Standard Laboratory Equipment Research Articles

Purification of Adeno-Associated Viral Vector Serotype 9 Using Ceramic Hydroxyapatite Chromatography and its Analysis.

Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic Protocols and optional methods for evaluating purified AAV vectors are also described. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of AAV9 vectors in 293EB cells Basic Protocol 2: Concentration and buffer exchange of AAV9 vectors from 293EB cell culture supernatants using tangential flow filtration Basic Protocol 3: Purification of AAV9 vectors from TFF samples using ceramic hydroxyapatite chromatography Basic Protocol 4: Analysis of the purified AAV9 vectors.

THE EFFICIENCY AND COEFFICIENT OF SPORTS BALLS RESTITUTION USING AN ACOUSTIC APPLICATION

Studying the efficiency of a sports ball and its coefficient of restitution is important for performance optimization, fairness, and consistency of play. This research paper is dedicated to studying the coefficient of restitution and bounce efficiency in sports ball. Ping Pong ball and tennis balls were used to show the relationship between the bounce height and drop height. This paper used standard laboratory equipment like meter ruler, retort stand, clamp and with the utilization of the acoustic program of the smartphone using PhyPhox. The result showed that the application was able to accurately measure the bounce height in relation to the drop height. This was showed in the straight-line graph plotted. There was a constant ball efficiency coefficient of restitution and have very small percent error from the standard. This experiment can boost the interest and confidence of students in the classroom. Being able to accurately measure the performance of different balls can help students understand the science and engineering principles behind how they work. This knowledge can benefit students by allowing them to make more informed decisions when purchasing sports equipment, as well as give them an opportunity to modify their playing methods and strategies.

Abstract 7622: ASPYRE-Lung: Validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in tissue

Abstract Genomic variant testing of tumors is critical for patients to access the full potential of personalized oncology therapeutics. Current methods such as NGS are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE® (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung® panel for NSCLC covers 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3) to robustly inform clinical management, based on practice guidelines. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously. We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with NSCLC, variant-negative FFPE tissue from tonsil tissue, and FFPE-based contrived samples with controllable variant allele fractions. The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, ≤ 100 copies for fusions, and ≤ 200 copies for MET exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant variants between ASPYRE-Lung and orthogonal testing (targeted enrichment NGS) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision. The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based analysis algorithm. ASPYRE-Lung has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with NSCLC. Citation Format: Ryan T. Evans, Elizabeth Gillon-Zhang, Julia Brown, Katherine Knudsen, Candace King, Amanda S. Green, Ana-Luisa Silva, Justyna M. Mordaka, Rebecca N. Palmer, Alessandro Tomassini, Alejandra Collazos, Christina Xyrafaki, Iyelola Turner, Chau Ha Ho, Dilyara Nugent, Jinsy Jose, Simonetta Andreazza, Kristine von Bargen, Eleanor R. Gray, Magdalena Stolarek-Januszkiewicz, AIshling Cooke, Wendy J. Levin, Honey V. Reddi, Barnaby W. Balmforth, Robert J. Osborne. ASPYRE-Lung: Validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7622.

Single-step immobilization of hydroxyapatite on fluoropolymer surfaces for enhanced cell adhesion

Herein, we report a single-step immobilization of hydroxyapatite (HAp) on the surface of poly(vinylidene fluoride) copolymer. This method utilizes the ability of fluoropolymer to undergo a limited swelling in acetone/water mixture followed by an entrapment of HAp on its surface. We found that while acetone/water ratio does not affect the thickness of HAp-containing layer, an increase in acetone content results in higher amounts of calcium and phosphorus found in modified area. HAp immobilization not only improved the hydrophilicity of the poly(vinylidene fluoride) copolymer surface but also contributed to enhanced cell adhesion (0.8 ± 0.4 % for the fluoropolymer surface vs. 86.9 ± 9.1 % for the modified fluoropolymer surface) and viability (increased by up to 30 %). Overall, this method represents a potent strategy for the modification of fluoropolymers which allows for the fabrication of bioactive implants using standard laboratory equipment.

PIV-MyoMonitor: an accessible particle image velocimetry-based software tool for advanced contractility assessment of cardiac organoids.

