Detection of microplastics in the human penis.

The proliferation of microplastics (MPs) represents a burgeoning environmental and health crisis. Measuring less than 5 mm in diameter, MPs have infiltrated atmospheric, freshwater, and terrestrial ecosystems, penetrating commonplace consumables like seafood, sea salt, and bottled beverages. Their size and surface area render them susceptible to chemical interactions with physiological fluids and tissues, raising bioaccumulation and toxicity concerns. Human exposure to MPs occurs through ingestion, inhalation, and dermal contact. To date, there is no direct evidence identifying MPs in penile tissue. The objective of this study was to assess for potential aggregation of MPs in penile tissue. Tissue samples were extracted from six individuals who underwent surgery for a multi-component inflatable penile prosthesis (IPP). Samples were obtained from the corpora using Adson forceps before corporotomy dilation and device implantation and placed into cleaned glassware. A control sample was collected and stored in a McKesson specimen plastic container. The tissue fractions were analyzed using the Agilent 8700 Laser Direct Infrared (LDIR) Chemical Imaging System (Agilent Technologies. Moreover, the morphology of the particles was investigated by a Zeiss Merlin Scanning Electron Microscope (SEM), complementing the detection range of LDIR to below 20 µm. MPs via LDIR were identified in 80% of the samples, ranging in size from 20-500 µm. Smaller particles down to 2 µm were detected via SEM. Seven types of MPs were found in the penile tissue, with polyethylene terephthalate (47.8%) and polypropylene (34.7%) being the most prevalent. The detection of MPs in penile tissue raises inquiries on the ramifications of environmental pollutants on sexual health. Our research adds a key dimension to the discussion on man-made pollutants, focusing on MPs in the male reproductive system.

An enzymatic continuous-flow reactor based on a pore-size matching nano- and isoporous block copolymer membrane.

Continuous-flow biocatalysis utilizing immobilized enzymes emerged as a sustainable route for chemical synthesis. However, inadequate biocatalytic efficiency from current flow reactors, caused by non-productive enzyme immobilization or enzyme-carrier mismatches in size, hampers its widespread application. Here, we demonstrate a general-applicable and robust approach for the fabrication of a high-performance enzymatic continuous-flow reactor via integrating well-designed scalable isoporous block copolymer (BCP) membranes as carriers with an oriented and productive immobilization employing material binding peptides (MBP). Densely packed uniform enzyme-matched nanochannels of well-designed BCP membranes endow the desired nanoconfined environments towards a productive immobilized phytase. Tuning nanochannel properties can further regulate the complex reaction process and fortify the catalytic performance. The synergistic design of enzyme-matched carriers and efficient enzyme immobilization empowers an excellent catalytic performance with >1 month operational stability, superior productivity, and a high space-time yield (1.05 × 105 g L-1 d-1) via a single-pass continuous-flow process. The obtained performance makes the designed nano- and isoporous block copolymer membrane reactor highly attractive for industrial applications.

Late Microaerobic Growth for Efficient Production of Human Cytochrome P450 3A4 in E. coli.

Detailed preclinical characterization of metabolites formed in vivo from candidate drug substances is mandatory prior to the initiation of clinical trials. Therefore, inexpensive and efficient methods for drug metabolite synthesis are of high importance for rapid advancement of the drug development process. A large fraction of small molecule drugs is modified by monooxygenase cytochrome P450 3A4 produced in the human liver and intestine. Therefore, this enzyme is frequently employed to catalyze metabolite synthesis in vitro, making 3A4 availability a critical requirement in early drug development. Unfortunately, the recombinant production of this enzyme in microbial hosts is notoriously difficult. Maintaining low oxygen transfer rates and the use of rich media for host cultivation are required for P450 3A4 production. However, detailed studies on the relationship between oxygen supply and P450 3A4 space-time yields are missing. We describe an improved biotechnological process for the heterologous expression of P450 3A4 together with its redox partner, cytochrome P450 reductase, in Escherichia coli. Enzyme production was most efficient under so-called "late microaerobic" growth conditions, in which the cells have just not yet made the switch to anaerobic metabolism, characterized by a limited oxygen supply leading to oxygen concentrations in the liquid phase that are far below the detection limit of standard oxygen electrodes. Furthermore, feeding the carbon source glycerol as well as controlling cellular acetate formation improved process productivity. The presented protocol resulted in the formation of functional recombinant 3A4 at concentrations up to 680 nmol L-1.

Signal Peptide Efficiency: From High-Throughput Data to Prediction and Explanation.

