Methods and Protocols — Open Access Journal

Lymphedema is a chronic skin disease that has many causes and leads to significant disfigurement and disability worldwide. Recommendations for lymphedema self-care vary by setting and the World Health Organization guidelines for people affected by lymphatic filariasis- and podoconiosis-related lymphedema are centered around a basic daily hygiene regimen. Research on cancer-related lymphedema in developed country settings suggests that deep-breathing exercises and self-massage can improve lymphedema status, but these exercises are not routinely taught to people affected by lymphedema in developing country settings. To determine if the activities proven in cancer-related lymphedema can improve outcomes for people affected by lymphatic filariasis- or podoconiosis-related lymphedema, an enhanced self-care protocol for lower limb lymphedema was developed and trialed in Nilphamari District in Bangladesh and Simada Woreda in Ethiopia. Enhanced self-care activities were chosen on the basis that they would not add financial burden to patients or their families and included recommendations to perform deep-breathing exercises and self-massage, drink clean water, and eat fresh fruits and vegetables. The enhanced-care protocol was developed in collaboration with implementing partners in both countries and may be applicable in other populations affected by lower-limb lymphedema. Trial methods and results will be submitted for peer reviewed publication. Current recommendations for lymphedema self-care may be less effective for people with more advanced disease and new or cross-cutting methods are needed to improve outcomes for these populations. Full article

Sphingolipid metabolism is an important process in sustaining the growth needs of rapidly dividing cancer cells. Enzymes that synthesize sphingolipids have become attractive targets in cancer pharmacology. Ceramide is a precursor for synthesizing sphingolipids such as sphingomyelin, sphingosine-1-phosphate, and glucosylceramide. Sphingomyelin synthase (SMS) is the enzyme that transfers a phosphatidylcholine to ceramide to generate sphingomyelin. To test the inhibition of SMS, scientists assess the buildup of ceramide in the cell, which is cytotoxic. Because ceramide is a small lipid molecule, there are limited tools like antibodies to detect its presence. Alternatively, designated machines for small-molecule separation coupled with mass spectrometry detection can be used; however, these can be cost-prohibitive. We used a commercially available NBD-ceramide to apply to human cancer cell lines in the presence or absence of a known SMS inhibitor, jaspine B. After short incubation times, we were able to collect cell lysates and using solvent extraction methods, run the cellular material on a thin-layer chromatography plate to determine the levels of intact fluorescently labeled ceramide. Brighter fluorescence on the TLC plate correlated to greater SMS inhibition. Small molecules can then be screened quantifiably to determine the biological impact of inhibiting the sphingolipid metabolism pathways involving ceramide. Full article

The use of alpha particles irradiation in clinical practice has gained interest in the past years, for example with the advance of radionuclide therapy. The lack of affordable and easily accessible irradiation systems to study the cell biological impact of alpha particles hampers broad investigation. Here we present a novel alpha particle irradiation set-up for uniform irradiation of cell cultures. By combining a small alpha emitting source and a computer-directed movement stage, we established a new alpha particle irradiation method allowing more advanced biological assays, including large-field local alpha particle irradiation and cell survival assays. In addition, this protocol uses cell culture on glass cover-slips which allows more advanced microscopy, such as super-resolution imaging, for in-depth analysis of the DNA damage caused by alpha particles. This novel irradiation set-up provides the possibility to perform reproducible, uniform and directed alpha particle irradiation to investigate the impact of alpha radiation on the cellular level. Full article

An accurate DNA damage response pathway is critical for the repair of DNA double-strand breaks. Repair may occur by homologous recombination, of which many different sub-pathways have been identified. Some recombination pathways are conservative, meaning that the chromosome sequences are preserved, and others are non-conservative, leading to some alteration of the DNA sequence. We describe an in vivo genetic assay to study non-conservative intra-chromosomal deletions at regions of non-tandem direct repeats in Schizosaccharomyces pombe. This assay can be used to study both spontaneous breaks arising during DNA replication and induced double-strand breaks created with the S. cerevisiae homothallic endonuclease (HO). The preliminary genetic validation of this assay shows that spontaneous breaks require rad52⁺ but not rad51⁺, while induced breaks require both genes, in agreement with previous studies. This assay will be useful in the field of DNA damage repair for studying mechanisms of intra-chromosomal deletions. Full article

