Analytical Workflows for Single-Cell Multiomic Data Using the BD Rhapsody Platform.

The conversion of raw sequencing reads to biologically relevant data in high-throughput single-cell RNA sequencing experiments is a complex and involved process. Drawing meaning from thousands of individual cells to provide biological insight requires ensuring not only that the data are of the highest quality but also that the signal can be separated from noise. In this article, we describe a detailed analytical workflow, including six pipelines, that allows high-quality data analysis in single-cell multiomics. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Image analysis Basic Protocol 2: Sequencing quality control and generation of a gene expression matrix Basic Protocol 3: Gene expression matrix data pre-processing and analysis Basic Protocol 4: Advanced analysis Basic Protocol 5: Conversion to flow cytometry standard (FCS) format Basic Protocol 6: Visualization using graphical interfaces.

Advances in TEER measurements of biological barriers in microphysiological systems.

Biological barriers are multicellular structures that precisely regulate the transport of ions, biomolecules, drugs, cells, and other organisms. Transendothelial/epithelial electrical resistance (TEER) is a label-free method for predicting the properties of biological barriers. Understanding the mechanisms that control TEER significantly enhances our knowledge of the physiopathology of different diseases and aids in the development of new drugs. Measuring TEER values within microphysiological systems called organ-on-a-chip devices that simulate the microenvironment, architecture, and physiology of biological barriers in the body provides valuable insight into the behavior of barriers in response to different drugs and pathogens. These integrated systems should increase the accuracy, reproducibility, sensitivity, resolution, high throughput, speed, cost-effectiveness, and reliable predictability of TEER measurements. Implementing advanced micro and nanoscale manufacturing techniques, surface modification methods, biomaterials, biosensors, electronics, and stem cell biology is necessary for integrating TEER measuring systems with organ-on-chip technology. This review focuses on the applications, advantages, and future perspectives of integrating organ-on-a-chip technology with TEER measurement methods for studying biological barriers. After briefly reviewing the role of TEER in the physiology and pathology of barriers, standard techniques for measuring TEER, including Ohm's law and impedance spectroscopy, and commercially available devices are described. Furthermore, advances in TEER measurement are discussed in multiple barrier-on-a-chip system models representing different organs. Finally, we outline future trends in implementing advanced technologies to design and fabricate nanostructured electrodes, complicated microfluidic chips, and membranes for more advanced and accurate TEER measurements.

Advanced models for respiratory disease and drug studies.

The global burden of respiratory diseases is enormous, with many millions of people suffering and dying prematurely every year. The global COVID-19 pandemic witnessed recently, along with increased air pollution and wildfire events, increases the urgency of identifying the most effective therapeutic measures to combat these diseases even further. Despite increasing expenditure and extensive collaborative efforts to identify and develop the most effective and safe treatments, the failure rates of drugs evaluated in human clinical trials are high. To reverse these trends and minimize the cost of drug development, ineffective drug candidates must be eliminated as early as possible by employing new, efficient, and accurate preclinical screening approaches. Animal models have been the mainstay of pulmonary research as they recapitulate the complex physiological processes, Multiorgan interplay, disease phenotypes of disease, and the pharmacokinetic behavior of drugs. Recently, the use of advanced culture technologies such as organoids and lung-on-a-chip models has gained increasing attention because of their potential to reproduce human diseased states and physiology, with clinically relevant responses to drugs and toxins. This review provides an overview of different animal models for studying respiratory diseases and evaluating drugs. We also highlight recent progress in cell culture technologies to advance integrated models and discuss current challenges and present future perspectives.

Corrigendum: The effects of COVID-19 on the placenta during pregnancy.

[This corrects the article DOI: 10.3389/fimmu.2021.743022.].

The Effects of COVID-19 on the Placenta During Pregnancy.

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. The virus primarily affects the lungs where it induces respiratory distress syndrome ranging from mild to acute, however, there is a growing body of evidence supporting its negative effects on other system organs that also carry the ACE2 receptor, such as the placenta. The majority of newborns delivered from SARS-CoV-2 positive mothers test negative following delivery, suggesting that there are protective mechanisms within the placenta. There appears to be a higher incidence of pregnancy-related complications in SARS-CoV-2 positive mothers, such as miscarriage, restricted fetal growth, or still-birth. In this review, we discuss the pathobiology of COVID-19 maternal infection and the potential adverse effects associated with viral infection, and the possibility of transplacental transmission.

A microfluidic approach to rapid sperm recovery from heterogeneous cell suspensions.

The isolation of sperm cells from background cell populations and debris is an essential step in all assisted reproductive technologies. Conventional techniques for sperm recovery from testicular sperm extractions stagnate at the sample processing stage, where it can take several hours to identify viable sperm from a background of collateral cells such as white bloods cells (WBCs), red blood cells (RBCs), epithelial cells (ECs) and in some cases cancer cells. Manual identification of sperm from contaminating cells and debris is a tedious and time-consuming operation that can be suitably addressed through inertial microfluidics. Microfluidics has proven an effective technology for high-quality sperm selection based on motility. However, motility-based selection methods cannot cater for viable, non-motile sperm often present in testicular or epididymal sperm extractions and aspirations. This study demonstrates the use of a 3D printed inertial microfluidic device for the separation of sperm cells from a mixed suspension of WBCs, RBCs, ECs, and leukemic cancer cells. This technology presents a 36-fold time improvement for the recovery of sperm cells (> 96%) by separating sperm, RBCS, WBCs, ECs and cancer cells into tight bands in less than 5 min. Furthermore, microfluidic processing of sperm has no impact on sperm parameters; vitality, motility, morphology, or DNA fragmentation of sperm. Applying inertial microfluidics for non-motile sperm recovery can greatly improve the current processing procedure of testicular sperm extractions, simplifying the fertility outcomes for severe forms of male infertility that warrant the surgery.

