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The T1w/T2w ratio is a novel magnetic resonance imaging (MRI) measure that is thought to be sensitive to cortical myelin. Using this novel measure requires developing novel pipelines for the data quality assurance, data analysis, and validation of the findings in order to apply the T1w/T2w ratio for classification of disorders associated with the changes in the myelin levels. In this article, we provide a detailed description of such a pipeline as well as the reference to the scripts used in our recent report that applied the T1w/T2w ratio and machine learning to classify individuals with depressive disorders from healthy controls., Graphical abstract Image, graphical abstract

The semenogelin 1 protein is secreted in the seminal vesicles. After ejaculation it is split into small peptide fragments using internal proteases. It was shown that the fragments SEM1(45-107), SEM1(49-107), SEM1(68-107) (SEM1(86-107) form amyloid fibrils, which increase the possibility of HIV infection. The article presents a protocol for the synthesis and purification of a 15N, 13C-labeled SEM1(68–107) peptide for further structural studies by high-resolution NMR spectroscopy. The work describes cloning, expression of fusion protein GB1-SEM1(68-107) in E.coli, its purification, removal of GB1 and purification of SEM1(68-107). The purity of SEM1(68-107) samples on each purification steps was evaluated by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) and tricine-SDS-PAGE. The developed protocol allows to obtain SEM1(68-107) peptide for NMR studies (using 3D experiments), instead of costly solid-phase synthesis., Graphical abstract Image, graphical abstract

Predisposition for a high alcohol intake and the impact of alcohol-abstinence-relapse may be reliable experimentally performed in conventional adult rat lines if animals received juvenile exposure to alcohol (e.g., by forced consumption) and selecting those individuals with high basal anxiety levels during juvenile periods. Importantly, a forced alcohol consumption phase must be followed by an imposed withdrawal period to form an exposure-abstinence cycle (at least two cycles are required) which allow to obtain animals with notorious alcohol relapses. The easier way to test alcohol relapses is through voluntary ethanol intake models. On the other hand, the anxiety classification may be performance by classical paradigms such as an elevated plus maze test, defensive burying behavior test or any other. Here, we provide a step-by-step protocol description to detect higher alcohol consumers animals from male Wistar rats. This protocol should be especially useful for those interested in studying the participation of specific brain nucleus [e.g., periaqueductal gray (PAG)] and/or the neurotransmitters involved [e.g., neuropeptide Y (NPY)] in the alcohol intake phenomena if it is combined with stereotaxic surgery. However, every administration route of treatments or experimental design is appropriate; the limit is the own imagination, and the resources., Graphical abstract Image, graphical abstract

At least three species of Pantoea are responsible for bacterial blight disease and grain discoloration of rice in Sub-Saharan Africa. Thus, measures need to be taken to limit the pathogens' dispersion and robust diagnostic tools are required for rapid and cheap diagnosis in the field as well as for routine seed certification or control. Therefore, several diagnostic tools such as simplex and multiplex PCR schemes and a semi-selective medium have been developed and are being used. However, the use of these tools is time-consuming, expensive and therefore limited to laboratories that can afford the chemicals. We have therefore developed two isothermal loop amplification (LAMP) protocols, one of which detects all Pantoea species in the genus and another one that is specific for P. ananatis.•The novel LAMP assays allow rapid and sensitive detection of these bacteria.•They will help plant protection services in routine field and laboratory tests especially for monitoring the phytosanitary status of rice seeds., Graphical abstract Image, graphical abstract

Single cardiomyocytes are widely used for investigations of the cellular and molecular mechanisms of regulation and modulation of cardiac performance. Intact cardiomyocytes allow one to study in detail cell function avoiding the effects of extracellular matrix and neighboring cells. The most established protocols of cardiomyocyte isolation are based on the isolated heart perfusion using a Langendorff-apparatus or on intraventricular perfusion using a syringe. However, the yield of single cardiomyocytes obtained by these methods may be low due to the cell injury following non-uniform enzyme digestion of connective tissue in different heart chambers. Moreover, isolation of atrial cardiomyocytes is challenging because of their small size and complex geometric shape. Here we present a new protocol for simultaneous isolation of high quality cardiomyocytes from the atria, ventricular free walls and interventricular septum. The protocol is based on the combination of the Langendorff perfusion method with the intraventricular and intra-atrial injection technique taking into account the collagen content variation between the different heart chambers. Obtained cells demonstrate rod-shaped morphology, a clear and regular sarcomere striation pattern and rat-specific frequency-dependence of contraction and calcium transient parameters. Our protocol provides gentle cell isolation that increases the yield of single cardiomyocytes suitable for biophysical researches ., Graphical abstract Image, graphical abstract

