A colorimetric biosensor for the detection of foodborne bacteria

We have synthesized 10,12-pentacosadyinoic acid (PCDA) + N-[(2-tetradecanamide)-ethyl]-ribonamide (TDER) vesicles to determine the colorimetric response induced by pathogenic bacteria (Staphylococcus aureus and Escherichia coli). The addition of bacterial supernatants caused a colorimetric transition in TDER/PCDA vesicles, even in diluted concentrations, indicating that chemical interactions occur between the vesicles and bacterial compounds. Bacterial substrates released from S. aureus induced a greater color change compared to the color change induced by E. coli. S. aureus metabolites also induced a greater color change when TDER/PCDA vesicles were incorporated into the cellulose strips. We determined the energy involved in the interaction between the bacterial substrates and the TDER/PCDA vesicles to be exothermic (and very high) for both bacterial supernatants. In addition, we analyzed the colorimetric transition in the presence of other molecules, using apple juice as a food matrix. Both apple juice and apple juice inoculated with bacterial substrates induced a TDER/PCDA color change; however, the S. aureus supernatant induced a slightly greater color change in the vesicles both in the suspension and in the cellulose strips. TDER/PCDA vesicles show great potential to be used as a biosensor to detect food pathogens in intelligent food packaging.     

Molecular modeling studies of l-arabinitol 4-dehydrogenase of Hypocrea jecorina: Its binding interactions with substrate and cofactor

l-Arabinitol 4-dehydrogenase (LAD1; EC 1.1.1.12) is an enzyme in the l-arabinose catabolic pathway of fungi that catalyzes the conversion of l-arabinitol into l-xylulose. The primary objective of this work is to identify the catalytic and coenzyme binding domains of LAD1 from Hypocrea jecorina in order to provide better insight into the possible catalytic events in these domains. The 3D structure of NAD⁺-dependent LAD1 was developed based on the crystal structure of human sorbitol dehydrogenase as a template. A series of molecular mechanics and dynamics operations were performed to find the most stable binding interaction for the enzyme and its ligands. Using the verified model, a docking study was performed with the substrate l-arabinitol, Zn²⁺ and NAD⁺. This study found a catalytic Zn²⁺ binding domain (Cys66, His91, Glu92 and Glu176) and a cofactor NAD⁺ binding domain (Gly202, ILeu204, Gly205, Cys273, Arg229 and Val298) with strong hydrogen bonding contacts with the substrate and cofactor. The binding pockets of the enzyme for l-arabinitol, NAD⁺, and Zn²⁺ have been explicitly defined. The results from this study should guide future mutagenesis studies and provide useful clues for engineering enzymes to improve the utilization of polyols for rare sugar production.     

Sensitive analysis of glutathione in bacteria and HaCaT cells by polydopamine/gold nanoparticle-coated microchip electrophoresis via online pre-concentration of field-amplified sample stacking

In this paper, polydopamine/gold nanoparticles (PDA/Au NPs) were used to construct a functional film on a glass microfluidic channel surface in microchip electrophoresis (MCE) for the separation of reduced glutathione (GSH) and oxidized glutathione (GSSH). The formation of the PDA/Au NPs was characterized by scanning electron microscopy, transmission electron microscope, UV–Vis spectra and ATR-FTIR. An online pre-concentration strategy involving field-amplified sample stacking was used to determine the sensitivity of the assay for measuring GSH and GSSH in bacteria (Escherichia coli, Staphylococcus aureus and Salmonella enterica serovar Typhimurium) and HaCaT cells samples by MCE with laser-induced fluorescence detection. The influences of the separation voltage, the concentration of the running buffer and the pH value of running buffer, were carefully investigated. Using this studied method, GSH and GSSH could be simultaneously pre-concentrated and separated within 70 s. The limits of detection of GSH and GSSH were as low as 0.81 and 0.91 nM, respectively (S/N = 3), which corresponded to approximately 180–301-fold improvements in peak height. Moreover, this method was successfully applied to the analysis of bacteria (E. coli, S. aureus and S. typhimurium) and HaCaT cell samples with a satisfactory recovery rate.     

