Support Protocol Research Articles

Sound absorption properties of the metamaterial curved microperforated panel

This paper proposes a metamaterial that utilizes resonators to enhance the sound absorption capacity of the curved microperforated panel (CMPP), and reliable theoretical analyses and experimental verification are comprehensively given. A metamaterial curved microperforated panel (MCMPP) absorber is formed by attaching local resonators on the CMPP. The displacements of the midplane of the curved panel are expanded into the improved Fourier series based on Spectral-Geometry Method (SGM), and the vibration equations of the MCMPP are derived by the Rayleigh-Ritz method. The sound absorption characteristics of the MCMPP are solved by the effective medium method and the acoustic-electric analogy method. Both a structure-acoustic coupling simulation model and an impedance tube test system are established to validate the accuracy of the present theoretical model. The essential parametric analyses are carried out in conjunction with mode splitting and acoustic-vibrational coupling, and the results show that the local resonance absorption peak within 1000 Hz increases from 1 to 5 with the absorption coefficients greater than 0.8 at these peaks under the installation options of the periodic gradient array and multiple resonances, realizing the effective absorption in multiple frequency bands. This paper provides theoretical support and experimental protocols for the design and application of the metamaterial curved microperforated panel.

Histodenz Separation of Lysosomal Subpopulations for Analysis of Chaperone-mediated Autophagy.

Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, in which proteins are individually selected for lysosomal degradation. CMA degradation targets bear a pentapeptide consensus motif that is recognized by the cytosolic chaperone HSPA8 (Hsc70), which participates in the trafficking of the target to the lysosomal surface. From there, it is translocated into the lysosomal lumen, independent of vesicle fusion, in a process dependent upon the lysosomal transmembrane protein LAMP2A. There are limited tools for studying CMA in whole cells and tissues, and many of the best techniques for studying CMA rely on the preparation of lysosome enriched fractions. Such experiments include (1) the in vitro evaluation of CMA substrate uptake activity, (2) the characterization of changes to lysosomal resident and CMA regulatory proteins, and (3) lysosomal targetomics, i.e., the use of quantitative proteomics to characterize lysosomal degradation targets. Previous studies using discontinuous metrizamide gradients have shown that a subpopulation of liver lysosomes is responsible for the majority of CMA activity ("CMA+ "). These CMA+ lysosomes are low density and have higher levels of MTORC2 relative to the "CMA- " lysosomes, which are high density and have higher levels of MTORC1. Because of safety concerns surrounding metrizamide, however, this compound is difficult to obtain, and it is impractically expensive. Here, we have provided protocols for isolation of lysosomal subpopulations for CMA-related analyses from mouse liver using Histodenz, a safe and affordable alternative to metrizamide. Supplementary protocols show how to perform CMA activity assays with appropriate statistical analysis, and how to analyze for lysosomal breakage/membrane integrity. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation of lysosomal subpopulations from mouse liver using discontinuous Histodenz gradients Alternate Protocol: Isolation of lysosomes from cultured cells using discontinuous Histodenz gradients Support Protocol 1: Verifying enrichment of lysosomal markers in lysosome-enriched fractions Support Protocol 2: Measuring in vitro uptake of CMA substrates Support Protocol 3: Measuring lysosomal membrane integrity by hexosaminidase assay.

E-therapists’ views on the acceptability and feasibility of an internet-administered, guided, low-intensity cognitive behavioural therapy intervention for parents of children treated for cancer: A qualitative study

Background Childhood cancer treatment completion can be a period of vulnerability for parents and is associated with mental health difficulties such as depression and anxiety. We developed an internet-administered, guided, low-intensity cognitive behavioural therapy-based self-help intervention (EJDeR) for parents delivered on the U-CARE-portal (Portal). The acceptability and feasibility of EJDeR and study procedures were examined using a single-arm feasibility trial (ENGAGE). Results indicated that EJDeR and ENGAGE study procedures are acceptable and feasible, however, a need for clinical and technical modifications to EJDeR and refinements to ENGAGE study procedures was identified. Objectives This study aimed to explore the acceptability and feasibility of EJDeR and ENGAGE study procedures from the perspective of e-therapists to inform clinical and technical modifications to EJDeR and refinements to study procedures prior to progression to a superiority randomised controlled trial. Methods We conducted semi-structured interviews with 10 e-therapists. Data were analysed using manifest content analysis. Results We identified three categories relating to the acceptability and feasibility of EJDeR: (a) Support to e-therapists (subcategories: Clinical supervision and Technical difficulties); (b) Guidance to parents (subcategories: Support protocols and Synchronous communication); and (c) Content (subcategories: Relevancy of the intervention and Pacing of the intervention). We identified four categories relating to the acceptability and feasibility of study procedures: (a) Recruitment and training of e-therapists (subcategories: Definition of the role and Training program); (b) Retention of parents (subcategories: Parent suitability and screening and Frequency of weekly Portal assessments); (c) Retention of e-therapists (subcategories: Administrative requirements and Communication with the research team); and (d) The Portal. Conclusions EJDeR and study procedures were considered acceptable and feasible, however, clinical and technical modifications and refinements to study procedures were suggested to enhance acceptability and feasibility. Results may also inform implementation considerations for both EJDeR and other similar digital psychological interventions. Trial registration number ISRCTN 57233429