Here, we describe the optimization and validation of five quantitative PCR (qPCR) assays by employing the SYBRGreen chemistry paired with melting curve analysis to detect and quantify clinically relevant antimicrobial resistance genes (ARGs) (i.e. ermB, blaCTXM1-like, blaCMY-2, qnrA and qnrS) from environmental samples (i.e. soil and manure). These five protocols accurately detected and quantified the aforementioned ARGs in complex environmental matrices and represent useful tools for both diagnostic and monitoring activities of resistant bacteria and ARGs into the environment., Graphical abstract Image, graphical abstract

Highlights • Following the step by step described in this protocol is it is possible to measure long-distance electrical signaling induced by several stimuli, including wounding, current application, irrigation, and others. • Extracellular electrical signal measurements were associated to gas exchange measurements using an infra-red gas analyzer (IRGA, model Li-6400, Li-Cor) and turgor pressure measurements using a patch clamp pressure probe (ZIM-probe, YARA ZIM-plant Technology). • The technique is interesting to study electrical signaling in association with other physiological parameters in plants. • We present results that validate the protocol and discuss the troubleshooting that may compromise measurements and data collection., We present a detailed protocol for measuring extracellular electrical signals in plants using the electrode insertion technique. Using this approach, it is possible to measure long-distance electrical signaling induced by several stimuli, including wounding, current application, irrigation, burning, and others. Additionally, we describe how to associate gas exchange measurements using an infra-red gas analyzer (IRGA, Model Li-6400, Li-Cor) and turgor pressure measurements using a patch clamp pressure probe (ZIM-probe, YARA ZIM-plant Technology) to measure extracellular electrical signals. • The method requires a complete electrical circuit that includes a measuring device (amplifier and voltmeter) and electrodes that provide a contact between the biological material and the equipment. • The infra-red gas analyzer (IRGA), needs to be grounded because it is an important source of noise for electrophysiological measurements. • The ZIM-probe did not cause any interference in electrical signal measure. • Our approach is useful for plant physiologists wishing to implement the technique of measuring electrical signals in plants, in association with other parameters of plant physiology. In addition, our text was written for agricultural and biological scientists who are not electronics specialists., Graphical abstract Image, graphical abstract

Hydrochar, as an energy-lean solid waste, is generated from an advanced biofuel conversion technique hydrothermal liquefaction (HTL) and always leads to environmental pollution without appropriate disposal. In this study, HTL-derived hydrochar is recycled and prepared as adsorbent used for Pb(Ⅱ) removal from wastewater. As the original porous structure of hydrochar is masked by oily volatiles remained after HTL, two types of oil-removal pretreatment (Soxhlet extraction and CO2 activation) are explored. The result shows that CO2 activation significantly enhances the adsorption capacity of Pb(Ⅱ), and the maximum adsorption capacity is 12.88 mg g−1, as evaluated using Langmuir adsorption model. Further, apart from oily volatiles, most inorganic compounds derived from wastewater-grown algae is enriched in hydrochar, causing a smaller surface area of hydrochar. An ash-removal alkali treatment following CO2 activation is introduced to dramatically increase the adsorption capacity to 25.00 mg g−1 with an extremely low Pb(II) equilibrium concentration of 5.1×10-4 mg L−1, which is much lower than the maximum level of Pb concentration in drinking water (set by World Health Organization). This work introduces an approach to reuse HTL-hydrochar as an inexpensive adsorbent in Pb-contaminated water treatment, which not only provides another possible renewable adsorbent candidate applied in the field of lead adsorption, but also finds an alternative route to reduce solid waste effluent from HTL process., Graphical abstract Image, graphical abstract

