Neutrophil spontaneous apoptosis, a process crucial for immune regulation, is mainly controlled by alterations in reactive oxygen species (ROS) and mitochondria integrity. Exercise has been proposed to be a physiological way to modulate immunity; while acute severe exercise (ASE) usually impedes immunity, chronic moderate exercise (CME) improves it. This study aimed to investigate whether and how ASE and CME oppositely regulate human neutrophil apoptosis. Thirteen sedentary young males underwent an initial ASE and were subsequently divided into exercise and control groups. The exercise group (n = 8) underwent 2 months of CME followed by 2 months of detraining. Additional ASE paradigms were performed at the end of each month. Neutrophils were isolated from blood specimens drawn at rest and immediately after each ASE for assaying neutrophil spontaneous apoptosis (annexin-V binding on the outer surface) along with redox-related parameters and mitochondria-related parameters. Our results showed that i) the initial ASE immediately increased the oxidative stress (cytosolic ROS and glutathione oxidation), and sequentially accelerated the reduction of mitochondrial membrane potential, the surface binding of annexin-V, and the generation of mitochondrial ROS; ii) CME upregulated glutathione level, retarded spontaneous apoptosis and delayed mitochondria deterioration; iii) most effects of CME were unchanged after detraining; and iv) CME blocked ASE effects and this capability remained intact even after detraining. Furthermore, the ASE effects on neutrophil spontaneous apoptosis were mimicked by adding exogenous H2O2, but not by suppressing mitochondrial membrane potential. In conclusion, while ASE induced an oxidative state and resulted in acceleration of human neutrophil apoptosis, CME delayed neutrophil apoptosis by maintaining a reduced state for long periods of time even after detraining.

Membrane proteins play vital roles in cellular signaling processes and serve as the most popular drug targets. A key task in studying cellular functions and developing drugs is to measure the binding kinetics of ligands with the membrane proteins. However, this has been a long-standing challenge because one must perform the measurement in a membrane environment to maintain the conformations and functions of the membrane proteins. Here, we report a new method to measure ligand binding kinetics to membrane proteins using self-assembled virion oscillators. Virions of human herpesvirus were used to display human G-protein-coupled receptors (GPCRs) on their viral envelopes. Each virion was then attached to a gold-coated glass surface via a flexible polymer to form an oscillator and driven into oscillation with an alternating electric field. By tracking changes in the oscillation amplitude in real-time with subnanometer precision, the binding kinetics between ligands and GPCRs was measured. We anticipate that this new label-free detection technology can be readily applied to measure small or large ligand binding to any type of membrane proteins and thus contribute to the understanding of cellular functions and screening of drugs.

Protein microarrays are crucial tools in the study of proteins in an unbiased, high-throughput manner, as they allow for characterization of up to thousands of individually purified proteins in parallel. The adaptability of this technology has enabled its use in a wide variety of applications, including the study of proteome-wide molecular interactions, analysis of post-translational modifications, identification of novel drug targets, and examination of pathogen-host interactions. In addition, the technology has also been shown to be useful in profiling antibody specificity, as well as in the discovery of novel biomarkers, especially for autoimmune diseases and cancers. In this review, we will summarize the developments that have been made in protein microarray technology in both in basic and translational research over the past decade. We will also introduce a novel membrane protein array, the GPCR-VirD array, and discuss the future directions of functional protein microarrays.

Human G protein-coupled receptors (GPCRs) respond to various ligands and stimuli. However, GPCRs rely on membrane for proper folding, making their biochemical properties difficult to study. By displaying GPCRs in viral envelopes, we fabricated a Virion Display (VirD) array containing 315 non-olfactory human GPCRs for functional characterization. Using this array, we found that 10 of 20 anti-GPCR mAbs were ultra-specific. We further demonstrated that those failed in the mAb assays could recognize their canonical ligands, suggesting proper folding. Next, using two peptide ligands on the VirD-GPCR array, we identified expected interactions and novel interactions. Finally, we screened the array with group B Streptococcus , a major cause of neonatal meningitis, and demonstrated that inhibition of a newly identified target, CysLTR1, reduced bacterial penetration both in vitro and in vivo. We believe that the VirD-GPCR array holds great potential for high-throughput screening for small molecule drugs, affinity reagents, and ligand deorphanization.

ASE and CME differentially affected neutrophil functions, whereas AME was ineffective. Moreover, the fact that CME improves neutrophil functions may partially explain why physically active subjects have a low risk of infection.

Coronavirus is an enveloped RNA virus that causes mild to severe respiratory diseases in humans, including HKU1-CoV, 229E-CoV, NL63-CoV, OC43-CoV, SARS-CoV, MERS-CoV, and SARS-CoV-2. Due to the outbreak of SARS-CoV-2, it is important to identify the patients and investigate their immune responses. Protein microarray is one of the best platforms to profile the antibodies in the blood because of its fast, multiplexed, and sensitive nature. To fully understand the immune responses and biological specificities, this study developed a human coronavirus (HCoV) protein microarray and included all seven human coronaviruses and three influenza viruses. Each protein was printed in triplicate and formed 14 identical blocks per array. The HCoV protein microarray showed high reproducibility and sensitivity to the monoclonal antibodies against spike and nucleocapsid protein with detection limits of 10–200 pg. The HCoV proteins that were immobilized on the array were properly folded and functional by showing interactions with a known human receptor, e.g., ACE2. By profiling the serum IgG and IgA from 32 COVID-19 patients and 36 healthy patients, the HCoV protein microarray demonstrated 97% sensitivity and 97% specificity with two biomarkers. The results also showed the cross-reactivity of IgG and IgA in COVID-19 patients to spike proteins from various coronaviruses, including that from SARS-CoV, HKU1-CoV, and OC43-CoV. Finally, an innate immune protein named surfactant protein D showed broad affinities to spike proteins in all human coronaviruses. Overall, the HCoV protein microarray is multiplexed, sensitive, and specific, which is useful in diagnosis, immune assessment, biological development, and drug screening.

SummaryKawasaki disease (KD) is a form of systemic vasculitis that generally occurs in children under 5 years old. Currently, KD is still diagnosed according to its clinical symptoms, including prolonged fever, skin rash, conjunctivitis, neck lymphadenopathy, palm erythema, and oral mucosa changes. Since KD is a type of inflammation without specific marker for diagnosis, we plan to profile the plasma antibodies by using E. coli proteome microarray and analyze the differences between KD and healthy subjects. Plasmas were collected from KD patient before intravenous immunoglobulin treatment (KD1), at least 3 weeks after treatment (KD3), non-fever This study is the first to profile plasma antibodies in KD and demonstrate that an E.coli proteome microarray can screen differences among patients with KD, non-fever controls, and fever controls.

ASE in sedentary but not active subjects facilitated NET formation via elevating the NADPH oxidase-generated ROS and suppressing the ΔΨm.