Can I benefit from laboratory automation? A decision aid for the successful introduction of laboratory automation.

The large volumes of samples to be analysed every day would be impossible to manage without laboratory automation. As laboratory procedures have progressed, so have the tasks of laboratory personnel. With this feature article, we would like to provide (bio)chemical practitioners with little or no knowledge of laboratory automation with a guide to help them decide whether to implement laboratory automation and find a suitable system. Especially in small- and medium-sized laboratories, operating a laboratory system means having bioanalytical knowledge, but also being familiar with the technical aspects. However, time, budget and personnel limitations allow little opportunity for personnel to get into the depths of laboratory automation. This includes not only the operation, but also the decision to purchase an automation system. Hasty investments do not only result in slow or non-existent cost recovery, but also occupy valuable laboratory space. We have structured the article as a decision tree, so readers can selectively read chapters that apply to their individual situation. This flexible approach allows each reader to create a personal reading flow tailored to their specific needs. We tried to address a variety of perspectives on the topic, including people who are either supportive or sceptical of laboratory automation, personnel who want or need to automate specific processes, those who are unsure whether to automate and those who are interested in automation but do not know which areas to prioritize. We also help to make a decision whether to reactivate or discard already existing and unused laboratory equipment.

An expandable voice user interface as lab assistant based on an improved version of Google's speech recognition.

Voice assistants are potentially helpful when working in a scientific laboratory. A big challenge is the extremely specific use of language in every laboratory. As with any voice assistant, another concern is data security. Here, we present Rainbow-an open source voice user interface (VUI) for scientific laboratories, that is adaptable to any Windows PC with Internet access. We used Google Translate Site (GTS) as a voice input and output system to ensure communication to the user. The scripting language AutoIt controls GTS, executes all actions and builds the VUI. Rainbow performs tasks from three different areas-general Microsoft Windows tasks, lab-specific tasks, and device-specific tasks. We achieved significantly higher speech recognition accuracy with our VUI than with GTS alone (91.3% versus 85.1%). Because of Rainbow's architecture, it is possible to improve the voice assistant in terms of functionality and accuracy, allowing each laboratory to optimize its own Rainbow system in a user-friendly way. In a test setup, this led to a speech recognition accuracy of 98.6%. Taken together, Rainbow provides an opportunity for every scientist to implement highly specific scientific terms and tasks to this open source voice assistant system in a very user-friendly way.

Establishment of low-cost laboratory automation processes using AutoIt and 4-axis robots.

In most small laboratories, many processes are not yet automated because existing laboratory automation solutions are usually expensive and inflexible to use. Examples of this are autosamplers that are only compatible with one specific laboratory instrument or larger liquid handling stations that are expensive and usually self-contained. A flexible and inexpensive way to automate laboratory processes would be to automate existing laboratory equipment with the help of suitable robotic arms. In this study, we investigate the feasibility of such a strategy based on a low-cost 4-axis robot and freely available software. We used the scripting language AutoIt that automates any Windows-based instrument control software. Using these tools, we automated three fundamentally different laboratory processes: a pipetting process, a use as an autosampler for an atomic absorption spectroscopy instrument, and a more complex process involving the inoculation of bacterial cultures. We also integrated a conventional webcam for 2D barcode recognition. Compared to a trained professional who performed all experiments manually, all setups showed no significant differences in accuracy and precision. In summary, the tested system consisting of a 4-axis robot and freely available software is suitable for flexible automation and has potential for even more complex laboratory processes. Limitations such as a lack of collaboration and speed will be addressed in follow-up studies. The system thus represents a well-suited flexible laboratory automation system for both research and teaching purposes.

Proline-Rich Antimicrobial Peptides in Medicinal Maggots of Lucilia sericata Interact With Bacterial DnaK But Do Not Inhibit Protein Synthesis.

