Methods and Protocols — Open Access Journal

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

Bacteriophages (phages) are intensely investigated as non-antibiotic alternatives to circumvent antibiotic resistance development as well as last resort therapeutic options against antibiotic resistant bacteria. As part of gaining a better understanding of phages and to determine if phages harbor putative virulence factors, whole genome sequencing is used, for which good quality phage DNA is needed. Traditional phage DNA extraction methods are tedious and time consuming, requiring specialized equipment e.g., an ultra-centrifuge. Here, we describe a quick and simple method (under four hours) to extract DNA from double stranded DNA (dsDNA) phages at titers above 1.0 × 10¹⁰ plaque-forming units (PFU)/mL. This DNA was suitable for library preparation using the Nextera XT kit and sequencing on the Illumina MiSeq platform. Full article

Bisphenol A (BPA) is a widely used chemical in several consumer products and a well-studied environmental toxicant, and therefore, its accurate measurement is highly demanded. However, the co-presence of nanoparticles as an emerging class of contaminants could result in inaccurate determination of BPA due to binding of BPA onto nanoparticle surface. In this study, mass spectrometry (MS) was used to investigate desorption of BPA bound on the surface of titania (TiO₂) nanoparticles in water. Ammonium acetate, fluoride, formate, and hydroxide were evaluated as chemical agents for their desorption capabilities. The percentages of recovery, adsorption, and desorption were determined by this new method without requiring any prior separation of nanoparticles from BPA. MS analysis demonstrated the desorption of BPA by 10–20 mM of ammonium hydroxide for a mixture of 5 µg/mL BPA and 10 µg/mL TiO₂ nanoparticles, with a desorption efficiency of 72 ± 1%. Due to adsorption of BPA onto the nanoparticle surface that was inefficient for electrospray ionization, the resulting abundance of target ions could be reduced in the detection of BPA by mass spectrometry. As such, these findings collectively promise an accurate determination of the total BPA concentration in water whether it exists in the free or bound form. Efficient desorption of contaminants from the surface of nanoparticles would improve the accuracy of the contaminant analysis by mass spectrometry. Full article

Ripple Effect Mapping (REM) is an evaluation approach that has traditionally been used in community settings to visually map the impact of programming and community interventions. This manuscript utilizes the Community Capitals Framework (CCF) to inform REM and to better highlight the changes and impact between various levels of a community, following a childhood obesity prevention intervention. The addition of in-depth qualitative analyses makes this approach particularly useful for the evaluation of interventions with a research–community partnership focus. The objective of this study was to describe a CCF-informed REM approach with detailed protocol, training, and application to the community-based, childhood obesity prevention intervention, iCook 4-H, which targeted youth and adult pairs. This protocol includes the steps required to prepare for REM sessions of, ideally, six youth and adult pairs, one facilitator, and one or two evaluators/note takers. REM sessions typically begin with an icebreaker and appreciative inquiry activities that inform the REM mapping process that follows. In-depth qualitative analysis of the notes and map images captured during REM sessions ensure the rigor required for research-related interventions. Researchers, community members, and participants can use CCF-informed REM collectively as a robust evaluation tool to demonstrate, through visual mapping, the positive effects of community-partnered research programs. Full article

Pseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). As we noticed that there was not a single primer pair available in the literature suitable for direct sequencing of this gene, we decided to design a unique oligonucleotide pair and to set up a polymerase chain reaction (PCR) protocol to obtain a single amplicon for the entire Pseudomonas genus. Based on the available gyrB sequence from 148 Pseudomonas species, we identified highly conserved regions to design oligonucleotides without fully degenerate positions. We then set up cycling conditions for achieving high specificity and yield of the PCR protocol. Then, we showed that the amplicons produced with this procedure were appropriate for direct sequencing with both primers, obtaining more than 95% of amplicons coverage. Finally, we demonstrated that a PCR-RFLP (restriction fragment length polymorphism) approach served to differentiate among Pseudomonas species, and even between members of the same species. Full article