The passage of proteins across biological membranes via the general secretory (Sec) pathway is a universally conserved process with critical functions in cell physiology and important industrial applications. Proteins are directed into the Sec pathway by a signal peptide at their N-terminus. Estimating the impact of physicochemical signal peptide features on protein secretion levels has not been achieved so far, partially due to the extreme sequence variability of signal peptides. To elucidate relevant features of the signal peptide sequence that influence secretion efficiency, an evaluation of ∼12,000 different designed signal peptides was performed using a novel miniaturized high-throughput assay. The results were used to train a machine learning model, and a post-hoc explanation of the model is provided. By describing each signal peptide with a selection of 156 physicochemical features, it is now possible to both quantify feature importance and predict the protein secretion levels directed by each signal peptide. Our analyses allow the detection and explanation of the relevant signal peptide features influencing the efficiency of protein secretion, generating a versatile tool for the de novo design and in silico evaluation of signal peptides.

Optimization of the antimicrobial peptide Bac7 by deep mutational scanning.

BACKGROUND: Intracellularly active antimicrobial peptides are promising candidates for the development of antibiotics for human applications. However, drug development using peptides is challenging as, owing to their large size, an enormous sequence space is spanned. We built a high-throughput platform that incorporates rapid investigation of the sequence-activity relationship of peptides and enables rational optimization of their antimicrobial activity. The platform is based on deep mutational scanning of DNA-encoded peptides and employs highly parallelized bacterial self-screening coupled to next-generation sequencing as a readout for their antimicrobial activity. As a target, we used Bac71-23, a 23 amino acid residues long variant of bactenecin-7, a potent translational inhibitor and one of the best researched proline-rich antimicrobial peptides. RESULTS: Using the platform, we simultaneously determined the antimicrobial activity of >600,000 Bac71-23 variants and explored their sequence-activity relationship. This dataset guided the design of a focused library of ~160,000 variants and the identification of a lead candidate Bac7PS. Bac7PS showed high activity against multidrug-resistant clinical isolates of E. coli, and its activity was less dependent on SbmA, a transporter commonly used by proline-rich antimicrobial peptides to reach the cytosol and then inhibit translation. Furthermore, Bac7PS displayed strong ribosomal inhibition and low toxicity against eukaryotic cells and demonstrated good efficacy in a murine septicemia model induced by E. coli. CONCLUSION: We demonstrated that the presented platform can be used to establish the sequence-activity relationship of antimicrobial peptides, and showed its usefulness for hit-to-lead identification and optimization of antimicrobial drug candidates.

Discovery of antimicrobials by massively parallelized growth assays (Mex).

The number of newly approved antimicrobial compounds has been steadily decreasing over the past 50 years emphasizing the need for novel antimicrobial substances. Here we present Mex, a method for the high-throughput discovery of novel antimicrobials, that relies on E. coli self-screening to determine the bioactivity of more than ten thousand naturally occurring peptides. Analysis of thousands of E. coli growth curves using next-generation sequencing enables the identification of more than 1000 previously unknown antimicrobial peptides. Additionally, by incorporating the kinetics of growth inhibition, a first indication of the mode of action is obtained, which has implications for the ultimate usefulness of the peptides in question. The most promising peptides of the screen are chemically synthesized and their activity is determined in standardized susceptibility assays. Ten out of 15 investigated peptides efficiently eradicate bacteria at a minimal inhibitory concentration in the lower µM or upper nM range. This work represents a step-change in the high-throughput discovery of functionally diverse antimicrobials.

Whole-cell screening of oxidative enzymes using genetically encoded sensors.

Biocatalysis is increasingly used for synthetic purposes in the chemical and especially the pharmaceutical industry. Enzyme discovery and optimization which is frequently needed to improve biocatalytic performance rely on high-throughput methods for activity determination. These methods should ideally be generic and applicable to entire enzyme families. Hydrogen peroxide (H2O2) is a product of several biocatalytic oxidations and its formation can serve as a proxy for oxidative activity. We designed a genetically encoded sensor for activity measurement of oxidative biocatalysts via the amount of intracellularly-formed H2O2. A key component of the sensor is an H2O2-sensitive transcriptional regulator, OxyR, which is used to control the expression levels of fluorescent proteins. We employed the OxyR sensor to monitor the oxidation of glycerol to glyceraldehyde and of toluene to o-cresol catalysed by recombinant E. coli expressing an alcohol oxidase and a P450 monooxygenase, respectively. In case of the P450 BM3-catalysed reaction, we additionally monitored o-cresol formation via a second genetically encoded sensor based on the phenol-sensitive transcriptional activator, DmpR, and an orthogonal fluorescent reporter protein. Single round screens of mutant libraries by flow cytometry or by visual inspection of colonies on agar plates yielded significantly improved oxidase and oxygenase variants thus exemplifying the suitability of the sensor system to accurately assess whole-cell oxidations in a high-throughput manner.

