IMPATIENT-qPCR: monitoring SELEX success during in vitro aptamer evolution.

SELEX (Systematic Evolution of Ligands by Exponential enrichment) processes aim on the evolution of high-affinity aptamers as binding entities in diagnostics and biosensing. Aptamers can represent game-changers as constituents of diagnostic assays for the management of instantly occurring infectious diseases or other health threats. Without in-process quality control measures SELEX suffers from low overall success rates. We present a quantitative PCR method for fast and easy quantification of aptamers bound to their targets. Simultaneous determination of melting temperatures (Tm) of each SELEX round delivers information on the evolutionary success via the correlation of increasing GC content and Tm alone with a round-wise increase of aptamer affinity to the respective target. Based on nine successful and published previous SELEX processes, in which the evolution/selection of aptamer affinity/specificity was demonstrated, we here show the functionality of the IMPATIENT-qPCR for polyclonal aptamer libraries and resulting individual aptamers. Based on the ease of this new evolution quality control, we hope to introduce it as a valuable tool to accelerate SELEX processes in general. IMPATIENT-qPCR SELEX success monitoring. Selection and evolution of high-affinity aptamers using SELEX technology with direct aptamer evolution monitoring using melting curve shifting analyses to higher Tm by quantitative PCR with fluorescence dye SYBR Green I. KEY POINTS: • Fast and easy analysis. • Universal applicability shown for a series of real successful projects.

Robust Fluorometric Aptamer Assay for Direct and Rapid Detection of Clinical Isolates of Candida spec.

Infections caused by yeasts of the genus Candida are likely to occur not only in immunocompromised patients but also in healthy individuals, leading to infections of the gastrointestinal tract, urinary tract, and respiratory tract. Due to the rapid increase in the frequency of reported Candidiasis cases in recent years, diagnostic research has become the subject of many studies, and therefore, we developed a polyclonal aptamer library-based fluorometric assay with high specificity and affinity towards Candida spec. to quantify the pathogens in clinical samples with high sensitivity. We recently obtained the specific aptamer library R10, which explicitly recognized Candida and evolved it by mimicking an early skin infection model caused by Candida using the FluCell-SELEX system. In the follow-up study presented here, we demonstrate that the aptamer library R10-based bioassay specifically recognizes invasive clinical Candida isolates, including not only C. albicans but also strains like C. tropcialis, C. krusei, or C. glabrata. The next-generation fluorometric bioassay presented here can reliably and easily detect an early Candida infection and could be used for further clinical research or could even be developed into a full in vitro diagnostic tool.

Robust Protocol for the Synthesis of BSA Nanohydrogels by Inverse Nanoemulsion for Drug Delivery.

In a highly efficient and reproducible process, bovine serum albumin (BSA) nanogels are prepared from inverse nanoemulsions. The concept of independent nanoreactors of the individual droplets in the nanoemulsions allows high protein concentrations of up to 0.6% in the inverse total system. The BSA gel networks are generated by the 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride coupling strategy widely used in protein chemistry. In a robust work-up protocol, the hydrophobic continuous phase of the inverse emulsion is stepwise replaced by water without compromising the colloidal stability and non-toxicity of the nanogel particles. Further, the simple process allows the loading of the nanogels with various cargos like a dye (Dy-495), a drug (ibuprofen), another protein [FMN-binding fluorescent protein (EcFbFP)], and oligonucleotides [plasmid DNA for enhanced GFP expression in mammalian cells (pEGFP c3) and a synthetic anti-Pseudomonas aeruginosa aptamer library]. These charged nanoobjects work efficiently as carriers for staining and transfection of cells. This is exemplarily shown for a phalloidin dye and a plasmid DNA as cargo with adenocarcinomic human alveolar basal epithelial cells (A549), a cell revertant of the SV-40 cancer rat cell line SV-52 (Rev2), and human breast carcinoma cells (MDA-MB-231), respectively.

Two quality and stability indicating imaged CIEF methods for mRNA vaccines.

Two imaged capillary isoelectric focusing methods were developed to provide insight into the quality and stability of messenger ribonucleic acid (mRNA) vaccines, specifically, mRNA encapsulated in lipid nanoparticles (LNPs). A variety of stressed and lipid composition-modified samples were measured and detected by their UV absorption. The results were supported by the data of an encapsulation assay and particle sizing. One method, using 9 M urea as an additive, shows two broad and jagged peaks in which the peak shape offers detailed information. The summed peak area of both peaks showed RSDs from 2% to 8% when one batch was measured in triplicate and apparently depends on the size of the LNPs. In the second method, a combination of 5.5 M urea and 2 M N-ethylurea was used. This method is characterized by a high repeatability of the isoelectric point (pI, <0.5%). The repeatable peak area (RSD of 2%-7%) correlates linearly with the mRNA content, which also applies to the first method, and added stress is evident by the change in pI and peak area. Furthermore, experiments with the addition of a fluorescent dye were performed (fluorescence detection), which tremendously increased the sensitivity of the methods. Both methods can be used to characterize the stability of mRNA-loaded LNPs, for example, when investigating various storage times at different temperatures and freeze-thaw cycles, as well as the ability of the methods to distinguish lipid compositions and measure batch-to-batch variability.

