Site-specific mutagenesis on Mnemiopsin 2: Calcium coordination and substrate binding properties of new variants.

We used site-specific mutagenesis by targeting E179 and F190 on the structure of photoprotein Mnemiopsin 2 (Mn2) from Mnemiopsis leidyi. The tertiary structure of E179S and F190L mutants was made by the MODELLER program. Far-ultraviolet circular dichroism data showed that the overall secondary structural content of photoprotein is not changed upon mutation, however the helicity and stabilizing interactions in helical structure decreases in mutants as compared with the wild-type (WT) photoprotein. Fluorescence spectra data revealed that the tertiary structure of the mutants is more compact than that of WT Mn2. According to the heat-induced denaturation experiments data, the melting temperature (Tm ) for the unfolding of tertiary structure of the F190L variant increases by 3°C compared with that of the WT and E179S mutant. Interestingly, the conformational enthalpy of the F190L mutant (86 kcal mol-1 ) is considerably lower than those in the WT photoprotein (102 kcal mol-1 ) and E179S mutant (106 kcal mol-1 ). The significant difference in the enthalpy of the thermal unfolding process could be explained by considering that the thermally denatured state of the F190L mutant is structurally less expanded than the WT and E179S variants. Bioluminescence activity data showed that the maximum characteristic wavelengths of the mutants undergo blue shift as compared with the WT protein. Initial intensity of the F190L and E179S variants was recorded to be 137.5% and 55.9% of the WT protein, respectively.

Isolation and Identification of Effective Probiotics on Drug-Resistant Acinetobacter baumannii Strains and Their Biofilms.

Introduction: This study aimed to identify, assess, and isolate strong lactobacilli demonstrating broad antibacterial and anti-biofilm activity against drug-resistant strains of Acinetobacter baumannii. Additionally, the mechanism of inhibition of these organisms was to be determined. Methods: Over a 6-month period (from December 2021 to June 2022), 53 clinical A. baumannii strains were collected from clinical samples. Twenty probiotic strains were isolated from local dairy products. Antibacterial activity of Lactobacillus strains' cell-free supernatant (CFS) was identified using the agar well diffusion method and the microbroth dilution test. Anti-biofilm effect was performed by the microtiter plate assay. The MTT assay was also used to look into the probiotics' cytotoxic effects on the L929 fibroblast cell line. Results: During the 6-month period, 53 clinical A. baumannii strains were obtained and identified. Out of 20 lactobacillus strains, the CFS of a lactobacillus strain (named L9) showed an inhibitory effect against all A. baumannii strains. Using the broth microdilution method, it was shown that the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of CFS extracts of L9 strains against A. baumannii strains were both » mg/mL. The result of the anti-biofilm showed that the selected probiotic could inhibit biofilm formation. The most common organic acid produced by all Lactobacillus strains, according to the HPLC method, was lactic acid, which was followed by acetic acid. The L929 fibroblast cell line was used in the cytotoxicity assay, which revealed that 100% of the cells in the L929 fibroblast cell line survived treatment with successive doses of CFSs for a full day. Conclusion: The probiotic strain isolated from local yogurt in this study showed potential anti-biofilm and antimicrobial properties against all drug-resistant Acinetobacter isolates. Given the increasing interest in probiotic microorganisms based on their high health benefits, further studies are recommended on the mechanisms of action between probiotics and A. baumannii strains to find new solutions for biological control and treatment of these infections without the use of antibiotics.

Application of Antimicrobial Peptides as Diagnostic Biosensors.

The COVID-19 pandemic has shown how emerging infectious diseases could quickly affect the global health and economy. New pathogens with pandemic potential are also expected to appear soon. Moreover, the large use of antibiotics has led to the development of different so-called "superbugs" capable of escaping all of the current antibiotics. In this context, the early and cost-effective detection of pathogens is crucial to avoid the spreading of new pathogens. Here, we present molecular sensors for the recognition of a broad panel of different bacterial species. The detection is based on the use of bacteria-binding peptides (BBPs) in combination with horseradish peroxidase (HRP). We developed a reliable ELISA-like assay that permits us to study the affinity of different BBPs toward some of the most important bacterial pathogens.

