Detailed Protocol Research Articles

Effect of Cue-Based Feeding on the Feeding Outcomes of Preterm Infants: A Systematic Review and Meta-Analysis.

To systematically evaluate the effect of cue-based feeding (CBF) on feeding outcomes in preterm infants. Databases including CNKI, Wanfang, PubMed, Embase, CINAHL and Cochrane Library were searched from the database's inception to November 2023. The literature screening and quality assessment were conducted by two researchers independently, and a meta-analysis was performed using Review Manager (RevMan) 5.4 software. A total of six randomised controlled trials and eight quasi-experimental studies involving 2239 preterm infants were included. The results indicated that CBF was effective in shortening the time to achieve full oral feeding (Z = 22.32, p < 0.01) and reducing the incidence of feeding intolerance (Z = 2.18, p < 0.05). Compared with scheduled feeding (SF), CBF was found to enhance parental participation and satisfaction. Health professionals such as paediatricians, nurses and feeding specialists, highly agreed on the positive effects of CBF. Cue-based feeding has the potential to shorten the time to achieve full oral feeding, reduce the incidence of feeding intolerance and promote parental involvement in feeding behaviour for preterm infants. Cue-based feeding is recommended for preterm infants, as it could enhance oral feeding competence, facilitate feeding progression and reduce the incidence of feeding complications. Our paper is a systematic review and meta-analysis, and such details do not apply to our work. The detailed study protocol can be found on the PROSPERO website.

Equine temporomandibular joint diseases: A systematic review.

The temporomandibular joint (TMJ) is a unique joint that enables mandibular movement. Temporomandibular diseases (TMDs) impair joint function, leading to more or less specific clinical signs. To compile and disseminate clinical data and research findings from existing publications on equine TMD. Systematic review. Following PRISMA 2020 guidelines, literature searches were conducted in PUBMED, Scopus, and Web of Knowledge. The inclusion criteria covered case reports and research articles on equine TMDs. The selected records were grouped considering septic TMJ arthritis, primary TMJ osteoarthritis (OA), and non-arthritic TMDs. A risk of bias assessment was performed for the research articles. Equine TMD has been described in 51 publications to date, with septic TMJ arthritis accounting for 41.2% of TMD cases, fractures/luxations for 29.4%, and primary TMJ OA for 21.6%. Trauma was confirmed or suspected in 54.9% of TMD cases. The severity of clinical signs was mild to moderate in primary TMJ OA and mild to severe in septic TMJ arthritis and non-arthritic TMDs. Clinical signs were related to the underlying cause, particularly in terms of TMJ swelling, pain, and masticatory problems. Among diagnostic imaging modalities, CT was utilised in 92.2% of horses, radiography in 84.4%, and ultrasonography in 21.6%. The lack of clinical data in some reports. A risk of bias due to missing data. TMDs are infrequently described, with trauma being the most common TMD cause. TMDs are associated with TMJ dysfunction; however, the diagnostic protocol is varied. Regardless of the TMD type, a detailed diagnostic protocol should include a thorough history, detailed TMJ examination, and radiography as the first imaging choice in field practice, with CT as the 'gold standard' in clinical settings. For septic TMJ arthritis, a bacterial culture is recommended, while for primary TMJ OA, functional tests and local analgesia.

A multifunctional sensor for cell traction force, matrix remodeling and biomechanical assays in self-assembled 3D tissues in vitro.