In the search for new antibiotics to combat multidrug-resistant microbes, insects offer a rich source of novel anti-infectives, including a remarkably diverse array of antimicrobial peptides (AMPs) with broad activity against a wide range of species. Larvae of the common green bottle fly Lucilia sericata are used for maggot debridement therapy, and their effectiveness in part reflects the large panel of AMPs they secrete into the wound. To investigate the activity of these peptides in more detail, we selected two structurally different proline rich peptides (Lser-PRP2 and Lser-PRP3) in addition to the α-helical peptide Lser-stomoxyn. We investigated the mechanism of anti-Escherichia coli action of the PRPs in vitro and found that neither of them interfered with protein synthesis but both were able to bind the bacterial chaperone DnaK and are therefore likely to inhibit protein folding. However, unlike Lser-stomoxyn that permeabilized the bacterial membrane by 1% at the low concentration (0.25 µM) neither of the PRPs alone was able to permeabilize E. coli membrane. In the presence of this Lser-stomoxyn concentration significant increase in anti-E. coli activity of Lser-PRP2 was observed, indicating that this peptide needs specific membrane permeabilizing agents to exert its antibacterial activity. We then examined the AMPs-treated bacterial surface and observed detrimental structural changes in the bacterial cell envelope in response to combined AMPs. The functional analysis of insect AMPs will help select optimal combinations for targeted antimicrobial therapy.

Rational design of a low-affinity peptide for the detection of cystatin C in a fast homogeneous immunoassay.

Immunoassays play an essential role in current research and diagnostics resulting in a variety of detection principles. Thereby, homogeneous assays are often used for a fast signal response as demanded for example in point-of-care diagnostics. These systems often rely on a competitive assay design where the sample analyte and the corresponding dye-labeled substance are competing for binding sites on an antibody present in limited amounts. Due to the similar affinities of the antibody towards the sample analyte and the competitor, both sensitivity and assay time are limited. As a consequence, a competitor with a slightly reduced affinity towards the antibody can potentially overcome these drawbacks. Here, we present the rational design of a low-affinity peptide (donor peptide) as a specific analyte competitor for a FRET-based homogeneous immunoassay for the analysis of the protein cystatin C. Thereby, the strategy of peptide-induced antibody generation was combined with the selective variation of the immunization sequence in order to achieve a lower affinity towards the antibody. We could show that shortened donor peptides improved the resulting quenching efficiency in the immunoassay. In addition, the substitution of small hydrophobic amino acids by those with a higher steric demand appeared to be the most promising strategy providing a fast assay response for cystatin C of only 90 s.

Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig.

Animal models of familial juvenile onset of Alzheimer's disease (AD) often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw). We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs) isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs) from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and ß-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation.

Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacteria.

Antimicrobial peptides (AMPs) and proteins are important components of innate immunity against pathogens in insects. The production of AMPs is costly owing to resource-based trade-offs, and strategies maximizing the efficacy of AMPs at low concentrations are therefore likely to be advantageous. Here, we show the potentiating functional interaction of co-occurring insect AMPs (the bumblebee linear peptides hymenoptaecin and abaecin) resulting in more potent antimicrobial effects at low concentrations. Abaecin displayed no detectable activity against Escherichia coli when tested alone at concentrations of up to 200 µM, whereas hymenoptaecin affected bacterial cell growth and viability but only at concentrations greater than 2 µM. In combination, as little as 1.25 µM abaecin enhanced the bactericidal effects of hymenoptaecin. To understand these potentiating functional interactions, we investigated their mechanisms of action using atomic force microscopy and fluorescence resonance energy transfer-based quenching assays. Abaecin was found to reduce the minimal inhibitory concentration of hymenoptaecin and to interact with the bacterial chaperone DnaK (an evolutionarily conserved central organizer of the bacterial chaperone network) when the membrane was compromised by hymenoptaecin. These naturally occurring potentiating interactions suggest that combinations of AMPs could be used therapeutically against Gram-negative bacterial pathogens that have acquired resistance to common antibiotics.

One-step assay for the quantification of T4 DNA ligase.

As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays.

A homogeneous assay principle for universal substrate quantification via hydrogen peroxide producing enzymes.

