Methods Protoc., Volume 2, Issue 2 (June 2019) – 29 articles

Next-generation sequencing (NGS) is a revolutionary advancement allowing large-scale discovery of functional molecular markers that has many applications, including plant breeding. High-quality genomic DNA (gDNA) is a prerequisite for successful NGS library preparation and sequencing; however, few reliable protocols to obtain such plant gDNA exist. A previously reported nuclear pellet (NP) method enables extraction of high-yielding gDNA from fresh leaf tissue of maize (Zea mays L.), but the quality does not meet the stringent requirements of NGS. In this study, we optimized the NP method for whole-genome sequencing of rice (Oryza sativa L.) through the integration of simple purification steps. The optimized NP method relied on initial nucleus enrichment, cell lysis, extraction, and subsequent gDNA purification buffers. The purification steps used proteinase K, RNase A, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1) treatments for protein digestion and RNA, protein, and phenol removal, respectively. Our data suggest that this optimized NP method allowed extraction of consistently high-yielding and high-quality undegraded gDNA without contamination by protein and RNA. Moreover, the extracted gDNA fulfilled the quality metrics of NGS library preparation for the Illumina HiSeq X Ten platform by the TruSeq DNA PCR-Free Library Prep Kit (Illumina). We provide a reliable step-by-step guide to the extraction of high-quality gDNA from fresh leaf tissues of rice for molecular biologists with limited resources. Full article

We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive to low level light signals than conventional epi-detection used in two-photon fluorescence microscopes. The DIVER detector efficiently collects scattered emission photons from a wide area of turbid samples at almost any entrance angle in a 2π spherical angle. Using an epi-detection scheme only photons coming from a relatively small area of a sample and at narrow acceptance angle can be detected. The transmission geometry of the DIVER imaging system makes it exceptionally suitable for Second and Third Harmonic Generation (SHG, THG) signal detection. It also has in-depth fluorescence lifetime imaging (FLIM) capability. Using special optical filters with sin-cos spectral response, hyperspectral analysis of images acquired in-depth in scattering media can be performed. The system was successfully employed in imaging of various biological tissues. The DIVER detector can be plugged into a standard microscope stage and used as an external detector with upright commercial two-photon microscopes. Full article

Stroke is the leading cause of death with over 5 million deaths worldwide each year. About 80% of strokes are ischemic strokes caused by blood clots. Tissue plasminogen activator (tPa) is the only FDA-approved drug to treat ischemic stroke with a wholesale price over $6000. tPa is now off patent although no biosimilar has been developed. The production of tPa is complicated by the 17 disulfide bonds that exist in correctly folded tPA. Here, we present an Escherichia coli-based cell-free protein synthesis platform for tPa expression and report conditions which resulted in the production of active tPa. While the activity is below that of commercially available tPa, this work demonstrates the potential of cell-free expression systems toward the production of future biosimilars. The E. coli-based cell-free system is increasingly becoming an attractive platform for low-cost biosimilar production due to recent developments which enable production from shelf-stable lyophilized reagents, the removal of endotoxins from the reagents to prevent the risk of endotoxic shock, and rapid on-demand production in hours. Full article

Modern optics offers several label-free microscopic and spectroscopic solutions which are useful for both imaging and pathological assessments of biological tissues. The possibility to obtain similar morphological and biochemical information with fast and label-free techniques is highly desirable, but no single optical modality is capable of obtaining all of the information provided by histological and immunohistochemical analyses. Integrated multimodal imaging offers the possibility of integrating morphological with functional-chemical information in a label-free modality, complementing the simple observation with multiple specific contrast mechanisms. Here, we developed a custom laser-scanning microscopic platform that combines confocal Raman spectroscopy with multimodal non-linear imaging, including Coherent Anti-Stokes Raman Scattering, Second-Harmonic Generation, Two-Photon Excited Fluorescence, and Fluorescence Lifetime Imaging Microscopy. The experimental apparatus is capable of high-resolution morphological imaging of the specimen, while also providing specific information about molecular organization, functional behavior, and molecular fingerprint. The system was successfully tested in the analysis of ex vivo tissues affected by urothelial carcinoma and by atherosclerosis, allowing us to multimodally characterize of the investigated specimen. Our results show a proof-of-principle demonstrating the potential of the presented multimodal approach, which could serve in a wide range of biological and biomedical applications. Full article

