Methods and Protocols — Open Access Journal

This report provides a broadly applicable and cost-effective method for the purification of mycosporine-like amino acids (MAAs) from cyanobacteria. As MAAs are known to have multiple bioactivities for health and beauty, a universal isolation method of MAAs from biomass is attractive. In particular, the biomass of photosynthetic microorganisms such as cyanobacteria is of interest as a natural source of useful compound production, because of their photoautotrophic property. The method presented here is applicable for the isolation of mycosporine-2-glycine (M2G), which is a rare MAA produced in a halotolerant cyanobacterium. This method also allowed for the isolation of two of the most common MAAs, shinorine (SHI) and porphyra-334 (P334). A three-step separation process using low pressure liquid chromatography yielded purified MAAs, which were characterized by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC/MS) analyses. The purified MAAs exhibited free radical scavenging activity in the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. The experimental parameters obtained in this report may allow for a scale-up of the MAA purification process for future industrial applications. Full article

Metabolite extraction is one of the critical steps in microbial metabolome analysis. It affects both the observed metabolite content and biological interpretation of the data. Several methods exist for metabolite extraction of microbes, but the literature is not consistent regarding the sample model, adequacy, and performance of each method. In this study, an optimal extraction protocol for Yersinia intracellular metabolites was investigated. The effect of five extraction protocols consisting of different extraction solvent systems (60% methanol, 100% methanol, acetonitrile/methanol/water (2:2:1), chloroform/methanol/water (2:1:1), and 60% ethanol) on Yersinia metabolic profiles were compared. The number of detected peaks, sample-to-sample variation, and metabolite yield were used as criteria. Extracted metabolites were analyzed by ¹H-NMR and principal component analysis (PCA), as well as partial least squares discriminant analysis (PLS-DA) multivariate statistics. The extraction protocol using 100% methanol as the extraction solvent provided the highest number of detected peaks for both Yersinia species analyzed, yielding more spectral information. Together with the reproducibility and spectrum quality, 100% methanol extraction was suitable for intracellular metabolite extraction from both species. However, depending on the metabolites of interest, other solvents might be more suitable for future studies, as distinct profiles were observed amongst the extraction methods. Full article

We propose an optical tweezers setup based on an annular-shaped laser beam that is efficient to trap 2.8 $\mathsf{\mu }$m-diameter superparamagnetic particles. The optical trapping of such particles was fully characterized, and a direct absolute comparison with a geometrical optics model was performed. With this comparison, we were able to show that light absorption by the superparamagnetic particles is negligible for our annular beam tweezers, differing from the case of conventional Gaussian beam tweezers, in which laser absorption by the beads makes stable trapping difficult. In addition, the trap stiffness of the annular beam tweezers increases with the laser power and with the bead distance from the coverslip surface. While this first result is expected and similar to that achieved for conventional Gaussian tweezers, which use ordinary dielectric beads, the second result is quite surprising and different from the ordinary case, suggesting that spherical aberration is much less important in our annular beam geometry. The results obtained here provide new insights into the development of hybrid optomagnetic tweezers, which can apply simultaneously optical and magnetic forces on the same particles. Full article

Segmentation is one of the most important steps in microscopy image analysis. Unfortunately, most of the methods use fluorescence images for this task, which is not suitable for analysis that requires a knowledge of area occupied by cells and an experimental design that does not allow necessary labeling. In this protocol, we present a simple method, based on edge detection and morphological operations, that separates total area occupied by cells from the background using only brightfield channel image. The resulting segmented picture can be further used as a mask for fluorescence quantification and other analyses. The whole procedure is carried out in open source software Fiji. Full article

Genetic engineering is considered to be an important tool for the improvement of cassava. Cassava is a highly heterozygous crop species for which conventional breeding is a lengthy and tedious process. Robust transformation is based on Agrobacterium-mediated transformation of friable embryogenic callus (FEC). Production of FEC is genotype-dependent and considered to be a major bottleneck for the genetic transformation of cassava. As a consequence, routine genetic transformation has only been established for a handful of cassava cultivars. Therefore, development of procedures enabling efficient production of high-quality cassava FEC is required to allow the translation of research from the model cultivar to farmer-preferred cassava cultivars. Here we study the FEC production capacity of Brazilian cassava cultivars and report the modification of the protocol for the genetic transformation of Verdinha (BRS 222), a recalcitrant cultivar with high potential for protein production that is extensively used by farmers in Brazil. Full article