Modeling antimicrobial effect of different grape pomace and extracts on S.aureus and E.coli in vegetable soup using artificial neural network and fuzzy logic system

In this study, antibacterial activities of the grape pomace powders (GPP) and grape pomace extracts (GPE) of Turkeys’ five different grape varieties (Emir, Gamay, Kalecik Karasi, Narince and Okuzgozu grape varieties) were determined against Staphylococcusaureus and Escherichiacoli in vegetable soup at 2, 5 and 10% concentrations. Antibacterial effects of GPE were more effective than those of the GPP against the bacteria. The bacterial counts decreased with increasing extract concentration. The highest antibacterial activity of the GPE was for Gamay and Kalecik Karasi against both bacteria. The 10% concentration of Gamay GPP completely inhibited S.aureus at the end of 120th h. Compared to E.coli, S.aureus was more sensitive to all GPP and GPE. This bacterium was inhibited by 10% concentrations of all the extracts at the initial storage time. Data were analyzed to predict antibacterial effects of the GPP and GPE against both bacteria by adaptive neuro fuzzy inference system (ANFIS) and artificial neural network (ANN) and multiple linear regression (MLR) models. As a result, ANFIS model was found to be better than the ANN and MLR for predicting antibacterial effects of GPP and GPE against S.aureus and E.coli in the soup.     

SERS driven cross-platform based multiplex pathogen detection

We demonstrate a cross-platform approach to simultaneously detect three different pathogens using Raman and UV-vis absorption spectroscopy. Gold (Au), silver (Ag), and Ag-Au core-shell nanoparticles were functionalized with anti-Salmonella typhimurium aptamers, anti-Staphylococcus aureus and anti-Escherichia coli O157:H7 antibodies respectively and labeled with unique Raman reporter molecules. A microfiltration step was used to consolidate a highly selective and specific detection platform, with total detection time under 45 min for both species (E. coli O157:H7 vs. S. typhimurium) and strain (E. coli O157:H7 vs. E. coli K12) level sensing at a limit of a detection ranging between 10² and 10³ CFU/ml. This simple yet robust multiplex detection platform has the potential to be developed into a rapid and portable pathogen sensor for ultrasensitive detection in liquid samples.     

Discerning the catalytic mechanism of Staphylococcus aureus sortase A with QM/MM free energy calculations

Sortases are key virulence factors in Gram-positive bacteria. These enzymes embed surface proteins in the cell wall through a transpeptidation reaction that involves recognizing a penta-peptide “sorting signal” in a target protein, cleaving it, and covalently attaching it to a second substrate that is later inserted into the cell wall. Although well studied, several aspects of the mechanism by which sortases perform these functions remains unclear. In particular, experiments have revealed two potential sorting signal binding motifs: a “Threonine-Out” (Thr-Out) structure in which the catalytically critical threonine residues protrudes into solution, and a “Threonine-In” (Thr-In) configuration in which this residue inserts into the binding site. To determine which of these is the biologically relevant state, we have performed a series of conventional and hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of the Staphylococcus aureus sortase A (SrtA) enzyme bound to a sorting signal substrate. Through the use of multi-dimensional metadynamics, our simulations were able to both map the acylation mechanism of SrtA in the Thr-In and Thr-Out states, as well as determine the free energy minima and barriers along these reactions. Results indicate that in both states the catalytic mechanisms are similar, however the free energy barriers are lower in the Thr-In configuration, suggesting that Thr-In is the catalytically relevant state. This has important implications for advancing our understanding of the mechanisms of sortase enzymes, as well we for future structure based drug design efforts aimed at inhibiting sortase function in vivo.     

In silico identification of novel inhibitors against Plasmodium falciparum dihydroorate dehydrogenase

Plasmodium falciparum causes the most fatal form of malaria and accounts for over 1 million deaths annually, yet currently used drug therapies are compromised by resistance. The malaria parasite cannot salvage pyrimidines and relies on de novo biosynthesis for survival. The enzyme dihydrooratate dehydrogenase (DHODH), a mitochondrial flavoenzyme, catalyzes the rate-limiting step of this pathway and is therefore an attractive anti-malarial chemotherapeutic target. In an effort to design new and potential anti-malarials, structure-based pharmacophore mapping, molecular docking, binding energy calculations and binding affinity predictions were employed in a virtual screening strategy to design new and potent P. falciparum dihydrooratate dehydrogenase (PfDHODH) inhibitors. A structure-based pharmacophore model was generated which consist of important interactions as observed in co-crystal of PfDHODH enzyme. The developed model was used to retrieve molecules from ChemBridge database, a freely available commercial database. A total of 87 molecules mapped on the modeled pharmacophore from the database. The retrieved hits were further screened by docking simulation, binding energy calculations and biding affinity predictions using genetic optimization for ligand docking (GOLD) and MOE. Based on these results, finally 26 chemo-types molecules were predicted as new, potential and structurally diverse PfDHODH inhibitors.     