Create, Analyze, and Visualize Phylogenomic Datasets Using PhyloFisher.

PhyloFisher is a software package written primarily in Python3 that can be used for the creation, analysis, and visualization of phylogenomic datasets that consist of protein sequences from eukaryotic organisms. Unlike many existing phylogenomic pipelines, PhyloFisher comes with a manually curated database of 240 protein-coding genes, a subset of a previous phylogenetic dataset sampled from 304 eukaryotic taxa. The software package can also utilize a user-created database of eukaryotic proteins, which may be more appropriate for shallow evolutionary questions. PhyloFisher is also equipped with a set of utilities to aid in running routine analyses, such as the prediction of alternative genetic codes, removal of genes and/or taxa based on occupancy/completeness of the dataset, testing for amino acid compositional heterogeneity among sequences, removal of heterotachious and/or fast-evolving sites, removal of fast-evolving taxa, supermatrix creation from randomly resampled genes, and supermatrix creation from nucleotide sequences. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Constructing a phylogenomic dataset Basic Protocol 2: Performing phylogenomic analyses Support Protocol 1: Installing PhyloFisher Support Protocol 2: Creating a custom phylogenomic database.

Thoracoscopic thymectomy for myasthenia gravis and non-invasive thymoma after COVID-19 pneumonia

Modern guidelines have identified thoracoscopic thymectomy as a preferable option for myasthenia gravis and non-invasive thymoma. In the era of the new coronavirus infection, it is relevant to develop protocols for anesthetic and perioperative support of patients undergoing thymectomy for myasthenia gravis after COVID-associated pneumonia (CAP). We present the results of thoracoscopic thymectomies in patients after CAP. Multidisciplinary team should determine therapeutic support, the need for plasmapheresis and thymectomy. Plasmapheresis and glucocorticosteroids are effective in addition to anticholinesterase therapy at the stages of perioperative support for correction of neurological status in patients with myasthenia combined with chronic obstructive pulmonary disease and pulmonary hypertension. Outpatient direct anticoagulants are advisable considering the need for prolonged postoperative prevention of thrombotic events.

Acute white-out lung in mechanically ventilated patient

ABSTRACT A 64-year-old male with a history of chronic kidney disease-5, type 2 diabetes mellitus (T2DM), and hypertension presented with syncope and weakness. Initial examination revealed a heart rate (HR) of 18/min and blood pressure (BP) of 140/90 mmHg. Intravenous (iv) atropine was administered, increasing HR to 85/min. Subsequently, the patient became pulseless, unconscious, and unresponsive, necessitating cardiopulmonary resuscitation (CPR) as per Advanced Cardiovascular Life Support (ACLS) protocol. Return of spontaneous circulation (ROSC) was achieved after three CPR cycles, and the patient regained consciousness. He was intubated and transferred to the intensive care unit. The patient, a smoker with a 4.5 pack-year history, experienced increased positive end-expiratory pressure (PEEP), elevated plateau pressure, and inadequate tidal volume generation a day later. Auscultation revealed significantly reduced air entry on the right side. Arterial blood gas (ABG) analysis indicated CO2 retention. Chest X-ray showed a white-out right lung, intercostal narrowing, and mediastinal shift toward the ipsilateral side.

Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines.