A robust method was developed to investigate the liposomal behavior of novel enzymatically-synthesized hydroxytyrosol and tyrosol phospholipids. Bilayer characteristic obtained by this method, including bilayer formation stability and adsorption properties, were explored using dynamic light scattering, zeta-potential measurements, and quartz crystal microbalance with dissipation monitoring (QCMD), respectively. Liposome diameters were found to typically increase from pH 5.5 to pH 10. Zeta potentials values, on the other hand, were found to be well below −25 mV at all pH conditions explored, with the lowest values (and thus, the best liposome stability) at pH 5.5 or pH 10. Quartz crystal microbalance with dissipation monitoring measurements demonstrated that 100% 1,2-dioloeoylphosphatidyl-hydroxytyrosol (DOPHT) liposomes adsorbed intact onto silica in buffer conditions at pH 5.5 and with no calcium, or at pH 7.5 with calcium (no adsorption was detected at pH 10). 1,2-Dioleoylphosphatidyl-tyrosol (DOPT) liposomes were shown to adsorb intact under buffer conditions only at pH 5.5 with and without calcium. 1,2-Dioleoylphosphatidyl-2-phenolethanol (DOPPE), in comparison, readily adsorbed intact at pH 7.5 without calcium and just slightly at pH 5.5 with calcium present, but formed a supported bilayer over hours at pH 5.5 in the absence of calcium ions. Full article

Prism-based total internal reflection fluorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum dots via evanescent field of a confined region of space, which is beneficial not only for single molecule detection but also for analysis of single molecule dynamics and for acquiring kinetics data. However, there is neither a commercial microscope available for purchase nor a detailed guide dedicated for building this microscope. Thus far, pTIRF microscopes are custom-built with the use of a commercially available inverted microscope, which requires high level of expertise in selecting and handling sophisticated instrument-parts. To directly address this technology gap, here we describe a step-by-step guide on how to build and characterize a pTIRF microscope for in vitro single-molecule imaging, nanostructure analysis and other life sciences research. Full article

The sick preterm infant monitoring is an intriguing job that medical staff in Neonatal Intensive Care Units (NICU) must deal with on a daily basis. As a standards monitoring procedure, preterm infants are monitored via sensors and electrodes that are firmly attached to their fragile and delicate skin and connected to processing monitors. However, an alternative exists in contactless imaging to record such physiological signals (we call it as Physio-Markers), detecting superficial changes and internal structures activities which can be used independently of, or aligned with, conventional monitors. Countless advantages can be gained from unobtrusive monitoring not limited to: (1) quick data generation; (2) decreasing physical and direct contact with skin, which reduces skin breakdown and minimizes risk of infection; and (3) reduction of electrodes and probes connected to clinical monitors and attached to the skin, which allows greater body surface-area for better care. This review is an attempt to build a solid ground for and to provide a clear perspective of the potential clinical applications of technologies inside NICUs that use contactless imaging modalities such as Visible Light Imaging (VLI), Near Infrared Spectroscopy (NIRS), and Infrared Thermography (IRT). Full article

