Stem-cell based, machine learning approach for optimizing natural killer cell-based personalized immunotherapy for high-grade ovarian cancer.

Advanced high-grade serous ovarian cancer continues to be a therapeutic challenge for those affected using the current therapeutic interventions. There is an increasing interest in personalized cancer immunotherapy using activated natural killer (NK) cells. NK cells account for approximately 15% of circulating white blood cells. They are also an important element of the tumor microenvironment (TME) and the body's immune response to cancers. In the present study, DeepNEU-C2Rx, a machine learning platform, was first used to create validated artificially induced pluripotent stem cell simulations. These simulations were then used to generate wild-type artificially induced NK cells (aiNK-WT) and TME simulations. Once validated, the aiNK-WT simulations were exposed to artificially induced high-grade serous ovarian cancer represented by aiOVCAR3. Cytolytic activity of aiNK was evaluated in presence and absence of aiOVCAR3 and data were compared with the literature for validation. The TME simulations suggested 26 factors that could be evaluated based on their ability to enhance aiNK-WT cytolytic activity in the presence of aiOVCAR3. The addition of programmed cell death-1 inhibitor leads to significant reinvigoration of aiNK cytolytic activity. The combination of programmed cell death-1 and glycogen synthase kinase 3 inhibitors showed further improvement. Further addition of ascitic fluid factor inhibitors leads to optimal aiNK activation. Our data showed that NK cell simulations could be used not only to pinpoint novel immunotherapeutic targets to reinvigorate the activity of NK cells against cancers, but also to predict the outcome of targeting tumors with specific genetic expression and mutation profiles.

Advances in understanding N-glycosylation structure, function, and regulation in health and disease.

N-linked glycosylation is a post-translational modification crucial for membrane protein folding, stability and other cellular functions. Alteration of membrane protein N-glycans is implicated in wide range of pathological conditions including cancer metastasis, chronic inflammatory diseases, and viral pathogenesis. Even though the roles of N-glycans have been studied extensively, our knowledge of their mechanisms remains unclear due to the lack of detailed structural analysis of the N-glycome. Mapping the N-glycome landscape will open new avenues to explore disease mechanisms and identify novel therapeutic targets. This review discusses the diverse structure of N-linked glycans, the function and regulation of N-glycosylation in health and disease, and ends with a focus on recent approaches to target N-glycans in rheumatoid arthritis and cancer metastasis.

Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern.

BACKGROUNDThe role of humoral immunity in COVID-19 is not fully understood, owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome.METHODSUsing SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients' plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution.RESULTSWe identified linear epitopes from the spike (S) and nucleocapsid (N) proteins and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S-811-825, S-881-895, and N-156-170 epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes.CONCLUSIONEpitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, but it also may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern in both the peptide array and latex agglutination formats.FUNDINGOntario Research Fund (ORF) COVID-19 Rapid Research Fund, Toronto COVID-19 Action Fund, Western University, Lawson Health Research Institute, London Health Sciences Foundation, and Academic Medical Organization of Southwestern Ontario (AMOSO) Innovation Fund.

Molecular mechanisms of cutis laxa- and distal renal tubular acidosis-causing mutations in V-ATPase a subunits, ATP6V0A2 and ATP6V0A4.

The a subunit is the largest of 15 different subunits that make up the vacuolar H+-ATPase (V-ATPase) complex, where it functions in proton translocation. In mammals, this subunit has four paralogous isoforms, a1-a4, which may encode signals for targeting assembled V-ATPases to specific intracellular locations. Despite the functional importance of the a subunit, its structure remains controversial. By studying molecular mechanisms of human disease-causing missense mutations within a subunit isoforms, we may identify domains critical for V-ATPase targeting, activity and/or regulation. cDNA-encoded FLAG-tagged human wildtype ATP6V0A2 (a2) and ATP6V0A4 (a4) subunits and their mutants, a2P405L (causing cutis laxa), and a4R449H and a4G820R (causing renal tubular acidosis, dRTA), were transiently expressed in HEK 293 cells. N-Glycosylation was assessed using endoglycosidases, revealing that a2P405L, a4R449H, and a4G820R were fully N-glycosylated. Cycloheximide (CHX) chase assays revealed that a2P405L and a4R449H were unstable relative to wildtype. a4R449H was degraded predominantly in the proteasomal pathway, whereas a2P405L was degraded in both proteasomal and lysosomal pathways. Immunofluorescence studies disclosed retention in the endoplasmic reticulum and defective cell-surface expression of a4R449H and defective Golgi trafficking of a2P405L Co-immunoprecipitation studies revealed an increase in association of a4R449H with the V0 assembly factor VMA21, and a reduced association with the V1 sector subunit, ATP6V1B1 (B1). For a4G820R, where stability, degradation, and trafficking were relatively unaffected, 3D molecular modeling suggested that the mutation causes dRTA by blocking the proton pathway. This study provides critical information that may assist rational drug design to manage dRTA and cutis laxa.