Background: Stroke is a leading cause of severe and long-term disability in developed countries. Around 15 million people suffer a stroke each year, being most of them ischemic due to modifiable risk factors. Adequate self-management abilities may help to manage the consequences of stroke, but it is unknown which specific intervention could be effective to booster these self-management abilities. Objective: To evaluate the improvement of self-management in chronic stroke survivors using decision support and self-management system (STARR). Methods: A randomized, prospective, parallel group, open, and the unicentric pilot trial will be performed. Stroke survivors and their caregivers will be randomly allocated to STARR management or standard of care. Main inclusion criteria are mild to moderate disabled first stroke adult survivor, living at home, able to cope and follow the guidelines and devices, without socio-familial exclusion. All will get a conventional treatment in the acute and subacute phase; however, in the chronic period, cases will use the developed STARR App and Decision Support System. Measurements will be performed at baseline, at 3 months, and at 6 months. Outcome measures are patient-report outcome measure of self-management competency, physical function, risk factor reduction, healthcare resource utilization, knowledge of the condition, mood, and social isolation. Discussion: If effective, the results of this study will enable stroke patients and their caregivers to deal better with the everyday life obstacles of stroke, improve the adherence of the treatment, improve the control of cardiovascular risk, and, in consequence, reduce the recurrence of secondary strokes, the number of complications, the number of consultations, and readmissions; to ultimately reduce the health systems costs. Taking into consideration that the number of stroke survivors is increasing around the world, a large number of individuals could profit from this intervention. Full article

Polarization sensitive second harmonic generation (pSHG) microscopy is an imaging technique able to provide, in a non-invasive manner, information related to the molecular structure of second harmonic generation (SHG) active structures, many of which are commonly found in biological tissue. The process of acquiring this information by means of pSHG microscopy requires a scan of the sample using different polarizations of the excitation beam. This process can take considerable time in comparison with the dynamics of in vivo processes. Fortunately, single scan polarization sensitive second harmonic generation (SS-pSHG) microscopy has also been reported, and is able to generate the same information at a faster speed compared to pSHG. In this paper, the orientation of second harmonic active supramolecular assemblies in starch granules is obtained on by means of pSHG and SS-pSHG. These results are compared in the forward and backward directions, showing a good agreement in both techniques. This paper shows for the first time, to the best of the authors’ knowledge, data acquired using both techniques over the exact same sample and image plane, so that they can be compared pixel-to-pixel. Full article

Amyloid plaques found in the brains of Alzheimer’s disease patients primarily consists of amyloid beta 1-42 (Aβ42). Commercially, Aβ42 is synthesized using high-throughput peptide synthesizers resulting in the presence of impurities and the racemization of amino acids that affects its aggregation properties. Furthermore, the repeated purchase of even a small quantity (~1 mg) of commercial Aβ42 can be expensive for academic researchers. Here, we describe a detailed methodology for robust expression of recombinant human Aβ(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli using standard molecular biology techniques with refined and rapid one-step analytical purification techniques. The peptide is isolated and purified from transformed cells using an optimized reverse-phase high-performance liquid chromatography (HPLC) protocol with commonly available C18 columns, yielding high amounts of peptide (~15–20 mg per 1 L culture) within a short period of time. The recombinant human Aβ(M1-42) forms characteristic aggregates similar to synthetic Aβ42 aggregates as verified by western blotting and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique produces pure recombinant human Aβ(M1-42) that may be used to synthesize chemical probes and in several downstream in vitro and in vivo assays to facilitate Alzheimer’s disease research. Full article