Multi-generational pharmacophore modeling for ligands to the cholane steroid-recognition site in the β1 modulatory subunit of the BKCa channel

Large conductance, voltage- and Ca²⁺-gated K⁺ (BK_Ca) channels play a critical role in smooth muscle contractility and thus represent an emerging therapeutic target for drug development to treat vascular disease, gastrointestinal, bladder and uterine disorders. Several compounds are known to target the ubiquitously expressed BK_Ca channel-forming α subunit. In contrast, just a few are known to target the BK_Ca modulatory β₁ subunit, which is highly expressed in smooth muscle and scarce in most other tissues. Lack of available high-resolution structural data makes structure-based pharmacophore modeling of β₁ subunit-dependent BK_Ca channel activators a major challenge. Following recent discoveries of novel BK_Ca channel activators that act via β₁ subunit recognition, we performed ligand-based pharmacophore modeling that led to the successful creation and fine-tuning of a pharmacophore over several generations. Initial models were developed using physiologically active cholane steroids (bile acids) as template. However, as more compounds that act on BK_Ca β₁ have been discovered, our model has been refined to improve accuracy. Database searching with our best-performing model has uncovered several novel compounds as candidate BK_Ca β₁ subunit ligands. Eight of the identified compounds were experimentally screened and two proved to be activators of recombinant BK_Ca β₁ complexes. One of these activators, sobetirome, differs substantially in structure from any previously reported activator.     

Molecular modeling and molecular dynamics simulation study of archaeal leucyl-tRNA synthetase in complex with different mischarged tRNA in editing conformation

Aminoacyl-tRNA synthetases (aaRSs) play important roles in maintaining the accuracy of protein synthesis. Some aaRSs accomplish this via editing mechanisms, among which leucyl-tRNA synthetase (LeuRS) edits non-cognate amino acid norvaline mainly by post-transfer editing. However, the molecular basis for this pathway for eukaryotic and archaeal LeuRS remain unclear. In this study, a complex of archaeal P. horikoshii LeuRS (PhLeuRS) with misacylated tRNA^Leu was modeled wherever tRNA’s acceptor stem was oriented directly into the editing site. To understand the distinctive features of organization we reconstructed a complex of PhLeuRS with tRNA and visualize post-transfer editing interactions mode by performing molecular dynamics (MD) simulation studies. To study molecular basis for substrate selectivity by PhLeuRS’s editing site we utilized MD simulation of the entire LeuRS complexes using a diverse charged form of tRNAs, namely norvalyl-tRNA^Leu and isoleucyl-tRNA^Leu. In general, the editing site organization of LeuRS from P.horikoshii has much in common with bacterial LeuRS. The MD simulation results revealed that the post-transfer editing substrate norvalyl-A76, binds more strongly than isoleucyl-A76. Moreover, the branched side chain of isoleucine prevents water molecules from being closer and hence the hydrolysis reaction slows significantly. To investigate a possible mechanism of the post-transfer editing reaction, by PhLeuRS we have determined that two water molecules (the attacking and assisting water molecules) are localized near the carbonyl group of the amino acid to be cleaved off. These water molecules approach the substrate from the opposite side to that observed for Thermus thermophilus LeuRS (TtLeuRS). Based on the results obtained, it was suggested that the post-transfer editing mechanism of PhLeuRS differs from that of prokaryotic TtLeuRS.     

An integrated microfluidic system using mannose-binding lectin for bacteria isolation and biofilm-related gene detection

Molecular diagnosis of biofilm-related genes (BRGs) in common bacteria that cause periprosthetic joint infections may provide crucial information for clinicians. In this study, several BRGs, including ica, fnbA, and fnbB, were rapidly detected (within 1 h) with a new integrated microfluidic system. Mannose-binding lectin (MBL)-coated magnetic beads were used to isolate these bacteria, and on-chip nucleic acid amplification (polymerase chain reaction, PCR) was then performed to detect BRGs. Both eukaryotic and prokaryotic MBLs were able to isolate common bacterial strains, regardless of their antibiotic resistance, and limits of detection were as low as 3 and 9 CFU for methicillin-resistant Staphylococcus aureus and Escherichia coli, respectively, when using a universal 16S rRNA PCR assay for bacterial identification. It is worth noting that the entire process including bacteria isolation by using MBL-coated beads for sample pre-treatment, on-chip PCR, and fluorescent signal detection could be completed on an integrated microfluidic system within 1 h. This is the first time that an integrated microfluidic system capable of detecting BRGs by using MBL as a universal capturing probe was reported. This integrated microfluidic system might therefore prove useful for monitoring profiles of BRGs and give clinicians more clues for their clinical judgments in the near future.     