Rolling Circle Amplification Tailored for Plasmonic Biosensors: From Ensemble to Single-Molecule Detection.

We report on the tailoring of rolling circle amplification (RCA) for affinity biosensors relying on the optical probing of their surface with confined surface plasmon field. Affinity capture of the target analyte at the metallic sensor surface (e.g., by using immunoassays) is followed by the RCA step for subsequent readout based on increased refractive index (surface plasmon resonance, SPR) or RCA-incorporated high number of fluorophores (in surface plasmon-enhanced fluorescence, PEF). By combining SPR and PEF methods, this work investigates the impact of the conformation of long RCA-generated single-stranded DNA (ssDNA) chains to the plasmonic sensor response enhancement. In order to confine the RCA reaction within the evanescent surface plasmon field and hence maximize the sensor response, an interface carrying analyte-capturing molecules and additional guiding ssDNA strands (complementary to the repeating segments of RCA-generated chains) is developed. When using the circular padlock probe as a model target analyte, the PEF readout shows that the reported RCA implementation improves the limit of detection (LOD) from 13 pM to high femtomolar concentration when compared to direct labeling. The respective enhancement factor is of about 2 orders of magnitude, which agrees with the maximum number of fluorophore emitters attached to the RCA chain that is folded in the evanescent surface plasmon field by the developed biointerface. Moreover, the RCA allows facile visualizing of individual binding events by fluorescence microscopy, which enables direct counting of captured molecules. This approach offers a versatile route toward a fast digital readout format of single-molecule detection with further reduced LOD.

Analyzing Criteria Affecting the Functionality of G-Quadruplex-Based DNA Aptazymes as Colorimetric Biosensors and Development of Quinine-Binding Aptazymes.

A DNA aptazyme consists of an aptamer domain and a DNAzyme module, in which the DNAzyme activity can be regulated by the aptamer-target interaction. The complex of G-quadruplex (GQ) and hemin is a peroxidase-mimicking DNAzyme and has become increasingly popular as a reporter system for biosensing applications. The development of GQ-based aptazymes is of high interest as they can be used as label-free biosensors for the real-time detection of pathogens. Herein, we rationally designed ca. 200 GQ-based aptazyme candidates and evaluated the suitability of 14 aptamers targeting quinine, Protein A, Staphylococcus enterotoxin B, and ATP for this detection concept. As a result, six novel aptazymes were developed for the specific detection of quinine based on two quinine-binding aptamers. The rest of designed probes, however, hardly showed significant functionality. To uncover the reasons, we performed enzyme-linked oligonucleotide assays to find how the affinity of aptamers is affected once conjugated to the DNAzyme sequence or upon integration into the aptazyme probe. Furthermore, we investigated the impact of the structure-switching functionality in the parent aptamer and the effect of the reaction matrix on the efficiency of probes.

Adenita: interactive 3D modelling and visualization of DNA nanostructures.

DNA nanotechnology is a rapidly advancing field, which increasingly attracts interest in many different disciplines, such as medicine, biotechnology, physics and biocomputing. The increasing complexity of novel applications requires significant computational support for the design, modelling and analysis of DNA nanostructures. However, current in silico design tools have not been developed in view of these new applications and their requirements. Here, we present Adenita, a novel software tool for the modelling of DNA nanostructures in a user-friendly environment. A data model supporting different DNA nanostructure concepts (multilayer DNA origami, wireframe DNA origami, DNA tiles etc.) has been developed allowing the creation of new and the import of existing DNA nanostructures. In addition, the nanostructures can be modified and analysed on-the-fly using an intuitive toolset. The possibility to combine and re-use existing nanostructures as building blocks for the creation of new superstructures, the integration of alternative molecules (e.g. proteins, aptamers) during the design process, and the export option for oxDNA simulations are outstanding features of Adenita, which spearheads a new generation of DNA nanostructure modelling software. We showcase Adenita by re-using a large nanorod to create a new nanostructure through user interactions that employ different editors to modify the original nanorod.

