Methods Protoc., Volume 3, Issue 1 (March 2020) – 24 articles

Trapped charge dating is a commonly used chronological tool in Earth Sciences and Archaeology. The two principle methods are luminescence dating and electron spin resonance. Both are based on stored energy produced by the absorption of natural radioactivity in common minerals such as quartz and feldspars and in some biological materials such as tooth enamel. Methodological developments in the last 20 years have substantially increased accuracy and precision. This essay introduces a compilation of papers that offers a taste of recent research into both method and application. Full article

In this paper, an analytical protocol was developed for the simultaneous determination of phthalates (di-methyl phthalate DMP, di-ethyl phthalate DEP, di-isobutyl phthalate DiBP, di-n-butyl phthalate DBP, bis-(2-ethylhexyl) phthalate DEHP, di-n-octyl phthalate DNOP) and bisphenol A (BPA). The extraction technique used was the ultrasound vortex assisted dispersive liquid–liquid microextraction (UVA-DLLME). The method involves analyte extraction using 75 µL of benzene and subsequent analysis by gas chromatography combined with ion trap mass spectrometry (GC-IT/MS). The method is sensitive, reliable, and reproducible with a limit of detection (LOD) below 13 ng g⁻¹ and limit of quantification (LOQ) below 22 ng g⁻¹ and the intra- and inter-day errors below 7.2 and 9.3, respectively. The method developed and validated was applied to six honey samples (i.e., four single-use commercial ones and two home-made ones. Some phthalates were found in the samples at concentrations below the specific migration limits (SMLs). Furthermore, the commercial samples were subjected to two different thermal stresses (24 h and 48 h at 40 °C) for evidence of the release of plastic from the containers. An increase in the phthalate concentrations was observed, especially during the first phase of the shock, but the levels were still within the limits of the regulations. Full article

The advent of next-generation sequencing has allowed for higher-throughput determination of which species live within a specific location. Here we establish that three analysis methods for estimating diversity within samples—namely, Operational Taxonomic Units; the newer Amplicon Sequence Variants; and a method commonly found in sequence analysis, minhash—are affected by various properties of these sequence data. Using simulations we show that the presence of Single Nucleotide Polymorphisms and the depth of coverage from each species affect the correlations between these approaches. Through this analysis, we provide insights which would affect the decisions on the application of each method. Specifically, the presence of sequence read errors and variability in sequence read coverage deferentially affects these processing methods. Full article

The assessment of minimum inhibitory concentration (MIC) is a conventional technique used for the screening of microbial resistance against antibiotics, biocides, and contaminants such as heavy metals. However, as part of our ongoing work, we have observed biases associated with using traditional liquid MIC method to screen microbial heavy metal resistance, including both bacterial and fungal strains. Specifically, the addition of uranium into synthetic media causes immediate precipitation prior to the initiation of microbial growth, thus hampering the optical density measurements, and the obtained MIC values are thus flawed and inaccurate. To address this discrepancy, we report the optimization and development of a serial-dilution-based MIC method conducted on solid growth media supplemented with uranium, which is more accurate, relative to the testing of MICs performed in liquid cultures. Notably, we report on the efficacy of this method to screen not only bacteria that are resistant to uranium but also demonstrate the successful application to yeast and fungal isolates, for their ability to resist uranium, is more accurate and sensitive relative to the liquid method. We believe that this newly developed method to screen heavy metal resistance, such as uranium, is far superior to the existing liquid MIC method and propose replacing the liquid assay with the solid plate MIC reported herein. Full article

The loess-paleosol archive from Mircea Vodă (Romania) represents one of the most studied sections in Europe. We are applying here the current state of the art luminescence dating protocols for revisiting the chronology of this section. Analysis were performed on fine (4–11 µm) and coarse (63–90 µm) quartz extracts using the single aliquot regenerative (SAR) optically stimulated luminescence (OSL) dating protocol. Laboratory generated SAR dose response curves in the high dose range (5 kGy for fine quartz and 2 kGy for coarse quartz) were investigated by employing a test dose of either 17 or 170 Gy. The results confirm the previously reported different saturation characteristics of the two quartz fractions, with no evident dependency of the equivalent dose (D_e) on the size of the test dose. The OSL SAR ages are discussed and compared to the previously obtained results on quartz and feldspars. The previous reports regarding the chronological discrepancy between the two quartz fractions are confirmed. However, while previous investigations on other sites concluded that this discrepancy appears only above equivalent doses of about 100 Gy, here fine grain quartz ages underestimate coarse quartz ages starting with equivalent doses as low as around 50 Gy. Full article