[(CH3 )Al(CH2 )]12 : Methylaluminomethylene (MAM-12).

The molecular structure of enigmatic "poly(aluminium-methyl-methylene)" (first reported in 1968) has been unraveled in a transmetalation reaction with gallium methylene [Ga8 (CH2 )12 ] and AlMe3 . The existence of cage-like methylaluminomethylene moieties was initially suggested by the reaction of rare-earth-metallocene complex [Cp*2 Lu{(µ-Me)2 AlMe2 }] with excess AlMe3 affording the deca-aluminium cluster [Cp*4 Lu2 (µ3 -CH2 )12 Al10 (CH3 )8 ] in low yield (Cp*=C5 Me5 ). Treatment of [Ga8 (CH2 )12 ] with excess AlMe3 reproducibly gave the crystalline dodeca-aluminium complex [(CH3 )12 Al12 (µ3 -CH2 )12 ] (MAM-12). Revisiting a previous approach to "poly(aluminium-methyl-methylene" by using a (C5 H5 )2 TiCl2 /AlMe3 (1 : 100) mixture led to amorphous solids displaying solubility behavior and spectroscopic features similar to those of crystalline MAM-12. The gallium methylene-derived MAM-12 was used as an efficient methylene transfer reagent for ketones.

High Plasticity of the Amicetin Biosynthetic Pathway in Streptomyces sp. SHP 22-7 Led to the Discovery of Streptcytosine P and Cytosaminomycins F and G and Facilitated the Production of 12F-Plicacetin.

A chemical reinvestigation of the Indonesian strain Streptomyces sp. SHP 22-7 led to the isolation of three new pyrimidine nucleosides, along with six known analogues and zincphyrin. The structures of the new compounds (6, 7, 10) were elucidated by employing spectroscopic techniques (NMR, MS, CD, and IR) as well as enantioselective analyses of methyl branched side chain configurations. Application of the precursor-directed feeding approach led to the production and partial isolation of nine further pyrimidine analogues. The new compounds 6, 7, and 11 and three of the known compounds (2-4) were found to possess antimycobacterial and cytotoxic properties.

Development and chromatographic exploration of stable-bonded cross-linked amino silica against classical amino phases.

The present work reports on a novel stable-bonded amino silica stationary phase obtained by crosslinking of surface aminopropyl moieties using triglycidyl isocyanurate. The obtained cross-linked amido-amino network silica material exhibited superior hydrolytic stability compared to classical 3-aminopropyl phases and showed, inter alia, excellent separation of nine therapeutically effective sulfonamides in hydrophilic interaction/weak anion exchange chromatography elution mode. Additionally, the separation of carbohydrates was investigated under classical hydrophilic interaction chromatography conditions as well proving the suitability of the novel phase for such applications. For the evaluation of the hydrolytic stability the prepared material, as well as two commercially available benchmark columns and a set of in-house synthesized amino-modified materials, were exposed to harsh aqueous mobile phase conditions for in total of 50 h at elevated temperature. In this context, the materials were examined by elemental analysis, (13 C and 29 Si cross-polarization/magic angle spinning) solid-state nuclear magnetic resonance, and a chromatographic test before and subsequent to the exposure to these stress conditions. Lastly, the new stationary phase was classified in comparison to a set of commercially available stationary phases by principal component analysis of resultant retention factors gained from chromatographic standard tests.

A Polyclonal Aptamer Library for the Specific Binding of the Gut Bacterium Roseburia intestinalis in Mixtures with Other Gut Microbiome Bacteria and Human Stool Samples.