Cell-matrix interactions, mediated by cellular force and matrix remodeling, result in dynamic reciprocity that drives numerous biological processes and disease progression. Currently, there is no available method for directly quantifying cell traction force and matrix remodeling in three-dimensional matrices as a function of time. To address this long-standing need, we developed a high-resolution microfabricated device that enables longitudinal measurement of cell force, matrix stiffness and the application of mechanical stimulation (tension or compression) to cells. Here a specimen comprising of cells and matrix self-assembles and self-integrates with the sensor. With primary fibroblasts, cancer cells and neurons we have demonstrated the feasibility of the sensor by measuring single or multiple cell force with a resolution of 1 nN and changes in tissue stiffness due to matrix remodeling by the cells. The sensor can also potentially be translated into a high-throughput system for clinical assays such as patient-specific drug and phenotypic screening. We present the detailed protocol for manufacturing the sensors, preparing experimental setup, developing assays with different tissues and for imaging and analyzing the data. Apart from microfabrication of the molds in a cleanroom (one time operation), this protocol does not require any specialized skillset and can be completed within 4-5 h.

Co-isolation of human donor eye cells and development of oncogene-mutated melanocytes to study uveal melanoma

BackgroundUveal melanoma (UM) is the most common intraocular tumor in adults, arises either de novo from normal choroidal melanocytes (NCMs) or from pre-existing nevi that stem from NCMs and are thought to harbor UM-initiating mutations, most commonly in GNAQ or GNA11. However, there are no commercially available NCM cell lines, nor is there a detailed protocol for developing an oncogene-mutated CM line (MutCM) to study UM development. This study aimed to establish and characterize premalignant CM models from human donor eyes to recapitulate the cell populations at the origin of UM.ResultsGiven the precious value of human donor eyes for studying multiple ocular cell types, we validated a co-isolation protocol of both human NCMs and retinal pigment epithelial (RPE) cells from a single eye. To this end, NCMs and RPE cells were sequentially isolated from 20 donors, with success rates of 95% and 75%, respectively. MutCMs were generated from 10 donors using GNAQQ209L-carried lentivirus with high mutant copies (up to 98.8% of total GNAQ copies being mutant). NCM growth and behavior were characterized under different culture conditions (i.e., supplementation with serum and 12-O-tetradecanoylphorbol-13-acetate) to determine optimized protocols. Particularly, Matrigel™ coating induced spheroid growth under certain coating thickness and cell seeding density but did not improve NCM metabolic activity. Current methodologies in NCM isolation, culture, and research applications were summarized. Proteomic profiling of 4 NCMs, 1 MutCM, and 3 UMs allowed to discover significant differences in UMs including a downregulation of proteins linked to melanocyte differentiation and an upregulation of proteins involved in RNA metabolism. RNA sequencing revealed enriched pathways related to cancer, notably PI3K-Akt and MAPK signaling pathways, in MutCMs and UM cells compared to NCMs, providing insights into molecular changes in GNAQQ209L-mutated pre-cancer cell models and UM cells.ConclusionsWe successfully isolated and established NCM, RPE, and MutCM cell lines. We describe efficient methods for the isolation and growth of NCMs and report their phenotypic, proteomic, and transcriptomic characteristics, which will facilitate the investigation of UM development and progression. The co-isolated RPE cells could benefit research on other ocular pathologies, such as age-related macular degeneration.Graphical Created with https://www.biorender.com/.

In Vitro Culturing Technique for Studying Cellular Dynamics in Zebrafish Scales.

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without the need for euthanasia, regenerate within a couple of weeks, and are translucent and small, allowing them to be viewed using a standard microscope. Zebrafish scales are especially useful in educational environments, as they provide a unique opportunity for students to engage in hands-on learning experiences, particularly in understanding cellular dynamics and in vitro culturing methods. The main objective of this protocol is to describe a method for collecting and maintaining zebrafish scales in culture for use in a variety of biological studies using basic laboratory equipment. Additionally, the protocol details their use in understanding bone homeostasis by examining the activity of bone cells involved in bone resorption and deposition. It also includes additional protocols for general techniques, such as the visualization of nuclei and apoptotic cells. The in vitro culturing protocol produces reliable results with minimal reagents and equipment. This article discusses the benefits of using in vitro cultures of zebrafish scales to foster scientific inquiry and outlines the resources needed to support their integration into educational settings.

