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Barium titanate (BaTiO3, hereafter BT) is an established ferroelectric material first discovered in the 1940s and still widely used because of its well-balanced ferroelectricity, piezoelectricity, and dielectric constant. In addition, BT does not contain any toxic elements. Therefore, it is considered to be an eco-friendly material, which has attracted considerable interest as a replacement for lead zirconate titanate (PZT). However, bulk BT loses its ferroelectricity at approximately 130 °C, thus, it cannot be used at high temperatures. Because of the growing demand for high-temperature ferroelectric materials, it is important to enhance the thermal stability of ferroelectricity in BT. In previous studies, strain originating from the lattice mismatch at hetero-interfaces has been used. However, the sample preparation in this approach requires complicated and expensive physical processes, which are undesirable for practical applications. In this study, we propose a chemical synthesis of a porous material as an alternative means of introducing strain. We synthesized a porous BT thin film using a surfactant-assisted sol-gel method, in which self-assembled amphipathic surfactant micelles were used as an organic template. Through a series of studies, we clarified that the introduction of pores had a similar effect on distorting the BT crystal lattice, to that of a hetero-interface, leading to the enhancement and stabilization of ferroelectricity. Owing to its simplicity and cost effectiveness, this fabrication process has considerable advantages over conventional methods.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 μm). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action. Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes. The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

Solid-state electrolytes are a promising replacement for current organic liquid electrolytes, enabling higher energy densities and improved safety of lithium-ion (Li-ion) batteries. However, a number of setbacks prevent their integration into commercial devices. The main limiting factor is due to nanoscale phenomena occurring at the electrode/electrolyte interfaces, ultimately leading to degradation of battery operation. These key problems are highly challenging to observe and characterize as these batteries contain multiple buried interfaces. One approach for direct observation of interfacial phenomena in thin film batteries is through the fabrication of electrochemically active nanobatteries by a focused ion beam (FIB). As such, a reliable technique to fabricate nanobatteries was developed and demonstrated in recent work. Herein, a detailed protocol with a step-by-step process is presented to enable the reproduction of this nanobattery fabrication process. In particular, this technique was applied to a thin film battery consisting of LiCoO2/LiPON/a-Si, and has further been previously demonstrated by in situ cycling within a transmission electron microscope.

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core. X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 °C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature. The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 °C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding 'tuft.' Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This research shows a protocol to assess the computational complexity of querying relational and non-relational (NoSQL (not only Structured Query Language)) standardized electronic health record (EHR) medical information database systems (DBMS). It uses a set of three doubling-sized databases, i.e. databases storing 5000, 10,000 and 20,000 realistic standardized EHR extracts, in three different database management systems (DBMS): relational MySQL object-relational mapping (ORM), document-based NoSQL MongoDB, and native extensible markup language (XML) NoSQL eXist. The average response times to six complexity-increasing queries were computed, and the results showed a linear behavior in the NoSQL cases. In the NoSQL field, MongoDB presents a much flatter linear slope than eXist. NoSQL systems may also be more appropriate to maintain standardized medical information systems due to the special nature of the updating policies of medical information, which should not affect the consistency and efficiency of the data stored in NoSQL databases. One limitation of this protocol is the lack of direct results of improved relational systems such as archetype relational mapping (ARM) with the same data. However, the interpolation of doubling-size database results to those presented in the literature and other published results suggests that NoSQL systems might be more appropriate in many specific scenarios and problems to be solved. For example, NoSQL may be appropriate for document-based tasks such as EHR extracts used in clinical practice, or edition and visualization, or situations where the aim is not only to query medical information, but also to restore the EHR in exactly its original form. This work was supported by Instituto de Salud Carlos III [grant numbers PI15/00321, PI15/00003, PI1500831, PI15CIII/00010 and RD16CIII]. Sí

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Reciprocal social behavior (RSB), an early-emerging capacity to engage in social contingency-which is foundational for both social learning and social competency-is hypothesized to be disrupted in autism spectrum disorder (ASD). The ability to quantify the full range of RSB during the toddler period, when core symptoms of ASD often arise, is pivotal for evaluating early risk for ASD, characterizing social development, and tracking response to early interventions. However, important parameters of variation in RSB-especially prior to the development of verbal language-can be nuanced and difficult to characterize using questionnaire-based methods. To address this challenge, we developed a system for measuring quantitative variation in RSB in toddlers (ages 18 - 30 months) that incorporated not only standard questionnaire data from caregivers but also a novel set of video-referenced items, through which a respondent compares the behavior of a subject to that observed in a short video of a young child manifesting a highly competent level of social communication. Testing of this measure in a general population sample of twins confirmed that both the video-referenced items and the RSB Total Score (video-referenced items plus non-video-referenced items) displayed unimodal, continuous distributions, strong internal consistency, marked preservation of individual differences, and extremely high heritability. In addition, video-referenced items were particularly sensitive to quantifying incremental changes in social communication, a major element of RSB, over the course of early childhood development. Scores on the vrRSB clearly differentiated children with and without ASD and these data comprise an initial validation of this promising method for quantifying early RSB-cross-sectionally, over time, and as a function of early intervention.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