Roseburia intestinalis has received attention as a potential probiotic bacterium. Recent studies have demonstrated that changes in its intestinal abundance can cause various diseases, such as obesity, enteritis and atherosclerosis. Probiotic administration or fecal transplantation alter the structure of the intestinal flora, offering possibilities for the prevention and treatment of these diseases. However, current monitoring methods, such as 16S rRNA sequencing, are complex and costly and require specialized personnel to perform the tests, making it difficult to continuously monitor patients during treatment. Hence, the rapid and cost-effective quantification of intestinal bacteria has become an urgent problem to be solved. Aptamers are of emerging interest because their stability, low immunogenicity and ease of modification are attractive properties for a variety of applications. We report a FluCell-SELEX polyclonal aptamer library specific for R. intestinalis isolated after seven evolution rounds, that can bind and label this organism for fluorescence microscopy and binding assays. Moreover, R. intestinalis can be distinguished from other major intestinal bacteria in complex defined mixtures and in human stool samples. We believe that this preliminary evidence opens new avenues towards aptamer-based electronic biosensors as new powerful and inexpensive diagnostic tools for the relative quantitative monitoring of R. intestinalis in gut microbiomes.

Polyclonal Aptamer Libraries from a FluRoot-SELEX for the Specific Labeling of the Apical and Elongation/Differentiation Zones of Arabidopsis thaliana Roots.

In more than 30 years of aptamer research, it has become widely accepted that aptamers are fascinating binding molecules for a vast variety of applications. However, the majority of targets have been proteins, although special variants of the so-called SELEX process for the molecular evolution of specific aptamers have also been developed, allowing for the targeting of small molecules as well as larger structures such as cells and even cellular networks of human (tumor) tissues. Although the provocative thesis is widely accepted in the field, that is, in principle, any level of complexity for SELEX targets is possible, the number of studies on whole organs or at least parts of them is limited. To pioneer this thesis, and based on our FluCell-SELEX process, here, we have developed polyclonal aptamer libraries against apices and the elongation/differentiation zones of plant roots as examples of organs. We show that dedicated libraries can specifically label the respective parts of the root, allowing us to distinguish them in fluorescence microscopy. We consider this achievement to be an initial but important evidence for the robustness of this SELEX variant. These libraries may be valuable tools for plant research and a promising starting point for the isolation of more specific individual aptamers directed against root-specific epitopes.

A Polyclonal Selex Aptamer Library Directly Allows Specific Labelling of the Human Gut Bacterium Blautia producta without Isolating Individual Aptamers.

Recent studies have demonstrated that changes in the abundance of the intestinal bacterium Blautia producta, a potential probiotic, are closely associated with the development of various diseases such as obesity, diabetes, some neurodegenerative diseases, and certain cancers. However, there is still a lack of an effective method to detect the abundance of B. producta in the gut rapidly. Especially, DNA aptamers are now widely used as biometric components for medical testing due to their unique characteristics, including high chemical stability, low production cost, ease of chemical modification, low immunogenicity, and fast reproducibility. We successfully obtained a high-affinity nucleic acid aptamer library (B.p-R14) after 14 SELEX rounds, which efficiently discriminates B. producta in different analysis techniques including fluorometric suspension assays or fluorescence microscopy from other major gut bacteria in complex mixtures and even in human stool samples. These preliminary findings will be the basis towards aptamer-based biosensing applications for the fast and reliable monitoring of B. producta in the human gut microbiome.

Optogenetic Approaches for the Spatiotemporal Control of Signal Transduction Pathways.

Biological signals are sensed by their respective receptors and are transduced and processed by a sophisticated intracellular signaling network leading to a signal-specific cellular response. Thereby, the response to the signal depends on the strength, the frequency, and the duration of the stimulus as well as on the subcellular signal progression. Optogenetic tools are based on genetically encoded light-sensing proteins facilitating the precise spatiotemporal control of signal transduction pathways and cell fate decisions in the absence of natural ligands. In this review, we provide an overview of optogenetic approaches connecting light-regulated protein-protein interaction or caging/uncaging events with steering the function of signaling proteins. We briefly discuss the most common optogenetic switches and their mode of action. The main part deals with the engineering and application of optogenetic tools for the control of transmembrane receptors including receptor tyrosine kinases, the T cell receptor and integrins, and their effector proteins. We also address the hallmarks of optogenetics, the spatial and temporal control of signaling events.

BSA Hydrogel Beads Functionalized with a Specific Aptamer Library for Capturing Pseudomonas aeruginosa in Serum and Blood.

Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide. Pseudomonas aeruginosa is a WHO-alerted multi-resistant pathogen of extreme importance as a cause of sepsis. Septicemia patients have significantly increased survival chances if sepsis is diagnosed in the early stages. Affinity materials can not only represent attractive tools for specific diagnostics of pathogens in the blood but can prospectively also serve as the technical foundation of therapeutic filtration devices. Based on the recently developed aptamers directed against P. aeruginosa, we here present aptamer-functionalized beads for specific binding of this pathogen in blood samples. These aptamer capture beads (ACBs) are manufactured by crosslinking bovine serum albumin (BSA) in an emulsion and subsequent functionalization with the amino-modified aptamers on the bead surface using the thiol- and amino-reactive bispecific crosslinker PEG4-SPDP. Specific and quantitative binding of P. aeruginosa as the dedicated target of the ACBs was demonstrated in serum and blood. These initial but promising results may open new routes for the development of ACBs as a platform technology for fast and reliable diagnosis of bloodstream infections and, in the long term, blood filtration techniques in the fight against sepsis.

