Long-Term Vaccination and Treatment Strategies for COVID-19 Disease and Future Coronavirus Pandemics.

The appearance of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased infectivity and immune escape capabilities has allowed continuation of the COVID-19 pandemic for the foreseeable future. This review describes the worldwide efforts aimed at developing new vaccination and treatment strategies to keep pace with these variants as they emerge. In the case of vaccines and monoclonal antibody-based therapeutics, we describe the development of variant-specific, multivalent, and universal coronavirus directed approaches. Existing treatment approaches consist of repurposed medicines, such as antiviral compounds and anti-inflammatory agents, although efforts are underway to develop new ways of preventing or minimizing the effects of infection with the use of small molecules that disrupt binding the SARS-CoV-2 virus to host cells. Finally, we discuss the preclinical and clinical testing of natural products from medicinal herbs and spices, which have demonstrated anti-inflammatory and antiviral properties and therefore show potential as novel and safe COVID-19 treatment approaches.

Rapid Detection of SARS-CoV-2 Variants of Concern by Genomic Surveillance Techniques.

This chapter describes the application of genomic, transcriptomic, proteomic, and metabolomic methods in the study of SARS-CoV-2 variants of concern. We also describe the important role of machine learning tools to identify the most significant biomarker signatures and discuss the latest point-of-care devices that can be used to translate these findings to the physician's office or to bedside care. The main emphasis is placed on increasing our diagnostic capacity and predictability of disease outcomes to guide the most appropriate treatment strategies.

X-ray-induced bio-acoustic emissions from cultured cells.

PURPOSE: We characterize for the first time the emission of acoustic waves from cultured cells irradiated with X-ray photon radiation. METHODS AND MATERIALS: Human cancer cell lines (MCF-7, HL-60) and control cell-free media were exposed to 1 Gy X-ray photons while recording the sound generated before, during and after irradiation using custom large-bandwidth ultrasound transducer. The effects of dose rate and cell viability were investigated. RESULTS: We report the first recorded acoustic signals captured from a collective pressure wave response to ionizing irradiation in cell culture. The acoustic signal was co-terminous with the radiation pulse, its magnitude was dependent on radiation dose rate, and live and dead cells showed qualitatively and quantitatively different acoustic signal characteristics. The signature of the collective acoustic peaks was temporally wider and with higher acoustic power for irradiated HL-60 than for irradiated MCF-7. CONCLUSIONS: We show that X-ray irradiation induces two cultured cancer cell types to emit a characteristic acoustic signal for the duration of the radiation pulse. The rapid decay of the signal excludes acoustic emissions themselves from contributing to the inter-organism bystander signal previously reported in intact animals, but they remain a potential component of the bystander process in tissues and cell cultures. This preliminary study suggests that further work on the potential role of radiation-induced acoustic emission (RIAE) in the inter-cellular bystander effect is merited.

Photosensitized and Photothermal Stimulation of Cellular Membranes by Organic Thin Films and Nanoparticles.

Conjugated polymers are increasingly exploited for biomedical applications. In this work, we explored the optical characteristics of conjugated polymers of variable chemical structures at multiple levels relevant to biological interfacing, from fluorescence yield to their influence on cellular membrane potential. We systematically compared the performance of conjugated polymer as cast thin films and as nanoparticles stabilized with amphiphilic polyethylene glycol-poly lactic acid-co-glycolic acid (PEG-PLGA). We assessed in both the dark and under illumination the stability of key optoelectronic properties in various environments, including air and biologically relevant physiological saline solutions. We found that photoreduction of oxygen correlates with nanoparticle and film degradation in physiologically relevant media. Using patch-clamp recordings in cell lines and primary neurons, we identified two broad classes of membrane potential response, which correspond to photosensitizer- and photothermal-mediated effects. Last, we introduced a metric named OED50 (optical energy for 50% depolarization), which conveys the phototoxic potency of a given agent and thereby its operational photo-safety profile.

Antibody-Based Affinity Capture Combined with LC-MS Analysis for Identification of COVID-19 Disease Serum Biomarkers.

Blood serum or plasma proteins are potentially useful in COVID-19 research as biomarkers for risk prediction, diagnosis, stratification, and treatment monitoring. However, serum protein-based biomarker identification and validation is complicated due to the wide concentration range of these proteins, which spans more than ten orders of magnitude. Here we present a combined affinity purification-liquid chromatography mass spectrometry approach which allows identification and quantitation of the most abundant serum proteins along with the nonspecifically bound and interaction proteins. This led to the reproducible identification of more than 100 proteins that were not specifically targeted by the affinity column. Many of these have already been implicated in COVID-19 disease.