Karst caves host most European Paleolithic sites. Near the Eurasian-Arabian Plate convergence in the Caucasus’ Lower Chegem Formation, Saradj-Chuko Grotto (SCG), a lava tube, contains 16 geoarchaeologically distinct horizons yielding modern to laminar obsidian-rich Middle Paleolithic (MP) assemblages. Since electron spin resonance (ESR) can date MP teeth with 2–5% uncertainty, 40 sediment samples were analyzed by neutron activation analysis to measure volumetrically averaged sedimentary dose rates. SCG’s rhyolitic ignimbrite walls produce very acidic clay-rich conglomeratic silts that retain 16–24 wt% water today. In Layers 6A-6B, the most prolific MP layers, strongly decalcified bones hinder species identification, but large ungulates inhabited deciduous interglacial forests. Unlike in karst caves, most SCG’s layers had sedimentary U concentrations >4 ppm and Th, >12 ppm, but Layer 6B2 exceeded 20.8 ppm U, and Layer 7, >5 ppm Th. Such high concentrations emit dose rates averaging ~1.9–3.7 mGy/y, but locally up to 4.1–5.0 mGy/y. Within Layer 6, dose rate variations reflect bone occurrence, necessitating that several samples must be geochemically analyzed around each tooth to ensure age accuracy. Coupled with dentinal dose rates up to 3.7–4.5 mGy/y, SCG’s maximum datable ages likely averages ~500–800 ka. Full article

The particularly unique composition of the gut microbiota has the potential to influence the health or disease status of animal and human hosts. Altering the homeostasis of the host-bacteria could lead to changes in gut flora that result in disease or activation of a specific immunological response, which could explain the variations observed in patient responses to current therapies. A standardized model is crucial for studying the influence of the gut microbiota on therapeutic modalities. A step by step mouse model and sterility management system that compares a control strain of C57BL/6 mice to the established C57BL/6 germ-free (GF) strain has been developed. The GF BL/6 mouse phenotype is well established, and the anatomical differences between the GF and control mice were evident in this model. This method could be applied to research studies investigating the microbiome impact, the response to various therapies, or disease transfer via fecal transplants. A standardized sterility maintenance method is crucial in this context. Full article

Dating fluvial terraces has long been a challenge for geologists and geomorphologists, because terrace straths and treads are not usually directly dated. In this study, the formation ages of the Yellow River terraces in the Baode area in China were determined by dating fluvial deposits overlying bedrock straths using optically stimulated luminescence (OSL) dating techniques. Seven terraces (from the lowest terrace T1 to the highest terrace T7) in the study area were recognized, and they are characterized by thick fluvial terrace deposits overlaid by loess sediments. Twenty-five samples from nine terrace sections were dated to about 2–200 ka. The OSL ages (120–190 ka) of the fluvial samples from higher terraces (T3–T6) seem to be reliable based on their luminescence properties and stratigraphic consistency, but the geomorphologic and stratigraphic evidence show that these ages should be underestimated, because they are generally similar to those of the samples from the lower terrace (T2). The formation ages of the terrace straths and treads for the T1 terrace were deduced to be about 44 ka and 36 ka, respectively, based on the deposition rates of the fluvial terrace deposits, and the T2 terrace has the same strath and tread formation age of about 135 ka. The incision rate was calculated to be about 0.35 mm/ka for the past 135 ka, and the uplift rate pattern suggests that the Ordos Plateau behaves as a rigid block. Based on our previous investigations on the Yellow River terraces and the results in this study, we consider that the formation ages of terrace straths and treads calculated using deposition rates of terrace fluvial sediments can overcome problems associated with age underestimation or overestimation of strath or fill terraces based on the single age of one fluvial terrace sample. The implication is that, for accurate dating of terrace formation, terrace sections should be systematically sampled and dated. Full article

Our objective is to review the scientific literature on the use of antimicrobial lock therapy (ALT). To achieve this result, our scoping review will address the following seven key questions: (1) Who are the patients who will benefit from this technique? (2) What are the techniques utilized? (3) What are the settings in which the technique is performed? (4) When the technique is performed? (5) Why the technique is performed? (6) How the technique is performed? (7) In how much amount, of such technique performed? This review considers all studies published in full and in peer-reviewed journals, with no restrictions on language, on the year of publication and age of the participants. Both randomized controlled trials and observational studies will be included. This scoping review has been planned on a five-stage framework: 1. Identifying the review question; 2. identifying relevant studies; 3. study selection; 4. charting the data; 5. collating, summarizing, and reporting the results. It is conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Guidelines. The databases utilized will include MEDLINE via PubMed, EMBASE and Cochrane Central Register of Controlled Trials and Grey Literature. SCOPING REVIEW REGISTRATION: Open Science Framework https://osf.io/vphwm/. Full article