[Orientation disorder, ophthalmoplegia and ataxia in a 48-year-old male]. / Orientierungsstörung, Ophthalmoplegie und Ataxie bei einem 48-Jährigen.

This case report concerns a 48-year-old male patient presenting the classic clinical picture of Wernicke encephalopathy with orientation disorder, ophthalmoplegia and ataxia. Wernicke encephalopathy is a neurological emergency that should be treated immediately with high-dose intravenous thiamine for a week, if suspected. Recognition of the disease in the emergency room is essential. Due to the limited compliance in patients with alcohol-use disorder, early diagnosis can be challenging.

Nocathioamides, Uncovered by a Tunable Metabologenomic Approach, Define a Novel Class of Chimeric Lanthipeptides.

The increasing number of available genomes, in combination with advanced genome mining techniques, unveiled a plethora of biosynthetic gene clusters (BGCs) coding for ribosomally synthesized and post-translationally modified peptides (RiPPs). The products of these BGCs often represent an enormous resource for new and bioactive compounds, but frequently, they cannot be readily isolated and remain cryptic. Here, we describe a tunable metabologenomic approach that recruits a synergism of bioinformatics in tandem with isotope- and NMR-guided platform to identify the product of an orphan RiPP gene cluster in the genomes of Nocardia terpenica IFM 0406 and 0706T . The application of this tactic resulted in the discovery of nocathioamides family as a founder of a new class of chimeric lanthipeptides I.

Migraine and rare neurological disorders.

Although migraine is generally considered an idiopathic and isolated neurological condition, it may also represent the presenting symptom of several uncommon heritable and acquired neurological diseases contributing to the recognition of such conditions. Migraine may indeed present with atypical characteristics or prolonged duration and may be associated with specific neuroradiological findings that may help in identifying the underlying condition. However, features of migraine in rare diseases are usually little known because of the lack of systematic studies. The aim of this paper is to provide clinicians with an updated review on specific clinical and neuroradiological features of migraine in uncommon neurological diseases that may be helpful to their diagnosis and treatment. Therefore, the early diagnosis of these uncommon diseases is crucial for patients' clinical management and for the implementation of therapeutic approaches aimed at targeting the underlying disease pathogenic mechanisms. Thus, when investigating patients affected by migraine, physicians should always be aware about rare causes of migraine that if misdiagnosed could seriously impact patients' outcome. Given these relevant implications, future studies specifically assessing features of migraine in uncommon diseases are mandatory.

Enzyme-Free Ligation of 5'-Phosphorylated Oligodeoxynucleotides in a DNA Nanostructure.

Multicomponent reactions are difficult synthetic transformations. For DNA, there is a special opportunity to align multiple strands in a folded nanostructure, so that they are preorganized to give a specific sequence. Multistrand reactions in DNA origami structures have previously been performed using photochemical crosslinking, 1,3-diploar cycloadditions or phosphoramidate-forming reactions. Here we report carbodiimide-driven phosphodiester formation in a small origami sheet that produces DNA strands up to 600 nucleotides in length in a single step. The method uses otherwise unmodified oligodeoxynucleotides with a 5'-terminal phosphate as starting materials. Compared to an enzymatic multistrand ligation involving linear duplexes, the carbodiimide-driven ligation gave fewer side products, as detected by gel electrophoresis. The full-length 600mer product was successfully amplified by polymerase chain reaction.

Phosphoramidate Ligation of Oligonucleotides in Nanoscale Structures.

The folding of long DNA strands into designed nanostructures has evolved into an art. Being based on linear chains only, the resulting nanostructures cannot readily be transformed into covalently linked frameworks. Covalently linking strands in the context of folded DNA structures requires a robust method that avoids sterically demanding reagents or enzymes. Here we report chemical ligation of the 3'-amino termini of oligonucleotides and 5'-phosphorylated partner strands in templated reactions that produce phosphoramidate linkages. These reactions produce inter-nucleotide linkages that are isoelectronic and largely isosteric to phosphodiesters. Ligations were performed at three levels of complexity, including the extension of branched DNA hybrids and the ligation of six scaffold strands in a small origami.