Liquid Chromatography-Mass Spectrometry Analysis of Peripheral Blood Mononuclear Cells from SARS-CoV-2 Infected Patients.

COVID-19 disease is caused by infection with the SARS-CoV-2 virus and is associated with a cytokine storm effect in some patients. This can lead to decreased ability of the host to cope with the infection and result in severe disease outcomes. Here, we present a protocol for isolation of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients followed by liquid chromatography-mass spectrometry (LC-MS) profiling to identify the affected molecules and molecular pathways. It is hoped that this will lead to the identification of potential biomarkers for monitoring the disease as well as treatment responses. This approach could also be used in the study of other respiratory viruses.

COVID-19 Detection Using the NHS Lateral Flow Test Kit.

Approximately one in three people infected with the SARS-CoV-2 virus have mild symptoms or are asymptomatic. However, these individuals can still spread the virus. Regular self-testing can help to detect these individuals and thereby slow the spread and protect the more vulnerable members of society. Here, we present a protocol for use of the COVID-19 rapid antigen test which was made freely available to residents of the United Kingdom in April of this year. This using the lateral flow technique for detection of antigens and is amenable to multiplexing.

Point-of-Care Device for Assessment of Blood Coagulation Status in COVID-19 Patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent driving the current COVID-19 pandemic. The acute respiratory distress that occurs in some severe COVID-19 cases has been linked with hypercoagulation or thrombotic events as well as a worse prognosis and increased risk of death. Thus, point-of-care devices that can be used for early detection of coagulation abnormalities would assist in COVID-19 management. This chapter describes the use of the Roche Diagnostics CoaguChek® XS test kit for potential use in COVID-19 personalized medicine approaches.

Size-tuneable and immunocompatible polymer nanocarriers for drug delivery in pancreatic cancer.

Nanocarriers have emerged as one of the most promising approaches for drug delivery. Although several nanomaterials have been approved for clinical use, the translation from lab to clinic remains challenging. However, by implementing rational design strategies and using relevant models for their validation, these challenges are being addressed. This work describes the design of novel immunocompatible polymer nanocarriers made of melanin-mimetic polydopamine and Pluronic F127 units. The nanocarrier preparation was conducted under mild conditions, using a highly reproducible method that was tuned to provide a range of particle sizes (<100 nm) without changing the composition of the carrier. A set of in vitro studies were conducted to provide a comprehensive assessment of the effect of carrier size (40, 60 and 100 nm) on immunocompatibility, viability and uptake into different pancreatic cancer cells varying in morphological and phenotypic characteristics. Pancreatic cancer is characterised by poor treatment efficacy and no improvement in patient survival in the last 40 years due to the complex biology of the solid tumour. High intra- and inter-tumoral heterogeneity and a dense tumour microenvironment limit diffusion and therapeutic response. The Pluronic-polydopamine nanocarriers were employed for the delivery of irinotecan active metabolite SN38, which is used in the treatment of pancreatic cancer. Increased antiproliferative effect was observed in all tested cell lines after administration of the drug encapsulated within the carrier, indicating the system's potential as a therapeutic agent for this hard-to-treat cancer.

Innate Immune Invisible Ultrasmall Gold Nanoparticles-Framework for Synthesis and Evaluation.

Nanomedicine is seen as a potential central player in the delivery of personalized medicine. Biocompatibility issues of nanoparticles have largely been resolved over the past decade. Despite their tremendous progress, less than 1% of applied nanosystems can hit their intended target location, such as a solid tumor, and this remains an obstacle to their full ability and potential with a high translational value. Therefore, achieving immune-tolerable, blood-compatible, and biofriendly nanoparticles remains an unmet need. The translational success of nanoformulations from bench to bedside involves a thorough assessment of their design, compatibility beyond cytotoxicity such as immune toxicity, blood compatibility, and immune-mediated destruction/rejection/clearance profile. Here, we report a one-pot process-engineered synthesis of ultrasmall gold nanoparticles (uGNPs) suitable for better body and renal clearance delivery of their payloads. We have obtained uGNP sizes of as low as 3 nm and have engineered the synthesis to allow them to be accurately sized (almost nanometer by nanometer). The synthesized uGNPs are biocompatible and can easily be functionalized to carry drugs, peptides, antibodies, and other therapeutic molecules. We have performed in vitro cell viability assays, immunotoxicity assays, inflammatory cytokine analysis, a complement activation study, and blood coagulation studies with the uGNPs to confirm their safety. These can help to set up a long-term safety-benefit framework of experimentation to reveal whether any designed nanoparticles are immune-tolerable and can be used as payload carriers for next-generation vaccines, chemotherapeutic drugs, and theranostic agents with better body clearance ability and deep tissue penetration.