It was hypothesized that executing repeated maximum apnea efforts would improve performance in a subsequent time to exhaustion test. Indeed, in young moderately fit male subjects without former experience in apnea has been shown that five repeated apnea maximal efforts with face immersion in cold water (PEA) have advantageous effect to consecutive performance in a time to exhaustion ride without being further affected by apnea training of two weeks. So, in the current article, we describe, in details, the protocol procedure and the technical steps of the five maximum-repeated apnea maneuvers prior to a middle-distance racing in order to improve performance, from our previous relevant published research., Graphical abstract Image, graphical abstract

Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue malignant tumor. Treatment of RMS usually includes primary tumor resection along with systemic chemotherapy. Two-dimensional (2D) cell culture systems and animal models have been extensively used for investigating the potential efficacy of new RMS treatments. However, RMS cells behave differently in 2D culture than in vivo, which has recently inspired the adoption of three-dimensional (3D) culture environments. In the current paper, we will describe the detailed methodology we have developed for fabricating a 3D engineered model to study alveolar RMS (ARMS) in vitro. This model consists of a thermally cross-linked collagen disk laden with RMS cells that mimics the structural and bio-chemical aspects of the tumor extracellular matrix (ECM). This process is highly reproducible and produces a 3D engineered model that can be used to analyze the cytotoxicity and autophagy induction of drugs on ARMS cells. The most improtant bullet points are as following:•We fabricated 3D model of ARMS.•The current ARMS 3D model can be used for screening of chemotherapy drugs.•We developed methods to detect apoptosis and autophagy in ARMS 3D model to detect the mechansims of chemotherapy agents., Graphical abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue malignant tumor. Despite numerous clinical trials that have investigated the efficacy of multidrug chemotherapy regimens, outcomes for high-risk RMS patients are still poor which has inspired research into new targeted therapies that molecularly target malignant RMS cells. In order to evaluate such therapies in vitro in a realistic tumor microenvironment, we have bioengineered a cell-laden 3D tissue-engineered model with uniform geometry and an uncomplicated and reproducible biofabrication process. The transparent collagen-I hydrogel promotes cell attachment and growth and allows for analysis of the cellular response to drugs through brightfield microscopy, immunocytochemistry, and live/dead viability assays.Image, graphical abstract

The number of studies reporting hormetic responses is rapidly increasing, and quantitative evaluations are needed to improve the understanding of hormetic dose responses. However, there is no standardized methodology to estimate the no-observed-adverse-effect-level (NOAEL) of hormetic dose-response relationships developed using data mined from the published literature. Here, we propose a protocol that can be followed to estimate NOAEL, a process that is illustrated using a specific example. This protocol can be used for maintaining a mutual language (since NOAEL can be defined in different ways), permitting comparisons among different studies, and facilitating cumulative science., Graphical abstract Image, graphical abstract

Single unit recording has an important application in neuroscience, especially in the vestibular system such as visual stabilization, posture maintenance, spatial orientation and cognition. However, single unit recording conducted in living animals is a demanding technique and non-ideal mechanical stability between the recording location of nerve tissues and the tip of microelectrode always results in failure to obtain successful recordings in the vestibular system. In order to improve the mechanical stability during single unit recording, we constructed a novel head fixation method based on skull cap. This article describes in detail how to construct this novel head fixation. Following the step-by-step procedure mentioned in this article will provide a high-quality mechanical stability for single unit recording in the vestibular system, allowing us to successfully record the nonlinear neural dynamic response over a big magnitude motion stimulation. This improvement of head fixation contributes to the in-depth understanding of the vestibular system., Graphical abstract Image, graphical abstract

We present concise results of method validation for trehalose quantitation by LC-MS/MS in spruce ectomycorrhizal roots in order to describe spruce health status, mainly in connection to contamination by a pathogenic fungus, Gemmamyces piceae. The procedure is based on Rogatsky et al. (2005) developed for human plasma. We found out that the best extraction yield was achieved with 80% methanol/water (v/v) solution and optimal extraction temperature was set between 50-60°C. In contrast to previous papers, we minimized the activity of trehalase enzyme by putting root samples into liquid N2 immediately after root excavation, followed by freeze-drying in order to stop trehalase activity. Higher content of trehalose was recorded in healthy trees, confirming the idea that ectomycorrhiza plays a significant role in plant-pathogen interactions., Graphical abstract Image, graphical abstract