Approaches are sought after to regulate ionotropic and chronotropic properties of the mammalian heart. Electrodes are commonly used for rapidly exciting cardiac tissue and resetting abnormal pacing. With the advent of optogenetics and the use of tissue-specific expression of light-activated channels, cardiac cells cannot only be excited but also inhibited with ion-selective conductance. As a proof of concept for the ability to slow down cardiac pacing, anion-conducting channelrhodopsins (GtACR1/2) and the anion pump halorhodopsin (eNpHR) were expressed in hearts of larval Drosophila and activated by light. Unlike body wall muscles in most animals, the equilibrium potential for Cl⁻ is more positive as compared to the resting membrane potential in larval Drosophila. As a consequence, upon activating the two forms of GtACR1 and 2 with low light intensity the heart rate increased, likely due to depolarization and opening of voltage-gated Ca²⁺ channels. However, with very intense light activation the heart rate ceases, which may be due to Cl^– shunting to the reversal potential for chloride. Activating eNpHR hyperpolarizes body wall and cardiac muscle in larval Drosophila and rapidly decreases heart rate. The decrease in heart rate is related to light intensity. Intense light activation of eNpHR stops the heart from beating, whereas lower intensities slowed the rate. Even with upregulation of the heart rate with serotonin, the pacing of the heart was slowed with light. Thus, regulation of the heart rate in Drosophila can be accomplished by activating anion-conducting channelrhodopsins using light. These approaches are demonstrated in a genetically amenable insect model. Full article

Elemental analysis of olive oils by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is challenging because of the high organic load in olive oil samples and the low analyte concentrations. However, conflicting operating procedures in the preparation of oils prior to analysis by ICP-MS have been reported to overcome these difficulties. This study compared three methods of inorganic elements’ extraction from olive oils: The two commonly used microwave-assisted, acid digestion, and liquid–liquid, ultrasound-assisted extraction methods; and an optimized method: The combined microwave digestion-evaporation. Overall, microwave digestion-based methods did not compare opportunely, and ultrasound-assisted extraction was found to provide the best accord between simplicity of use, detection limits and precision improvement. The detection limits were in the range of 0.3–160 µg·kg-¹, 0.012–190 µg·kg⁻¹ and 0.00061–1.5 µg·kg⁻¹, while repeatabilities were in the range of 5–21%, 5.4–99% and 5.1–40% for the microwave digestion, the combined digestion-evaporation and the ultrasound assisted extraction, respectively. The ultrasound-assisted extraction is therefore recommended as a preparation method for olive oils prior to analysis by ICP-MS. The broader range of elements that can be accurately detected is expected to help increase the discriminatory power and performance of geographical traceability models. Full article

The cancer stem cell (CSC) hypothesis suggests that tumors are sustained exclusively by a small population of the cells with stem cell properties. CSCs have been identified in most tumors and are responsible for the initiation, recurrence, and resistance of different cancers. In vitro CSC models will be of great help in revisiting the mechanism of cancer development, as well as the tumor microenvironment and the heterogeneity of cancer and metastasis. Our group recently described the generation of CSCs from induced pluripotent stem cells (iPSCs), which were reprogrammed from normal cells, and/or embryonic stem cells (ESCs). This procedure will improve the understanding of the essential niche involved in cancer initiation. The composition of this cancer-inducing niche, if identified, will let us know how normal cells convert to malignant in the body and how, in turn, cancer prevention could be achieved. Further, once developed, CSCs demonstrate the ability to differentiate into endothelial cells, cancer-associated fibroblasts, and other phenotypes establishing the CSC niche. These will be good materials for developing novel cancer treatments. In this protocol, we describe how to handle mouse iPSCs/ESCs and how to choose the critical time for starting the conversion into CSCs. This CSC generation protocol is essential for understanding the role of CSC in cancer initiation and progress. Full article

