Methods Protoc., Volume 1, Issue 4 (December 2018) – 12 articles

Cell culture is an excellent alternative for the maintenance of cell lines and to obtain quality chromosome preparations of fishes. However, this methodology is still little employed, mainly because of the difficulty of standardization of cell cultures. In this study, we describe a methodology for the rapid acquisition of cell lineages and mitotic chromosomes for cytogenetic studies of fish species from muscle tissue cells. Our methodology is based on the use of a gelatin film, which provides better adhesion of a large number of cells and appropriate conditions for multiplication. The cells of Astyanax altiparanae, used as an experimental model, with fibroblast-like morphology, showed rapid cellular proliferation, resulting in a great number of cells. Chromosomal preparations of cultured cells showed the diploid number of the species, 2n = 50 chromosomes, in 80% of the cells examined, with chromosomes intact and distended. Cell populations were cryopreserved and after being recovered, these cells maintained their proliferative capacity. The development of this methodology represents an innovation for the fish cytogenetics area and it may bring a significant contribution to the conservation and study of several groups due to the difficulty of obtaining good-quality chromosome preparations. Full article

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