Engineering Metastability into a Virus-like Particle to Enable Triggered Dissociation.

For a virus-like particle (VLP) to serve as a delivery platform, the VLP must be able to release its cargo in response to a trigger. Here, we use a chemical biology approach to destabilize a self-assembling capsid for a subsequent triggered disassembly. We redesigned the dimeric hepatitis B virus (HBV) capsid protein (Cp) with two differentially addressable cysteines, C150 for reversibly crosslinking the capsid and C124 to react with a destabilizing moiety. The resulting construct, Cp150-V124C, assembles into icosahedral, 120-dimer VLPs that spontaneously crosslink via the C-terminal C150, leaving C124 buried at a dimer-dimer interface. The VLP is driven into a metastable state when C124 is reacted with the bulky fluorophore, maleimidyl BoDIPY-FL. The resulting VLP is stable until exposed to modest, physiologically relevant concentrations of reducing agent. We observe dissociation with FRET relaxation of polarization, size exclusion chromatography, and resistive-pulse sensing. Dissociation is slow, minutes to hours, with a characteristic lag phase. Mathematical modeling based on the presence of a nucleation step predicts disassembly dynamics that are consistent with experimental observations. VLPs transfected into hepatoma cells show similar dissociation behavior. These results suggest a generalizable strategy for designing a VLP that can release its contents in an environmentally responsive reaction.

Ganoderma microsporum immunomodulatory protein induces apoptosis and potentiates mitomycin C-induced apoptosis in urinary bladder urothelial carcinoma cells.

Current chemotherapy and immunotherapy treatments followed by transurethral resection for urinary bladder urothelial carcinoma (UC) usually suffer from poor prognosis and high recurrence rate. Design and modification of current formulation with the novel adjuvants are needed. A recombinant protein derived from Ganoderma microsporum named as Ganoderma microsporum immunomodulatory protein (GMIP) was used to treat UC cells. We found GMIP elicits a dose-dependent and time-dependent anti-UC cell proliferation effect, with a half-maximal inhibition concentration (IC50 ) comparable to mitomycin C (MMC), a commonly used chemotherapy agent. After GMIP treatment, UC cells showed apoptotic phenomenon including cell cycle arrest in the G1 phase, elevated sub-G1 population, mitochondrial membrane potential loss, up-regulated p21 expression, p21 nuclear translocation, caspase activation, and PARP cleavage in a p53-independent but p21-mediated pathways. Unlike lung cancer cells, GMIP treated UC cells showed no autophagic scheme including Beclin-1, an autophagy to apoptosis switch marker, was not cleaved by caspase 3 and slight LC3B-II accumulation. Also, the classic autophagic inhibitor, chloroquine had no effect in GMIP-mediated cell death made us conclude that GMIP induced apoptosis through caspase activation but not autophagy in UC cells. Additionally, GMIP showed synergistic effects with MMC in killing UC cells and thus decreased the concentration of MMC usage to reach the comparable apoptotic effects. Our results delineate novel strategies for treatment of UC by GMIP alone or in combination with MMC application and provide a promising therapeutic cocktail for better treatment of urinary bladder urothelial carcinoma.

Stable laminar shear stress induces G1 cell cycle arrest and autophagy in urothelial carcinoma by a torque sensor-coupled cone-and-plate device.

The microenvironments of urinary systems play crucial roles in the development and metastasis of cancers due to their generation of complex temporal and spatial fluidic profiles. Because of their versatility in creating desired biomimetic flow, cone-and-plate bioreactors offer great potential for bladder cancer research. In this study, we construct a biocompatible cone-and-plate device coupled with a torque sensor, enabling the application and real-time monitoring of stable shear stress up to 50 dyne/cm². Under a stable shear stress stimulation at 12 dyne/cm2, bladder cancer cell BFTC-905 is arrested at the G1 phase with decreased cell proliferation after 24-hour treatment. This effect is associated with increased cyclin-dependent kinase inhibitors p21 and p27, inhibiting cyclin D1/CDK4 complex with dephosphorylation of serine 608 on the retinoblastoma protein. Consequently, an increase in cyclin D3 and decreases in cyclin A2 and cyclin E2 are observed. Moreover, we demonstrate that the shear stress stimulation upregulates the expression of autophagy-related proteins Beclin-1, LC3B-I and LC3B-II, while caspase cleavages are not activated under the same condition. The design of this system and its application shed new light on flow-induced phenomena in the study of urothelial carcinomas.

Heterogeneity of HPV16 virus-like particles indicates a complex assembly energy surface.