Induced pluripotent stem cell (iPSC)-derived cardiac organoids offer a versatile platform for personalized cardiac toxicity assessment, drug screening, disease modeling, and regenerative therapies. While previous image-based contractility analysis techniques allowed the assessment of contractility of two-dimensional cardiac models, they face limitations, including encountering high noise levels when applied to three-dimensional organoid models and requiring expensive equipment. Additionally, they offer fewer functional parameters compared to commercial software. To address these challenges, we developed an open-source, particle image velocimetry-based software (PIV-MyoMonitor) and demonstrated its capacity for accurate contractility analysis in both two- and three-dimensional cardiac models using standard lab equipment. Comparisons with four other open-source software programs highlighted the capability of PIV-MyoMonitor for more comprehensive quantitative analysis, providing 22 functional parameters and enhanced video outputs. We showcased its applicability in drug screening by characterizing the response of cardiac organoids to a known isotropic drug, isoprenaline. In sum, PIV-MyoMonitor enables reliable contractility assessment across various cardiac models without costly equipment or software. We believe this software will benefit a broader scientific community.

A Modular Robotic Platform for Biological Research: Cell Culture Automation and Remote Experimentation

Robotic arms are now commonplace in diverse settings and are poised to play a crucial role in automating laboratory tasks. However, biological experiments remain challenging for automation due to their dependence on human factors, such as researchers’ skills and experience. This article introduces robotic automation and remote control for both general and biological research tasks through a modularized platform comprising a robotic arm, auxiliary tools, and software. This platform facilitates fully automated or remote execution of key experiments in chemistry and biology, including liquid handling, mixing, cell seeding, culturing, and genetic manipulation. The robot interfaces seamlessly with standard laboratory equipment and operates remotely in real time through an online program. Integration of a vision system via robotic arm webcams ensures precise positioning and object localization, enhancing accuracy. This modularized robotic platform signifies a substantial advancement in lab automation, promising enhanced efficiency, reproducibility, and scientific progress compared to human‐led experiments.

A protocol for a turbidimetric assay using a Saccharomyces cerevisiae thiamin biosynthesis mutant to estimate total vitamin B1 content in plant tissue samples

BackgroundUnderstanding thiamin (thiamine; vitamin B1) metabolism in plants is crucial, as it impacts plant nutritional value as well as stress tolerance. Studies aimed at elucidating novel aspects of thiamin in plants rely on adequate assessment of thiamin content. Mass spectrometry-based methods provide reliable quantification of thiamin as well as closely related biomolecules. However, these techniques require expensive equipment and expertise. Microbiological turbidimetric assays can evaluate the presence of thiamin in a given sample, only requiring low-cost, standard lab equipment. Although these microbiological assays do not reach the accuracy provided by mass spectrometry-based methods, the ease with which they can be deployed in an inexpensive and high-throughput manner, makes them a favorable method in many circumstances. However, the thiamin research field could benefit from a detailed step-by-step protocol to perform such assays as well as a further assessment of its potential and limitations.ResultsHere, we show that the Saccharomyces cerevisiae thiamin biosynthesis mutant thi6 is an ideal candidate to be implemented in a turbidimetric assay aimed at assessing the content of thiamin and its phosphorylated equivalents (total vitamer B1). An optimized protocol was generated, adapted from a previously established microbiological assay using the thi4 mutant. A step-by-step guidance for this protocol is presented. Furthermore, the applicability of the assay is illustrated by assessment of different samples, including plant as well as non-plant materials. In doing so, our work provides an extension of the applicability of the microbiological assay on top of providing important considerations upon implementing the protocol.ConclusionsAn inexpensive, user-friendly protocol, including step-by-step guidance, which allows adequate estimation of vitamer B1 content of samples, is provided. The method is well-suited to screen materials to identify altered vitamer B1 content, such as in metabolic engineering or screening of germplasm.