Evaluating Therapeutic and Chemical Toxicity Using Organ-Cultured Porcine Corneas and Epithelial Wound Healing.

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular toxicity assessment. An air/liquid corneal culture system using porcine eyes was developed, and ex vivo epithelial wound healing was utilized as a critical parameter for these studies. Fresh pig corneas were processed for organ culture, with or without epithelial wounding. The corneas were cultured in a humidified 5% CO2 incubator at 37 °C in MEM, with or without testing agents. Corneal permeability and wound healing rates were measured, and epithelial cells and/or whole corneas can be processed for immunohistochemistry, western blotting, and qPCR for molecular and cellular analyses. This study describes a detailed protocol and presents two studies using this ex vivo system. The data show that porcine corneal organ culture, combined with epithelial wound healing, is a suitable ex vivo model for chemical toxicity testing, studying diabetic keratopathy, and identifying potential therapies.

Voices from the Rainbow: Exploring Participants' Experiences in an Online LGBTIQ+-Affirmative Psychoeducation Program.

Background/Objectives: This article highlights the importance of addressing the mental health of LGBTQ people, specifically through psychoeducation/intervention programs. The primary objective was to understand the effects of participating in an online affirmative program. The theoretical frameworks of the minority stress model and the concept of decompensation were used to understand and address disparities with the general population. Methods: A detailed protocol was designed to understand the participants' experiences. An analysis of the results was carried out using a content analysis of the data collected from a convenience sample of participants from two groups. Results: The findings show that the participants' experiences were satisfactory, especially with developing coping skills and changing their attitudes towards homophobia, also showing improvements in mental health indicators. Conclusions: This study concludes that online LGBTQ-affirmative psychoeducation programs can be positive in enhancing the mental health and well-being of the LGBTQ population. The research highlights the importance of extending these programs to LGBTQ family members. It is also important to acknowledge the specificities of each subgroup.

3D Cell Culture Models as a Platform for Studying Tumor Progression, Testing Treatment Responses, and Discovering Biomarkers.

In this chapter, we present a detailed protocol for establishing a three-dimensional (3D) multicellular tumor spheroids (MCTSs) model to simulate the tumor microenvironment (ME) associated with metabolic dysfunction-associated steatotic liver disease (MASLD) for the study of hepatocellular carcinoma (HCC) and colorectal cancer (CRC) cell aggressiveness, growth, and metastasis potential. The MASLD microenvironment (MASLD-ME) is recreated by embedding hepatic stellate cells in a collagen I matrix within a Boyden chamber system. The metabolic medium mimics MASLD conditions, enriched with high glucose, fructose, insulin, and fatty acids, to simulate metabolic stresses associated with the disease.In the protocol, cancer cells are loaded in the upper compartment to analyze their migration toward the MASLD-ME, thereby facilitating studies on cancer cell invasiveness and metastatic capacity. This method offers an adaptable, reproducible model to research disease progression and investigate therapeutic interventions, contributing to preclinical research on MASLD-related liver cancer pathophysiology and potential drug responses.

Reproducibility and Consistency of Isolation Protocols for Fibroblasts, Smooth Muscle Cells, and Epithelial Cells from the Human Vagina.