The evolving landscape of predictive biomarkers in immuno-oncology with a focus on spatial technologies.

Immunotherapies have shown long-lasting and unparalleled responses for cancer patients compared to conventional therapy. However, they seem to only be effective in a subset of patients. Therefore, it has become evident that a greater understanding of the tumor microenvironment (TME) is required to understand the nuances which may be at play for a favorable outcome to therapy. The immune contexture of the TME is an important factor in dictating how well a tumor may respond to immune checkpoint inhibitors. While traditional immunohistochemistry techniques allow for the profiling of cells in the tumor, this is often lost when tumors are analysed using bulk tissue genomic approaches. Moreover, the actual cellular proportions, cellular heterogeneity and deeper spatial distribution are lacking in characterisation. Advances in tissue interrogation technologies have given rise to spatially resolved characterisation of the TME. This review aims to provide an overview of the current methodologies that are used to profile the TME, which may provide insights into the immunopathology associated with a favorable outcome to immunotherapy.

Metal-Organic Framework-Enhanced ELISA Platform for Ultrasensitive Detection of PD-L1.

The programmed cell death ligand 1 (PD-L1) protein has emerged as a predictive cancer biomarker and sensitivity to immune checkpoint blockade-based cancer immunotherapies. Current technologies for the detection of protein-based biomarkers, including the enzyme-linked immunosorbent assay (ELISA), have limitations such as low sensitivity and limit of detection (LOD) in addition to degradation of antibodies in exposure to environmental changes such as temperature and pH. To address these issues, we have proposed a metal-organic framework (MOF)-based ELISA for the detection of the PD-L1. A protective coating based on Zeolitic Imidazolate Framework 8 (ZIF-8) MOF thin film and polydopamine-polyethylenimine (PDA-PEI) was introduced on an ELISA plate for the improvement of antibody immobilization. Sensitivity and LOD of the resulting platform were compared with a conventional ELISA kit, and the bioactivity of the antibody in the proposed immunoassay was investigated in response to various pH and temperature values. The LOD and sensitivity of the MOF-based PD-L1 ELISA were 225 and 15.12 times higher, respectively, compared with those of the commercial ELISA kit. The antibody@ZIF-8/PDA-PEI was stable up to 55 °C and the pH range 5-10. The proposed platform can provide sensitive detection for target proteins, in addition to being resistant to elevated temperature and pH. The proposed MOF-based ELISA has significant potential for the clinical and diagnostic studies.

Estimation of the optimum number and location of nanoparticle injections and the specific loss power for ideal hyperthermia.

Hyperthermia is one of the most appealing methods of cancer treatment in which the temperature of tumor is elevated to reach a desired temperature. One of the methods of increasing tissue temperature is injection of nanoparticle fluids to tumor and applying alternative magnetic field, which is called magnetic nanoparticle hyperthermia method. The total number of injection points, as well as the their location within a tissue play a significant role in this method. Furthermore, the power of heating of a magnetic material per gram or specific loss power (SLP) is another important factor which needs to be investigated. As the uniform temperature of 43 °C is effective enough for a tumor regression in certain specific tissues, the inverse method is applied to find out both the number of injection points and their location. Furthermore, the effective amount of heat generated by nanoparticles is investigated by this technique. Two-dimensional cancerous brain tissue was considered, zero gradients on boundary conditions were assumed, and diffusion equation and Pennes equation, which is regarded as energy equation, were solved, respectively. Conjugate gradient technique as a one way of inverse methods is applied, and unknowns are investigated. The results illustrate that three-point injection with the best injection sites cannot induce a uniform temperate distribution of 43 °C, and although four-point injection can create a uniform temperature elevation, the amount of it cannot reach the 43 °C. Finally, the optimum locations of five-point injection which are ((0.80,3.24), (0.80,0.84), (2.00,2.00), (3.20,3.24), (3.32,0.84)) (all dimensions are in mm) in the studied domain with special loss power of 420 W/g, all of which are obtained after 36 iterations, demonstrate that these conditions can meet the requirements of the magnetic fluid hyperthermia and can be considered for the future usage of researchers and investigators.

A hybrid micromixer with planar mixing units.

The application of microfluidic systems in chemical and biological assays has progressed dramatically in recent years. One of the fundamental operations that microfluidic devices must achieve is a high mixing index. Of particular importance is the role of planar mixing units with repetitive obstacles (MURO) in the formation of micromixers. To date, a myriad of planar passive micromixers has been proposed. However, a strategy for the combination of these units to find an efficient planar mixer has not been investigated. As such, five different MURO have been selected to form a "hybrid micromixer," and their combination was evaluated via numerical and experimental methods. These mixing units include ellipse-like, Tesla, nozzle and pillar, teardrop, and obstruction in a curved mixing unit. Since these units have distinctive dimensions, dynamic and geometric similarities were used to scale and connect them. Afterwards, six slots were designated to house each mixing unit. Since the evaluation of all possible unit configurations is not feasible, the design of experiment method is applied to reduce the total number of experiments from 15 625 to 25. Following this procedure, the "hybrid" micromixer proposed here, comprising Tesla, nozzle and pillar, and obstruction units, shows improved performance for a wide range of Re (i.e., mixing index of >90% for Re 0.001-0.1, 22-45) over existing designs. The use of velocity profiles, concentration diagrams, vorticity and circulation plots assist in the analysis of each unit. Comparison of the proposed "hybrid" micromixer with other obstacle-based planar micromixers demonstrates improved performance, indicating the combination of planar mixing units is a useful strategy for building high-performance micromixers.