Flavanones [2-aryl-2,3-dihydrochromen-4(1H)ones] and 2-aryl-2,3-dihydroquinolin-4(1H)-ones are valuable precursors in the synthesis of important pharmacological scaffolds, so efficient methodologies towards their synthesis are important in the medicinal chemistry context. Their synthesis also involves theoretical concepts such as aldol condensation, isomerization, and catalysis that make it useful in an undergraduate organic chemistry laboratory. The use of both microwave irradiation as a source of energy to promote reactions and efficient catalysts are considered within green chemistry principles, mostly because the reaction yields are improved and reaction time decreased. In this paper, the efficiency of microwave irradiation use in the synthesis of chalcone derivatives and efficient catalyst systems to promote their isomerization into flavanones and 2-aryl-2,3-dihydroquinolin-4(1H)-ones is demonstrated. Full article

Available protocols for the synthesis of ketocyanine dyes precursor 2,5-bis((dimethylamino)methylene)cyclopentanone are not straightforward and the reported yields are low to moderate. The important feature in the synthesis of this product through organocatalyzed condensation of cyclopentanone and N,N-Dimethylformamide dimethyl acetal is the removal of methanol produced during the reaction. By studying the reaction profile, in particular the selectivity for the formation of mono- and bis-condensation products, a high yield of the desired product can be obtained through an operationally simple and solvent-free protocol. Full article

With the advancement of synthetic biology, the cell-free protein synthesis (CFPS) system has been receiving the spotlight as a versatile toolkit for engineering natural and unnatural biological systems. The CFPS system reassembles the materials necessary for transcription and translation and recreates the in vitro protein synthesis environment by escaping a physical living boundary. The cell extract plays an essential role in this in vitro format. Here, we propose a practical protocol and method for Escherichia coli-derived cell extract preparation and optimization, which can be easily applied to both commercially available and genomically engineered E. coli strains. The protocol includes: (1) The preparation step for cell growth and harvest, (2) the thorough step-by-step procedures for E. coli cell extract preparation including the cell wash and lysis, centrifugation, runoff reaction, and dialysis, (3) the preparation for the CFPS reaction components and, (4) the quantification of cell extract and cell-free synthesized protein. We anticipate that the protocol in this research will provide a simple preparation and optimization procedure of a highly active E. coli cell extract. Full article

A new home-made UV photochemical reactor (95 cm of irradiation zone) consisting of a 12 parallel quartz tubes flow reactor, PQT6 (95 cm under irradiation and an internal diameter of 0.6 cm) was assembled to perform photochemical transformations in continuous-flow. PQT6 was evaluated for the photoreaction of 1-allylpyridinium bromide (1a) to 6-allyl-6-azabicyclo[3.1.0]hex-3-en-2-ol (2a), in a continuous process. This technology provides reduced reaction times, continuous production of 2a, and a productivity of 129 mg h⁻¹, corresponding to 1.94 g of isolated 2a after 15 h of irradiation. Full article

All terrestrial organisms are subject to evolutionary pressures associated with natural sources of ionizing radiation (IR). The legacy of human-induced IR associated with energy, weapons production, medicine, and research has changed the distribution and magnitude of these evolutionary pressures. To date, no study has systematically examined the effects of environmentally relevant doses of radiation exposure across an organismal proteome. This void in knowledge has been due, in part, to technological deficiencies that have hampered quantifiable environmentally relevant IR doses and sensitive detection of proteomic responses. Here, we describe a protocol that addresses both needs, combining quantifiable IR delivery with a reliable method to yield proteomic comparisons of control and irradiated Medaka fish. Exposures were conducted at the Savannah River Ecology Laboratory (SREL, in Aiken, SC), where fish were subsequently dissected into three tissue sets (carcasses, organs and intestines) and frozen until analysis. Tissue proteins were extracted, resolved by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and each sample lane was divided into ten equal portions. Following in-gel tryptic digestion, peptides released from each gel portion were identified and quantified by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain the most complete, comparative study to date of proteomic responses to environmentally relevant doses of IR. This method provides a simple approach for use in ongoing epidemiologic studies of chronic exposure to environmentally relevant levels of IR and should also serve well in physiological, developmental, and toxicological studies. Full article