High-resolution structure determination by electron cryo-microscopy underwent a step change in recent years. This now allows study of challenging samples which previously were inaccessible for structure determination, including membrane proteins. These developments shift the focus in the field to the next bottlenecks which are high-quality sample preparations. While the amounts of sample required for cryo-EM are relatively small, sample quality is the key challenge. Sample quality is influenced by the stability of complexes which depends on buffer composition, inherent flexibility of the sample, and the method of solubilization from the membrane for membrane proteins. It further depends on the choice of sample support, grid pre-treatment and cryo-grid freezing protocol. Here, we discuss various widely applicable approaches to improve sample quality for structural analysis by cryo-EM.

Super-Resolution Stimulated Raman Scattering Microscopy with Graphical User Interface-Supported A-PoD.

Raman microscopy is a vibrational imaging technology that can detect molecular chemical bond vibrational signals. Since this signal is originated from almost every vibrational mode of molecules with different vibrational energy levels, it provides spatiotemporal distribution of various molecules in living organisms without the need for any labeling. The limitations of low signal strength in Raman microscopy have been effectively addressed by incorporating a stimulated emission process, leading to the development of stimulated Raman scattering (SRS) microscopy. Furthermore, the issue of low spatial resolution has been resolved through the application of computational techniques, specifically image deconvolution. In this article, we present a comprehensive guide to super-resolution SRS microscopy using an Adam-based pointillism deconvolution (A-PoD) algorithm, complemented by a user-friendly graphical user interface (GUI). We delve into the crucial parameters and conditions necessary for achieving super-resolved images through SRS imaging. Additionally, we provide a step-by-step walkthrough of the preprocessing steps and the use of GUI-supported A-PoD. This complete package offers a user-friendly platform for super-resolution SRS microscopy, enhancing the versatility and applicability of this advanced microscopy technique to reveal nanoscopic multimolecular nature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Super-resolution stimulated Raman scattering microscopy with graphical user interface-supported A-PoD Support Protocol: Deuterium labeling on cells with heavy water for metabolic imaging.

Effect of a cervical collar on optic nerve sheath diameter in trauma patients.

As advocated in advanced trauma life support and prehospital trauma life support protocols, cervical immobilization is applied until cervical spine injury is excluded. This study aimed to show the difference in optic nerve sheath diameter (ONSD) between patients with and without a cervical collar using computed tomography (CT). This was a single-center, retrospective study examining trauma patients who presented to the emergency department between January 1, 2021, and December 31, 2021. The ONSD on brain CT of the trauma patients was measured and analyzed to determine whether there was a difference between the ONSD with and without the cervical collar. The study population consisted of 169 patients. On CT imaging of patients with (n=66) and without (n=103) cervical collars, the mean ONSD in the axial plane were 5.43 ± 0.50 mm and 5.04 ± 0.46 mm respectively for the right eye and 5.50 ± 0.52 mm and 5.11 ± 0.46 mm respectively for the left eye. The results revealed an association between the presence of a cervical collar and the mean ONSD, which was statistically significant (P<0.001) for both the right and left eyes. A cervical collar may be associated with increased ONSD. The effect of this increase in the ONSD on clinical outcomes needs to be investigated, and the actual need for cervical collar in the emergency department should be evaluated on a case-by-case basis.

Comparison Between the Advanced Cardiac Life Support and Adult Advanced Life Support Protocols: A Simulation-Based Pilot Study.

Introduction: Cardiac arrest is a public health concern associated with unfavorable disease outcomes. Cardiopulmonary resuscitation (CPR) of optimal quality is widely acknowledged as an indispensable technique in restoring spontaneous circulation. In order to perform advanced cardiac life support (ACLS), chest compression must be paused twice: once to assess the rhythm and again to administer the shock. Australian advanced life support (ALS) recommends that the defibrillator needs to be precharged in order to administer the shock during a solitary interval in chest compressions. While performing chest compressions, precharging defibrillators can decrease hands-off time without posing a risk of injury. Aim: To compare chest compression fraction (CCF)-which is the cumulative time spent providing chest compressions divided by the total time taken for the entire resuscitation-by calculating the hands-off time duration in cardiac arrest between the Australian Resuscitation Council (ARC) and American Heart Association (AHA) protocols for CPR. Methods: A simulation-based pilot study was designed using a Laerdal Resusci Anne mannequin and a LIFEPACK 20 defibrillator. The study included six participants recruited from King Khalid University Hospital in Riyadh, Saudi Arabia, where three participants were certified ACLS providers and there were certified ALS providers. Participants were divided into two groups, ALS and ACLS, each following one protocol. For each scenario, a random job was assigned to each participant, regardless of their role as assistant, team leader, or performer of CPR. Each case's shockable and nonshockable rhythms were hidden from the team leader and the chest compressor. Ten trials of CPR were performed, each for four cycles with a total time of 8 min. The simulation was video recorded for hands-off time counting. Comparison between CCF (seconds) per cycle between the two protocols was performed using an independent sample t-test. A p value of 0.05 was used to measure statistical significance. Results: Comparing CCF in shockable rhythms between ARC and AHA protocols, it was observed that the CCF of ALS-ARC was significantly higher than ACLS-AHA in all cycles; the first cycle: t = 3.782, p=0.004; the second cycle: t = 3.380, p=0.007; the third cycle: t = 3.803, p=0.003; and the fourth cycle: t = 4.341, p=0.001. Conclusion: Precharging a defibrillator before a rhythm check during chest compression, in anticipation of a potentially shockable rhythm, reduces the time required for defibrillation and limits interruptions in chest compression during CPR. This practice effectively enhances the CCF. Enhancing the continuity of chest compressions can potentially improve survival rates in ARC.

