by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

Geraniol is a fragrance which occurs in natural terpene oil or is chemically synthesized on a large scale. It is used in a wide variety of consumer products such as perfumes, deodorants, household products and cosmetics. Hence, not only industry workers in the production of geraniol, but also consumers can come into contact with the substance. Human biomonitoring (HBM), i.e. the analytical determination of substances and their metabolites in human biological material, is a key element in the analysis and assessment of the distribution and intensity of occupational and environmental exposure of humans. Therefore, a procedure for the quantitative determination of the urinary metabolites Hildebrandt acid, geranic acid, 3-hydroxycitronellic acid and 8-carboxygeraniol as potential biomarkers of geraniol exposure was developed and validated. The method is based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) after enzymatic hydrolysis and liquid-liquid extraction (LLE) of the target analytes. The limit of quantification (LOQ) is 1.5 µg L-1 for 8-carboxygeraniol, 2.7 µg L-1 each for Hildebrandt acid and geranic acid, and 1.8 µg L-1 for 3-hydroxycitronellic acid. The method was applied to urine samples of 41 persons without occupational exposure to geraniol. Hildebrandt acid and geranic acid were detected in all samples, 8-carboxygeraniol in 83% and 3-hydroxycitronellic acid in 81% of the samples. Hildebrandt acid (median: 313 µg L-1, range: 37-1966 µg L-1) was the most abundant metabolite, followed by geranic acid (93 µg L-1; 9-477 µg L-1), 3-hydroxycitronellic acid (18 µg L-1;

A Supramolecular Stabilizer of the 14-3-3ζ/ERα Protein-Protein Interaction with a Synergistic Mode of Action.

We report on a stabilizer of the interaction between 14-3-3ζ and the Estrogen Receptor alpha (ERα). ERα is a driver in the majority of breast cancers and 14-3-3 proteins are negative regulators of this nuclear receptor, making the stabilization of this protein-protein interaction (PPI) an interesting strategy. The stabilizer (1) consists of three symmetric peptidic arms containing an arginine mimetic, previously described as the GCP motif. 1 stabilizes the 14-3-3ζ/ERα interaction synergistically with the natural product Fusicoccin-A and was thus hypothesized to bind to a different site. This is supported by computational analysis of 1 binding to the binary complex of 14-3-3 and an ERα-derived phosphopeptide. Furthermore, 1 shows selectivity towards 14-3-3ζ/ERα interaction over other 14-3-3 client-derived phosphomotifs. These data provide a solid support of a new binding mode for a supramolecular 14-3-3ζ/ERα PPI stabilizer.

In Vitro and In Vivo Short-Term Pulmonary Toxicity of Differently Sized Colloidal Amorphous SiO2.

In vitro prediction of inflammatory lung effects of well-dispersed nanomaterials is challenging. Here, the in vitro effects of four colloidal amorphous SiO2 nanomaterials that differed only by their primary particle size (9, 15, 30, and 55 nm) were analyzed using the rat NR8383 alveolar macrophage (AM) assay. Data were compared to effects of single doses of 15 nm and 55 nm SiO2 intratracheally instilled in rat lungs. In vitro, all four elicited the release of concentration-dependent lactate dehydrogenase, ß-glucuronidase, and tumor necrosis factor alpha, and the two smaller materials also released H2O2. All effects were size-dependent. Since the colloidal SiO2 remained well-dispersed in serum-free in vitro conditions, effective particle concentrations reaching the cells were estimated using different models. Evaluating the effective concentration-based in vitro effects using the Decision-making framework for the grouping and testing of nanomaterials, all four nanomaterials were assigned as "active." This assignment and the size dependency of effects were consistent with the outcomes of intratracheal instillation studies and available short-term rat inhalation data for 15 nm SiO2. The study confirms the applicability of the NR8383 AM assay to assessing colloidal SiO2 but underlines the need to estimate and consider the effective concentration of such well-dispersed test materials.

Simultaneous Rayleigh/Mie and Raman/Fluorescence Characterization of Molecularly Functionalized Colloids by Correlative Single-Particle Real-Time Imaging in Suspension.

Many applications of nano- and microparticles require molecular functionalization. Assessing the heterogeneity of a colloidal sample in terms of its molecular functionalization is highly desirable but not accessible by conventional ensemble experiments. Retrieving this information necessitates single-particle experiments which simultaneously detect both functionalized and nonfunctionalized particles via two separate imaging channels. In this contribution, we present an optical setup for performing correlative single-particle imaging using laser light-sheet illumination: the first detection channel records elastic light scattering (Rayleigh/Mie), while the second channel detects inelastic light scattering (Raman) or fluorescence. The instrument is tested with Raman reporter-functionalized SERS-active metal nanoparticles (core/satellite silver nanoparticles, dimers and monomers of gold nanoparticles) and fluorophore-functionalized colloids (fluorescent polymer microparticles, dye-labeled protein on gold nanoparticles).