H2O2 is a widely occurring molecule which is also a byproduct of a number of enzymatic reactions. It can therefore be used to quantify the corresponding enzymatic substrates. In this study, the time-resolved fluorescence emission of a previously described complex consisting of phthalic acid and terbium (III) ions (PATb) is used for H2O2 detection. In detail, glucose oxidase and choline oxidase convert glucose and choline, respectively, to generate H2O2 which acts as a quencher for the PATb complex. The response time of the PATb complex toward H2O2 is immediate and the assay time only depends on the conversion rate of the enzymes involved. The PATb assay quantifies glucose in a linear range of 0.02-10 mmol L(-1), and choline from 1.56 to 100 µmol L(-1) with a detection limit of 20 µmol L(-1) for glucose and 1.56 µmol L(-1) for choline. Both biomolecules glucose and choline could be detected without pretreatment with good precision and reproducibility in human serum samples and infant formula, respectively. Furthermore, it is shown that the detected glucose concentrations by the PATb system agree with the results of a commercially available assay. In principle, the PATb system is a universal and versatile tool for the quantification of any substrate and enzyme reaction where H2O2 is involved.

Protein surface charge of trypsinogen changes its activation pattern.

BACKGROUND: Trypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. In this study, human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein-protein interactions. Trypsinogen is active only after its eight-amino-acid-long activation peptide has been cleaved off by another protease, enteropeptidase. Trypsinogen can also be autoactivated without the involvement of enteropeptidase. This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and therefore is based on a protein-protein interaction. RESULTS: Based on a rational protein design based on autoactivation-defective guinea pig trypsinogen, several amino acid residues, all located far away from the active site, were changed to modify the surface charge of human trypsinogen. The influence of the surface charge on the activation pattern of trypsinogen was investigated. The autoactivation properties of mutant trypsinogen were characterized in comparison to the recombinant wild-type enzyme. Surface-charged trypsinogen showed practically no autoactivation compared to the wild-type but could still be activated by enteropeptidase to the fully active trypsin. The kinetic parameters of surface-charged trypsinogen were comparable to the recombinant wild-type enzyme. CONCLUSION: The variant with a modified surface charge compared to the wild-type enzyme showed a complete different activation pattern. Our study provides an example how directed modification of the protein surface charge can be utilized for the regulation of functional protein-protein interactions, as shown here for human trypsinogen.

Improvement of an antibody-enzyme coupling yield by enzyme surface supercharging.

BACKGROUND: Protein cross-coupling reactions demand high yields, especially if the products are intended for bioanalytics, like enzyme-linked immunosorbent assays. Amongst other factors, the coupling yield depends on the concentration of the proteins being used for coupling. Protein supercharging of enzymes can increase the solubility dramatically, which could promote enzyme-antibody coupling reactions. A highly soluble, supercharged variant of the enzyme human enteropeptidase light chain was created by a site-directed mutagenesis of surface amino acids, used for the production of an antibody-enzyme conjugate and compared to the wild type enzyme. RESULTS: Wild type and mutant enzyme could successfully be cross-coupled to an antibody to give antibody-enzyme conjugates suitable for ELISA. Their assay performances and the analysis of the enzyme activities in solution demonstrate that the supercharged version could be coupled to a higher extent, which resulted in better assay sensitivities. The generated conjugate, based on the supercharged enzyme, was feasible as a reporter molecule in a sandwich ELISA and allowed the detection of epidermal growth factor with a detection limit of 15.63 pg (25 pmol/L). CONCLUSION: The highly soluble, surface supercharged, human enteropeptidase light chain mutant provided better yields in coupling the enzyme to an antibody than the wild type. This is most likely related to the higher protein concentration during the coupling. The data suggest that supercharging can be applied favourably to other proteins which have to be covalently linked to other polymers or surfaces with high yields without losses in enzyme activity or specificity.

His-tag protein monitoring by a fast mix-and-measure immunoassay.