(1) Background: For the reconstruction of a human vagina, various surgical procedures are available that are often associated with complications due to their failure to mimic the physiology of the human vagina. We recently developed a vascularized, organ-specific matrix from healthy human vaginal wall tissue with suitable biomechanical properties. A superior graft would require further extensive colonization with autologous vaginal cells to reduce complications upon implantation. However, reports on isolation of vaginal cells from biopsies are scarce, and published protocols rarely contain sufficient details. In this study, we aimed to examine protocols for inconsistencies and identify (where possible) the optimal protocol in terms of reproducibility and efficiency for isolation of human vaginal fibroblasts (FBs), epithelial cells (VECs), and smooth muscle cells (SMCs). Overall, this study aims to guide other researchers and aid future tissue engineering solutions that rely on autologous cells. (2) Methods: A total of 41 isolation protocols were tested: four protocols specific to FBs, 13 protocols for VECs, and 24 protocols for SMCs. Protocols were derived from published reports on cell isolation by enzymes, with exclusion criteria including the need for specialized equipment, surgical separation of tissue layers, or missing protocol details. Enzymatic digestion with collagenase-I, collagenase-IV, and dispase-II was used for isolation of VECs, collagenase-IV for isolation of SMCs, and collagenase-IA for isolation of FBs. Fluorescent immunostaining was applied to identify VECs with cytokeratin, SMCs with desmin, endothelial cells with UEA-1, and FBs with vimentin. Protocols were assessed based on (>95%) homogeneity, duplicate consistency, cell viability, and time to first passage. (3) Results: A total of 9 out of the 41 protocols resulted in isolation and expansion of vaginal FBs. This involved 1 out of 13 VEC protocols, 6 out of 24 SMC protocols, and 2 out of 2 FB protocols. Isolation of vaginal SMCs or VECs was not achieved. The best results were obtained after digestion with 0.1% collagenase-IV, where pure FB colonies formed with high cell viability. (4) Conclusions: Today, vaginoplasty is considered the gold standard for surgically creating a neovagina, despite its considerable drawbacks and limitations. Tissue-engineered solutions carry great potential as an alternative, but cell seeding is desired to prevent complications upon implantation of grafts. In this study, we examined isolation of human vaginal FBs, SMCs, and VECs, and identified the most efficient and reliable protocol for FBs. We further identified inconsistencies and irreproducible methods for isolation of VECs and SMCs. These findings aid the clinical translation of cell-based tissue engineering for the reconstruction and support of vaginas, fulfilling unmet medic needs.

Fluorescence Analysis of Quinine in Commercial Tonic Waters.

Quinine is known for treating malaria, muscle cramps, and, more recently, has been used as an additive in tonic water due to its bitter taste. However, it was shown that excessive consumption of quinine can have severe side effects on health. In this work, we utilized fluorescence spectroscopy to measure the concentration of quinine in commercial tonic water samples. An external standard method was used to calculate the concentrations of quinine in two commercially available tonic water brands, namely Canada Dry and Schweppes, and compare them to the maximum allowable concentration of quinine in beverages. Upon analysis of the data collected by five different groups, the levels of quinine were found to be above the average concentration in most commercial tonic water samples, but below the maximum permitted concentration. Moreover, the five replicate sets of data demonstrated high reproducibility of the method employed in this study. The simple yet instructive protocol that we developed can be adapted to determine the concentration of other fluorescent compounds in foods and beverages. Further, the presented method and detailed protocol can be easily adopted for undergraduate labs and in chemical education.

The diagnostic accuracy of point-of-care nucleic acid-based isothermal amplification assays for scrub typhus: a systematic review and meta-analysis.