Maternal tobacco smoking is a recognized risk behavior that has adverse impacts on maternal and fetal health. However, in some populations, the use of smokeless tobacco exceeds the use of smoked tobacco. In central Australia, Aboriginal populations utilize wild tobacco plants (Nicotiana spp.) as a smokeless product. These plants are known by a variety of names, one of which is pituri. The plants are masticated and retained in the oral cavity for extended periods of time and their use continues throughout pregnancy, birth, and lactation. In contrast to the evidence related to combusted tobacco use, there is no evidence as to the effects of pituri use in pregnancy. Central Australian Aboriginal women who were at least 28 weeks pregnant were stratified into three tobacco exposure groups: (a) Pituri chewers, (b) smokers, and (c) non-tobacco users. Routine antenatal and birth information, pre-existing and pregnancy-related maternal characteristics, fetal characteristics, and biological samples were collected and compared. The biological samples were analysed for tobacco and nicotine metabolite concentrations. Samples from the mother included venous blood, urine, hair and colostrum and/or breast milk. From the neonate, this included Day 1 and Day 3 urine and meconium, and from the placenta, arterial and venous cord blood following delivery. This is the first study to correlate the pregnancy outcomes of central Australian Aboriginal women with different tobacco exposures. The findings will provide the foundation for epidemiological data collection in related studies. Note to readers: In this article, the term “Aboriginal” was chosen by central Australian women to refer to both themselves and the Aboriginal people in their communities. “Indigenous” was chosen to refer to the wider Australian Aboriginal and Torres Strait Islander people. Full article

The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform (M/C) precipitation and in-solution trypsin digestion in LC-MS/MS-based urine proteomics. The urine proteins prepared by M/C precipitation showed around 80% of the protein recovery rate. The samples showed the largest number of identified proteins, which were over 1000 on average compared with other precipitation methods in LC-MS/MS-based urine proteomics. For further improvement of the workflow, the essences were arranged in protein dissolving and trypsin digestion step for the extraction of urine proteins. Addition of Ethylene diamine tetraacetic acid (EDTA) dramatically enhanced the dissolution of protein and promoted the trypsin activity in the digestion step because the treatment increased the number of identified proteins with less missed cleavage sites. Eventually, an optimized workflow was established by a well-organized strategy for daily use in the LC-MS/MS-based urine proteomics. The workflow will be of great help for several aims based on urine proteomics approaches, such as diagnosis and biomarker discovery. Full article

Th17 cells are a subset of effector T helper cells that produce interleukin (IL)-17A, IL-17F, IL-22, and IL-26, which can promote tissue inflammation and contribute to the pathogenesis of rheumatic, fibrosing, and other diseases. Research into these diseases is often limited by a lack of an animal model that closely mimics human disease and the paucity of patient clinical tissues. Therefore, the development of relevant experimental models is crucial. Three media formulations of Th17-skewing cocktail (CT) were evaluated for the ability to induce a Th17 signature in an ex vivo human skin model: CT9 contained αCD3, αCD28, IL-23, IL-1β, IFNγ, IL-4, IL-6, IL-21, and TGFβ; CT8 lacked IL-1β; and CT4 only contained αCD3, αCD28, IL-23, and IL-1β. Healthy donor skin was defatted, distributed as 3 mm punch biopsies, and incubated with one of the cocktail formulations or vehicle for 48 h. All of the cocktail formulations independently significantly stimulated the expression of each gene examined. CT4 induced IL-17A expression 1024-fold, significantly higher than CT9 and CT8. IL-17F was robustly stimulated by CT4 (1557-fold), CT9 (622-fold), and CT8 (111-fold), with significant differences between the CT groups. All of the formulations significantly induced IL-22 (16–42-fold). CT9 stimulated the highest IL-26 response (41-fold), which was significantly higher than CT4 and CT8. IL-10 was stimulated significantly higher with CT8 (10-fold) than CT4 or CT9. The secretion of IL-17A was significantly elevated with all cocktail formulations. Robust IL-17A/IL-17F cytokine induction was preferentially mediated by CT4, which suggested that its components are the minimal constituents necessary for the full induction of these genes in this human skin explant model, while the downstream cytokines were preferentially upregulated by CT4 (IL-22), CT9 (IL-26), or CT8 (IL-10). In summary, our findings suggest that the induction of a Th17 phenotype in human skin is feasible and can be used as a model for rheumatic and fibrosing diseases where Th17 skewing is observed. Full article