Here, we present a fast mix-and-measure immunoassay for the specific semiquantitative detection of His-tagged proteins, for example in E. coli cell lysate. The assay is based on Förster resonance energy transfer (FRET) between a lanthanide dye-labeled low-affinity His-peptide and an acceptor-labeled anti-His-tag antibody. The targeted His-tag protein in the sample displaces the donor-labeled peptide and leads to a concentration-dependent time-resolved fluorescence signal. The assay has a total assay time of less than two minutes including sample preparation. The assay recognizes both, N- and C-terminally tagged proteins. The detection limit is comparable to those obtained in SDS-PAGE or Western Blot, which are used as standard methods for the characterization of His-tag protein expression. Additionally, we demonstrate a full compatibility of the developed assay to cell lysate, and a correlation to detectable bands in a western blot application. In conclusion, this fast, sensitive, specific and affordable mix-and-measure assay provides a timesaving and user-friendly way to quantify recombinant protein expression. It substantially reduces the workload for recombinant protein detection, especially when His-tag-protein-containing fractions in manual chromatographic purifications have to be identified.

Aggregation behavior of chemically synthesized, full-length huntingtin exon1.

Repeat length disease thresholds vary among the 10 expanded polyglutamine (polyQ) repeat diseases, from about 20 to about 50 glutamine residues. The unique amino acid sequences flanking the polyQ segment are thought to contribute to these repeat length thresholds. The specific portions of the flanking sequences that modulate polyQ properties are not always clear, however. This ambiguity may be important in Huntington's disease (HD), for example, where in vitro studies of aggregation mechanisms have led to distinctly different mechanistic models. Most in vitro studies of the aggregation of the huntingtin (HTT) exon1 fragment implicated in the HD mechanism have been conducted on inexact molecules that are imprecise either on the N-terminus (recombinantly produced peptides) or on the C-terminus (chemically synthesized peptides). In this paper, we investigate the aggregation properties of chemically synthesized HTT exon1 peptides that are full-length and complete, containing both normal and expanded polyQ repeat lengths, and compare the results directly to previously investigated molecules containing truncated C-termini. The results on the full-length peptides are consistent with a two-step aggregation mechanism originally developed based on studies of the C-terminally truncated analogues. Thus, we observe relatively rapid formation of spherical oligomers containing from 100 to 600 HTT exon1 molecules and intermediate formation of short protofibril-like structures containing from 500 to 2600 molecules. In contrast to this relatively rapid assembly, mature HTT exon1 amyloid requires about one month to dissociate in vitro, which is similar to the time required for neuronal HTT exon1 aggregates to disappear in vivo after HTT production is discontinued.

A luminescence-based probe for sensitive detection of hydrogen peroxide in seconds.

Here, we present a fast and simple hydrogen peroxide assay that is based on time-resolved fluorescence. The emission intensity of a complex consisting of terbium ions (Tb(3+)) and phthalic acid (PA) in HEPES buffer is quenched in the presence of H2O2 and this quenching is concentration-dependent. The novel PATb assay detects hydrogen peroxide at a pH range from 7.5 to 8.5 and with a detection limit of 150 nmol L(-1) at pH 8.5. The total assay time is less than 1 min. The linear range of the assay can be adapted by a pH adjustment of the aqueous buffer and covers a concentration range from 310 nmol L(-1) to 2.56 mmol L(-1) in total which encompasses four orders of magnitude. The assay is compatible with high concentrations of all 47 tested inorganic and organic compounds. The PATb assay was applied to quantify H2O2 in polluted river water samples. In conclusion, this fast and easy-to-use assay detects H2O2 with high sensitivity and precision.

Highly efficient förster resonance energy transfer in a fast, serum-compatible immunoassay.

Highly efficient FRET leads to important enhancements for homogeneous immunoassays. By using the novel phosphorescent dye EuLH and BHQ-10 as a donor-acceptor pair, the FRET efficiency increases to >99.5 %, leading to significantly improved signal-to-background ratio, precision and linear range. The phosphorescence detection enabled full compatibility to serum samples for this fast-responding immunoassay.

Crystal structure of a supercharged variant of the human enteropeptidase light chain.