Expansion of the active site of the azoreductase from Shewanella oneidensis MR-1

Azoreductase from Shewanella oneidensis MR-1 (soAzoR) possesses great potential in cleaving azo bond of azo dyes during degradation progress. However, detailed information on interaction of soAzoR with either prosthetic group or substrate remains unavailable, mainly due to the absence of crystallization of soAzoR. In order to unravel these mechanisms, we firstly built the tertiary structure of soAzoR and then computationally predicted the binding mode of FMN, NADH and a model dye, methyl red (MR). Ten residues of soAzoR, which are predicted to participate in ligands binding, were separately substituted for either alanine or phenylalanine to confirm their function. The homologous modeling result reveals soAzoR employs a typical Rossmann fold. In terms of ligand binding modes, the isoalloxazine ring of FMN is stabilized in planar conformation by amino acids in the loop L6 and L9 region. NADH and MR is superposed against the isoalloxazine ring with an angle and the distance from C4 atom of NADH and azo bond of MR to N5 atom of FMN is 4.3 Å and 4.6 Å, respectively. The result of predicted interaction and enzyme kinetic analysis suggests that Asn96, Gly140 and Gly141 are crucial for FMN and MR binding; Tyr119 and Phe161 are more meaningful for NADH binding; Ser16 plays an important role in appropriately binding of both FMN and NADH.     

Branching Time Logics \mathcal {BTL}^{\mathrm {U,S}}_{\mathrm {N},\mathrm {N}^{-1}}(\mathcal {Z})_{\alpha } with Operations Until and Since Based on Bundles of Integer Numbers, Logical Consecutions, Deciding Algorithms

This paper is intended as an attempt to describe logical consequence in branching time logics. We study temporal branching time logics $\mathcal {BTL}^{\mathrm {U,S}}_{\mathrm {N},\mathrm {N}^{-1}}(\mathcal {Z})_{\alpha }$ which use the standard operations Until and Next and dual operations Since and Previous (LTL, as standard, uses only Until and Next). Temporal logics $\mathcal {BTL}^{\mathrm {U,S}}_{\mathrm {N},\mathrm {N}^{-1}}(\mathcal {Z})_{\alpha }$ are generated by semantics based on Kripke/Hinttikka structures with linear frames of integer numbers $\mathcal {Z}$ with a single node (glued zeros). For $\mathcal {BTL}^{\mathrm {U,S}}_{\mathrm {N},\mathrm {N}^{-1}}(\mathcal {Z})_{\alpha }$ , the permissible branching of the node is limited by α (where 1≤α≤ω). We prove that any logic $\mathcal {BTL}^{\mathrm {U,S}}_{\mathrm {N},\mathrm {N}^{-1}}(\mathcal {Z})_{\alpha }$ is decidable w.r.t. admissible consecutions (inference rules), i.e. we find an algorithm recognizing consecutions admissible in $\mathcal {BTL}^{\mathrm {U,S}}_{\mathrm {N},\mathrm {N}^{-1}}(\mathcal {Z})_{\alpha }$ . As a consequence, it implies that $\mathcal {BTL}^{\mathrm {U,S}}_{\mathrm {N},\mathrm {N}^{-1}}(\mathcal {Z})_{\alpha }$ itself is decidable and solves the satisfiability problem.     