A Systematic Structure-Activity Study of a New Type of Small Peptidic Transfection Vector Reveals the Importance of a Special Oxo-Anion-Binding Motif for Gene Delivery.

We discovered a new class of artificial peptidic transfection vectors based on an artificial anion-binding motif, the guanidiniocarbonylpyrrole (GCP) cation. This new type of vector is surprisingly smaller than traditional systems, and our previous work suggested that the GCP group was important for promoting critical endosomal escape. We now present here a systematic comparison of similar DNA ligands featuring our GCP oxo-anion-binding motif with DNA ligands only consisting of naturally occurring amino acids. Structure-activity studies showed that the artificial binding motif clearly outperformed natural amino acids such as histidine, lysine, and arginine. It improved the ability to shuttle foreign genetic material into cells, yet successfully mediated endosomal escape. Also, plasmids that were complexed by our artificial ligands were stabilized against cytosolic degradation to some extent. This resulted in the successful expression of plasmid information (comparable to gold standards such as polyethyleneimine). Hence, our study clearly demonstrates the importance of the tailor-made GCP anion-binding site for efficient gene transfection.

Site-Specific SERS Assay for Survivin Protein Dimer: From Ensemble Experiments to Correlative Single-Particle Imaging.

An assay for Survivin, a small dimeric protein which functions as modulator of apoptosis and cell division and serves as a promising diagnostic biomarker for different types of cancer, is presented. The assay is based on switching on surface-enhanced Raman scattering (SERS) upon incubation of the Survivin protein dimer with Raman reporter-labeled gold nanoparticles (AuNP). Site-specificity is achieved by complexation of nickel-chelated N-nitrilo-triacetic acid (Ni-NTA) anchors on the particle surface by multiple histidines (His6 -tag) attached to each C-terminus of the centrosymmetric protein dimer. Correlative single-particle analysis using light sheet laser microscopy enables the simultaneous observation of both elastic and inelastic light scattering from the same sample volume. Thereby, the SERS-inactive AuNP-protein monomers can be directly discriminated from the SERS-active AuNP-protein dimers/oligomers. This information, i.e. the percentage of SERS-active AuNP in colloidal suspension, is not accessible from conventional SERS experiments due to ensemble averaging. The presented correlative single-particle approach paves the way for quantitative site-specific SERS assays in which site-specific protein recognition by small chemical and in particular supramolecular ligands can be tested.

Efficient Gene Transfection through Inhibition of ß-Sheet (Amyloid Fiber) Formation of a Short Amphiphilic Peptide by Gold Nanoparticles.

The effect of citrate-stabilized gold nanoparticles (AuNPs) on the secondary structure of an artificial ß-sheet-forming cationic peptide has been studied. The AuNPs inhibited ß-sheet formation and led to fragmented fibrils and spherical oligomers with assembled AuNPs on their surface. Besides this structural change, the functional properties of the peptide are also different. Whereas the peptide was unable to act as a vector for gene delivery, formation of a complex with AuNPs allowed successful gene delivery into cells.

Guanidiniocarbonyl pyrrole (GCP) conjugated PAMAM-G2, a highly efficient vector for gene delivery: the importance of DNA condensation.

A novel hybrid compound 1 efficiently shuttles genetic material into HeLa cells at concentrations as low as 0.6 µM, whereas the parent compound PAMAM-G2 is ineffective even at 200 µM. The high efficiency of 1 stems from its capabiliy to form highly condensed ligand-DNA polyplexes. Its binding affinity is actually lower than for the parent dendrimer. Compound 1 is even 200 times more effective and less cytotoxic than PEI, the current gold standard in gene transfection with cationic polymers.

Evolutionary divergence of Threonine Aspartase1 leads to species-specific substrate recognition.