Advanced non-small-cell lung cancer (NSCLC) is generally linked with a poor prognosis and is one of the leading causes of cancer-related deaths worldwide. Since only a minority of the patients respond well to chemotherapy and/or targeted therapies, immunotherapy might be a valid alternative in the lung cancer treatment field, as immunotherapy attempts to strengthen the body’s own immune response to recognize and eliminate malignant tumor cells. However, positive response patterns to immunotherapy remain unclear. In this study, we demonstrate how immune-related factors could be visualized from single NSCLC tissue sections (Biobank@UZA) while retaining their spatial information by using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), in order to unravel the molecular profile of NSCLC patients. In this way, different regions in lung cancerous tissues could be discriminated based on the molecular composition. In addition, we linked visualization (MALDI MSI) and identification (based on liquid chromatography higher resolution mass spectrometry) of the molecules of interest for the correct biological interpretation of the observed molecular differences within the area in which these molecules are detected. This is of major importance to fully understand the underlying molecular profile of the NSCLC tumor microenvironment. Full article

Mechanical characterization of living cells undergoing substantial external strain promises insights into material properties and functional principles of mechanically active tissues. However, due to the high strains that occur in physiological situations (up to 50%) and the complex structure of living cells, suitable experimental techniques are rare. In this study, we introduce a new system composed of an atomic force microscope (AFM), a cell stretching system based on elastomeric substrates, and light microscopy. With this system, we investigated the influence of mechanical stretch on monolayers of keratinocytes. In repeated indentations at the same location on one particular cell, we found significant stiffening at 25% and 50% strain amplitude. To study the contribution of intermediate filaments, we used a mutant keratinocyte cell line devoid of all keratins. For those cells, we found a softening in comparison to the wild type, which was even more pronounced at higher strain amplitudes. Full article

Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks. Full article

Early diagnosis is a critical component of the global response to the human immunodeficiency virus (HIV). In Australia, more than two-thirds of women from Southeast Asia are diagnosed late with HIV. There is limited evidence regarding the barriers to HIV testing and which interventions work to increase an uptake among migrants living in high-income countries. This participatory action research (PAR) project will work with women from Indonesia to co-design an intervention to increase HIV testing uptake in Western Australia. The project will involve trained community researchers, representatives from relevant organizations, and community women born in Indonesia. We will conduct three PAR cycles. Phase one will use focus groups to understand enablers for HIV testing among community members. In phase two, data will be presented back to members of the participating communities who will be invited to co-design an intervention to increase HIV testing. The final cycle will focus on implementing and evaluating the resulting intervention. This project will add to the small body of literature on pathways and enablers to HIV testing, and to new insights regarding interventions that work for women from migrant communities and why. Full article

Tetrahymena pyriformis (protozoa) is intensely investigated as a model organism, offering numerous advantages in comprehensive and multidisciplinary studies using morphologic or molecular methods. Since DNA extraction is a vital step of any molecular experiment, here a new mixed surfactant (Sodium dodecyl sulfate (SDS) 20%/Triton X-100) was adopted for effective DNA extraction from Tetrahymena pyriformis under an easy, fast protocol. The efficiency of this technique was then compared with three widely-used alternative techniques, namely the Chelex 100 matrix, Ammonium pyrrolidine dithiocarbamate (APD) complex and SDS–chloroform methods. DNA extraction was analyzed by pulsed-field gel electrophoresis, spectral measurement, fluorometry (Qubit), restriction enzyme digestion, and polymerase chain reaction. Data analysis revealed that the quantity and quality of the recovered DNA varied depending on the applied DNA extraction method. The new method (SDS 20%/Triton X-100) was the most efficient for extracting DNA from Tetrahymena pyriformis with high integrity and purity, affordable cost, less time, and suitability for molecular applications. Full article