To study the biological function of uncharacterized proteins, specific antibodies are in high demand. However, the production of desirable antibodies such as highly specific or high affinity is not always successful. Furthermore, even if commercially available antibodies exist, the cost, quality, and accessibility often differ from country to country. In comparison, epitope tags are reliable and economical options since good antibodies against major epitope tags are commercially available. Although exogenously expressed epitope-tagged protein appears as a timely method, the excessive protein production may not faithfully recapitulate its biology. Thanks to the recent advances in genome editing by CRISPR-Cas9, HDR-mediated endogenous protein tagging has become an accessible approach for many labs. However, currently the synthesis of long (>100 bp), chemically modified oligos can be time-consuming and costly. To develop a reliable, simple, and cost-effective epitope-tagging method that requires minimal materials and apparatus, we focus on an approach utilizing two non-chemically modified shorter-annealed oligos (semi-dsODNs) mediated HDR for epitope tags insertion. We also use a cationic lipid chemical, polyethyleneimine (PEI), for plasmid delivery to minimize the cost and materials used while a considerable success rate could be achieved. • This protocol provides a more economical way to generate CRISPR-Cas9 mediated gene knock-in. • This protocol provides a simplified design of semi-dsODN without chemical modification on the oligos. • This protocol provides a simplified experimental procedure. In vitro assembled Cas9 complex and electroporation are not required., Graphical abstract Image, graphical abstract

Bioprinting is a rapidly expanding technology with the ability to fabricate in vitro three-dimensional (3D) tissues in a layer-by-layer manner to ultimately produce a living tissue which physiologically resembles native in vivo tissue functionality. Unfortunately, large costs associated with commercially available bioprinters severely limit access to the technology. We investigated the potential for modifying a low-cost commercially available RepRap Prusa iteration 3 (i3) 3D printer with an open-source syringe-housed microextrusion print-head unit (universal paste extruder by Richard Horne, RichRap), that allowed for controlled deposition of cell-laden bioinks and Freeform Reversible Embedding of Suspended Hydrogels (FRESH) method-based printing., Graphical abstract Image, graphical abstract

Highlights • TRI reagent modified protocol for RNA isolation is cost-effective compared to the kit method. • The Quality and quantity of isolated RNA is better in the TRI reagent modified protocol compared to the kit method. • The TRI reagent modified procedure has fewer steps than the kit method without requiring any additional training or time., Approximately 93% of the human genome is translated into RNAs, of which only 2% code for proteins and the rest 98% are noncoding RNAs. Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs of > 200 nucleotides length that are emerging as novel players in the field of cancer diagnostics or prognostics. Recently, lncRNAs are known to be associated with oral squamous cell carcinomas (OSCC). The demonstration of stable lncRNA has been a challenge in formalin-fixed paraffin-embedded tissues (FFPE). The survivability and expression level of lncRNA in FFPE tissues compared with fresh tissues is not well documented in the literature. Hence, we designed the current pilot study with the main aim to optimize modified TRI (Total RNA isolation) reagent RNA isolation protocol to identify the lncRNA expression in archived FFPE tissues of OSCC in comparison to the standard RNA isolation kit method. The findings of our study demonstrated that the RNA quantity and quality were comparatively better with the optimized TRI reagent modified protocol than the standard RNA isolation kit method. Furthermore, ct (cycle threshold) values after reverse-transcription and qRT-PCR (Quantitative Real time PCR) were comparable and almost equal in both the methods for normal mucosa (control) and OSCC samples., Graphical abstract Image, graphical abstract

Highlights • A new method for microplastic extraction from lake sediments is proposed. • Potassium formate was applied for density separation. • Repeated wet peroxide oxidation was applied to digest organic matter. • Quality control procedures are proposed. • The effectiveness of the microplastic extraction with potassium formate is 98.0 ± 2%., In recent years, microplastics (MPs) contamination has been emphasized as one of the most global ecological problems. Bottom sediments were identified as one of the major sinks of MPs, while river-lake systems - as the main transport routs of MPs into the coastal seas and the World Ocean. Thus, the MPs determination in freshwater sediments is an important task for identifying sedimentation processes and contamination sources. Density separation is commonly used to extract anthropogenic particles from bottom sediments. The proposed method is dedicated to determining the abundance of MPs in freshwater organic-rich sediments. Repeated wet peroxide oxidation was applied to digest organic matter that can hamper the microplastic determination in a sample. Potassium formate with a density 1.5 g/mL was used as a separation solution. This method is intended to identify various types of MPs (fibers, films, fragments and beads) ranging in size from 0.174 to 5 mm. The effectiveness of the MPs extraction with potassium formate was 98.0 ± 2 % (р = 0.05; n = 16), while the extraction of MPs when analyzed under a microscope was 92.0 ± 4% (р = 0.05; n = 20)., Graphical abstract Image, graphical abstract