The impact of dust in filter materials of respiratory protective devices on the microorganisms viability

Plant biomass processed in a heat and power plant is a source of airborne dust present in the working environment. The aim of this study was to assess the influence of various levels of organic dust content in the filter material on the viability of microorganisms in model conditions, taking into consideration the workplace environment and the physico-chemical as well as the microbiological characteristics of the dust particles. The possibility of a biofilm developing on the reusable filtering facepiece respirators (FFR) in standard use by heat and power plant workers for respiratory tract protection was also investigated.Standard reusable filtering half masks FFP3R and high-performance melt-blown fabrics were used in the study. The biomass dust characteristics were determined using an elementary analyser and a condensation particle counter with an electrostatic classifier. For quantitative analysis of microbiological pollution we used the plate count method with the microorganisms identified either using the API biochemical tests (for bacteria and yeasts) and the taxonomic key (for moulds). Biofilm development on the filtering half masks was assessed using SEM scanning microscopy. The viability of microorganisms (namely Escherichia coli ATCC 10536, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231, Aspergillus niger ATCC 16404; Bacillus subtilis NCAIM 01644) on the filter materials with different dust content was measured using the quantitative method AATCC 100-2004 (2008).In the plant biomass processed at the heat and power plant, a high concentration of bacteria (2.30 × 10⁷ CFU g⁻¹) and a lower concentration of fungi (4.46 × 10⁵ CFU g⁻¹) were detected. It was determined to be a good environment for the growth of microorganisms (the carbon to nitrogen ratio was 48:1). The presence of dust and the development of microbiological biofilms on reusable FFR, which is used by the heat and power plant workers for processing the plant biomass in increased humidity conditions, were also confirmed.It was found that the viability of microorganisms on a filter material depends on the kind of microorganism and the dust content on the material. Model research showed that biomass dust in filter materials stimulates the growth of E. coli. Moreover, dust concentration above 21% inhibited the growth of S. aureus and C. albicans but had no significant influence on the growth of B. subtilis and A. niger.     

Memetic Pareto Evolutionary Artificial Neural Networks to determine growth/no-growth in predictive microbiology

The main objective of this work is to automatically design neural network models with sigmoid basis units for binary classification tasks. The classifiers that are obtained achieve a double objective: a high classification level in the dataset and a high classification level for each class. We present MPENSGA2, a Memetic Pareto Evolutionary approach based on the NSGA2 multiobjective evolutionary algorithm which has been adapted to design Artificial Neural Network models, where the NSGA2 algorithm is augmented with a local search that uses the improved Resilient Backpropagation with backtracking—IRprop+ algorithm. To analyze the robustness of this methodology, it was applied to four complex classification problems in predictive microbiology to describe the growth/no-growth interface of food-borne microorganisms such as Listeria monocytogenes, Escherichia coli R31, Staphylococcus aureus and Shigella flexneri. The results obtained in Correct Classification Rate (CCR), Sensitivity (S) as the minimum of sensitivities for each class, Area Under the receiver operating characteristic Curve (AUC), and Root Mean Squared Error (RMSE), show that the generalization ability and the classification rate in each class can be more efficiently improved within a multiobjective framework than within a single-objective framework.     

Structural insights into the camel milk lactoperoxidase: Homology modeling and molecular dynamics simulation studies

Lactoperoxodase (LPO) is a heme peroxidase enzyme present in mammalian milk. It is an antimicrobial protein with wide range of industrial applications. Although the three dimensional structure of LPO from various mammalian species has been reported, but its structure from camel source is still unknown. So far, the crystallization attempts have not been successful in determining camel LPO (cLPO) structure. Herein, we developed the three dimensional structure of cLPO by homology modeling approach using prime module available in Schrodinger suite. The developed model in complex with ligand hypothiocyanate (OSCN⁻) was further validated by Ramachandran plot followed by molecular dynamics (MD) simulation studies using Desmond module of Schrodinger. cLPO model exhibited overall structural similarity with template crystal structure, however, it displayed different interaction pattern of amino acid residues with ligand OSCN⁻ in comparison to template crystal structure. Moreover, the ligand binding site environment in cLPO is more polar, less hydrophobic, and harbours more number of charged residues than template crystal structure. The substrate binding pocket environment of cLPO shows a considerable difference from template crystal structure. This subsequently resulted in dissimilar behaviour of ligand during the course of MD simulation studies.     

A PDMS microfluidic impedance immunosensor for E. coli O157:H7 and Staphylococcus aureus detection via antibody-immobilized nanoporous membrane

In this article, a PDMS microfluidic immunosensor integrated with specific antibody immobilized alumina nanoporous membrane was developed for rapid detection of foodborne pathogens Escherichia coli O157:H7 and Staphylococcus aureus with electrochemical impedance spectrum. Firstly, antibodies to the targeted bacteria were covalently immobilized on the nanoporous alumina membranes via self assembled (3-glycidoxypropyl)trimethoxysilane (GPMS) silane. Then, the impedance spectrum was recorded for bacteria detection ranging from 1 Hz to 100 kHz. The maximum impedance amplitude change for these two food pathogens was around 100 Hz. This microfluidic immunosensor based on nanoporous membrane impedance spectrum could achieve rapid bacteria detection within 2 h with a high sensitivity of 10² CFU/ml. Cross-bacteria experiments for E. coli O157:H7 and S. aureus were also explored to testify the specificity. The results showed that impedance amplitude at 100 Hz had a significant reduction in binding of bacteria when the membrane was exposed to non-specific bacteria.     