The diagnosis and detection of pathogens such as Rickettsia and Orientia is a cause of major concern among the public health community. Unavailability of rapid, cost-effective diagnostic assays contributes to delayed diagnosis and timely treatment. Using the methodology of systematic reviewing and meta-analysis, the study aimed to synthesize and compare the diagnostic performances of all the available isothermal assays for the detection of classical rickettsial diseases. Studies were retrieved from PubMed and Scopus, and selection and screening were conducted using pre-determined inclusion and exclusion criteria. Analysis was performed using Meta-DiSc 2.0 for the assessment and comparison of diagnostic performance of the isothermal assays. Overall, six studies were selected as a part of this systematic review. All the selected studies (n = 6) optimized LAMP as their index test to detect scrub typhus. The quality assessment of the selected studies revealed only (n = 1) study to be of poor quality with a QUADAS-2 score of (<2). Meta-analysis revealed the pooled sensitivity of LAMP to be 66% [95% CI (0.40-0.85)] with a pooled specificity of 94% [95% CI (0.81-0.98)]. LAMP was estimated with a positive likelihood ratio of 8.3 [95% CI (3.8-18.1)] and a negative likelihood ratio of 0.3 [95% CI (0.2-0.7)] with a false positivity rate of 0.07 [95% CI (0.02-0.2)]. The diagnostic odds ratio was reported to be 21.96 [95% CI (10.2-47.3)]. Due to severe heterogeneity in the body of evidence (I 2 = 0.77), a meta-regression was performed with certain covariates to explore the potential causes. A case-control design was found to exaggerate the sensitivity {0.84 [95% CI (0.5-0.9)]} and specificity {0.73 [95% CI (0.6-0.8)]}. The findings reveal subpar performance of LAMP for the detection of scrub typhus. Active research and development focused on optimization of novel molecular diagnosis that are efficient, rapid, and cost-effective shall foster timely diagnosis and aid in reduction of the overall burden of scrub typhus. A detailed protocol of this review is registered and available in Prospero at: https://www.crd.york.ac.uk/prospero/. (registration number CRD42024511706).

Differential DNA Extraction from Lateral Flow Immunochromatographic Tests via the EZ1® Advanced XL System.

This differential extraction protocol details the steps for isolating DNA from sample pads used in lateral flow immunochromatographic (LFI) tests, particularly for cases involving mixed biological samples such as semen and menstrual blood, or other evidence related to sexual assault. This procedure utilizes a differential extraction technique applied to sample pads from immunochromatographic tests, where the sample pads serve as the substrate. The method involves two sequential lysis steps to effectively separate non-sperm and sperm fractions, enabling the targeted isolation of distinct cell types for downstream DNA analysis. The efficiency of this procedure is demonstrated by the results within this paper, which highlights the successful recovery of both male autosomal and Y-STR profiles, even in mixed samples with a high female presence. Overall, this protocol demonstrates the effective recovery of DNA from sample pads, which is beneficial for forensic practitioners dealing with limited sample quantities, underscoring the value of using these pads in forensic analysis.

Using HBmito Crimson to Observe Mitochondrial Cristae Through STED Microscopy.

Mitochondrial cristae, formed by folding the mitochondrial inner membrane (IM), are essential for cellular energy supply. However, the observation of the IM is challenging due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vitro probes specifically targeting the IM. Here, we describe a detailed imaging protocol for the mitochondrial inner membrane using the Si-rhodamine dye HBmito Crimson, which has excellent photophysical properties, to label live cells for imaging via stimulated emission depletion (STED) microscopy. This allows for STED imaging over more than 500 frames (approximately one hour), with a spatial resolution of 40 nm, enabling the observation of cristae dynamics during various mitochondrial processes. The protocol includes detailed steps for cell staining, image acquisition, image processing, and resolution analysis. Utilizing the superior resolution of STED microscopy, the structure and complex dynamic changes of cristae can be visualized. Key features • The protocol is designed to visualize mitochondrial cristae in living cells using STED microscopy. • The protocol enables nanoscale observation of dynamic mitochondrial cristae. • Real-time observation of mitochondrial morphological changes, fusion, and fission events.

Home Care Patient's Experiences and Medication Burden Related to High-Risk Medication Use: A Cross-Sectional Study.