Proteases are key regulators of life. Human Threonine Aspartase1 processes substrates, such as the mixed-lineage leukemia (MLL) protein, containing two cleavage sites, CS1 and CS2. Likewise, MLL's Drosophila ortholog trithorax is cleaved by Drosophila Threonine Aspartase1 (dTasp), suggesting a mechanistic coevolution. However, a detailed analysis of dTasp's function was missing so far. Here, active and inactive dTasp mutants allowed to compare substrate recognition and cleavage site selectivity of human and Drosophila enzymes. In contrast to the human protease, our cell-based assay revealed a preferential processing of CS2-like (QLD↓Gx[xD/Dx]) targets for dTasp, whereas cleavage of CS1-like targets (QVD↓Gx[xD/Dx]) was significantly impaired. Systematic mutagenesis of the CS2 sequence defined the motif x[FILMW]D↓Gx[xD/Dx] as the consensus cleavage sequence for dTasp. Substrate species selectivity of the enzymes was uncovered by demonstrating that dTasp cleaves Drosophila TFIIA, but not the human ortholog, suggesting evolutionary divergence of TFIIA downstream networks. Also, Drosophila USF2 was neither predicted nor cleaved by dTasp. Moreover, we found that dTasp cleavage site selectivity is independent of heterocomplex formation, as dTasp exists predominantly as an αß-monomer. Collectively, we provide novel insights into evolutionary similarities and divergence concerning Threonine Aspartase1 function in different species, which may aid to dissect and better target human Threonine Aspartase1 in malignancies.

Fly versus man: evolutionary impairment of nucleolar targeting affects the degradome of Drosophila's Taspase1.

Human Taspase1 is essential for development and cancer by processing critical regulators, such as the mixed-lineage leukemia protein. Likewise, its ortholog, trithorax, is cleaved by Drosophila Taspase1 (dTaspase1), implementing a functional coevolution. To uncover novel mechanism regulating protease function, we performed a functional analysis of dTaspase1 and its comparison to the human ortholog. dTaspase1 contains an essential nucleophile threonine(195), catalyzing cis cleavage into its α- and ß-subunits. A cell-based assay combined with alanine scanning mutagenesis demonstrated that the target cleavage motif for dTaspase1 (Q(3)[F/I/L/M](2)D(1)↓G(1')X(2')X(3')) differs significantly from the human ortholog (Q(3)[F,I,L,V](2)D(1)↓G(1')x(2')D(3')D(4')), predicting an enlarged degradome containing 70 substrates for Drosophila. In contrast to human Taspase1, dTaspase1 shows no discrete localization to the nucleus/nucleolus due to the lack of the importin-α/nucleophosmin1 interaction domain (NoLS) conserved in all vertebrates. Consequently, dTaspase1 interacts with neither the Drosophila nucleoplasmin-like protein nor human nucleophosmin1. The impact of localization on the protease's degradome was confirmed by demonstrating that dTaspase1 did not efficiently process nuclear substrates, such as upstream stimulatory factor 2. However, genetic introduction of the NoLS into dTaspase1 restored its nucleolar localization, nucleophosmin1 interaction, and efficient cleavage of nuclear substrates. We report that evolutionary functional divergence separating vertebrates from invertebrates can be achieved for proteases by a transport/localization-regulated mechanism.

Simultaneous measurement of kidney function by dynamic contrast enhanced MRI and FITC-sinistrin clearance in rats at 3 tesla: initial results.

Glomerular filtration rate (GFR) is an essential parameter of kidney function which can be measured by dynamic contrast enhanced magnetic resonance imaging (MRI-GFR) and transcutaneous approaches based on fluorescent tracer molecules (optical-GFR). In an initial study comparing both techniques in separate measurements on the same animal, the correlation of the obtained GFR was poor. The goal of this study was to investigate if a simultaneous measurement was feasible and if thereby, the discrepancies in MRI-GFR and optical-GFR could be reduced. For the experiments healthy and unilateral nephrectomised (UNX) Sprague Dawley (SD) rats were used. The miniaturized fluorescent sensor was fixed on the depilated back of an anesthetized rat. A bolus of 5 mg/100 g b.w. of FITC-sinistrin was intravenously injected. For dynamic contrast enhanced perfusion imaging (DCE-MRI) a 3D time-resolved angiography with stochastic trajectories (TWIST) sequence was used. By means of a one compartment model the excretion half-life (t1/2) of FITC-sinistrin was calculated and converted into GFR. GFR from DCE-MRI was calculated by fitting pixel-wise a two compartment renal filtration model. Mean cortical GFR and GFR by FITC-sinistrin were compared by Bland-Altman plots and pair-wise t-test. Results show that a simultaneous GFR measurement using both techniques is feasible. Mean optical-GFR was 4.34 ± 2.22 ml/min (healthy SD rats) and 2.34 ± 0.90 ml/min (UNX rats) whereas MRI-GFR was 2.10 ± 0.64 ml/min (SD rats) and 1.17 ± 0.38 ml/min (UNX rats). Differences between healthy and UNX rats were significant (p<0.05) and almost equal percentage difference (46.1% and 44.3%) in mean GFR were assessed with both techniques. Overall mean optical-GFR values were approximately twice as high compared to MRI-GFR values. However, compared to a previous study, our results showed a higher agreement. In conclusion, the possibility to use the transcutaneous method in MRI may have a huge impact in improving and validating MRI methods for GFR assessment in animal models.