Ionic liquids (ILs) have advanced a variety of applications, including matrix-assisted laser desorption/ionization–mass spectrometry (MALDI–MS). ILs can be used as matrices and solvents for analyte extraction and separation prior to analysis using laser desorption/ionization–mass spectrometry (LDI–MS). Most ILs show high stability with negligible sublimation under vacuum, provide high ionization efficiency, can be used for qualitative and quantitative analyses with and without internal standards, show high reproducibility, form homogenous spots during sampling, and offer high solvation efficiency for a wide range of analytes. Ionic liquids can be used as solvents and pseudo-stationary phases for extraction and separation of a wide range of analytes, including proteins, peptides, lipids, carbohydrates, pathogenic bacteria, and small molecules. This review article summarizes the recent advances of ILs applications using MALDI–MS. The applications of ILs as matrices, solvents, and pseudo-stationary phases, are also reviewed. Full article

The ability of a photocatalyst to degrade a target pollutant is a commonly used method to assess its effectiveness for environmental applications, while ultraviolet-visible (UV-vis) spectroscopy and spectroscopic ellipsometry are conventional techniques for the estimation of a semiconductor band gap. In this work, an array of six light-emitting diodes (LEDs), characterized by different emission peaks between 470–370 nm and absorbed power of 3 W, was implemented into an existing standard testing apparatus for the testing of nitrogen oxides degradation in air. The abatement indexes, obtained under different LEDs irradiation, were firstly compared to the ones determined according the standard and, secondly, correlated with the measured LED emission spectrum, in order to estimate the photocatalyst band gap. Results suggest that this expeditious technique can be easily implemented into existing testing apparatus for the estimation of the band gap and for the appraisal of photocatalytic materials under realistic conditions. Full article

The prevalence of non-alcoholic fatty liver disease (NAFLD) and its advanced form, nonalcoholic steatohepatitis (NASH), is increasing, and as such its contribution to the development of hepatocellular carcinoma is also rising. NAFLD has been shown to influence the immune tumor microenvironment. Therefore, development of pre-clinical mouse models in the context of NAFLD are increasingly important. Here, we describe a mouse model designed to recapitulate the findings of NAFLD followed by rapid induction of orthotopic liver tumors with intrahepatic tumor injection. Additionally, we utilized bioluminescent imaging to monitor tumor growth and response to therapy. The development of one dominant tumor nodule allows precise separation of tumor and liver tissue. This is useful for immunotherapy studies as mononuclear cells from the tumor and the surrounding liver tissue can be analyzed separately. Full article

Immunofluorescence staining has become an essential tool in pathology and biomedical sciences to identify rare cells, cell–cell interactions, and submicroscopic cellular components. Many experimental settings, however, suffer from the fact that traditional widefield fluorescence microscopy is usually restricted to imaging three or four fluorophores only. Due to a lack of morphological information and a high detection limit, even flow cytometry—which is capable of staining 20 or more fluorophores at the same time—is limited in its applicability, especially in areas such as rare cell detection. Other advanced imaging approaches, such as confocal laser scanning microscopy and imaging flow cytometry, may be addressing these shortcomings, but in turn require sophisticated downstream data processing and high capital outlay. Here, we describe a new method and filter set-up to routinely employ up to seven fluorophores on a traditional widefield fluorescence microscope equipped with a standard high-pressure mercury light source. Quantification of crosstalk between channels and actual seven-color imaging of cancer cells spiked into leukocytes demonstrate that there is no need for digital compensation correction algorithms. Our set-up thus permits a detailed analysis of rare cell populations, co-localization of antigens, and cell morphology in a standard research or routine laboratory setting. Full article

The chick chorioallantoic membrane (CAM) is an extra-embryonic membrane, comprised of a high density of blood and lymphatic vessels. CAM has a dense capillary network and is commonly used to study in vivo angiogenesis and anti-angiogenesis in response to potential biomolecules and drugs. Most of the earlier reported CAM assays described the in-ovo method—where the viability of the embryo is higher, but accessibility to the CAM is limited. Ex-ovo CAM methods were previously described that employed shell-less cultures of chick embryos, but the low viability of embryos reduced the overall robustness of the angiogenesis assays. We described a method (named as cup-CAM method) which is more economical, has better accessibility and has significantly improved the viability of the embryo till advanced developmental stages. We could perform this simple yet useful experimentation with the common tools available in the laboratory. We successfully used the cup-CAM method for showing the paracrine effects of conditioned media from tumor cells, on the angiogenesis. This method can be used to assay the angiogenic potential of a drug or protein and to observe the embryonic development of the chick embryo and other related scientific applications. Full article