Three-dimensional gait analysis (3DGA) is shown to be a useful clinical tool for the evaluation of gait abnormality due to movement disorders. However, the use of 3DGA in actual clinics remains uncommon. Possible reasons could include the time-consuming measurement process and difficulties in understanding measurement results, which are often presented using a large number of graphs. Here we present a clinician-friendly 3DGA method developed to facilitate the clinical use of 3DGA. This method consists of simplified preparation and measurement processes that can be performed in a short time period in clinical settings and intuitive results presentation to facilitate clinicians' understanding of results. The quick, simplified measurement procedure is achieved by the use of minimum markers and measurement of patients on a treadmill. To facilitate clinician understanding, results are presented in figures based on the clinicians' perspective. A Lissajous overview picture (LOP), which shows the trajectories of all markers from a holistic viewpoint, is used to facilitate intuitive understanding of gait patterns. Abnormal gait pattern indices, which are based on clinicians' perspectives in gait evaluation and standardized using the data of healthy subjects, are used to evaluate the extent of typical abnormal gait patterns in stroke patients. A graph depicting the analysis of the toe clearance strategy, which depicts how patients rely on normal and compensatory strategies to achieve toe clearance, is also presented. These methods could facilitate implementation of 3DGA in clinical settings and further encourage development of measurement strategies from the clinician's point of view.

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

Oxygen deprivation in animals can result from exposure to low atmospheric oxygen levels or from internal tissue damage that interferes with oxygen distribution. It is also possible that aberrant behavior of oxygen-sensing neurons could induce hypoxia-like behavior in the presence of normal oxygen levels. In D. melanogaster, development at low oxygen levels results in inhibition of growth and sluggish behavior during the larval phases. However, these established manifestations of oxygen deficit overlap considerably with the phenotypes of many mutations that regulate growth, stress responses or locomotion. As result, there is currently no assay available to identify i) cellular hypoxia induced by a mutation or ii) hypoxia-like behavior when induced by abnormal neuronal behavior. We have recently identified two distinctive behaviors in D. melanogaster larvae that occur at normal oxygen levels in response to internal detection of hypoxia. First, at all stages, such larvae avoid burrowing into food, often straying far away from a food source. Second, tunneling into a soft substratum, which normally occurs during the wandering third instar stage is completely abolished if larvae are hypoxic. The assay described here is designed to detect and quantitate these behaviors and thus to provide a way to detect hypoxia induced by internal damage rather than low external oxygen. Assay plates with an agar substratum and a central plug of yeast paste are used to support animals through larval life. The positions and state of the larvae are tracked daily as they proceed from first to third instar. The extent of tunneling into the agar substratum during wandering phase is quantitated after pupation using NIH ImageJ. The assay will be of value in determining when hypoxia is a component of a mutant phenotype and thus provide insight into possible sites of action of the gene in question.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use products. Based on their chemical structure and estradiol-like biological properties, they have been classified as endocrine disrupting compounds (EDC). Long-term exposure to EDCs, even at low doses, has been linked to various health defects including cancer, behavioral disorders and infertility, with greater vulnerability indicated during early developmental periods. Cellular and molecular studies with the genetically tractable nematode model Caenorhabditis elegans have demonstrated that exposure to BPA causes apoptosis, embryonic lethality and disruption in the DNA repair mechanisms. We have previously reported that exposure of C. elegans embryos to low doses of different bisphenols decreases fecundity. In addition, we have shown that the effects of exposure during the very early stages of development persist into adulthood as assayed by quantifying habituation behavior, a form of non-associative learning. Here, we provide detailed protocols for embryonic exposure to low-dose EDCs as well as the associated fecundity and anterior touch habituation assays, along with representative results.

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro-and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4(+) and CD8(+) T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori's modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking γδ T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 °C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 °C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

International audience; The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good's buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera. QC 20180920

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. degrees degrees degrees This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic-and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a high surface area carbon, and to test their performance in thin film rotating disk electrode (TF-RDE) measurements. The TF-RDE methodology is widely used for catalyst screening; nevertheless, the measured performance sometimes considerably differs among research groups. These uncertainties impede the advancement of new catalyst materials and, consequently, several authors discussed possible best practice methods and the importance of benchmarking. The visual tutorial highlights possible pitfalls in the TF-RDE testing of Pt/C catalysts. A synthesis and testing protocol to assess standard Pt/C catalysts is introduced that can be used together with polycrystalline Pt disks as benchmark catalysts. In particular, this study highlights how the properties of the catalyst film on the glassy carbon (GC) electrode influence the measured performance in TF-RDE testing. To obtain thin, homogeneous catalyst films, not only the catalyst preparation, but also the ink deposition and drying procedures are essential. It is demonstrated that an adjustment of the ink's pH might be necessary, and how simple control measurements can be used to check film quality. Once reproducible TF-RDE measurements are obtained, determining the Pt loading on the catalyst support (expressed as Pt wt%) and the electrochemical surface area is necessary to normalize the determined reaction rates to either surface area or Pt mass. For the surface area determination, so-called CO stripping, or the determination of the hydrogen underpotential deposition (Hupd) charge, are standard. For the determination of the Pt loading, a straightforward and cheap procedure using digestion in aqua regia with subsequent conversion of Pt(IV) to Pt(II) and UV-vis measurements is introduced.