Synthetic biology integrates diverse engineering disciplines to create novel biological systems for biomedical and technological applications. The substantial growth of the synthetic biology field in the past decade is poised to transform biotechnology and medicine. To streamline design processes and facilitate debugging of complex synthetic circuits, cell-free synthetic biology approaches has reached broad research communities both in academia and industry. By recapitulating gene expression systems in vitro, cell-free expression systems offer flexibility to explore beyond the confines of living cells and allow networking of synthetic and natural systems. Here, we review the capabilities of the current cell-free platforms, focusing on nucleic acid-based molecular programs and circuit construction. We survey the recent developments including cell-free transcription–translation platforms, DNA nanostructures and circuits, and novel classes of riboregulators. The links to mathematical models and the prospects of cell-free synthetic biology platforms will also be discussed. Full article

Pharmacokinetic alterations of medications administered during surgeries involving cardiopulmonary bypass (CPB) and extracorporeal membrane oxygenation (ECMO) have been reported. The impact of CPB on the cytochrome P450 (CYP) enzymes’ activity is the key factor. The metabolic rates of caffeine, dextromethorphan, midazolam, omeprazole, and Losartan to the CYP-specific metabolites are validated measures of in vivo CYP 1A2, 2D6, 3A4, 2C19, and 2C9 activities, respectively. The study aim is to assess the activities of major CYPs in patients on extracorporeal circulation (EC). This is a pilot, prospective, open-label, observational study in patients undergoing surgery using EC and patients undergoing laparoscopic cholecystectomy as a control group. CYP activities will be measured on the day, and 1–2 days pre-surgery/3–4 days post-surgery (cardiac surgery and Laparoscopic cholecystectomy) and 1–2 days after starting ECMO, 1–2 weeks after starting ECMO, and 1–2 days after discontinuation from ECMO. Aforementioned CYP substrates will be administered to the patient and blood samples will be collected at 0, 1, 2, 4, and 6 h post-dose. Major CYP enzymes’ activities will be compared in each participant on the day, and before/after surgery. The CYP activities will be compared in three study groups to investigate the impact of CYPs on EC. Full article

The nacreous layer of shells and pearls is composed of aragonite crystals arranged in an organic matrix. The organic matrix contains chitin and several proteins that regulate the formation of the nacreous layer. Owing to their strong interactions in the organic matrix, the current method for extraction of insoluble proteins from the pre-powdered nacreous layer involves heating to high temperatures in the presence of a detergent (e.g., sodium dodecyl sulfate, SDS) and reductant (e.g., dithiothreitol, DTT), which is likely to induce protein degradation. Therefore, we have developed an electroextraction method to isolate proteins from the organic matrix of a nacreous organic sheet, that was obtained following the decalcification of shells in their original shape. Our electroextraction method employs milder conditions without heating or detergent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the electro-extracted proteins (EEPs) under non-reduced and reduced conditions revealed that this method yielded a greater number of different proteins compared with the conventional extraction method and the isolated EEPs retained their disulfide bonds. Our method is able to easily extract insoluble proteins from the nacreous layer under mild conditions and will undoubtedly aid future analyses into the functions of the nacreous layer proteins. Full article

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca²⁺ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca²⁺ has been reported for the extraction of DNA from sand flies. Full article

Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications. Full article

The discovery of optogenetics has revolutionized research in neuroscience by providing the tools for noninvasive, cell-type selective modulation of membrane potential and cellular function in vitro and in vivo. Rhodopsin-based optogenetics has later been introduced in experimental cardiology studies and used as a tool to photoactivate cardiac contractions or to identify the sites, timing, and location most effective for defibrillating impulses to interrupt cardiac arrhythmias. The exploitation of cell-selectivity of optogenetics, and the generation of model organisms with myocardial cell type targeted expression of opsins has started to yield novel and sometimes unexpected notions on myocardial biology. This review summarizes the main results, the different uses, and the prospective developments of cardiac optogenetics. Full article