Hybrid Organic-Inorganic-Organic Isoporous Membranes with Tunable Pore Sizes and Functionalities for Molecular Separation.

Accomplishing on-demand molecular separation with a high selectivity and good permeability is very desirable for pollutant removal and chemical and pharmaceutical processing. The major challenge for sub-10 nm filtration of particles and molecules is the fabrication of high-performance membranes with tunable pore size and designed functionality. Here, a versatile top-down approach is demonstrated to produce such a membrane using isoporous block copolymer membranes with well-defined pore sizes combined with growth of metal oxide using sequential infiltration synthesis and atomic layer deposition (SIS and ALD). The pore size of the membranes is tuned by controlled metal oxide growth within and onto the polymer channels, enabling up to twofold pore diameter reduction. Following the growth, the distinct functionalities are readily incorporated along the membrane nanochannels with either hydrophobic, cationic, or anionic groups via straightforward and scalable gas/liquid-solid interface reactions. The hydrophilicity/hydrophobicity of the membrane nanochannel is significantly changed by the introduction of hydrophilic metal oxide and hydrophobic fluorinated groups. The functionalized membranes exhibit a superior selectivity and permeability in separating 1-2 nm organic molecules and fractionating similar-sized proteins based on size, charge, and hydrophobicity. This demonstrates the great potential of organic-inorganic-organic isoporous membranes for high-performance molecular separation in numerous applications.

Directed Evolution of a Surface-Displayed Artificial Allylic Deallylase Relying on a GFP Reporter Protein.

Artificial metalloenzymes (ArMs) combine characteristics of both homogeneous catalysts and enzymes. Merging abiotic and biotic features allows for the implementation of new-to-nature reactions in living organisms. Here, we present the directed evolution of an artificial metalloenzyme based on Escherichia coli surface-displayed streptavidin (SavSD hereafter). Through the binding of a ruthenium-pianostool cofactor to SavSD, an artificial allylic deallylase (ADAse hereafter) is assembled, which displays catalytic activity toward the deprotection of alloc-protected 3-hydroxyaniline. The uncaged aminophenol acts as a gene switch and triggers the overexpression of a fluorescent green fluorescent protein (GFP) reporter protein. This straightforward readout of ADAse activity allowed the simultaneous saturation mutagenesis of two amino acid residues in Sav near the ruthenium cofactor, expediting the screening of 2762 individual clones. A 1.7-fold increase of in vivo activity was observed for SavSD S112T-K121G compared to the wild-type SavSD (wt-SavSD). Finally, the best performing Sav isoforms were purified and tested in vitro (SavPP hereafter). For SavPP S112M-K121A, a total turnover number of 372 was achieved, corresponding to a 5.9-fold increase vs wt-SavPP. To analyze the marked difference in activity observed between the surface-displayed and purified ArMs, the oligomeric state of SavSD was determined. For this purpose, crosslinking experiments of E. coli cells overexpressing SavSD were carried out, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The data suggest that SavSD is most likely displayed as a monomer on the surface of E. coli. We hypothesize that the difference between the in vivo and in vitro screening results may reflect the difference in the oligomeric state of SavSD vs soluble SavPP (monomeric vs tetrameric). Accordingly, care should be applied when evolving oligomeric proteins using E. coli surface display.

An alginate-confined peroxygenase-CLEA for styrene epoxidation.

Oxyfunctionalisation reactions in neat substrate still pose a challenge for biocatalysis. Here, we report an alginate-confined peroxygenase-CLEA to catalyse the enantioselective epoxidation of cis-ß-methylstyrene in a solvent-free reaction system achieving turnover numbers of 96 000 for the biocatalyst and epoxide concentrations of 48 mM.

Real-Time Respiration Changes as a Viability Indicator for Rapid Antibiotic Susceptibility Testing in a Microfluidic Chamber Array.