An innovative charge‐based extracellular vesicle isolation method for highly efficient extraction of EV‐miRNAs from liquid samples: miRQuick

AbstractExtracellular vesicle‐derived microRNAs (EV‐miRNAs) are promising biomarkers for early cancer diagnosis. However, existing EV‐miRNA extraction technologies have a complex two‐step process that results in low extraction efficiency and inconsistent results. This study aimed to develop and evaluate a new single‐step extraction method, called miRQuick, for efficient and high‐recovery extraction of EV‐miRNAs from samples. The miRQuick method involves adding positively charged substances to the sample, causing negatively charged EVs to quickly aggregate and precipitate. A membrane lysate is then added to extract only miRNA. The entire process can be completed within an hour using standard laboratory equipment. We validated the miRQuick method using various analytical techniques and compared its performance to other methods for plasma, urine and saliva samples. The miRQuick method demonstrated significantly higher performance than other methods, not only for blood plasma but also for urine and saliva samples. Furthermore, we successfully extracted and detected nine biomarker candidate miRNAs in the plasma of breast cancer patients using miRQuick. Our results demonstrate that miRQuick is a rapid and efficient method for EV‐miRNA extraction with excellent repeatability, making it suitable for various applications including cancer diagnosis.

Sit-to-walk strategy classification in healthy adults using hip and knee joint angles at gait initiation

Forward continuation, balance, and sit-to-stand-and-walk (STSW) are three common movement strategies during sit-to-walk (STW) executions. Literature identifies these strategies through biomechanical parameters using gold standard laboratory equipment, which is expensive, bulky, and requires significant post-processing. STW strategy becomes apparent at gait-initiation (GI) and the hip/knee are primary contributors in STW, therefore, this study proposes to use the hip/knee joint angles at GI as an alternate method of strategy classification. To achieve this, K-means clustering was implemented using three clusters corresponding to the three STW strategies; and two feature sets corresponding to the hip/knee angles (derived from motion capture data); from an open access online database (age: 21–80 years; n = 10). The results identified forward continuation with the lowest hip/knee extension, followed by balance and then STSW, at GI. Using this classification, strategy biomechanics were investigated by deriving the established biomechanical quantities from literature. The biomechanical parameters that significantly varied between strategies (P < 0.05) were time, horizontal centre of mass (COM) momentum, braking impulse, centre of pressure (COP) range and velocities, COP–COM separation, hip/knee torque and movement fluency. This alternate method of strategy classification forms a generalized framework for describing STW executions and is consistent with literature, thus validating the joint angle classification method.

The spinning toilet brush–a classroom experiment on the mechanical equivalent of Joule’s heat

Although Joule’s experiment on the mechanical equivalent of heat is well known and discussed in many introductory physics courses and textbooks, we are not aware of any version of this experiment that can be set up by lecturers or teachers using standard laboratory equipment. To fill this gap, we present a setup quite similar to Joule’s original experiment and show what kind of quantitative results can be obtained with it. It will be shown that temperature control is a major challenge, making Joule’s work even more impressive.

Making Every Single Puff Count-Simple and Sensitive E-Cigarette Aerosol Sampling for GCxIMS and GC-MS Analysis.

The analysis of the aerosol from tobaccoless electronic cigarettes (e-cigarettes) is an important part of understanding their impact on human health, yet sampling aerosol from e-cigarettes is still considered a challenge. It lacks a standard method for research and quality control and there are a variety of methods. However, few are simple and inexpensive, and none have been suggested for the use with gas chromatography coupled ion mobility spectrometry (GCxIMS). This work presents and evaluates such a setup made from standard lab equipment to quickly collect a quantitative sample from the aerosol of a single puff (5 s totaling 125 mL). The aerosol condensates directly in the cooled headspace (HS) vial, which is analyzed in the HS-GCxIMS or mass spectrometer (HS-GC-MS). The combined use of GC-MS and GCxIMS allows the simple and sensitive identification of unknown substances in complex mixtures and the identification of degradation products in the aerosols. A calibration of 26 flavor compounds (0.2-20 µg/g) was created using single puffs of a spiked, flavorless commercial refill solution and 2-alkanones as internal standards. This sensitive but easily reproducible setup enables a wide range of further investigations, even for labs that were previously unable to afford it.