A simple method for the identification of brush-border membrane α-glucosidases is described. The proteins were first solubilized and separated in a gel under native, non-denaturing, conditions. The gel was then incubated in substrate solutions (maltose or sucrose), and the product (glucose) exposed in situ by the oxidation of o-dianisidine, which yields a brown-orange color. Nano-liquid chromatography coupled to mass spectrometry analyses of proteins (nano LC-MS/MS) present in the colored bands excised from the gels, was used to confirm the presence of the enzymes. The stain is inexpensive and the procedure permits testing several substrates in the same gel. Once enzymes are identified, their abundance, relative to that of other proteins in the brush border, can be semi-quantified using nano LC-MS/MS. Full article

Slow waves (SWs) are spatio-temporal patterns of cortical activity that occur both during natural sleep and anesthesia and are preserved across species. Even though electrophysiological recordings have been largely used to characterize brain states, they are limited in the spatial resolution and cannot target specific neuronal population. Recently, large-scale optical imaging techniques coupled with functional indicators overcame these restrictions, and new pipelines of analysis and novel approaches of SWs modelling are needed to extract relevant features of the spatio-temporal dynamics of SWs from these highly spatially resolved data-sets. Here we combined wide-field fluorescence microscopy and a transgenic mouse model expressing a calcium indicator (GCaMP6f) in excitatory neurons to study SW propagation over the meso-scale under ketamine anesthesia. We developed a versatile analysis pipeline to identify and quantify the spatio-temporal propagation of the SWs. Moreover, we designed a computational simulator based on a simple theoretical model, which takes into account the statistics of neuronal activity, the response of fluorescence proteins and the slow waves dynamics. The simulator was capable of synthesizing artificial signals that could reliably reproduce several features of the SWs observed in vivo, thus enabling a calibration tool for the analysis pipeline. Comparison of experimental and simulated data shows the robustness of the analysis tools and its potential to uncover mechanistic insights of the Slow Wave Activity (SWA). Full article

Early European plucked instruments have recently experienced a great revival, but a few aspects remain unknown (e.g., the gauge of gut strings). Here we report, for the first time, that the electron spin resonance (ESR) signal intensity of oxidized iron, Fe(III), from gut strings at g = 2 increases linearly with age within a few hundred years. The signal increase in the remaining old strings on early instruments can be used to judge if they are as old as or younger than the instrument. Obtaining the authenticity information of gut strings contributes to the revival of the old instruments and the music. Full article

Monitoring movements of larval Drosophila with electrical detection allows one to record the behaviors without the use of lights and cameras. This is a suitable technique when studying the use of light-sensitive proteins in optogenetic studies. Electrical measures are feasible to use in determining when a larva starts to move or continues to move after a light induced activation of channelrhodopsin. We have developed a technique using an electrical measure of the media as an index of larval movement. As a proof of concept, recordings with an infrared camera of the larval movement were simultaneous made with electrical measures. The two techniques parallel each other in their ability to index larval movements. Bright light-emitting diode (LED) lights used in optogenetic experiments tend to saturate the detectors of the camera unless filters are used and different filters maybe necessary depending on the LED spectrum and sensitivity of the camera. Impedance measures are independent of the type of LED or brightness. We also assessed the use of a non-solvent based glue (3M Vetbond) to hold larvae in place while measuring synaptic function of neuromuscular junctions, cardiac function and influence of modulators, or activation of light-sensitive channels. Full article

Bioluminescent tumor cell lines are used extensively in vivo to monitor tumor growth and metastasis but rarely used in vitro to follow tumor cell behavior. Tumor cell migration is frequently studied in vitro using transwell assays, however, current methods do not permit the co-incubation of tumor cells with different stromal cell types for analysis of the effects of intercellular cross-talk on tumor cell migration. We describe a novel migration assay using bioluminescent tumor cell lines that is rapid, accurate, and permits the study of the effects of tumor cell-stromal cell interactions on tumor cell migratory behavior. Full article