Rapid identification of a pathogen and the measurement of its antibiotic susceptibility are key elements in the diagnostic process of bacterial infections. Microfluidic technologies offer great control over handling and manipulation of low sample volumes with the possibility to study microbial cultures on the single-cell level. Downscaling the dimensions of cultivation systems directly results in a lower number of bacteria required for antibiotic susceptibility testing (AST) and thus in a reduction of the time to result. The developed platform presented in this work allows the reading of pathogen resistance profiles within 2-3 h based on the changes of dissolved oxygen levels during bacterial cultivation. The platform contains hundreds of individual growth chambers prefilled with a hydrogel containing oxygen-sensing nanoprobes and different concentrations of antibiotic compounds. The performance of the developed platform is tested using quality control Escherichia coli strains (ATCC 25922 and ATCC 35218) in response to clinically relevant antibiotics. The results are in agreement with values given in reference guidelines and independent measurements using a clinical AST protocol. Finally, the platform is successfully used for the AST of an E. coli clinical isolate obtained from a patient blood culture.

Multicomponent Network Formation in Selective Layer of Composite Membrane for CO2 Separation.

As a promising material for CO2/N2 separation, PolyActiveTM can be used as a separation layer in thin-film composite membranes (TFCM). Prior studies focused on the modification of PolyActiveTM using low-molecular-weight additives. In this study, the effect of chemical crosslinking of reactive end-groups containing additives, forming networks within selective layers of the TFCM, has been studied. In order to understand the influence of a network embedded into a polymer matrix on the properties of the resulting materials, various characterization methods, including Fourier transform infrared spectroscopy (FTIR), gas transport measurements, differential scanning calorimetry (DSC) and atomic force microscopy (AFM), were used. The characterization of the resulting membrane regarding individual gas permeances by an in-house built "pressure increase" facility revealed a twofold increase in CO2 permeance, with insignificant losses in CO2/N2 selectivity.

RAFT Emulsion Polymerization of Styrene Using a Poly((N,N-dimethyl acrylamide)-co-(N-isopropyl acrylamide)) mCTA: Synthesis and Thermosensitivity.

Thermoresponsive poly((N,N-dimethyl acrylamide)-co-(N-isopropyl acrylamide)) (P(DMA-co-NIPAM)) copolymers were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The monomer reactivity ratios were determined by the Kelen-Tüdos method to be rNIPAM = 0.83 and rDMA = 1.10. The thermoresponsive properties of these copo-lymers with varying molecular weights were characterized by visual turbidimetry and dynamic light scattering (DLS). The copolymers showed a lower critical solution temperature (LCST) in water with a dependence on the molar fraction of DMA in the copolymer. Chaotropic and kosmotropic salt anions of the Hofmeister series, known to affect the LCST of thermoresponsive polymers, were used as additives in the aqueous copolymer solutions and their influence on the LCST was demonstrated. Further on, in order to investigate the thermoresponsive behavior of P(DMA-co-NIPAM) in a confined state, P(DMA-co-NIPAM)-b-PS diblock copolymers were prepared via polymerization induced self-assembly (PISA) through surfactant-free RAFT mediated emulsion polymerization of styrene using P(DMA-co-NIPAM) as the macromolecular chain transfer agent (mCTA) of the polymerization. As confirmed by cryogenic transmission electron microscopy (cryoTEM), this approach yielded stabilized spherical micelles in aqueous dispersions where the PS block formed the hydrophobic core and the P(DMA-co-NIPAM) block formed the hydrophilic corona of the spherical micelle. The temperature-dependent behavior of the LCST-type diblock copolymers was further studied by examining the collapse of the P(DMA-co-NIPAM) minor block of the P(DMA-co-NIPAM)-b-PS diblock copolymers as a function of temperature in aqueous solution. The nanospheres were found to be thermosensitive by changing their hydrodynamic radii almost linearly as a function of temperature between 25 °C and 45 °C. The addition of kosmotropic salt anions, as a potentially useful tuning feature of micellar assemblies, was found to increase the hydrodynamic radius of the micelles and resulted in a faster collapse of the micelle corona upon heating.

High-Throughput Optimization of Recombinant Protein Production in Microfluidic Gel Beads.

Efficient production hosts are a key requirement for bringing biopharmaceutical and biotechnological innovations to the market. In this work, a truly universal high-throughput platform for optimization of microbial protein production is described. Using droplet microfluidics, large genetic libraries of strains are encapsulated into biocompatible gel beads that are engineered to selectively retain any protein of interest. Bead-retained products are then fluorescently labeled and strains with superior production titers are isolated using flow cytometry. The broad applicability of the platform is demonstrated by successfully culturing several industrially relevant bacterial and yeast strains and detecting peptides or proteins of interest that are secreted or released from the cell via autolysis. Lastly, the platform is applied to optimize cutinase secretion in Komagataella phaffii (Pichia pastoris) and a strain with 5.7-fold improvement is isolated. The platform permits the analysis of >106 genotypes per day and is readily applicable to any protein that can be equipped with a His6 -tag. It is envisioned that the platform will be useful for large screening campaigns that aim to identify improved hosts for large-scale production of biotechnologically relevant proteins, thereby accelerating the costly and time-consuming process of strain engineering.