Suitability of Low-Cost Sensors for Submicron Aerosol Particle Measurement

The measurement and assessment of indoor air quality in terms of respirable particulate constituents is relevant, especially in light of the COVID-19 pandemic and associated infection events. To analyze indoor infectious potential and to develop customized hygiene concepts, the measurement monitoring of the anthropogenic aerosol spreading is necessary. For indoor aerosol measurements usually standard lab equipment is used. However, these devices are time-consuming, expensive and unwieldy. The idea is to replace this standard laboratory equipment with low-cost sensors widely used for monitoring fine dust (particulate matter—PM). Due to the low acquisition costs, many sensors can be used to determine the aerosol load, even in large rooms. Thus, the aim of this work is to verify the measurement capability of low-cost sensors. For this purpose, two different models of low-cost sensors are compared with established laboratory measuring instruments. The study was performed with artificially prepared NaCl aerosols with a well-defined size and morphology. In addition, the influence of the relative humidity, which can vary significantly indoors, on the measurement capability of the low-cost sensors is investigated. For this purpose, a heating stage was developed and tested. The results show a discrepancy in measurement capability between low-cost sensors and laboratory measuring instruments. This difference can be attributed to the partially different measuring method, as well as the different measuring particle size ranges. The determined measurement accuracy is nevertheless good, considering the compactness and the acquisition price of the low-cost sensors.

Investigating mass transfer coefficients in lean methane combustion reaction through the morphological and geometric analysis of structured open cell foam catalysts

In this work, we characterized ceramic open cell foams made of zirconia, silicon carbide and alumina of different nominal pore densities (30–45 ppi) using X-ray computed microtomography. This technique allowed the comprehensive and quantitative extraction of morphological and geometrical characteristics of the structures (pore size, strut diameter and length, node diameter, open porosity and specific geometrical surface area). An empirical model was proposed to determine the specific surface area from parameters easily accessible with standard laboratory equipment. Moreover, lean CH4 combustion tests were performed on ceramic foams coated with PdO/Co3O4 at 3 wt%. Mass transfer coefficients of the foams were measured by monitoring the oxidation reaction under diffusional control conditions. We derived a dimensionless correlation of the type Sh = A∙Rem∙Sc1/3. Finally, the pressure drop across the foams as a function of surface velocity was evaluated and compared with various experimental data and models available in the literature.

Large-scale growth of C. elegans and isolation of membrane protein complexes.

Purification of membrane proteins for biochemical and structural studies is commonly achieved by recombinant overexpression in heterologous cell lines. However, many membrane proteins do not form a functional complex in a heterologous system, and few methods exist to purify sufficient protein from a native source for use in biochemical, biophysical and structural studies. Here, we provide a detailed protocol for the isolation of membrane protein complexes from transgenic Caenorhabditis elegans. We describe how to grow a genetically modified C. elegans line in abundance using standard laboratory equipment, and how to optimize purification conditions on a small scale using fluorescence-detection size-exclusion chromatography. Optimized conditions can then be applied to a large-scale preparation, enabling the purification of adequate quantities of a target protein for structural, biochemical and biophysical studies. Large-scale worm growth can be accomplished in ~9 d, and each optimization experiment can be completed in less than 1 d. We have used these methods to isolate the transmembrane channel-like protein 1 complex, as well as three additional protein complexes (transmembrane-like channel 2, lipid transfer protein and 'Protein S'), from transgenic C. elegans, demonstrating the utility of this approach in purifying challenging, low-abundance membrane protein complexes.

Performance of a manually operated salad spinner centrifuge for serum separation in the healthy domestic horse (Equus caballus) and southern white rhinoceros (Ceratotherium simum).

