Live Organoid Cyclic Imaging.

Organoids are becoming increasingly relevant in biology and medicine for their physiological complexity and accuracy in modeling human disease. To fully assess their biological profile while preserving their spatial information, spatiotemporal imaging tools are warranted. While previously developed imaging techniques, such as four-dimensional (4D) live imaging and light-sheet imaging have yielded important clinical insights, these technologies lack the combination of cyclic and multiplexed analysis. To address these challenges, bioorthogonal click chemistry is applied to display the first demonstration of multiplexed cyclic imaging of live and fixed patient-derived glioblastoma tumor organoids. This technology exploits bioorthogonal click chemistry to quench fluorescent signals from the surface and intracellular of labeled cells across multiple cycles, allowing for more accurate and efficient molecular profiling of their complex phenotypes. Herein, the versatility of this technology is demonstrated for the screening of glioblastoma markers in patient-derived human glioblastoma organoids while conserving their viability. It is anticipated that the findings and applications of this work can be broadly translated into investigating physiological developments in other organoid systems.

Design of a flexing organ-chip to model in situ loading of the intervertebral disc.

The leading cause of disability of all ages worldwide is severe lower back pain. To address this untreated epidemic, further investigation is needed into the leading cause of back pain, intervertebral disc degeneration. In particular, microphysiological systems modeling critical tissues in a degenerative disc, like the annulus fibrosus (AF), are needed to investigate the effects of complex multiaxial strains on AF cells. By replicating these mechanobiological effects unique to the AF that are not yet understood, we can advance therapies for early-stage degeneration at the cellular level. To this end, we designed, fabricated, and collected proof-of-concept data for a novel microphysiological device called the flexing annulus-on-a-chip (AoC). We used computational models and experimental measurements to characterize the device's ability to mimic complex physiologically relevant strains. As a result, these strains proved to be controllable, multi-directional, and uniformly distributed with magnitudes ranging from - 10 % to 12% in the axial, radial, and circumferential directions, which differ greatly from applied strains possible in uniaxial devices. Furthermore, after withstanding accelerated life testing (66 K cycles of 10% strain) and maintaining 2000 bovine AF cells without loading for more than three weeks the AoC proved capable of long-term cell culture. Additionally, after strain (3.5% strain for 75 cycles at 0.5 Hz) was applied to a monolayer of AF cells in the AoC, a population remained adhered to the channel with spread morphology. The AoC can also be tailored for other annular structures in the body such as cardiovascular vessels, lymphatic vessels, and the cervix.

Development of multi-epitope peptide-based vaccines against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic involving so far more than 22 million infections and 776,157 deaths. Effective vaccines are urgently needed to prevent SARS-CoV-2 infections. No vaccines have yet been approved for licensure by regulatory agencies. Even though host immune responses to SARS-CoV-2 infections are beginning to be unravelled, effective clearance of virus will depend on both humoral and cellular immunity. Additionally, the presence of Spike (S)-glycoprotein reactive CD4+ T-cells in the majority of convalescent patients is consistent with its significant role in stimulating B and CD8+ T-cells. The search for immunodominant epitopes relies on experimental evaluation of peptides representing the epitopes from overlapping peptide libraries which can be costly and labor-intensive. Recent advancements in B- and T-cell epitope predictions by bioinformatic analysis have led to epitope identifications. Assessing which peptide epitope can induce potent neutralizing antibodies and robust T-cell responses is a prerequisite for the selection of effective epitopes to be incorporated in peptide-based vaccines. This review discusses the roles of B- and T-cells in SARS-CoV-2 infections and experimental validations for the selection of B-, CD4+ and CD8+ T-cell epitopes which could lead to the construction of a multi-epitope peptide vaccine. Peptide-based vaccines are known for their low immunogenicity which could be overcome by incorporating immunostimulatory adjuvants and nanoparticles such as Poly Lactic-co-Glycolic Acid (PLGA) or chitosan.

Identification and selection of immunodominant B and T cell epitopes for dengue multi-epitope-based vaccine.

Dengue virus (DENV) comprises four serotypes (DENV1-4) which cause 390 million global infections with 500,000 hospitalizations and 25,000 fatalities annually. Currently, the only FDA approved DENV vaccine is the chimeric live-attenuated vaccine, Dengvaxia®, which is based on the yellow fever virus (YFV) genome that carries the prM and E genes of the respective DENV 1, 2, 3, and 4 serotypes. However, it has lower efficacies against serotypes DENV1 (51%) and DENV2 (34%) when compared with DENV3 (75%) and DENV4 (77%). The absence of T cell epitopes from non-structural (NS) and capsid (C) proteins of the yellow fever vaccine strain might have prevented Dengvaxia® to elicit robust cellular immune responses, as CD8+ T cell epitopes are mainly localized in the NS3 and NS5 regions. Multi-epitope-based peptide vaccines carrying CD4+, CD8+ T cell and B cell epitopes represent a novel approach to generate specific immune responses. Therefore, assessing and selecting epitopes that can induce robust B and T cell responses is a prerequisite for constructing an efficient multi-epitope peptide vaccine. Potent B and T cell epitopes can be identified by utilizing immunoinformatic analysis, but the immunogenicity of the epitopes have to be experimentally validated. In this review, we presented T cell epitopes that have been predicted by bioinformatic approaches as well as recent experimental validations of CD4+ and CD8+ T cell epitopes by ex-vivo stimulation of PBMCs with specific peptides. Immunoproteomic analysis could be utilized to uncover HLA-specific epitopes presented by DENV-infected cells. Based on various approaches, immunodominant epitopes capable of inducing strong immune responses could be selected and incorporated to form a universally applicable multi-epitope-based peptide dengue vaccine.