Identification of novel allosteric modulator binding sites in NMDA receptors: A molecular modeling study

The dysfunction of N-methyl-d-Aspartate receptors (NMDARs), a subtype of glutamate receptors, is correlated with schizophrenia, stroke, and many other neuropathological disorders. However, not all NMDAR subtypes equally contribute towards these disorders. Since NMDARs composed of different GluN2 subunits (GluN2A-D) confer varied physiological properties and have different distributions in the brain, pharmacological agents that target NMDARs with specific GluN2 subunits have significant potential for therapeutic applications. In our previous research, we have identified a family of novel allosteric modulators that differentially potentiate and/or inhibit NMDARs of differing GluN2 subunit composition. To further elucidate their molecular mechanisms, in the present study, we have identified four potential binding sites for novel allosteric modulators by performing molecular modeling, docking, and in silico mutations. The molecular determinants of the modulator binding sites (MBS), analysis of particular MBS electrostatics, and the specific loss or gain of binding after mutations have revealed modulators that have strong potential affinities for specific MBS on given subunits and the role of key amino acids in either promoting or obstructing modulator binding. These findings will help design higher affinity GluN2 subunit-selective pharmaceuticals, which are currently unavailable to treat psychiatric and neurological disorders.     

Radial scan of the molecular electrostatic potential of RNA double helices: an application to the enzyme-tRNA recognition

We introduced a method to characterize quantitatively the molecular electrostatic potential (MEP) of the minor and major grooves of base pairs located at nucleic acid double helices. By means of a radial MEP scan, we obtained a n-tuple of potential values corresponding to each groove, which can be analyzed by plotting the MEP values as a function of the angle in the radial scan. We studied base pairs of two different tRNAs, relevant in the recognition process with their cognate aminoacyl tRNA synthetases (aaRSs), in order to correlate their electrostatic behavior with the corresponding aminoacylation activity. We analyzed the first three base pairs of the Escherichia coli tRNA(Ala) acceptor stem, finding several cases where the MEP profiles obtained from the plots are in agreement with the reported aminoacylation activities. Additionally, a non-hierarchical clustering performed over the MEP n-tuples resulted in meaningful classifications that correlate with the activity and with the predicted stereochemistry of the reaction. We also studied the first two base pairs of the E. coli tRNA(Thr) acceptor stem but constraining the analysis to the angle intervals that seem relevant for the binding sites of the enzyme. These intervals were deduced from the ThrRS-tRNA(Thr) complex crystal structure. In this case, we also found a good agreement between the MEP profiles and the activity, supporting the idea that the tRNA identity elements function is to allow an optimal electrostatic complementarity between the aminoacyl-tRNA synthetase and the tRNA.     

Design of an empirical study for comparing the usability of concurrent programming languages

Context: Developing concurrent software has long been recognized as a difficult and error-prone task. To support developers, a multitude of language proposals exist that promise to make concurrent programming easier. Empirical studies are needed to support the claim that a language is more usable than another.Objective: This paper presents the design of a study to compare concurrent programming languages with respect to comprehending and debugging existing programs and writing correct new programs. The design is applied to a comparison of two object-oriented languages for concurrency, multithreaded Java and SCOOP.Method: A critical challenge for such a study is avoiding the bias that might be introduced during the training phase and when interpreting participants’ solutions. We address these issues by the use of self-study material and an evaluation scheme that exposes any subjective decisions of the corrector, or eliminates them altogether.Results: The study template consisting of the experimental design and the structure of the self-study and evaluation material is demonstrated to work successfully in an academic setting. The concrete instantiation of the study template shows results in favor of SCOOP even though the study participants had previous training in writing multithreaded Java programs.Conclusion: It is concluded that the proposed template of a small but therefore easy-to-implement empirical study with a focus on core language constructs is helpful in characterizing the usability of concurrent programming paradigms. Applying the template to further languages could shed light on which approaches are promising and hence drive language research into the right direction.