Age, polypharmacy and comorbidity are examples of known factors that increase the risk of adverse drug reactions in patients. The use of high-risk medication also entails a heightened risk of harm. There is currently no information available on the home care patients' experiences and medication burden experienced due to their high-risk medication use and how they manage their medication. Further investigation with regard to this combination is necessary. The patient's experiences and medication burden related to high-risk medication use can be taken into account when drawing up guidelines and standards of care for healthcare professionals. To describe home care patients' experiences and medication burden related to high-risk medication use, more specifically how patients manage their high-risk medication use, which professional support they receive and which potential adverse drug reactions they experience. A cross-sectional study of home care patients in Belgium, aged 65 years and older who took at least one high-risk medication. In our population of 106 home care patients, a median use of 8 medications per patient is reported, of which 2 can be considered high-risk medication. Metformin, insulin and lormetazepam are the most frequently used high-risk medications. Home care patients believe their medication is important to them, are able to manage the intake and seem to have a high level of therapy adherence. Most patients do not believe their medication intake implies a certain risk. Most patients are supported by a home care nurse for the preparation of their medication. A mean number of 5 symptoms/potential adverse drug reactions is reported out of the 21 potential adverse drug reactions questioned. The potential adverse drug reaction most frequently attributed to medication use was bleeding. Practice guidelines with detailed medicine-specific protocols are needed to enhance (high-risk) medication-related care in an overall high-risk medication policy. Understanding the patient's risk experiences and communicating with the patient is important to ensure safe medication care but also to identify patients at risk for nonadherence and adverse reactions. The patient's experiences with their medication intake provide rich information for healthcare providers and should therefore be included in patient observations. Home care nurses should closely follow up on the home care patient's medication therapy with respect for the patient's autonomy.

A Sensitive Radioimmunoassay for T3 and T4 Determinations in Plasma and Tissues.

This chapter details protocols for determining plasma thyroid hormone (TH) levels and tissue TH content by competitive radioimmunoassays (RIAs). These protocols include: an initial test of the chromatographic performance, isotopic labeling to produce high activity 125I-T3 and 125I-T4, free iodide estimation of the labeled products, purification of tracers from iodide by paper electrophoresis, extraction of THs from plasma and tissue samples, and the RIA procedures. The RIA involves the competition between radioactive labeled and unlabeled hormones for specific antibody binding, and due to its high sensitivity is capable of detecting a minimum of 2.5pg of T4 and 0.4pg of T3.Drs. María Jesús Obregon and Gabriella Morreale de Escobar improved these protocols in the 1970s, enhancing sensitivity and accuracy. Their improvements enabled the detection of TH content in tissues, providing crucial insights into maternal THs role in fetal development and the importance of iodine intake during gestation. Their research also facilitated the early detection of congenital hypothyroidism, preventing neurological impairments in newborns. Internationally and in Spain, their contributions are widely acknowledged, leading to substantial public health impacts, including the implementation of nationwide neonatal screening programs. Despite progress, thyroid diseases remain prevalent, underscoring the need for continued research into thyroid physiology and associated disorders, employing highly sensitive techniques like the RIA outlined herein that we continue using in our research.

Systematic review meta-analysis protocol preparation and registration – A narrative review

Systematic reviews and meta-analyses (SRMAs) help in decision making, especially on conflicting publication findings and quality. SRMA identifies the source of heterogeneity, reduces subjectivity and identifies and fills gaps in existing knowledge. However, conducting an SRMA requires a well-written protocol, which acts as a clear roadmap for the researcher and defines objectives, methods and expected outcomes. Predefined research questions and methods minimise bias, thus increasing the reliability of the results. The protocol ensures that the study team stays consistent in data collection, synthesis and interpretation. The SRMA protocol includes rationale, hypothesis, aims and methodology. A detailed protocol improves the overall quality of the research. All SRMA protocols must be registered and made available publicly. Protocol registration ensures transparency in the research process, which avoids the wastage of resources and prevents duplication. A well-planned protocol facilitates faster peer review and enhances the chances for publication. This narrative review primarily focuses on SRMA protocol preparation and its registration.

Maistero-2, a Novel Probe for Sterols: Application for Visualizing Cellular Cholesterol.