Protein Isolation from 3D Hydrogel Scaffolds.

Protein isolation is an essential tool in cell biology to characterize protein abundance under various experimental conditions. Several protocols exist, tailored to cell culture or tissue sections, and have been adapted to particular downstream analyses (e.g., western blotting or mass spectrometry). An increasing trend in bioengineering and cell biology is to use three-dimensional (3D) hydrogel-based scaffolds for cell culture. In principle, the same protocols can be used to extract protein from hydrogel-based cell and tissue constructs. However, in practice the yield and quality of the recovered protein pellet is often substantially lower when using standard protocols and requires tuning of multiple steps, including the selected lysis buffer and the scaffold homogenization strategy, as well as the methods for protein purification and reconstitution. We present here specific protocols tailored to common 3D hydrogels to help researchers using hydrogel-based 3D cell culture improve the quantity and quality of their extracted protein. We focus on three materials: protease-degradable PEG-based hydrogels, collagen hydrogels, and alginate hydrogels. We discuss how the protein extraction procedure can be adapted to the scaffold of interest (degradable or non-degradable gels), proteins of interests (soluble, matrix-bound, or phosphoproteins), and downstream biochemical assays (western blotting or mass spectrometry). With the growing interest in 3D cell culture, the protocols presented should be useful to many researchers in cell biology, protein science, biomaterials, and bioengineering communities. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolating proteins from PEG-based hydrogels Basic Protocol 2: Isolating proteins from collagen hydrogels Basic Protocol 3: Isolating proteins from alginate hydrogels Alternate Protocol: Isolating protein from alginate gels using EDTA to dissolve the gel Support Protocol: Isolating protein and RNA simultaneously from the same samples.

Palliative Care for Infants in the Neonatal Intensive Care Unit: A Scoping Review.

This scoping review aimed to explore the characteristics of neonatal palliative care in the neonatal intensive care unit, including the features, contents, and experiences of infants, parents, and nurses during palliative care. Five databases (PubMed, Cochrane, CINAHL, Research Information Sharing Service, and Korean Studies Information Service System) were searched to identify relevant articles published between 2011 and 2020. From the systematic search and review process, 13 studies that met the eligibility criteria were selected for the analysis. From the literature review, 2 key principles were found to facilitate neonatal palliative care: family-centered care and integrative care in the neonatal intensive care unit. In addition, the themes found in this review included (1) providing comfortable care to dying infants with respect to infants and offering parents choices, (2) therapeutic communication, (3) support with respect, and (4) bereavement care for parents of dying infants in the neonatal intensive care unit. Caregivers require effective communication, manpower support, emotional support, educational programs, and well-defined protocols. The evidence mapped and synthesized in this review indicates the need to facilitate the provision of palliative care in the neonatal intensive care unit in line with the unique needs of infants, parents, and nurses.

Reproducible Differentiation of Human Pluripotent Stem Cells into Two-Dimensional Cortical Neuron Cultures with Checkpoints for Success.