Ultra-high throughput screening for novel protease specificities.

The screening of large libraries of enzyme variants remains an essential tool in evolving biocatalysts toward improved properties for applications in medicine, chemistry, and a broad variety of other fields. Over the last decades, the technology for conducting systematic screens of arrayed members of a library of enzyme variants has made great strides in terms of increasing throughput and reducing assay volume. Here, we describe in detail an alternative to arrayed analysis, which is a screen based on density shifts in result of changed enzyme function, which allows highly parallelized screening. Specifically, we link changes in protease substrate specificity in vivo to the production of an alternative reporter protein, catalase. Depending on the catalase expression level, microcolonies of library bacteria with active protease variants contained in polymeric droplets generate an oxygen bubble, which causes a density shift in the droplet and enables it to float.

Salzburg Database of Polygonal Data: Polygons and Their Generators.

The Salzburg Database is a repository of polygonal areas of various classes and sizes, with and without holes. Positive weights are assigned to all edges of all polygons. We introduce this collection and describe the generators that produced its polygons. The source codes for all generators as well as the polygons generated are publicly available.

In vivo directed enzyme evolution in nanoliter reactors with antimetabolite selection.

Scoring changes in enzyme or pathway performance by their effect on growth behavior is a widely applied strategy for identifying improved biocatalysts. While in directed evolution this strategy is powerful in removing non-functional catalysts in selections, measuring subtle differences in growth behavior remains difficult at high throughput, as it is difficult to focus metabolic control on only one or a few enzymatic steps over the entire process of growth-based discrimination. Here, we demonstrate successful miniaturization of a growth-based directed enzyme evolution process. For cultivation of library clones we employed optically clear gel-like microcarriers of nanoliter volume (NLRs) as reaction vessels and used fluorescence-assisted particle sorting to estimate the growth behavior of each of the gel-embedded clones in a highly parallelized fashion. We demonstrate that the growth behavior correlates with the desired improvements in enzyme performance and that we can fine-tune selection stringency by including an antimetabolite in the assay. As a model enzyme reaction, we improve the racemization of ornithine, a possible starting block for the large-scale synthesis of sulphostin, by a broad-spectrum amino acid racemase and confirm the discriminatory power by showing that even moderately improved enzyme variants can be readily identified.

"Basicles": Microbial Growth and Production Monitoring in Giant Lipid Vesicles.

We present an optimized protocol to encapsulate bacteria inside giant unilamellar lipid vesicles combined with a microfluidic platform for real-time monitoring of microbial growth and production. The microfluidic device allows us to immobilize the lipid vesicles and record bacterial growth and production using automated microscopy. Moreover, the lipid vesicles retain hydrophilic molecules and therefore can be used to accumulate products of microbial biosynthesis, which we demonstrate here for a riboflavin-producing bacterial strain. We show that stimulation as well as inhibition of bacterial production can be performed through the liposomal membrane simply by passive diffusion of inducing or antibiotic compounds, respectively. The possibility to introduce as well as accumulate compounds in liposomal cultivation compartments represents great advantage over the current state of the art systems, emulsion droplets, and gel beads. Additionally, the encapsulation of bacteria and monitoring of individual lipid vesicles have been accomplished on a single microfluidic device. The presented system paves the way toward highly parallel microbial cultivation and monitoring as required in biotechnology, basic research, or drug discovery.

Analysis of modular bioengineered antimicrobial lanthipeptides at nanoliter scale.

The rise of antibiotic resistance demands the acceleration of molecular diversification strategies to inspire new chemical entities for antibiotic medicines. We report here on the large-scale engineering of ribosomally synthesized and post-translationally modified antimicrobial peptides carrying the ring-forming amino acid lanthionine. New-to-nature variants featuring distinct properties were obtained by combinatorial shuffling of peptide modules derived from 12 natural antimicrobial lanthipeptides and processing by a promiscuous post-translational modification machinery. For experimental characterization, we developed the nanoFleming, a miniaturized and parallelized high-throughput inhibition assay. On the basis of a hit set of >100 molecules, we identified variants with improved activity against pathogenic bacteria and shifted activity profiles, and extrapolated design guidelines that will simplify the identification of peptide-based anti-infectives in the future.

Corrigendum: Identification and Characterisation of a pH-stable GFP.

This corrects the article DOI: 10.1038/srep28166.