Glioblastoma is a heterogeneous glial cell malignancy with extremely high morbidity and mortality. Current treatment is limited and provide minimal therapeutic efficacy. Previous studies were reliant on cell lines that do not accurately reflect the heterogeneity of the glioma microenvironment. Developing reliable models of human glioblastoma is therefore essential. Direct culture of human brain tumours is often difficult and there is a limited number of protocols available. Hence, we have developed an effective method for the primary culture of human glioblastoma samples obtained during surgical resection. Culturing tumour tissue direct from human brain is advantageous in that cultures (1) more closely resemble true human disease, relative to the use of cell lines; (2) comprise a range of cellular components present in the natural tumour microenvironment; and (3) are free of added antibodies and reagents. Additionally, primary glioblastoma cultures are valuable in studies examining the effects of anti-cancer pharmaceuticals and therapeutic agents, and can be further used in live cell imaging, immunocytochemistry, flow cytometry and immunoassay experiments. Via this protocol, cells are maintained in supplemented medium at 37 °C (5% CO₂) and are expected to achieve sufficient confluency within 7 days of initial culture. Full article

Question prompt lists (QPL) are an instrument to promote patient participation in medical encounters by providing a set of questions patients can use during consultations. QPL have predominantly been examined in oncology. Less is known about their use in other contexts. Therefore, we plan to conduct a scoping review to provide an overview of the fields of healthcare in which QPL have been developed and evaluated. MEDLINE/PUBMED, PSYCINFO, PSYNDEX, WEB OF SCIENCE, and CINAHL will be systematically searched. Primary studies from different healthcare contexts that address the following participants/target groups will be included: persons with an acute, chronic, or recurring health condition other than cancer; healthy persons in non-oncological primary preventive measures. There will be no restrictions in terms of study design, sample size, or outcomes. However, only published studies will be included. Studies that were published in English and German between 1990 and 2019 will be examined. Two independent reviewers will apply defined inclusion/exclusion criteria and determine study eligibility in the review process guided by the PRISMA statement. Full article

Ethyl ferulate was transesterified with Enova Oil (a soy-based vegetable oil containing 80–85% diacylglycerol) using Novozym 435 at 60 °C. The resultant feruloylated vegetable oil reaction product produced a precipitate (96.4 g, 4.02 wt%) after 7 d of standing at room temperature. Preliminary characterization of the precipitate identified the natural phenylpropenoids 1,3-diferuloyl-sn-glycerol (F₂G) and 1-feruloyl-sn-glycerol (FG) as the major components. A flash chromatography method was developed and optimized (e.g., mass of sample load, flow rate, binary solvent gradient slope, and separation run length) using a binary gradient of hexane and acetone mobile phase and silica gel stationary phase to separate and isolate F₂G and FG. The optimized parameters afforded F₂G (1.188 ± 0.052 g, 39.6 ± 1.7%) and FG (0.313 ± 0.038 g, 10.4 ± 1.3%) from 3.0 g of the transesterification precipitate, n = 10 trials. Overall, all flash chromatography separations combined, F₂G (39.1 g, 40.6%) and FG (9.4 g, 9.8%) were isolated in a combined yield of 48.5 g (51.4%), relative to the 96.4 g of transesterification precipitate collected. The optimized flash chromatography method was a necessary improvement over previously reported preparative HPLC and column chromatography methods used to purify milligram to low gram quantities of F₂G and FG to be able to process ~100 g of material in a timely, efficient manner. Full article

Compared to quartz, the infrared stimulated luminescence (IRSL) of K-feldspar saturates at higher dose, which has great potential for extending the dating limit. However, dating applications with K-feldspar has been hampered due to anomalous fading of the IRSL signal. The post-IR IRSL (pIRIR) signal of K-feldspar stimulated at a higher temperature after a prior low-temperature IR stimulation has significantly lower fading rate. Different dating protocols have been proposed with the pIRIR signals and successful dating applications have been made. In this study, we review the development of various pIRIR dating protocols, and compare their performance in estimating the equivalent dose (D_e). Standard growth curves (SGCs) of the pIRIR signals of K-feldspar are introduced. Single-grain K-feldspar pIRIR dating is presented and the existing problems are discussed. Full article

Finding the source or provenance of quartz grains occurring in a specific location allows us to constrain their transport pathway, which is crucial information to solve diverse problems in geosciences and related fields. The optically stimulated luminescence (OSL) sensitivity (light intensity per unit mass per unit radiation dose) has a high capacity for discrimination of quartz sediment grains and represents a promising technique for provenance analysis. In this study, we tested the use of quartz OSL sensitivity (ultraviolet emission) measured under different preheating temperatures and with blue light stimulation at room temperature (~20 °C) for sediment provenance analysis. Quartz OSL sensitivity measured at 20 °C is positively correlated with the sensitivity of an OSL signal measured using procedures (preheat at 190 °C for 10 s, blue stimulation at 125 °C and initial 1 s of light emission) to increase the contribution of the fast OSL component, which has been successfully applied for sediment provenance analysis. The higher OSL signal intensity measured without preheating and with light stimulation at room temperature allows the use of lower given doses, thus reducing measurement time. Additionally, the OSL sensitivity measured at 20 °C in polymineral silt samples of a marine sediment core is also suitable for provenance analysis, as demonstrated by comparison with other independent proxies. OSL signals obtained through light stimulation at room temperature have thus the potential to considerably expand measurement possibilities, including in situ measurements using portable OSL readers. Full article