Noncoding RNAs (ncRNAs) comprise a class of versatile transcripts that are highly involved in the regulation of a wide range of biological processes. Functional long ncRNAs (> 200 nts in length) often adopt secondary structures that arise co-transcriptionally. To maintain the secondary structure elements as well as preparation homogeneity of such transcripts, native-like conditions should be maintained throughout the in vitro synthesis, purification and chemical tagging processes. In this optimized protocol, we describe a simple method for obtaining homogenous samples followed by chemically tagging the 3′ termini of natively-purified structured ncRNA domains that are longer than 200 nts. This protocol replaces traditional hazardous radioactive labeling with fluorescence tagging, and eliminates laborious and time consuming RNA purification and concentration steps and replaces them with straightforward recovery of RNA through centrifugal filtration, preserving the homogeneity and mono-dispersion of the preparations. The protocol provides:•An integrative, simple and straightforward approach for synthesis, purification and labeling of structured ncRNAs whilst maintaining their secondary structure intact.•Replacing hazardous, laborious and time-consuming radioactive labeling of RNA with much simpler fluorescence tagging, thereby facilitating potential downstream applications such as electrophoretic mobility shift assay (EMSA).•A versatile protocol that could be applicable to a wide-range of chemical tags and in principle could be used to label DNA or RNA., Graphical abstract Image, graphical abstract

Graphical abstract, This study was carried out to investigate Pb (II) and Zn (II) removal from aqueous solutions by Red Earth (RE) as a new local natural adsorbent in using the batch method. The chemical structure of RE adsorbent was characterized by XRF. Giles, Langmuir, and Freundlich isotherms were used to describe the adsorption data. The effect of metals concentration, initial pH, adsorbent dosage, and agitation time were studied. The results showed that RE contains of SiO2 (58 %) and Al2O3 (15.2 %) as major compounds. The equilibrium time was reached following 30 min and the maximum adsorption capacities (mg/g), based on Langmuir equation were 10.31 and 8.74 for Pb (II) and Zn (II), respectively. By increasing the initial metal ions concentration, the adsorption efficiencies were decreased and adsorption capacity of RE increased with an increase in the initial pH.

This research evaluated Moringa oleifera seed powder (MOSP) as an antibacterial agent, and a coagulant. In the former, clinical isolates of Salmonella sp., Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were used, and in the latter, river and stream water were used. Both the isolates and water samples were treated with MOSP at varying final concentrations of 0.001, 0.002, 0.004 and 0.017 g/ml. For the antibacterial assay, a dose of 0.017 g/ml of MOSP was effective on all three isolates with CFU/ml reduction of 99.4, 78.8 and 57.3% on Salmonella sp., E. coli and S. aureus respectively. An ANOVA confirmed this finding at P, Graphical abstract Image, graphical abstract

In the present paper, we provide a protocol for experimentally measuring and calculating individual inhibitory control capacity index in adult participants in an online Inquisit-based setting. We believe that this method can serve other researchers in the standardized assessment of individual inhibitory control capacity that can be used in studies on the possible role of inhibitory control in many every-day cognitive phenomena. Thus, the paper focuses mainly on the calculation of a composite inhibitory control capacity score from two inhibitory control tasks. Specifically, it is calculated on the basis of participants' performance in two well-established experimental paradigms: the Stroop Task and the Eriksen Flanker Task. The methods described in this protocol have already been successfully applied in both lab and online settings. In the first part of the article, we provide a short theoretical background and a brief description of the previous usage of this method in our two original studies. In the following parts, we provide step-by-step instructions for measuring and calculating the individual inhibitory control capacity index using web-based methodology. The protocol is supplemented with slides of original experimental tasks and instructions for participants (both translated from Polish)., Graphical abstract Image, graphical abstract