The Brownian and Flow-Driven Rotational Dynamics of a Multicomponent DNA Origami-Based Rotor.

Nanomechanical devices are becoming increasingly popular due to the very diverse field of potential applications, including nanocomputing, robotics, and drug delivery. DNA is one of the most promising building materials to realize complex 3D structures at the nanoscale level. Several mechanical DNA origami structures have already been designed capable of simple operations such as a DNA box with a controllable lid, bipedal walkers, and cargo sorting robots. However, the nanomechanical properties of mechanically interlinked DNA nanostructures that are in general highly deformable have yet to be extensively experimentally evaluated. In this work, a multicomponent DNA origami-based rotor is created and fully characterized by electron microscopy under negative stain and cryo preparations. The nanodevice is further immobilized on a microfluidic chamber and its Brownian and flow-driven rotational behaviors are analyzed in real time by single-molecule fluorescence microscopy. The rotation in previous DNA rotors based either on strand displacement, electric field or Brownian motion. This study is the first to attempt to manipulate the dynamics of an artificial nanodevice with fluidic flow as a natural force.

Bone Scintigraphy in Staging of Newly Diagnosed Prostate Cancer in Regard of Different Risk Groups.

OBJECTIVES: Prostate cancer (PC) is the most common cancer in men over 50 years of age. Bone scintigraphy is still performed in many institutions at the time of primary diagnosis. We aimed to evaluate the role of bone scan in the primary staging of PC in regard of different risk groups. METHODS: A retrospective analysis of bone scans in 296 patients (mean age 64±6 y) acquired at the time of primary diagnosis was performed in our institution. The median prostate specific antigen (PSA) was 6.73 ng/ml, all patients had a Gleason score of >5. RESULTS: Only 11/296 patients had a positive bone scan, 1 being in the intermediate risk group, 10 in the high-risk group and none in the low-risk group according to D'Amico classification. CONCLUSION: Our results support the few published studies that less than 10% of patients with newly diagnosed PC by biopsy would develop bone metastasis, all in the intermediate or high-risk groups. Therefore, a staging by bone scan can only be recommended in patients with intermediate or high-risk, or symptomatic patients only.

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures.

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

(Poly)cation-induced protection of conventional and wireframe DNA origami nanostructures.

DNA nanostructures hold immense potential to be used for biological and medical applications. However, they are extremely vulnerable towards salt depletion and nucleases, which are common under physiological conditions. In this contribution, we used chitosan and linear polyethyleneimine for coating and long-term stabilization of several three-dimensional DNA origami nanostructures. The impact of the degree of polymerization and the charge density of the polymer together with the N/P charge ratio (ratio of the amines in polycations to the phosphates in DNA) on the stability of encapsulated DNA origami nanostructures in the presence of nucleases and in low-salt media was examined. The polycation shells were compatible with enzyme- and aptamer-based functionalization of the DNA nanostructures. Additionally, we showed that despite being highly vulnerable to salt depletion and nucleolytic digestion, self-assembled DNA nanostructures are stable in cell culture media up to a week. This was contrary to unassembled DNA scaffolds that degraded in one hour, showing that placing DNA strands into a spatially designed configuration crucially affect the structural integrity. The stability of naked DNA nanostructures in cell culture was shown to be mediated by growth media. DNA origami nanostructures kept in growth media were significantly more resistant towards low-salt denaturation, DNase I and serum-mediated digestion than when in a conventional buffer. Moreover, we confirmed that DNA origami nanostructures remain not only structurally intact but also fully functional after exposure to cell media. Agarose gel electrophoresis and negative stain transmission electron microscopy analysis revealed the hybridization of DNA origami nanostructures to their targets in the presence of serum proteins and nucleases. The structural integrity and functionality of DNA nanostructures in physiological fluids validate their use particularly for short-time biological applications in which the shape and structural details of DNA nanodevices are functionally critical.

Multiscale Visualization and Scale-Adaptive Modification of DNA Nanostructures.