The patterning of excitatory cortical neurons from human pluripotent stem cells (hPSCs) is a desired technique for the study of neurodevelopmental disorders, as neurons can be created and compared from control hPSC lines, hPSC lines generated from patients, and CRISPR-modified hPSC lines. Therefore, this technique allows for the examination of disease phenotypes and assists in the development of potential new therapeutics for neurodevelopmental disorders. Many protocols, however, are optimized for use with specific hPSC lines or within a single laboratory, and they often provide insufficient guidance on how to identify positive stages in the differentiation or how to troubleshoot. Here, we present an efficient and reproducible directed differentiation protocol to generate two-dimensional cultures of hPSC-derived excitatory cortical neurons without intermediary embryoid body formation. This novel protocol is supported by our data generated with five independent hPSC lines and in two independent laboratories. Importantly, as neuronal differentiations follow a long time course to reach maturity, we provide extensive guidance regarding morphological and flow cytometry checkpoints allowing for early indications of successful differentiation. We also include extensive troubleshooting tips and support protocols to assist the operator. The goal of this protocol is to assist others in the successful differentiation of excitatory cortical neurons from hPSCs. © 2023 Wiley Periodicals LLC. Basic Protocol: Directed differentiation of hPSCs into excitatory cortical neurons Support Protocol 1: Harvesting and fixing cells for flow cytometry analyses Support Protocol 2: Performing flow cytometry analyses Support Protocol 3: Thawing NPCs from a cryopreserved stock Alternate Protocol 1: Continuing Expansion of NPCs Alternate Protocol 2: Treatment of neurons with Ara-C to ablate radial glia Support Protocol 4: Experimental methods for validation of excitatory cortical neurons.

Quantitative Analysis of Siglec Ligands by Flow Cytometry.

Siglecs (sialic acid-binding, immunoglobulin superfamily, lectins) are a family of transmembrane receptor-type glycan recognition proteins in vertebrates that are primarily expressed on leukocytes and regulate immune responses. Siglecs are involved in several diseases, such as cancer and neurodegenerative diseases. Most Siglecs suppress the activation of leukocytes by recognizing ligands containing sialic acid, a group of acidic sugars commonly found in vertebrate glycans, but rare among microbes. Siglec ligands are critical in the interaction between leukocytes and target cells. The abundance of the Siglec ligand is influenced by both the abundance of the glycoconjugate carrier (glycoprotein or glycolipid) and that of the terminal glycan epitope directly recognized by the Siglec. Therefore, a direct approach to evaluate the expression level of a Siglec ligand on cells of interest is to analyze the binding of recombinant Siglec protein to these cells. In this article, we describe a protocol for semi-quantitatively analyzing the expression level of Siglec ligands via flow cytometry using recombinant Siglec-Fc fusion protein. Support protocols describe how to remove sialic acids from the cell surface with sialidase under mild conditions to demonstrate the sialic acid dependence of Siglec binding, and the preparation of recombinant Siglec-Fc fusion proteins by transient transfection of mammalian cells. © 2023 Wiley Periodicals LLC. Basic Protocol: Quantitative analysis of Siglec ligands on mammalian cells via flow cytometry with recombinant Siglec-Fc fusion protein Support Protocol 1: Sialidase treatment of mammalian cells Support Protocol 2: Preparation of recombinant Siglec-Fc fusion protein via transient transfection of mammalian cells.

Production of CRISPR-Cas9 Transgenic Cell Lines for Knocksideways Studies.

Protein activity is generally functionally integrated and spatially restricted to key locations within the cell. Knocksideways experiments allow researchers to rapidly move proteins to alternate or ectopic regions of the cell and assess the resultant cellular response. Briefly, individual proteins to be tested using this approach must be modified with moieties that dimerize under treatment with rapamycin to promote the experimental spatial relocalizations. CRISPR technology enables researchers to engineer modified protein directly in cells while preserving proper protein levels because the engineered protein will be expressed from endogenous promoters. Here we provide straightforward instructions to engineer tagged, rapamycin-relocalizable proteins in cells. The protocol is described in the context of our work with the microtubule depolymerizer MCAK/Kif2C, but it is easily adaptable to other genes and alternate tags such as degrons, optogenetic constructs, and other experimentally useful modifications. Off-target effects are minimized by testing for the most efficient target site using a split-GFP construct. This protocol involves no proprietary kits, only plasmids available from repositories (such as addgene.org). Validation, relocalization, and some example novel discoveries obtained working with endogenous protein levels are described. A graduate student with access to a fluorescence microscope should be able to prepare engineered cells with spatially controllable endogenous protein using this protocol. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Choosing a target site for gene modification Basic Protocol 2: Design of gRNA(s) for targeted gene modification Basic Protocol 3: Split-GFP test for target efficiency Basic Protocol 4: Design of the recombination template and analytical primers Support Protocol 1: Design of primers for analytical PCR Basic Protocol 5: Transfection, isolation, and validation of engineered cells Support Protocol 2: Stable transfection of engineered cells with binding partners.