Acceptance and commitment therapy (ACT) has been reported to be effective in the treatment of some psychiatric disorders. It remains uncertain, however, whether ACT is safe and effective in treating schizophrenia spectrum and other psychotic disorders (e.g., psychosis). This protocol describes the methodology for a systematic review and meta-analysis of the safety and efficacy of ACT in the treatment of psychosis. The review will be guided by the standards set by the Cochrane Collaboration. We will search the Allied and Complementary Medicine Database (AMED), Cumulative Index to Nursing and Allied Health Literature (CINAHL), Cochrane Central Register of Controlled Trials (CENTRAL), Excerpta Medica database (EMBASE), EMCARE, Education Resources Information Center (ERIC), MEDLINE, and PsycINFO databases for randomized controlled trials, whose arms are ACT and any comparator, as well as ClinicalTrials.gov, Australian New Zealand Clinical Trials Registry (ANZCTR), and Current Controlled Trials (ISRCTN), for unpublished and ongoing trials. The primary outcome will be any standard (or surrogate) measure of psychotic pathology. The meta-analysis will summarize short-term and long-term effects and different control conditions with or without treatment as usual or comparative to other interventions. In cases where heterogeneity is detected (via χ² and I²), we will adopt the random effects model for computation. Full article

Tuberculosis (TB) remains as a major public health issue in developing countries. Accurate detection is essential for the proper management of patients with active disease. Here, we present a simple DNAzol-LAMP (loop-mediated isothermal amplification) procedure for the detection of Mycobacterium tuberculosis in sputum specimens. Twenty smear-positive sputum samples were analyzed as follows: (i) Genetic material was extracted by a standard DNAzol protocol, and (ii) mycobacterial DNA was detected by a typical TB-specific loop-mediated isothermal amplification method. Results and diagnostic test performance attests to the suitability of the proposed procedure. Full article

Test methods for efficacy assessment of antimicrobial coatings are not modelled on a hospital environment, and instead use high humidity (>90%) high temperature (37 °C), and no airflow. Therefore, an inoculum will not dry, resulting in an antimicrobial surface exhibiting prolonged antimicrobial activity, as moisture is critical to activity. Liquids will dry quicker in a hospital ward, resulting in a reduced antimicrobial efficacy compared to the existing test, rendering the test results artificially favourable to the antimicrobial claim of the product. This study aimed to assess how hospital room environmental conditions can affect the drying time of an inoculum, and to use this data to inform test parameters for antimicrobial efficacy testing based on the hospital ward. The drying time of different droplet sizes, in a range of environmental conditions likely found in a hospital ward, were recorded (n = 630), and used to create a model to inform users of the experimental conditions required to provide a drying time similar to what can be expected in the hospital ward. Drying time data demonstrated significant (p < 0.05) variance when humidity, temperature, and airflow were assessed. A mathematical model was created to select environmental conditions for in vitro antimicrobial efficacy testing. Drying time in different environmental conditions demonstrates that experimental set-ups affect the amount of time an inoculum stays wet, which in turn may affect the efficacy of an antimicrobial surface. This should be an important consideration for hospitals and other potential users, whilst future tests predict efficacy in the intended end-use environment. Full article

The accurate detection and quantification of pathogenic viruses in water is essential to understand and reduce the risk of human infection. This paper describes a two-step method suitable for concentrating viruses in water and wastewater samples. The method involves a tangential flow ultrafiltration step that reduces the sample volume of 1–10 L to approximately 50 mL, followed by secondary precipitation using polyethylene glycol 6000, which reduces the volume to 1–4 mL. For method validation, water samples were spiked with different concentrations of enteric viruses, and viral recovery in the concentrates exceeded 10% in all experiments. The method is suitable for water samples with high and low salinity and turbidity, allowing an accurate comparison of viral titers in a diverse range of water types. Furthermore, the method has the potential to concentrate other pathogens, e.g., bacteria or protozoa. Hence, the use of this method can improve the holistic assessment of risks associated with wastewater-contaminated environments. Full article

Several studies have shown the potential of using Ectomycorrhizal (ECM) fungi in conifer micropropagation to overcome the cessation of adventitious root development. In vitro inoculation promotes the re-growth of the root system induced previously by auxin treatments, facilitating acclimation and diminishing the losses of plants because of a weak root system that is incapable of water and nutrient absorption. During a series of mycorrhization experiments, cryostat and ultrafine cuts were used to study the morpho-histological transformation of the symbiotic roots. To obtain cryostat cuts from pine roots a method frequently used for animal tissue was adopted. Molecular methods allowed fungi identification in all the mycorrhization phases and in the acclimation of derived plants. Mycorrhizal-like-structures derived from in vitro culture and axenic liquid cultures of roots were microscopically analyzed and compare with mycorrhizal roots. Full article

