Ratiometric Afterglow Luminescent Imaging of Matrix Metalloproteinase-2 Activity via an Energy Diversion Process.

Ratiometric afterglow luminescent (AGL) probes are attractive for in vivo imaging due to their high sensitivity and signal self-calibration function. However, there are currently few ratiometric AGL probes available for imaging enzymatic activity in living organisms. Here, we present an energy diversion (ED) strategy that enables the design of an enzyme-activated ratiometric AGL probe (RAG-RGD) for in vivo afterglow imaging. The ED process provides RAG-RGD with a radiative transition for an 'always on' 520-nm AGL signal (AGL520) and a cascade three-step energy transfer (ET) process for an 'off-on' 710-nm AGL signal (AGL710) in response to a specific enzyme. Using matrix metalloproteinase-2 (MMP-2) as an example, RAG-RGD shows a significant ~11-fold increase in AGL710/AGL520 toward MMP-2. This can sensitively detect U87MG brain tumors through ratiometric afterglow imaging of MMP-2 activity, with a high signal-to-background ratio and deep imaging depth. Furthermore, by utilizing the self-calibration effect of ratiometric imaging, RAG-RGD demonstrated a strong negative correlation between the AGL710/AGL520 value and the size of orthotopic U87MG tumor, enabling accurate monitoring of orthotopic glioma growth in vivo. This ED process may be applied for the design of other enzyme-activated ratiometric afterglow probes for sensitive afterglow imaging.

Pseudotyped Virus for Bandavirus.

The genus Bandavirus, belonging to family Phenuiviridae, order Bunyavirales, consists of eight tick-borne bunyaviruses. The Dabie bandavirus, formerly known as severe fever with thrombocytopenia virus (SFTSV), belongs to the genus Bandavirus. This emerging pathogen was first identified in central China in 2009. In recent years, the disease has been reported to cause several outbreaks in eastern Asia areas, including China, Japan, Korea, and Vietnam. Tick-to-human transmission is the main route of infection in humans, and transmission via the contact of body fluids from person-to-person was also reported. Despite its high fatality rate, there is currently no vaccine or antiviral therapy available. The therapeutic efficacies of several antiviral agents against Dabie bandavirus are still being evaluated. However, the virus is a potent pathogen with high biosafety experimental conditions. Therefore, replication-incompetent pseudotyped viruses play an important role. In this chapter, we succinctly summarize the basic features concerning Dabie bandavirus, including virion structure, genome characteristics, especially the characteristics of glycoprotein, and probable pathogenic mechanism. And, we put an important part in expounding the construction of pseudoviruses and its application.

Pseudotyped Viruses for Mammarenavirus.

Mammarenaviruses are classified into New World arenaviruses (NW) and Old World arenaviruses (OW). The OW arenaviruses include the first discovered mammarenavirus-lymphocytic choriomeningitis virus (LCMV) and the highly lethal Lassa virus (LASV). Mammarenaviruses are transmitted to human by rodents, resulting in severe acute infections and hemorrhagic fever. Pseudotyped viruses have been widely used as a tool in the study of mammarenaviruses. HIV-1, SIV, FIV-based lentiviral vectors, VSV-based vectors, MLV-based vectors, and reverse genetic approaches have been applied in the construction of pseudotyped mammarenaviruses. Pseudotyped mammarenaviruses are commonly used in receptor research, neutralizing antibody detection, inhibitor screening, viral virulence studies, functional analysis of N-linked glycans, and studies of viral infection, endocytosis, and fusion mechanisms.

Rigidity Bridging Flexibility to Harmonize Three Excited-State Deactivation Pathways for NIR-II-Fluorescent-Imaging-Guided Phototherapy.