Disaccahrides-Based Cryo-Formulant Effect on Modulating Phospho/Mitochondrial Lipids and Biological Profiles of Human Leukaemia Cells.

BACKGROUND/AIMS: The use of novel cryo-additive agents to increase cell viability post-cryopreservation is paramount to improve future cell based-therapy treatments. We aimed to establish the Human Leukemia (HL-60) cells lipidomic and biological patterns when cryo-preserved in DMSO alone and with 300 µM Nigerose (Nig), 200 µM Salidroside (Sal) or a combination of Nig (150 µM) and Sal (100 µM). METHODS: HL-60 cells were pre-incubated with Nig/Sal prior, during and post cryopreservation, and subjected to global lipidomic analysis. Malondialdeyhde (MDA), released lactate dehydrogenase (LDH) and reactive oxygen scavenger (ROS) measurements were also carried out to evaluate levels of lipid peroxidation and cytotoxicity. RESULTS: Cryopreserving HL-60 cells in DMSO with Nig and Sal provided optimal protection against unsaturated fatty acid oxidation. Post-thaw, cellular phospholipids and mitochondrial cardiolipins were increased by Nig/Sal as the ratio of unsaturated to saturated fatty acids 2.08 +/- 0.03 and 0.95 +/- 0.09 folds respectively in comparison to cells cryopreserved in DMSO alone (0.49 +/- 0.05 and 0.86 +/- 0.10 folds). HL-60 lipid peroxidation levels in the presence of DMSO + Nig and Sal combined were significantly reduced relative to pre-cryopreservation levels (10.91 +/- 2.13 nmole) compared to DMSO (17.1 +/- 3.96 nmole). DMSO + Nig/Sal combined also significantly reduced cell cytotoxicity post-thaw (0.0128 +/- 0.00182 mU/mL) in comparison to DMSO (0.0164 +/- 0.00126 mU/mL). The combination of Nig/Sal also reduced significantly ROS levels to the levels of prior cryopreservation of HL-60. CONCLUSION: Overall, the establishment of the cryopreserved HL-60 cells lipidomic and the corresponding biological profiles showed an improved cryo-formulation in the presence of DMSO with the Nig/Sal combination by protecting the, mitochondrial inner membrane, unsaturated fatty acid components (i. e. Cardiolipins) and total phospholipids.

Early Diagnosis and Targeted Treatment Strategy for Improved Therapeutic Outcomes in Alzheimer's Disease.

There have been repeated failures of clinical studies in the development of new efficacious treatments for Alzheimer's disease. This may be due to the fact that Alzheimer's disease is a heterogeneous disorder caused by person-to-person differences in genetic background, epigenetic profiles, environmental triggers, or the presence of other diseases. Furthermore, most Alzheimer's disease patients are diagnosed in the middle to late stages of the illness, when irreversible damage to the brain has already occurred. With this in mind, a strategy is presented involving identification and implementation of biomarker tests for diagnosis during the prodromal or early stages of the disease. In addition, it is proposed that targeting specific components of the amyloid deposition, tau oligomerization and neuroinflammation pathways may lead to improved outcomes in clinical studies.

Proteomic Analysis of Rat Hippocampus for Studies of Cognition and Memory Loss with Aging.

This chapter describes a protocol for proteomic profiling of the rat hippocampal proteome using a combination of liquid chromatography tandem mass spectrometry (LC-MS/MS) and data analysis to determine the cellular location of the identified proteins. In the example given, many of these proteins were localized in the plasma membrane and nucleus. These could be of interest in further studies of neurological and neurodegenerative disorders linked with hippocampal dysfunction, such as aging, major depression, and Alzheimer's disease.

Human leukemia cells (HL-60) proteomic and biological signatures underpinning cryo-damage are differentially modulated by novel cryo-additives.