We present an approach to represent DNA nanostructures in varying forms of semantic abstraction, describe ways to smoothly transition between them, and thus create a continuous multiscale visualization and interaction space for applications in DNA nanotechnology. This new way of observing, interacting with, and creating DNA nanostructures enables domain experts to approach their work in any of the semantic abstraction levels, supporting both low-level manipulations and high-level visualization and modifications. Our approach allows them to deal with the increasingly complex DNA objects that they are designing, to improve their features, and to add novel functions in a way that no existing single-scale approach offers today. For this purpose we collaborated with DNA nanotechnology experts to design a set of ten semantic scales. These scales take the DNA's chemical and structural behavior into account and depict it from atoms to the targeted architecture with increasing levels of abstraction. To create coherence between the discrete scales, we seamlessly transition between them in a well-defined manner. We use special encodings to allow experts to estimate the nanoscale object's stability. We also add scale-adaptive interactions that facilitate the intuitive modification of complex structures at multiple scales. We demonstrate the applicability of our approach on an experimental use case. Moreover, feedback from our collaborating domain experts confirmed an increased time efficiency and certainty for analysis and modification tasks on complex DNA structures. Our method thus offers exciting new opportunities with promising applications in medicine and biotechnology.

In vivo magnetic resonance imaging of pancreatic tumors using iron oxide nanoworms targeted with PTR86 peptide.

To cut the high mortality rate of malignant disease such as pancreatic cancer, development of newly diagnostic probes for early stage detection of tumor lesions is required. Multimodal imaging nanoprobes allowing targeted and real time functional/anatomical imaging of tumors meet the demands. For this purpose, a MRI/optical dual-modality probe based on biodegradable magnetic iron oxide nanoworms has been developed. The cross-linked surface of nanoworms were anchored to fluorescent dyes and to FITC.PTR86; a novel synthetic peptide with high affinity towards somatostatin receptors. Combination of various in vitro techniques including Prussian blue staining, fluorescent microscopy and fluorescence activated cell sorting (FACS) have been performed to explore the interaction of developed nanoprobe with pancreatic tumor cell lines. Together with in vivo studies in a xenograft mouse model of human pancreatic adenocarcinoma and ex vivo investigations, the results show the efficient imaging and targeting of pancreatic tumors by our newly developed nanosystem using both MRI and optical imaging modalities.

64Cu-PSMA-617 PET/CT Imaging of Prostate Adenocarcinoma: First In-Human Studies.

AIM: The prostate-specific membrane antigen (PSMA) is a cell surface protein, which is overexpressed in nearly all cases of prostate cancer (PCa). PET imaging with 68Ga-PSMA-HBED-CC has recently found widespread application in the diagnosis of recurrent PCa. In this study, the diagnostic potential of 64Cu-labeled PSMA ligand (PSMA-617) PET in patients with PCa has been investigated. MATERIALS AND METHODS: The study was conducted simultaneously at two nuclear medicine centers, Austria (Vienna, Center 1) and Germany (Bad Berka, Center 2). The patients (n = 29) included in this study were referred for PET (Center 1, 21 patients) or PET/CT (Center 2, 8 patients) imaging with either a high suspicion of recurrent disease or for possible surgical or PSMA radioligand therapy planning. PET images of the whole body were performed at 1 hour p.i. and additional images of the pelvis at 2 hours p.i. RESULTS: In 23 of 29 patients, at least one focus of pathological tracer uptake suspicious for primary disease in the prostate lobe or recurrent disease was detected. Among healthy organs, the salivary glands, kidneys, and liver showed the highest radiotracer uptake. Lesions suspicious for PCa were detected with excellent contrast as early as 1 hour p.i. with high detection rates even at low prostate-specific antigen (PSA) levels. CONCLUSION: The preliminary results of this study demonstrate the high potential of 64Cu-PSMA ligand PET/CT imaging in patients with recurrent disease and in the primary staging of selected patients with progressive local disease. The acquired PET images showed an excellent resolution of the detected lesions with very high lesion-to- background contrast. Furthermore, the long half-life of 64Cu allows distribution of the tracer to clinical PET centers that lack radiochemistry facilities for the preparation of 68Ga-PSMA ligand (satellite concept).