Microplastics (MP) are a pollutant that can be found in all marine ecosystems. Currently one of the most used forms to classify them is through their size. However, the current size categories in use cover an extremely wide range of sizes and are not based on a biological or physical basis. Thus, here we propose to harmonize the MP size categories with the ones already in used on plankton research for more than 120 years. This will allow the implementation of more refined MP size classes that are connected to a biological reality and will also enable the comparison of a myriad of literature on plankton research with the new work on MP., Graphical abstract Image, graphical abstract

This study aimed to introduce a new method for eye lens thermo-luminescent dosimetry and also estimate the dose associated with induced cancer risk due to the ionizing radiation exposure received by physicians and other staff cooperating in interventional cardiology (IC) procedures. The measurements were performed with six TLDs (thermoluminescent dosimeters): four TLDs for eye lens dosimetry (2 positioned on respiratory/surgical mask under the eye region as the new method; and 2 near the outside border of the eye as the common method) and two TLDs for whole-body dosimetry. Whole-body doses were used to calculate the cancer risks induced by IC procedures. The results of the new proposed method for eye lens dosimetry were similar to common TLD positioning (mean differences, Graphical abstract Image, graphical abstract

Primary human vulvovaginal fibroblast cell lines are useful for studying biological mechanisms underlying genital pain, pelvic organ prolapse, and the spread of sexually transmitted infections. However, the vaginal biopsies necessary for establishing these cell lines are invasive and relatively difficult to obtain. Primary mouse fibroblast cell lines derived from pre-clinical animal models of these conditions can be used for better controlled experiments that can help us dissect disease mechanisms. To our knowledge, there are no published protocols for establishing primary murine vaginal fibroblast cell lines to date. Here, we describe a protocol for the establishment of murine vaginal fibroblast cell lines via enzymatic digestion of vaginal canal tissue. Cell lines generated using this method can be used for in vitro studies of these important structural cells in a variety of pre-clinical mouse models; such studies are required to identify and characterize relevant regulatory and therapeutic targets in a wide array of diseases of interest. As shown in our representative data, this protocol yields viable cell lines from ND4 Swiss outbred mice. These cells bear surface markers characteristic of fibroblasts and are capable of producing inflammatory cytokines in response to treatment with bacterial and yeast antigens in vitro., Graphical abstract Graphical Representation of Murine Vaginal Fibroblast Cell Line EstablishmentTo generate murine vaginal fibroblast cell lines, extract murine vaginal canal tissue and soften, mince, and enzymatically digest tissue with Collagenase D. Plate digested tissue pieces and allow two weeks for fibroblasts to migrate out of tissue pieces onto the plate. After 3 passages, cell lines exhibit surface markers characteristic of fibroblasts and spindle-like morphology, and produce cytokines following in vitro treatment with bacterial and yeast antigens. These cell lines represent a novel tool for exploring disease mechanisms in murine models.Image, graphical abstract

Baculovirus expression vector systems (BEVS) have been widely used for production of recombinant proteins in insect cells. However, baculoviruses superinfection and repeated passages originate defective interfering particle (DIP) mutants, which is a limitation to a continuous large-scale production. Accordingly, a classical chemical transfection method performed on monolayer of Spodoptera frugiperda insect cells (Sf9) was modified to produce recombinant baculoviruses with high efficiency. Modifications consist to transfect exponentially growing cells in suspension after concentration by tenfold through centrifugation. Ten different constructions of recombinant baculoviruses with insert varying in size from 180 bp to 2,395 bp, were obtained through employment of the Bac-to-Bac expression system (ThermoFisher/Invitrogen). The transfection efficiency of the modified protocol varied from 45 to 57%, independent of the insert size, while classical method present transfection efficiency of 2 to 20%. After transfection of 6 × 106 cells, the recombinant baculoviruses titer obtained with modified method was about 2 × 107 pfu/ mL in a total volume of 12 mL, which is scalable to 24 liters of 1 × 108 pfu/ mL, after only two amplification rounds, contributing to improve large scale heterologous protein production in insect cells, with low amplification passages., Graphical abstract Image, graphical abstract