Computational Pipeline for Analysis of Biomedical Networks with BioNAR.

In a living cell, proteins interact to assemble both transient and constant molecular complexes, which transfer signals/information around internal pathways. Modern proteomic techniques can identify the constituent components of these complexes, but more detailed analysis demands a network approach linking the molecules together and analyzing the emergent architectural properties. The Bioconductor package BioNAR combines a selection of existing R protocols for network analysis with newly designed original methodological features to support step-by-step analysis of biological/biomedical . Critically, BioNAR supports a pipeline approach whereby many networks and iterative analyses can be performed. Here we present a network analysis pipeline that starts from initiating a network model from a list of components/proteins and their interactions through to identifying its functional components based solely on network topology. We demonstrate that BioNAR can help users achieve a number of network analysis goals that are difficult to achieve anywhere else. This includes how users can choose the optimal clustering algorithm from a range of options based on independent annotation enrichment, and predict a protein's influence within and across multiple subcomplexes in the network and estimate the co-occurrence or linkage between metadata at the network level (e.g., diseases and functions across the network, identifying the clusters whose components are likely to share common function and mechanisms). The package is freely available in Bioconductor release 3.17: https://bioconductor.org/packages/3.17/bioc/html/BioNAR.html. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Creating and annotating the network Support Protocol 1: Installing BioNAR from RStudio Support Protocol 2: Building the sample network from synaptome.db Basic Protocol 2: Network properties and centrality Basic Protocol 3: Network communities Basic protocol 4: Choosing the optimal clustering algorithm based on the enrichment with annotation terms Basic Protocol 5: Influencing network components and bridgeness Basic Protocol 6: Co-occurrence of the annotations.

PRO-seq: Precise Mapping of Engaged RNA Pol II at Single-Nucleotide Resolution.

Gene regulation is dependent on the production of mRNAs and a repertoire of non-coding RNAs by RNA polymerase II (RNAPII). Precision run-on sequencing (PRO-seq) maps the position of engaged RNAPII complexes at single-nucleotide resolution and can reveal direct targets of regulation, locations of enhancers, and transcription mechanisms that are difficult or impossible to measure by analysis of total cellular RNA. Briefly, this method first involves permeabilizing cells with mild detergents to remove intracellular NTPs and halt transcription. Transcription is then resumed in the presence of biotin-NTPs and sarkosyl to allow transcriptional incorporation of a single biotinylated NTP by RNAPII. The biotin moiety is then bound to streptavidin beads to stringently enrich for nascent RNAs. Sequencing libraries are then generated such that the first base read corresponds to the 3' end of the nascent transcript. Here, we describe our current protocol for generating PRO-seq libraries from metazoan cells, including adaptations of previously published protocols to incorporate unique molecular identifiers, reduce ligation bias, and improve library yields. Additional commentary describes quality control and processing of PRO-seq data and references for more advanced downstream analysis such as gene and enhancer identification. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cell permeabilization for PRO-seq Basic Protocol 2: Construction of PRO-seq libraries Support Protocol: Adenylation of 3' adapter.

Plant Sample Collection and Shipment for Multi-omic Analyses and Phytosanitary Evaluation.

Plant sample preparation for analyses is a fundamental step in high-throughput omics strategies. Especially for plant metabolomics, quenching of hydrolytic enzymes able to affect metabolite concentrations is crucial for the accuracy of results. Given that DNA is usually less labile than metabolites, most sampling and shipment procedures able to preserve the metabolome are also suitable for preventing the degradation of plant DNA or of DNA of pathogens in the plant tissue. In this article, we describe all the steps of sample collection, shipment (including the phytosanitary issues of moving plant samples), and processing for combined genomics and metabolomics from a single sample, as well as the protocols used in our laboratories for downstream approaches for crop plants, allowing collection of multi-omic datasets in large experimental setups. The protocols have been adjusted to apply to both freeze-dried and fresh-frozen material to allow the processing of crop plant samples that will require long-distance transport. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of freeze-dried leaf disks for multiplexed PCR or DArT-Seq genotyping Basic Protocol 2: Medium-throughput preparation of pathogen-free nucleic acids for most genotyping-resequencing applications or pathogen detection Alternate Protocol: Low-throughput extraction of high-quality DNA for resequencing using commercial kits Support Protocol: DNA quality control Basic Protocol 3: Preparation of freeze-dried plant material for metabolomics Basic Protocol 4: Preparation of fresh-frozen plant material for metabolomics Basic Protocol 5: Preparation and shipment of metabolite extracts for metabolomic analyses Basic Protocol 6: Sample shipping and long-term storage.