Wide-field temporal focused (WF-TeFo) two-photon microscopy allows for the simultaneous imaging of a large planar area, with a potential order of magnitude enhancement in the speed of volumetric imaging. To date, low repetition rate laser sources with over half a millijoule per pulse have been required in order to provide the high peak power densities for effective two-photon excitation over the large area. However, this configuration suffers from reduced signal intensity due to the low repetition rate, saturation effects due to increased excitation fluences, as well as faster photobleaching of the fluorescence probe. In contrast, with the recent advent of high repetition rate, high pulse energy laser systems could potentially provide the advantages of high repetition rate systems that are seen in traditional two-photon microscopes, while minimizing the negatives of high fluences in WF-TeFo setups to date. Here, we use a 100 microjoule/high repetition rate (50–100 kHz) laser system to investigate the performance of a WF-TeFo two-photon microscope. While using micro-beads as a sample, we demonstrate a proportionate increase in signal intensity with repetition rate, at no added cost in photobleaching. By decreasing pulse intensity, via a corresponding increase in repetition rate to maintain fluorescence signal intensity, we find that the photobleaching rate is reduced by ~98.4%. We then image live C. elegans at a high repetition rate for 25 min. as a proof-of-principle. Lastly, we identify the steady state temperature increase as the limiting process in further increasing the repetition rate, and we estimate that repetition rate in the range between 0.5 and 5 MHz is ideal for live imaging with a simple theoretical model. With new generation low-cost fiber laser systems offering high pulse energy/high repetition rates in what is essentially a turn-key solution, we anticipate increased adoption of this microscopy technique by the neuroscience community. Full article

This study was aimed to extract rosmarinic acid from Orthosiphon stamineus Benth. (Lamiaceae) in high yield. The mixture of chloroform–ethyl acetate (70:30) was chosen as the solvent system because rosmarinic acid gave the lowest solvation free energy in that solvent system based on the computational solubility prediction. The crude extract of the plant was fractionated by C18 reversed phase absorbent to recover rosmarinic acid. The content of rosmarinic acid was increased from 4.0% w/w to 6.7% w/w after fractionation. The radical scavenging activity of rosmarinic acid rich fraction (IC₅₀ = 38.3 μg/mL) was higher than the crude extract (IC₅₀ = 58.85 μg/mL) based on the DPPH assay. Several phytochemicals were also identified based on the detection of fragment ions of target compounds. Fractions 1 to 3 could be combined to be a rosmarinic acid rich fraction. Simultaneously, the combination of fractions 4 to 6 could obtain a plant fraction rich in rosmarinic acid, sinensetin and eupatorin, whereas fractions 7 to 9 could be combined as a sinensetin rich fraction. The preparation of known phytochemical profile of O. stamineus fraction is highly required for value added product formulation and pharmacological studies, particularly for anti-diabetes and kidney related diseases which had previously been reported attributed to this herbal plant. This is the first study using solvation free energy to predict the suitable solvent system for rosmarinic acid extraction from highly complex herbal sample using the technology of solid phase extraction. The use of solvation free energy simulation is convenient and reliable before wet experiments for time and cost saving. Full article

In eukaryotes, cellular functions are tightly controlled by diverse post-translational modifications (PTMs) of proteins. One such PTM affecting many proteins is the deimination of arginine to citrulline. This process, called citrullination is catalyzed by a group of hydrolases called protein arginine deiminases (PADs), of which five isoforms have been identified. Hypercitrullination, as a result of increased PAD expression or activity, is associated with autoimmune diseases e.g., rheumatoid arthritis, lupus, Alzheimer’s disease, ulcerative colitis, multiple sclerosis, and certain cancers. Three common single nucleotide polymorphisms (SNPs) in the PADI4 gene have been described, namely rs874881, rs11203366, and rs11203367, which are thought to affect PAD4 expression and activity. We here compared the suitability of four methods for the screening of SNPs in the PADI4 gene: (i) SYBR-green based real-time polymerase chain reaction followed by high resolution melting curve analysis (HRM-PCR); (ii) PCR followed by detection of restriction fragment length polymorphisms (PCR-RFLP); (iii) conventional tetra-primer amplification refractory mutation system PCR (ARMS-PCR); and (iv) real-time PCR based on the one-step ARMS-PCR. Of these, ARMS-PCR proved to be the most suitable method regarding handling, duration, and cost of experiments. Using the method with SYBR-green based real-time PCR reagents further diminished handling steps and thus potential sources of error. Full article