Human Papillomavirus serotype 16 (HPV16) capsid protein (L1) pentamers canonically assemble into T = 7 icosahedral capsids. Such virus-like particles are the basis of the HPV vaccine. We examined assembly of L1 pentamers in response to pH, mild oxidants, and ionic strength and found a mixture of closed, roughly spherical structures from ∼20 to ∼70 nm in diameter, indicating the presence of many kinetically accessible energy minima. Using bulk and single particle techniques we observed that the size distribution changes but does not reach homogeneity. Though heterogenous in size, particles showed uniform responses to low ionic strength dissociation, thermal unfolding, and susceptibility to protease digestion. These assays suggest maturation over time, but at different rates. Cysteine oxidation further stabilized particles at early, but not late, times without changing general characteristics including thermal stability and protease digestion. These data show complex assembly paths to species of different sizes, but with locally similar interactions.

The lymphatic system: a therapeutic target for central nervous system disorders.

The lymphatic vasculature forms an organized network that covers the whole body and is involved in fluid homeostasis, metabolite clearance, and immune surveillance. The recent identification of functional lymphatic vessels in the meninges of the brain and the spinal cord has provided novel insights into neurophysiology. They emerge as major pathways for fluid exchange. The abundance of immune cells in lymphatic vessels and meninges also suggests that lymphatic vessels are actively involved in neuroimmunity. The lymphatic system, through its role in the clearance of neurotoxic proteins, autoimmune cell infiltration, and the transmission of pro-inflammatory signals, participates in the pathogenesis of a variety of neurological disorders, including neurodegenerative and neuroinflammatory diseases and traumatic injury. Vascular endothelial growth factor C is the master regulator of lymphangiogenesis, a process that is critical for the maintenance of central nervous system homeostasis. In this review, we summarize current knowledge and recent advances relating to the anatomical features and immunological functions of the lymphatic system of the central nervous system and highlight its potential as a therapeutic target for neurological disorders and central nervous system repair.

Increasingly successful highly active antiretroviral therapy delays the emergence of new HLA class I-associated escape mutations in HIV-1.

BACKGROUND: HLA class I-restricted cytotoxic T lymphocytes and highly active antiretroviral therapy (HAART) exert strong selective pressures on human immunodeficiency virus type 1 (HIV-1), leading to escape mutations compromising virologic control. Immune responses continue to shape HIV-1 evolution after HAART initiation, but the extent and rate at which this occurs remain incompletely quantified. Here, we characterize the incidence and clinical correlates of HLA-associated evolution in HIV-1 Pol after HAART initiation in a large, population-based observational cohort. METHODS: British Columbia HAART Observational, Medical Evaluation and Research cohort participants with available HLA class I types and longitudinal posttherapy protease/reverse transcriptase sequences were studied (n = 619; median, 5 samples per patient and 5.2 years of follow-up). HLA-associated polymorphisms were defined according to published reference lists. Rates and correlates of immune-mediated HIV-1 evolution were investigated using multivariate Cox proportional hazard models incorporating baseline and time-dependent plasma viral load and CD4 response data. RESULTS: New HLA-associated escape events were observed in 269 (43%) patients during HAART and occurred at 49 of 63 (78%) investigated immune-associated sites in Pol. In time-dependent analyses adjusting for baseline factors, poorer virologic, but not immunologic, response to HAART was associated with increased risk of immune escape of 1.9-fold per log(10) viral load increment (P < .0001). Reversion of escape mutations following HAART initiation was extremely rare. CONCLUSIONS: HLA-associated HIV-1 evolution continues during HAART to an extent that is inversely related to the virologic success of therapy. Minimizing the degree of immune escape could represent a secondary benefit of effective HAART.

Corrigendum: Blood Immune Cell Composition Associated With Obesity and Drug Repositioning Revealed by Epigenetic and Transcriptomic Conjoint Analysis.

[This corrects the article DOI: 10.3389/fphar.2021.714643.].

Blood Immune Cell Composition Associated with Obesity and Drug Repositioning Revealed by Epigenetic and Transcriptomic Conjoint Analysis.

This research was designed to analyze the composition of immune cells in obesity and identify novel and potent drugs for obesity management by epigenetic and transcriptomic conjoint analysis. DNA methylation data set (GSE166611) and mRNA expression microarray (GSE18897) were obtained from the Gene Expression Omnibus database. A total of 72 objects (35 obese samples and 37 controls) were included in the study. Immune cell composition analysis, drug repositioning, and gene set enrichment analysis (GSEA) were performed using CIBERSORT, connectivity map (CMap), and GSEA tools. Besides, we performed a single-cell RNA-seq of the immune cells from whole blood samples obtained from one obese patient and one healthy control. mRNA levels of drug target genes were analyzed by qPCR assay in blood samples from six patients and six healthy controls. Immune cell composition analysis found that CD8 + T cells and NK cells were significantly lower in the obese group. 11 drugs/compounds are considered to possess obesity-control potential, such as atorvastatin. Moreover, the expression of drug targets (STAT3, MCL1, PMAIP1, SOD2, FOX O 3, FOS, FKBP5) in obese patients were higher than those in controls. In conclusion, immune cells are potential therapeutic targets for obesity. Our results also contribute to accelerate research on drug development of obesity.