Characterisation of peptides containing intact disulphide bonds (DSBs) via mass spectrometry is challenging. Our study demonstrates that the addition of aniline to alpha-cyano-4-hydroxycinnamic acid improves detection and fragmentation of complex DSB peptides by matrix-assisted laser desorption/ionization, tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS). This improved assignment will be a significant new tool when a simple screening to confirm the DSB existence is required. Full article

We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains (“mirror” products) using a template containing 8-oxoG. The misincorporation of dAMP in the “mirror” product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of “mirror” product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded “mirror” products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered. Full article

A method for the measurement of total mercury (T-Hg) in environmental samples using cold vapour atomic absorption spectrometry (CV AAS) has been validated yielding a dynamic range (0.04–10.00 μg/kg) and high certified reference material (CRM) recovery (>90%). The validation was carried out according to International Union of Pure and Applied Chemistry (IUPAC) validation and Eurachem Guides. A freeze-dried and homogenised sample was weighed and then digested using Suprapur acids (HNO₃, H₂SO₄, and HF) with potassium dichromate solution in a hot block digestion system. A calibration curve was constructed (R² > 0.999). Two CRMs (Marine Sediment Reference Material (PACS-3) and Trace Elements in Muscle Tissue (Trace Elements and Methylmercury in Mussel Tissue (NIST2976)) were utilised for quality assurance and control. The limit of quantification (LOQ) calculated as 0.04 µg/kg, and uncertainty (U) calculated as 2%. The obtained results showed the suitability of this method for direct mercury measurement in environmental samples. Additionally, the proficiency of this method was recognised by accreditation under the standard of International Organization for Standardization (ISO/IEC 17025:2017) for competence of testing and calibration laboratories. Full article

Neonatal screening for phenylketonuria (PKU, OMIM: 261600) was introduced at the end of the 1960s. We developed a rapid and simple molecular test for the most frequent phenylalanine hydroxylase (PAH, Gene ID: 5053) mutations. Using this method to detect the 18 most frequent mutations, it is possible to achieve a 75% detection rate in Italian population. The variants selected also reach a high detection rate in other populations, for example, 70% in southern Germany, 68% in western Germany, 76% in Denmark, 68% in Sweden, 63% in Poland, and 60% in Bulgaria. We successfully applied this confirmation test in neonatal screening for hyperphenylalaninemias using dried blood spots and obtained the genotype in approximately 48 h. The method was found to be suitable as second tier test in neonatal screening for hyperphenylalaninemias in neonates with a positive screening test. This test can also be useful for carrier screening because it can bypass the entire coding sequence and intron–exon boundaries sequencing, thereby overcoming the questions that this approach implies, such as new variant interpretations. Full article

The emergence in recent years of DNA editing technologies—Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors—have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis. Full article

The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells and make clonal lines hugely expands their utility as the insertion of clinically relevant mutations allows study of potentially every genetic disease affecting red blood cell development. Additionally, insertion of sequences encoding short protein tags such as Strep, FLAG and Myc permits study of protein behaviour in the normal and disease state. This approach is useful to augment the analysis of patient cells as large cell numbers are obtainable with the additional benefit that the need for specific antibodies may be circumvented. This approach is likely to lead to insights into disease mechanisms and provide reagents to allow drug discovery. HUDEP-2 cells provide a favourable alternative to the existing immortalised erythroleukemia lines as their karyotype is much less abnormal. These cells also provide sufficient material for a broad range of analyses as it is possible to generate in vitro-differentiated erythroblasts in numbers 4–7 fold higher than starting cell numbers within 9–12 days of culture. Here we describe an efficient, robust and reproducible plasmid-based methodology to introduce short (<20 bp) DNA sequences into the genome of HUDEP-2 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 Cas9 system combined with single-stranded oligodeoxynucleotide (ssODN) donors. This protocol produces genetically modified lines in ~30 days and could also be used to generate knock-out and knock-in mutations. Full article