Maistero-2 is a novel, non-toxic cholesterol-binding protein derived from an edible mushroom Grifola frondosa mRNA. Maistero-2 specifically binds to lipid membranes containing 3-hydroxy sterols with a lower cholesterol concentration threshold than cholesterol-binding domain 4 (D4) of perfringolysin O (PFO) and anthrolysin O (ALO). Maistero-2 binding is particularly sensitive to the size and conformation of the A-, B-, and D-ring of sterols but not very sensitive to modifications of the isooctyl side chain commonly found in phytosterols. EGFP-maistero-2 has been demonstrated to be a suitable tool to visualize the increase of cell surface cholesterol during the differentiation of Neuro 2a cells and heterogeneous cholesterol distribution between CD63-positive and LAMP1-positive late endosomes/lysosomes in HeLa cells. This chapter describes the detailed protocols for examining the binding of maistero-2 to lipids and model membranes and the labeling of cell surface and intracellular cholesterol by EGFP-maistero-2.

Generation of a Mouse Model for the Study of Thyroid Hormones Regulatory Effect on the Immune System.

The generation of hypothyroid and hyperthyroid mouse models is one of the approaches used to investigate the complex interplay between thyroid hormones and the immune system. We present a detailed protocol describing how to induce endotoxic shock by lipopolysaccharide (LPS) administration, and how to investigate the role of immune populations, specifically macrophages, responding to endotoxemia.This book chapter provides the use of different molecular techniques, such as Western Blotting, Immunohistochemistry, q-PCR, Luciferase assays, or ChIP assays, with which researchers can gain valuable insights into the immune system's interaction with hormonal signaling pathways, for instance, examining the effect of thyroid hormones on signaling of STAT3, NF-κB, and ERK in response to LPS, and inflammatory mediators, such as interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNFα) within these cells. The signaling pathways involved and the exploration of the relationship between thyroid hormones and the immune system can be analyzed using several molecular biology technologies in order to clarify their interplay in various disease states.

Flow-based In Vivo Method to Enumerate Translating Ribosomes and Translation Elongation Rate.

Protein synthesis is by far the most energetically costly cellular process in rapidly dividing cells. Quantifying translating ribosomes in individual cells and their average mRNA transit rate is arduous. Quantitating assembled ribosomes in individual cells requires electron microscopy and does not indicate ribosome translation status. Measurement of average transit rates entails in vitro pulse-chase radiolabeling of isolated cells or ribosome profiling after ribosome runoff, which is expensive and extremely demanding technically. Here, we detail protocols based on ribosome-mediated nascent chain puromycylation, harringtonine to stall initiating ribosomes while allowing ribosome elongation to continue normally, and cycloheximide to freeze translating ribosomes in place. Each compound is delivered intravenously to mice in the appropriate order, and after ex vivo cell fixation and permeabilization, translating ribosome numbers and transit rates are measured by flow cytometry using a directly conjugated puromycin-specific antibody. Key features • Measure relative numbers of translating ribosomes in mixed single-cell preparations. • Quantitate relative in vivo ribosome transit rates in mixed single-cell preparations. • Detect ribosome stalling in vivo.

Golden Gate Cloning of MoClo Standard Parts.

Efficient DNA assembly methods are an essential prerequisite in the field of synthetic biology. Modular cloning systems, which rely on Golden Gate cloning for DNA assembly, are designed to facilitate assembly of multigene constructs from libraries of standard parts through a series of streamlined one-pot assembly reactions. Standard parts consist of the DNA sequence of a genetic element of interest such as a promoter, coding sequence, or terminator, cloned in a plasmid vector. Standard parts for the modular cloning system MoClo, also called level 0 modules, must be flanked by two BsaI restriction sites in opposite orientations and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of level 0 modules. This protocol requires the following steps: (1) defining the type of part that needs to be cloned, (2) designing primers for amplification, (3) performing polymerase chain reaction (PCR) amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large standard parts, it is preferable to first clone sub-parts as intermediate level -1 constructs. These sub-parts are sequenced individually and are then further assembled to make the final level 0 module.