BACKGROUND: Cryopreservation is a routinely used methodology for prolonged storage of viable cells. The use of cryo-protective agents (CPAs) such as dimethylsulfoxide (DMSO), glycerol, or trehalose is paramount to reducing cellular cryo-injury, but their effectiveness is still limited. The current study focuses on establishing and modulating the proteomic and the corresponding biological profiles associated with the cryo-injury of human leukemia (HL-60) cells cryopreserved in DMSO alone or DMSO +/- novel CPAs (e.g., nigerose [Nig] or salidroside [Sal]). FINDINGS: To reduce cryo-damage, HL-60 cells were cultured prior and post cryopreservation in malondialdehyde Roswell Park Memorial Institute medium-1640 media +/- Nig or Sal. Shotgun proteomic analysis showed significant alterations in the levels of proteins in cells cryopreserved in Nig or Sal compared to DMSO. Nig mostly affected cellular metabolism and energy pathways, whereas Sal increased the levels of proteins associated with DNA repair/duplication, RNA transcription, and cell proliferation. Validation testing showed that the proteome profile associated with Sal was correlated with a 2.8-fold increase in cell proliferative rate. At the functional level, both Nig and Sal increased glutathione reductase (0.0012±6.19E-05 and 0.0016±3.04E-05 mU/mL, respectively) compared to DMSO controls (0.0003±3.7E-05 mU/mL) and reduced cytotoxicity by decreasing lactate dehydrogenase activities (from -2.5 to -4.75 fold) and lipid oxidation (-1.6 fold). In contrast, only Nig attenuated protein carbonylation or oxidation. CONCLUSIONS: We have identified key molecules and corresponding functional pathways underpinning the effect of cryopreservation (+/- CPAs) of HL-60 cells. We also validated the proteomic findings by identifying the corresponding biological profiles associated with promoting an anti-oxidative environment post cryopreservation. Nig or Sal in comparison to DMSO showed differential or additive effects in regard to reducing cryo-injury and enhancing cell survival/proliferation post thaw. These results can provide useful insight to cryo-damage and the design of enhanced cryomedia formulation.

Characterization of the db/db Mouse Model of Type 2 Diabetes.

This chapter presents characterization of the db/db mouse model of type 2 diabetes. The protocol describes generation of the mice along with measurements of physical characteristics such as body weight and body composition, and behaviours including food and water consumption. Such a procedure can also be used for omics-based analyses of brain tissues to investigate the potential use of db/db mouse model in neuropsychiatric research. In addition, we present measurement of blood glucose and plasma insulin levels which tend to be elevated in this model and can modulated post treatment with anti-diabetic agents. All of these characteristics can be used for studying the pathophysiology and for monitoring response to potential therapeutics.

Cryopreservation of Red Blood Cells.

Glycerol and trehalose are widely used protective agents in the cryopreservation of red blood cells (RBCs). This chapter presents a protocol for use of these agents as cryoprotectants of RBCs, with post-thaw assessment of cell survival and cellular oxidative-reductive status. The main aim is to provide a framework for further studies aimed at improving RBC survival and function and to supply improved biomaterials for studies on RBC biochemistry, major operations, as well as those for urgent use in emergency room situations.

Molecular Characterization of Human Leukemia 60 (HL-60) Cells as a Model of Acute Myelogenous Leukemia Post Cryopreservation.

The use of human leukemic (HL)-60 cells is important for studies of acute myeloid leukemia (AML) and as a model system for investigating how specific types of blood cells are formed during the process of hematopoiesis. Here, we present a protocol for growth of HL-60 cells along with molecular and functional profiles associated with their cryostorage. We also elucidate the effects of these procedures on cell viability and functions. This method can be used to provide biomarkers as readouts for testing the efficacy and/or toxicity of novel compounds in AML research as well as in a number of other experimental manipulations.

Studies of Isolated Peripheral Blood Cells as a Model of Immune Dysfunction.

Peripheral blood mononuclear cells (PBMCs) have been used as a surrogate model of immune function in studies of multiple medical areas, such as metabolic diseases and immune dysfunction. This chapter describes a standardized technique for blood draw and preparation of PBMCs from whole blood using density gradient centrifugation, followed by cell culture. The main focus is on collection of the PBMC culture media and extraction of cellular proteins in order to provide the materials for biomarker studies.

Mass Spectrometry Profiling of Pituitary Glands.

Many chronic diseases are associated with hypothalamic-pituitary-adrenal axis dysfunction. Therefore, proteomic profiling of the pituitary gland has potential to uncover new information on the underlying pathways affected in these conditions. This could lead to identification of new biomarkers or drug targets for development of novel therapeutics. Here we present a protocol for preparation of pituitary protein extracts and analysis of the major hormones and accessory proteins using liquid chromatography tandem mass spectrometry (LC-MS/MS). The same methods can be applied in the study of other tissues of the diffuse neuroendocrine system.

Application of Proteomic Approaches to Accelerate Drug Development for Psychiatric Disorders.

Proteomic-based biomarkers are now an integral part of the drug development process. This chapter covers the role of proteomic biomarker tests as useful tools for improving preclinical research and clinical development. One medical area that has been lagging behind this process is the study of psychiatric disorders, and this is most likely due to the complexity of these diseases. The potential of incorporating biomarkers in the clinical pipeline to improve decision-making, accelerate drug development, improve translation and reduce development costs is also discussed, with a focus on psychiatric diseases like schizophrenia. This chapter will also discuss the next steps that must be taken to keep moving this process forwards.