Transient Luciferase reporter assays are widely used in the study of gene regulation and intracellular cell signaling. In order to control for sample-to-sample variation in luminescence arising from variability in transfection efficiency and other sources, an internal control reporter is co-transfected with the experimental reporter. The luminescence of the experimental reporter is normalized against the control by taking the ratio of the two. Here we show that this method of normalization, “ratiometric”, performs poorly when the transfection efficiency is low and leads to biased estimates of relative activity. We propose an alternative methodology based on linear regression that is much better suited for the normalization of reporter data, especially when transfection efficiency is low. We compare the ratiometric method against three regression methods on both simulated and empirical data. Our results suggest that robust errors-in-variables (REIV) regression performs the best in normalizing Luciferase reporter data. We have made the R code for Luciferase data normalization using REIV available on GitHub. Full article

Zymography is a widely used electrophoretic method to determine proteolytic activities in samples from various sources. The method is based on copolymerizing a suitable protein substrate within a sodium dodecyl sulfate-polyacrylamide gel. Following electrophoretic separation of the protease containing samples and a suitable incubation period, degradation of the substrate can be visualized through staining with Coomassie blue. Sites of proteolysis become visible as white bands on a dark blue background. However, this staining protocol requires considerable amounts of ethanol and acetic acid to remove unbound dye molecules. In this report, we describe a new staining protocol using Ponceau S which offers substantial advantages in terms of assay usability and cost reduction, especially when performing large quantities of zymograms or in resource-limited settings. Fast and reproducible staining of zymograms with our protocol is demonstrated, and reliable quantitation of proteolytic activity in comparison to the standard Coomassie staining procedure is shown. Full article

Efficient reaction monitoring is crucial for data acquisition in kinetic and mechanistic studies. However, for conversions of nucleosides to their corresponding nucleobases, as observed in enzymatically catalyzed nucleoside phosphorylation reactions, the current analytical arsenal does not meet modern requirements regarding cost, speed of analysis and high throughput. Herein, we present a UV/Vis spectroscopy-based assay employing an algorithm for spectral unmixing in a 96-well plate format. The algorithm relies on fitting of reference spectra of nucleosides and their bases to experimental spectra and allows determination of nucleoside/nucleobase ratios in solution with high precision. The experimental procedure includes appropriate dilution of a sample into aqueous alkaline solution, transfer to a multi-well plate, measurement of a UV/Vis spectrum and subsequent in silico spectral unmixing. This enables data collection in a high-throughput fashion and reduces costs compared to state-of-the-art HPLC analyses by approximately 5-fold while being 20-fold faster and offering comparable precision. Additionally, the method is robust regarding dilution and sample transfer errors as it only considers spectral form and not absolute intensity. It can be applied to all natural nucleosides and nucleobases and even unnatural ones as demonstrated by several examples. Full article

High-quality in-depth imaging of three-dimensional samples remains a major challenge in modern microscopy. Selective plane illumination microscopy (SPIM) is a widely used technique that enables imaging of living tissues with subcellular resolution. However, scattering, absorption, and optical aberrations limit the depth at which useful imaging can be done. Adaptive optics (AOs) is a method capable of measuring and correcting aberrations in different kinds of fluorescence microscopes, thereby improving the performance of the optical system. We have incorporated a wavefront sensor adaptive optics scheme to SPIM (_WAOSPIM) to correct aberrations induced by optically-thick samples, such as multi-cellular tumor spheroids (MCTS). Two-photon fluorescence provides us with a tool to produce a weak non-linear guide star (NGS) in any region of the field of view. The faintness of NGS; however, led us to develop a high-sensitivity Shack–Hartmann wavefront sensor (SHWS). This paper describes this newly developed SHWS and shows the correction capabilities of _WAOSPIM using NGS in thick, inhomogeneous samples like MCTS. We report improvements of up to 79% for spatial frequencies corresponding to cellular and subcellular size features. Full article