Discriminating Between Apoptosis, Necrosis, Necroptosis, and Ferroptosis by Microscopy and Flow Cytometry.

Apoptosis is a mode of programmed cell death that plays important roles in tissue sculpting during development, in the maintenance of tissue homeostasis in the adult, and in the eradication of injured or infected cells during pathological processes. Numerous physiological as well as pathological stimuli trigger apoptosis, such as engagement of plasma-membrane-associated Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia, radiation, and exposure to diverse cytotoxic drugs. Apoptosis is coordinated by members of the caspase family of cysteine proteases, which, upon activation, trigger a series of dramatic morphological and biochemical changes including retraction from the substratum, cell shrinkage, extensive and protracted plasma membrane blebbing, chromatin condensation, DNA hydrolysis, nuclear fragmentation, and proteolytic cleavage of numerous caspase substrates. These dramatic structural and biochemical alterations result not only in the controlled dismantling of the cell, but also in the rapid recognition and removal of apoptotic cells by phagocytes through the cell surface display of phagocytotic triggers such as phosphatidylserine. Necrosis, which is typically nonprogrammed or imposed upon the cell by overwhelming membrane or organelle damage, is characterized by high-amplitude cell swelling, followed by rapid plasma membrane rupture and release of cellular contents into the extracellular space. Necrosis is often provoked by infectious agents or severe departure from physiological conditions due to toxins, temperature extremes, or physical injury. However, forms of programmed necrosis (necroptosis, pyroptosis, ferroptosis) can also occur in specific circumstances. Nonprogrammed and programmed necrosis can be distinguished from apoptosis by morphological features, based on the rapid uptake of vital dyes, and through the application of specific inhibitors of key molecules associated with the latter modes of cell death. This unit describes protocols for the measurement of apoptosis and necrosis and for distinguishing apoptosis from programmed as well as conventional necrosis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Analysis of cell morphology by phase-contrast microscopy Alternative Protocol 1: Assessment of morphological changes using eosin-methylene blue staining Alternative Protocol 2: Analysis of nuclear morphology by fluorescence microscopy Support Protocol: Preparation of cytospins Basic Protocol 2: Measurement of plasma membrane composition with annexin V and propidium iodide Basic Protocol 3: Measurement of DNA fragmentation by flow cytometry Alternative Protocol 3: Analysis of DNA fragmentation by the TUNEL assay Basic Protocol 4: Measurement of caspase activation by flow cytometry Basic Protocol 5: Discriminating between apoptosis, necrosis, necroptosis, and ferroptosis.

Machine Perfusion Deters Ischemia-Related Derangement of a Rodent Free Flap: Development of a Model

IntroductionMachine perfusion can enable isolated support of composite tissues, such as free flaps. The goal of perfusion in this setting is to preserve tissues prior to transplantation or provide transient support at the wound bed. This study aimed to establish a rodent model of machine perfusion in a fasciocutaneous-free flap to serve as an affordable testbed and determine the potential of the developed support protocol to deter ischemia-related metabolic derangement. MethodsRat epigastric-free flaps were harvested and transferred to a closed circuit that provides circulatory and respiratory support. Whole rat blood was recirculated for 8 h, while adjusting the flow rate to maintain arterial-like perfusion pressures. Blood samples were collected during support. Extracellular tissue lactate and glucose levels were characterized with a microdialysis probe and compared with warm ischemic, cold ischemic, and anastomosed-free flap controls. ResultsMaintenance of physiologic arterial pressures (85-100 mmHg) resulted in average pump flow rates of 360–430 μL/min. Blood-based measurements showed maintained glucose and oxygen consumption throughout machine perfusion. Average normalized lactate to glucose ratio for the perfused flaps was 5-32-fold lower than that for the warm ischemic flap controls during hours 2-8 (P < 0.05). ConclusionsWe developed a rat model of ex vivo machine perfusion of a fasciocutaneous-free flap with maintained stable flow and tissue metabolic activity for 8 h. This model can be used to assess critical elements of support in this setting as well as explore other novel therapies and technologies to improve free tissue transfer.