Due to the ongoing crises of fossil fuel depletion, climate change, and environmental pollution, microbial processes are increasingly considered as a potential alternative for cleaner and more efficient production of the diverse chemicals required for modern civilization. However, many issues, including low efficiency of raw material conversion and unintended release of genetically modified microorganisms into the environment, have limited the use of bioprocesses that rely on recombinant microorganisms. Cell-free metabolic engineering is emerging as a new approach that overcomes the limitations of existing cell-based systems. Instead of relying on metabolic processes carried out by living cells, cell-free metabolic engineering harnesses the metabolic activities of cell lysates in vitro. Such approaches offer several potential benefits, including operational simplicity, high conversion yield and productivity, and prevention of environmental release of microorganisms. In this article, we review the recent progress in this field and discuss the prospects of this technique as a next-generation bioconversion platform for the chemical industry. Full article

While single-molecule force spectroscopy has greatly advanced the study of protein folding, there are limitations to what can be learned from studying the effect of force alone. We developed a novel technique, chemo-mechanical unfolding, that combines multiple perturbants—force and chemical denaturant—to more fully characterize the folding process by simultaneously probing multiple structural parameters—the change in end-to-end distance, and solvent accessible surface area. Here, we describe the theoretical background, experimental design, and data analysis for chemo-mechanical unfolding experiments probing protein folding thermodynamics and kinetics. This technique has been applied to characterize parallel protein folding pathways, the protein denatured state, protein folding on the ribosome, and protein folding intermediates. Full article

Current antifungal interventions have often limited efficiency in treating fungal pathogens, particularly those resistant to commercial drugs or fungicides. Antifungal drug repurposing is an alternative intervention strategy, whereby new utility of various marketed, non-antifungal drugs could be repositioned as novel antifungal agents. In this study, we investigated “chemosensitization” as a method to improve the efficiency of antifungal drug repurposing, wherein combined application of a second compound (viz., chemosensitizer) with a conventional, non-antifungal drug could greatly enhance the antifungal activity of the co-applied drug. Redox-active natural compounds or structural derivatives, such as thymol (2-isopropyl-5-methylphenol), 4-isopropyl-3-methylphenol, or 3,5-dimethoxybenzaldehyde, could serve as potent chemosensitizers to enhance antifungal activity of the repurposed drug bithionol. Of note, inclusion of fungal mutants, such as antioxidant mutants, could also facilitate drug repurposing efficiency, which is reflected in the enhancement of antifungal efficacy of bithionol. Bithionol overcame antifungal (viz., fludioxonil) tolerance of the antioxidant mutants of the human/animal pathogen Aspergillus fumigatus. Altogether, our strategy can lead to the development of a high efficiency drug repurposing design, which enhances the susceptibility of pathogens to drugs, reduces time and costs for new antifungal development, and abates drug or fungicide resistance. Full article

In the biopharmaceutical pipeline, protein expression systems are of high importance not only for the production of biotherapeutics but also for the discovery of novel drugs. The vast majority of drug targets are proteins, which need to be characterized and validated prior to the screening of potential hit components and molecules. A broad range of protein expression systems is currently available, mostly based on cellular organisms of prokaryotic and eukaryotic origin. Prokaryotic cell-free systems are often the system of choice for drug target protein production due to the simple generation of expression hosts and low cost of preparation. Limitations in the production of complex mammalian proteins appear due to inefficient protein folding and posttranslational modifications. Alternative protein production systems, so-called eukaryotic cell-free protein synthesis systems based on eukaryotic cell-lysates, close the gap between a fast protein generation system and a high quality of complex mammalian proteins. In this study, we show the production of druggable target proteins in eukaryotic cell-free systems. Functional characterization studies demonstrate the bioactivity of the proteins and underline the potential for eukaryotic cell-free systems to significantly improve drug development pipelines. Full article

Doxorubicin (DOXO) is an adjuvant chemotherapy agent and is also commonly used in cell biology research. Cytotoxic assays in cell culture are frequently used in order to stablish drug concentrations that are useful for controlling cell proliferation. One common cytotoxic method used is 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). Our present research aims to support future studies in engaging MTT assay using DOXO that exhibits a strong red coloration and fluorescence, and so it is assumed that DOXO may interfere with commonly used colorimetric assays such as MTT. The interference of DOXO in the MTT determination was evaluated in a Breast Cancer cell line Michigan Cancer Foundation-7 (MCF-7). The interference was evaluated by means of spectroscopic methods in particular spectrophometry and fluorescence spectroscopy of MTT and DOXO. We postulate that the medium and the MTT reagent itself can interfere on the metabolic activity method, so in order to achieve better results, DMEM was replaced by a neutral buffer like Phosphate-buffered saline (PBS). This protocol may be extremely useful in future studies involving DOXO. Full article