The colorful wings of butterflies are emerging as model systems for evolutionary and developmental studies. Some of these studies focus on localizing gene transcripts and proteins in wings at the larval and pupal stages using techniques such as immunostaining and in situ hybridization. Other studies quantify mRNA expression levels or identify regions of open chromatin that are bound by proteins at different stages of wing development. All these techniques require dissection of the wings from the animal but a detailed video protocol describing this procedure has not been available until now. Here, we present a written and accompanying video protocol where we describe the tools and the method we use to remove the larval and pupal wings of the African Squinting Bush Brown butterfly Bicyclus anynana. This protocol should be easy to adapt to other species. Full article

Female cyclists are prone to a variety of injuries and illnesses that occur as a result of prolonged contact with a bicycle saddle. Saddle sores are a range of skin ailments on the buttocks, genitals and inner thigh that result from a combination of friction, heat, pressure, moisture and bacteria in the saddle area. Whilst saddle sores are reportedly common, for some cyclists, the condition may cause only mild discomfort. However, for female competitive cyclists, the condition can be an ongoing source of pain and illness affecting participation and performance in the sport. Despite many online sources for health information and products for saddle sores, it is unknown what empirical evidence exists for the prevalence and severity of saddle sores, and for the effectiveness of prevention and treatment methods. This paper outlines the protocol for a scoping review, which aims to describe the empirical evidence for the prevalence, prevention and treatment of saddle sores among female competitive cyclists. Ethics approval has been obtained for this study from Curtin University’s Human Research Ethics Committee no: HRE2019-0120. The findings from this study will contribute to the literature for injury in female sport. Full article

We propose an improved earlier described “mirror” method for detecting in cell nuclear extracts mutations that arise in DNA during its replication due to the misincorporation of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). This method is based on the synthesis of a complementary chain (“mirror”) by nuclear extracts of different mice organs on a template containing 8-oxoG and dideoxycytidine residue (ddC) at the 3′‑end. The “mirror” was amplified by PCR using primers part of which was non-complementary to the template. It allowed obtaining the “framed mirror” products. The misincorporation of dAMP in “framed mirror” products forms an EcoRI restriction site. The restriction analysis of double-stranded “framed mirror” products allows a quantification of the mutation frequency in nuclear extracts. The data obtained show that the mutagenic potential of 8-oxoG markedly varied in different organs of adult mice and embryos. Full article

We studied the characteristics of the optically stimulated luminescence (OSL) signal of single-grain quartz from three sites in China, Italy, and Libya, including the brightness, decay curve and dose response curve (DRC) shapes, recuperation, and reproducibility. We demonstrate the large variation in OSL behaviors for individual quartz grains of different samples from different regions, and show that recuperation, sensitivity change, and reproducibility are independent of the brightness and decay curve shape of the OSL signals. The single-grain DRCs can be divided into at least eight groups with different characteristic saturation doses (D₀), and a standardized growth curve (SGC) can be established for each of the DRC groups. There is no distinctive difference in the shape of OSL decay curves among different DRC groups, but samples from different regions have a difference in the OSL sensitivities and decay shapes for different groups. Many of the quartz grains have low D₀ values (30–50 Gy), and more than 99% of the grains have D₀ values of <200 Gy. Our results raise caution against the dating of samples with equivalent dose values higher than 100 Gy, if there are many low-D₀ and ‘saturated’ grains. Full article

In order to increase the metabolic activity of human hepatocytes and liver cancer cell lines, many approaches have been reported in recent years. The metabolic activity could be increased mainly by cultivating the cells in 3D systems or co-cultures (with other cell lines). However, if the system becomes more complex, it gets more difficult to quantify the number of cells (e.g., on a 3D matrix). Until now, it has been impossible to quantify different cell types individually in 3D co-culture systems. Therefore, we developed a PCR-based method that allows the quantification of HepG2 cells and 3T3-J2 cells separately in a 3D scaffold culture. Moreover, our results show that this method allows better comparability between 2D and 3D cultures in comparison to the often-used approaches based on metabolic activity measurements, such as the conversion of resazurin. Full article