Experimental verification of the individual energy dependencies of the partial L-shell photoionization cross sections of Pd and Mo.

An experimental method for the verification of the individually different energy dependencies of L(1)-, L(2)-, and L(3)- subshell photoionization cross sections is described. The results obtained for Pd and Mo are well in line with theory regarding both energy dependency and absolute values, and confirm the theoretically calculated cross sections by Scofield from the early 1970 s and, partially, more recent data by Trzhaskovskaya, Nefedov, and Yarzhemsky. The data also demonstrate the questionability of quantitative x-ray spectroscopical results based on the widely used fixed jump ratio approximated cross sections with energy independent ratios. The experiments are carried out by employing the radiometrically calibrated instrumentation of the Physikalisch-Technische Bundesanstalt at the electron storage ring BESSY II in Berlin; the obtained fluorescent intensities are thereby calibrated at an absolute level in reference to the International System of Units. Experimentally determined fixed fluorescence line ratios for each subshell are used for a reliable deconvolution of overlapping fluorescence lines. The relevant fundamental parameters of Mo and Pd are also determined experimentally in order to calculate the subshell photoionization cross sections independently of any database.

Identification and quantification of protein adducts formed by metabolites of 1-methoxy-3-indolylmethyl glucosinolate in vitro and in mouse models.

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate (GLS) occurring in cabbage, broccoli, and other cruciferous plants is a potent mutagen requiring metabolic activation by myrosinase present in plant cells and intestinal bacteria. We previously reported that 1-MIM-GLS and its alcoholic breakdown product 1-MIM-OH, which requires additional activation by sulfotransferases, form DNA adducts in mice. In the present study, the formation of protein adducts was investigated. First, two major adducts obtained after incubation of individual amino acids, serum albumin, or hemoglobin with 1-MIM-GLS in the presence of myrosinase were identified as τN-(1-MIM)-His and πN-(1-MIM)-His using MS and NMR spectroscopy. After the development of a specific detection method using isotope-dilution UPLC-ESI-MS/MS, adduct formation was confirmed in mice after oral treatment with 1-MIM-GLS. Adduct levels were highest in the cecum and colon, somewhat lower in serum albumin and the liver, and also readily detectable in the lung and hemoglobin. On the contrary, oral treatment with 1-MIM-OH produced the highest adduct levels in the liver. The higher ratio of albumin to hemoglobin adducts in 1-MIM-OH- compared to 1-MIM-GLS-treated animals (8.1 versus 3.5) suggests that in 1-MIM-OH-treated animals albumin adducts were produced mostly in the liver, the site of albumin synthesis. The formation of adducts was approximately linear over a range of single oral doses from 20 to 600 µmol/kg body mass. Repeated oral administration of 1-MIM-OH (up to 40 treatments, thrice per week) led to continuous accumulation of hemoglobin adducts, whereas the level of serum albumin adducts remained rather constant, which reflects the different turnover rates of these proteins (t1/2 nearly 1.9 d for serum albumin and 25 d for hemoglobin in the mouse). Accumulation of adducts was also noticed in the lung. Adduct levels were higher, but their accumulation was weaker in the liver and kidney. The method developed will be useful to assess the exposure of humans to reactive metabolites formed from 1-MIM-GLS present in many foods.

The Designed Pore-Forming Antimicrobial Peptide C14R Combines Excellent Activity against the Major Opportunistic Human Pathogen Pseudomonas aeruginosa with Low Cytotoxicity.

The diminishing portfolio of mankind's available antibiotics urges science to develop novel potent drugs. Here, we present a peptide fitting the typical blueprint of amphipathic and membrane-active antimicrobial peptides, denominated C14R. This 2 kDa peptide consists of 16 amino acid residues, with seven being either hydrophobic, aromatic, or non-polar, and nine being polar or positively charged, strictly separated on opposite sides of the predicted α-helix. The affinity of the peptide C14R to P. aeruginosa membranes and its intrinsic tendency to productively insert into membranes of such composition were analyzed by dynamic simulations. Its biological impact on the viability of two different P. aeruginosa reference strains was demonstrated by determining the minimal inhibitory concentrations (MICs), which were found to be in the range of 10-15 µg/mL. C14R's pore-forming capability was verified in a permeabilization assay based on the peptide-triggered uptake of fluorescent dyes into the bacterial cells. Finally, the peptide was used in radial diffusion assays, which are commonly used for susceptibility testing of antimicrobial peptides in clinical microbiology. In comparison to reference strains, six clinical P. aeruginosa isolates were clearly affected, thereby paving the way for further in-depth analyses of C14R as a promising new AMP drug in the future.