Colicins are antimicrobial proteins produced by Escherichia coli that hold great promise as viable complements or alternatives to antibiotics. Cell-free protein synthesis (CFPS) is a useful production platform for toxic proteins because it eliminates the need to maintain cell viability, a common problem in cell-based production. Previously, we demonstrated that colicins produced by CFPS based on crude Escherichia coli lysates are effective in eradicating antibiotic-tolerant bacteria known as persisters. However, we also found that some colicins have poor solubility or low cell-killing activity. In this study, we improved the solubility of colicin M from 16% to nearly 100% by producing it in chaperone-enriched E. coli extracts, resulting in enhanced cell-killing activity. We also improved the cytotoxicity of colicin E3 by adding or co-expressing the E3 immunity protein during the CFPS reaction, suggesting that the E3 immunity protein enhances colicin E3 activity in addition to protecting the host strain. Finally, we confirmed our previous finding that active colicins can be rapidly synthesized by observing colicin E1 production over time in CFPS. Within three hours of CFPS incubation, colicin E1 reached its maximum production yield and maintained high cytotoxicity during longer incubations up to 20 h. Taken together, our findings indicate that colicin production can be easily optimized for improved solubility and activity using the CFPS platform. Full article

BACKGROUND: The Physical Literacy in the Early Years (PLEY) intervention is a randomized mixed-methods controlled trial focused on embedding loose parts materials into the outdoor play spaces of regulated child care centres across Nova Scotia. The aim is to evaluate the efficacy of the PLEY intervention versus standard regulated childcare practice in influencing thoughts and behaviors of children, parents, and educators. METHODS: Participating early child care centres (n = 19) were randomly assigned to intervention or control sites. Intervention sites received loose parts kits at the beginning of the project while control sites received kits upon project completion. The kits included items such as rocks, tree cookies, balls, wood planks, tubes, tires, ropes, and pulleys. Children (n = 183 at baseline) had their physical activity (accelerometers) and movement skills (TGMD-3 and PGMQ) measured before and after the intervention. All centres provided responses to environmental surveys (Go NAP SACC and Site Context Questionnaire), and educators in intervention sites participated in focus group and photovoice sessions. Educators were also provided with a full day professional development opportunity (plus ongoing mentoring) focused on physical activity, physical literacy, outdoor play, risk-taking, and loose parts. Parents participated in an interview addressing active outdoor play, physical literacy, and attitudes towards risk taking during play. DISCUSSION: This study will provide a better understanding of how integrating loose parts materials into outdoor play spaces impacts children’s health, and the impact on educator and parent attitudes, beliefs, and understanding around physical literacy, active outdoor play and risk-taking during play. Full article

Industrial fermentation in aerobic processes is plagued by high costs due to gas transfer limitations and substrate oxidation to CO₂. It has been a longstanding challenge to engineer an obligate aerobe organism, such as Pseudomonas putida, into an anaerobe to facilitate its industrial application. However, the progress in this field is limited, due to the poor understanding of the constraints restricting its anoxic phenotype. In this paper, we provide a methodological description of a novel cultivation technology for P. putida under anaerobic conditions, using the so-called microbial electrochemical technology within a bioelectrochemical system. By using an electrode as the terminal electron acceptor (mediated via redox chemicals), glucose catabolism could be activated without oxygen present. This (i) provides an anoxic-producing platform for sugar acid production at high yield and (ii) more importantly, enables systematic and quantitative characterizations of the phenotype of P. putida in the absence of molecular oxygen. This unique electrode-based cultivation approach offers a tool to understand and in turn engineer the anoxic phenotype of P. putida and possibly also other obligate aerobes. Full article