Qβ is a positive (+) single-stranded RNA bacteriophage covered by a 25 nm icosahedral shell. Qβ belongs to the family of Leviviridae and is found throughout the world (bacterial isolates and sewage). The genome of Qβ is about 4.2 kb, coding for four proteins. This genome is surrounded by 180 copies of coat proteins (capsomers) each comprised of 132 residues of amino acids. The other proteins, the subunit II (β) of a replicase, the maturation protein (A₂) and the read-through or minor coat protein (A₁), play a key role in phage infection. With the replicase protein, which lacks proofreading activity, as well as its short replication time, and high population size, Qβ phage has attractive features for in vitro evolution. The A₁ protein gene shares the same initiation codon with the coat protein gene and is produced during translation when the coat protein’s UGA stop codon triplet (about 400 nucleotides from the initiation) is suppressed by a low level of ribosome misincorporation of tryptophan. Thus, A₁ is termed the read-through protein. This RNA phage platform technology not only serves to display foreign peptides but is also exceptionally suited to address questions about in vitro evolution. The C-terminus of A₁ protein confers to this RNA phage platform an exceptional feature of not only a linker for foreign peptide to be displayed also a model for evolution. This platform was used to present a peptide library of the G-H loop of the capsid region P1 of the foot-and-mouth disease virus (FMDV) called VP1 protein. The library was exposed on the exterior surface of Qβ phages, evolved and selected with the monoclonal antibodies (mAbs) SD6 of the FMDV. These hybrid phages could principally be good candidates for FMDV vaccine development. Separately, the membrane proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) epitopes was fused with the A₁ proteins and exposed on the Qβ phage exterior surface. The engineered phages with MPER epitopes were recognized by anti-MPER specific antibodies. This system could be used to overcome the challenge of effective presentation of MPER to the immune system. A key portion of this linear epitope could be randomized and evolved with the Qβ system. Overall, antigens and epitopes of RNA viruses relevant to public health can be randomized, evolved and selected in pools using the proposed Qβ model to overcome their plasticity and the challenge of vaccine development. Major epitopes of a particular virus can be engineered or displayed on the Qβ phage surface and used for vaccine efficacy evaluation, thus avoiding the use of live viruses. Full article

Cas9 is a site-specific RNA-guided endonuclease (RGEN) that can be used for precise genome editing in various cell types from multiple species. Ribonucleoprotein (RNP) complexes, which contains the Cas9 protein in complex with a guide RNA, are sufficient for the precise editing of genomes in various cells. This DNA-free method is more specific in editing the target sites and there is no integration of foreign DNA into the genome. Also, there are ongoing studies into the interactions of Cas9 protein with modified guide RNAs, as well as structure-activity studies of Cas9 protein and its variants. All these investigations require highly pure Cas9 protein. A single-step metal affinity enrichment yielding impure Cas9 is the most common method of purification described. This is sufficient for many gene editing applications of this protein. However, to obtain Cas9 of higher purity, which might be essential for biophysical characterization, chemical modifications, and structural investigations, laborious multi-step protocols are employed. Here, we describe a two-step Cas9 purification protocol that uses metal affinity enrichment followed by cation exchange chromatography. This simple method can yield a milligram of highly pure Cas9 protein per liter of culture in a single day. Full article

CRISPR-Cas9 is revolutionizing the field of genome editing in non-model organisms. The robustness, ease of use, replicability and affordability of the technology has resulted in its widespread adoption among researchers. The African butterfly Bicyclus anynana is an emerging model lepidopteran species in the field of evo-devo, with a sequenced genome and amenable to germ line transformation. However, efficient genome editing tools to accelerate the pace of functional genetic research in this species have only recently become available with CRISPR-Cas9 technology. Here, we provide a detailed explanation of the CRISPR-Cas9 protocol we follow in the lab. The technique has been successfully implemented to knock-out genes associated with eyespot development and melanin pigmentation. Full article