Field veterinarians and researchers studying wild species, such as the southern white rhinoceros, often work in remote areas with limited access to standard laboratory equipment, hindering the ability to measure serum analytes. The first objective was to produce an inexpensive, manually operated centrifuge that could accept standard laboratory tubes by modifying a consumer-grade salad spinner with low-cost materials. The second objective was to compare biochemistry analysis results obtained from equine and southern white rhinoceros serum separated by traditional laboratory and manual salad spinner centrifugation. We optimized the design and serum separation protocol using non-anticoagulated equine blood. Equine and rhinoceros serum samples were separated by manual salad spinner or traditional laboratory centrifugation. Measured analytes included sodium, potassium, chloride, urea nitrogen, creatinine, phosphorous, total calcium, magnesium, glucose, total protein, albumin, globulin, creatinine kinase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, total bilirubin, bicarbonate, sorbitol dehydrogenase, and triglycerides. Results obtained from serum separated by each centrifugation technique were compared by Deming regression and Bland-Altman analyses. A tube adaptor insert modeled after a swing angle rotor and a two-step salad spinner centrifugation yielded serum comparable to traditional laboratory centrifugation. For the majority of analytes, no proportional or constant biases were detected between centrifugation methods. A positive proportional bias in the measurement of ALP in serum separated by manual centrifugation was detected in both equine and rhinoceros samples. Manual centrifugation with a modified salad spinner yields diagnostic quality serum suitable for the measurement of most standard biochemistry analytes.

Validation of In-Shoe Force Sensors during Loaded Walking in Military Personnel.

The loadsol® wireless in-shoe force sensors can be useful for in-field measurements. However, its accuracy is unknown in the military context, whereby soldiers have to carry heavy loads and walk in military boots. The purpose of this study was to establish the validity of the loadsol® sensors in military personnel during loaded walking on flat, inclined and declined surfaces. Full-time Singapore Armed Forces (SAF) personnel (n = 8) walked on an instrumented treadmill on flat, 10° inclined, and 10° declined gradients while carrying heavy loads (25 kg and 35 kg). Normal ground reaction forces (GRF), perpendicular to the contact surface, were simultaneously measured using both the loadsol® sensors inserted in the military boots and the Bertec instrumented treadmill as the gold standard. A total of eight variables of interest were compared between loadsol® and treadmill, including four kinetic (impact peak force, active peak force, impulse, loading rate) and four spatiotemporal (stance time, stride time, cadence, step length) variables. Validity was assessed using Bland-Altman plots and 95% Limits of Agreement (LoA). Bias was calculated as the mean difference between the values obtained from loadsol® and the instrumented treadmill. Results showed similar force-time profiles between loadsol® sensors and the instrumented treadmill. The bias of most variables was generally low, with a narrow range of LoA. The high accuracy and good agreement with standard laboratory equipment suggest that the loadsol® system is a valid tool for measuring normal GRF during walking in military boots under heavy load carriage.

Single-cell m6A mapping in vivo using picoMeRIP–seq

Current N6-methyladenosine (m6A) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio, we developed picogram-scale m6A RNA immunoprecipitation and sequencing (picoMeRIP–seq) for studying m6A in vivo in single cells and scarce cell types using standard laboratory equipment. We benchmark m6A mapping on titrations of poly(A) RNA and embryonic stem cells and in single zebrafish zygotes, mouse oocytes and embryos.

O-006 Novel and scalable non-invasive metabolic imaging of early embryos using a lab-on-a-chip approach