The highly specific serine protease human enteropeptidase light chain cleaves the Asp4Lys recognition sequence and represents an interesting enzyme for biotechnological applications. The human enzyme shows 10 times faster kinetics compared to other animal sources but low solubility under low salt conditions, which hampers protein production and crystallization. Therefore, a supercharged variant (N6D/G21D/G22D/N142D/K210E/C112S) with increased solubility was used for crystallization. The structure (resolution, 1.9 Å) displays a typical α/ß trypsin-like serine protease-fold. The mutations introduced for protein supercharging generate larger clusters of negative potential on both sites of the active cleft but do not affect the structural integrity of the protein.

Highly adaptable and sensitive protease assay based on fluorescence resonance energy transfer.

Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.

Homogeneous fluorescence-based immunoassay detects antigens within 90 seconds.

Homogeneous immunoassays are prevalent tools for the detection of antigens. The major advantage over heterogeneous immunoassays is the absence of numerous incubation and washing steps, reducing the assay time and allowing rapid on-site detection of antigens (e.g., toxins and pollutants). The simple experimental setup of a homogeneous immunoassay also allows a robust analysis even when performed by non-laboratory-trained personnel. Here we present a homogeneous immunoassay for the rapid determination of antigens. As a proof of concept, a phosphorylation-specific anti-human tau monoclonal antibody was labeled with an acceptor and the corresponding peptide probe with a donor fluorophore. The analyte sample is spiked with a fixed amount of donor peptide before acceptor-labeled antibody is added leading to a donor fluorescence quenching. Thus the intensity of the fluorescence signal of the donor peptide probe depends on the concentration of the target antigen. The sequence of the donor peptide was optimized to lower its affinity to the antibody giving a higher response for the analyte antigen compared to the native epitope. This allowed a semiquantitative analysis of the antigen within only 90 s.

Surface supercharged human enteropeptidase light chain shows improved solubility and refolding yield.

Enteropeptidase is a serine protease used in different biotechnological applications. For many applications the smaller light chain can be used to avoid the expression of the rather large holoenzyme. Recombinant human enteropeptidase light chain (hEPL) shows high activity but low solubility and refolding yields, currently limiting its use in biotechnological applications. Here we describe several protein modifications that lead to improved solubility and refolding yield of human hEPL whilst retaining the enzyme activity. Specifically, protein surface supercharging (N6D, G21D, G22D, N141D, K209E) of the protein increased the solubility more than 100-fold. Replacement of a free cysteine residue with serine (C112S) improved the refolding yield by 50%. The heat stability of this C112S variant was also significantly improved by supercharging. This study shows that even mild protein surface supercharging can have pronounced effects on protein solubility and stability.

Synthesis of pathological and nonpathological human exon 1 huntingtin.

Huntington's disease (HD) is a neurodegenerative disorder that affects approximately 1 in 10 000 individuals. The underlying gene mutation was identified as a CAG-triplet repeat expansion in the gene huntingtin. The CAG sequence codes for glutamine, and in HD, an expansion of the polyglutamine (poly-Q) stretch above 35 glutamine residues results in pathogenicity. It has been demonstrated in various animal models that only the expression of exon 1 huntingtin, a 67-amino acid-long polypeptide plus a variable poly-Q stretch, is sufficient to cause full HD-like pathology. Therefore, a deeper understanding of exon 1 huntingtin, its structure, aggregation mechanism and interaction with other proteins is crucial for a better understanding of the disease. Here, we describe the synthesis of a 109-amino acid-long exon 1 huntingtin peptide including a poly-Q stretch of 42 glutamines. This microwave-assisted solid phase peptide synthesis resulted in milligram amounts of peptide with high purity. We also synthesized a nonpathogenic version of exon 1 huntingtin (90-amino acid long including a poly-Q stretch of 23 glutamine residues) using the same strategy. In circular dichroism spectroscopy, both polypeptides showed weak alpha-helical properties with the longer peptide showing a higher helical degree. These model peptides have great potential for further biomedical analyses, e.g. for large-scale pre-screenings for aggregation inhibitors, further structural analyses as well as protein-protein interaction studies.