Abstract Study question Is it feasible to safely determine metabolic imaging signatures to measure nicotinamide adenine dinucleotide (NADH) associated auto-fluorescence in early embryos using a scalable lab-on-a-chip approach? Summary answer We developed an optofluidic device capable of non-invasively obtain high-resolution 3D images of the metabolic activity of live mouse embryos using a lab-on-a-chip approach. What is known already Selecting the most suitable embryos for implantation and subsequent healthy live birth is crucial to the success rate of assisted reproduction and offspring health. Thus, developing non-invasive methods that are reliable to assess oocyte and embryo quality has been a significant aim for assisted reproduction. Besides morphological evaluation using optical microscopy, a promising alternative is the non-invasive imaging of live embryos to establish metabolic activity performance. However, metabolic imaging has been only achieved using highly complex advanced microscopy such as Fluorescence-lifetime imaging (FLIM) and hyperspectral microscopy, methods that are costly and challenging, limiting the potential for deployment in fertility clinics. Study design, size, duration The non-invasive nature of the system was investigated by assessing the development and viability of live embryos after embryo culture for 67hrs post metabolic imaging at the 2-cell embryo stage (n = 115), including a control for culture conditions and sham controls (system no-illuminated). Embryo quality of developed blastocysts was assessed by immunocytochemistry to quantify trophectoderm and inner mass cells (n = 75). Furthermore, inhibition of metabolic activity (FK866 inhibitor) during embryo culture was also assessed (n = 18). Participants/materials, setting, methods Optofluidic devices were manufactured by cast-moulding using a negative photoresist (SU8-2075; MicroChemicals GmbH, Ulm, Germany) by a standard UV-photolithography process. The microstructures fabricated of polydimethylsiloxane (PDSM) integrated Light Sheet Fluorescence Microscopy into a microfluidic system, including on-chip micro-lenses to generate a light sheet at the center of a microchannel. Super-ovulated F1 (CBA/C57Bl6) mice were used to produce 2-cell embryos and embryo culture experiments. Blastocyst formation rates and embryo quality (immunocytochemistry) were compared between study groups. Main results and the role of chance The optofluidic device was capable of non-invasively obtaining high-resolution 3D images of the metabolic activity in live mouse embryos. The system’s design allowed continuous tracking of the embryo location, including high control displacement through the light-sheet, fast imaging of the embryos (&amp;lt;2 second) and keeping a low dose of light exposure (16 J cm-2 and 8 J cm-2). Optimum settings for keeping sample viability showed that a modest light dosage was capable to obtain 30 times higher signal-noise-ratio images than images obtained with a confocal system (p &amp;lt; 0.00001; t-test). The results showed no significant differences between the control, illuminated and non-illuminated embryos (sham control) for embryo development as well as embryo quality at the blastocyst stage (p &amp;gt; 0.05; Yate’s chi squared test). Additionally, embryos with inhibited metabolic activity showed decreased blastocyst formation rates as well as 47% reduction in metabolic activity measured by non-invasive metabolic imaging (p &amp;lt; 0.0001; t-test). This study reports an optofluidic device capable of non-invasive metabolic imaging of live embryos using a similar concept as previously reported using FLIM technology and hyperspectral microscopy, but allowing a novel, scalable and affordable system with implementation potential in standard IVF laboratory equipment. Limitations, reasons for caution The study was conducted using a mouse model focused on early embryo development. Further safety studies are required to assess embryonic health by investigating the impact of the use of light during embryo development, live birth, embryo gene expression and epigenome stability. Wider implications of the findings This lab-on-a-chip is novel, scalable and has the potential to be used in fertility clinics as a technology platform to enable real-time monitoring of the metabolic function of embryos prior embryo transfer. Future applications include potential integration into existing IVF laboratory equipment such as bench and time-lapse incubators. Trial registration number N/A

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration.

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Rapid Diagnostics of Joint Infections Using IS-Pro.

Diagnosis of bone and joint infections (BJI) relies on microbiological culture which has a long turnaround time and is challenging for certain bacterial species. Rapid molecular methods may alleviate these obstacles. Here, we investigate the diagnostic performance of IS-pro, a broad-scope molecular technique that can detect and identify most bacteria to the species level. IS-pro additionally informs on the amount of human DNA present in a sample, as a measure of leukocyte levels. This test can be performed in 4 h with standard laboratory equipment. Residual material of 591 synovial fluid samples derived from native and prosthetic joints from patients suspected of joint infections that were sent for routine diagnostics was collected and subjected to the IS-pro test. Bacterial species identification as well as bacterial load and human DNA load outcomes of IS-pro were compared to those of culture. At sample level, percent positive agreement (PPA) between IS-pro and culture was 90.6% (95% CI 85.7- to 94%) and negative percent agreement (NPA) was 87.7% (95% CI 84.1 to 90.6%). At species level PPA was 80% (95% CI 74.3 to 84.7%). IS-pro yielded 83 extra bacterial detections over culture for which we found supporting evidence for true positivity in 40% of the extra detections. Missed detections by IS-pro were mostly related to common skin species in low abundance. Bacterial and human DNA signals measured by IS-pro were comparable to bacterial loads and leukocyte counts reported by routine diagnostics. We conclude that IS-pro showed an excellent performance for fast diagnostics of bacterial BJI.