Small organic phototherapeutic molecules of the second near-infrared (NIR-II) window (1000-1700 nm) serve as promising candidates for theranostics. However, developing such versatile agents for fluorescence-guided photodynamic/photothermal therapy remains a demanding task stirred by competitive energy dissipation pathways, including radiative decay, internal conversion, and intersystem crossing. To the best of current knowledge, the current paradigm for addressing the issue has deliberately approached the optimum balance among three deactivation processes through offsetting from each other, possibly leading to a comprehensively compromised theranostic efficacy. Few reports aim to modulate the three deactivation pathways excluding sacrificing any one of them. Herein, a molecular design strategy to construct a phototherapeutic organic fluorophore CCNU-1060, armed with NIR-II luorescence-guided phototherapeutic properties, is rationally developed. With a flexible motor, tetraphenylethene, bridged to the rigidified coplanar core boron-azadipyrromethene, the desired CCNU-1060 is subsequently encapsulated into an amphiphilic matrix to form CCNU-1060 nanoparticles (NPs), which match or transcend its precursor NJ-1060 NPs in the three energy dissipation processes. CCNU-1060 NPs are utilized to realize high-spatial vessel imaging and effective NIR-II fluorescence-guided phototherapeutic tumor ablation. This study unlocks a viewpoint of molecular engineering that simultaneously regulates multiple energy dissipation pathways for the construction of versatile phototherapy agents.

Design of Activatable NIR-II Molecular Probe for In Vivo Elucidation of Disease-Related Viscosity Variations.

A clear elucidation of a disease-related viscosity change in vivo is significant yet highly challenging as well. Fluorescence imaging in the second near-infrared region (NIR-II, 1000-1700 nm) has gained increasing attention for observation in living organisms, but a viscosity-activatable fluorescent probe emitting at this region remains a vacancy. Herein, we report the first panel of a viscosity-activated NIR-II emissive fluorescent probe WD-X. By embedding different substituents into the WD-X platform and screening, we obtained an ideal probe, WD-NO2, which displayed the best combination of properties, including a 31-fold fluorescence enhancement in response to viscosity, insensitivity to environments (pH, polarity), and relatively high quantum yield (1.6% in glycerol). WD-NO2 was successfully applied to track the variation of viscosity in diabetes-induced liver injury in vivo.

In Situ Imaging of Cysteine in the Brains of Mice with Epilepsy by a Near-Infrared Emissive Fluorescent Probe.

Epilepsy is characterized by oxidative stress in the brain. As the crucial reductive biothiol, cysteine (Cys) directly regulates the occurrence of oxidative stress in the brain. Observations suggest that the decreased cysteine in plasma could potentially serve as a redox biomarker in temporal lobe epilepsy. However, due to the complexity of the brain and lack of appropriate in situ detecting tools, the concentration change and regulation of Cys in epileptic brains remain unclear. Here, we report a near-infrared imaging probe (named Mito-CP) for tracking endogenous Cys in brains of pentylenetetrazole (PTZ)-induced epileptic seizures with high sensitivity and selectivity. Using this probe, we achieved an in situ visualization of the increased Cys in PC12 cells under dithiothreitol stimulation. In addition, Mito-CP was able to real-time monitor Cys fluctuation in lipopolysaccharide-mediated oxidative stress in zebrafish. Ultimately, we directly visualized the precipitous reduction of Cys in epileptic brains for the first time. Mito-CP also revealed the fluctuation of Cys in epileptic brains during the treatment by an antiepileptic drug, curcumin. This work provides an effective tool for Cys imaging in brains and will help to expand the understanding of the pathogenesis of epilepsy.

Sequential treatment with aT19 cells generates memory CAR-T cells and prolongs the lifespan of Raji-B-NDG mice.

Treatment with chimeric antigen receptor (CAR)-modified T cells targeting CD19 has proved successful in patients with relapsed/refractory B cell malignancies. However, long-term follow-up indicates that remission in a substantial proportion of patients is not sustainable. Most patients that experience recurrence have tumors and lost the CAR-T cells. To maintain the activity of CAR-T cells, Raji-B-NDG mice were treated sequentially with CAR-T-19 cells and homologous cells expressing human CD19 to promote expansion of CAR-T cells. Sequential treatment of mice with CAR-T-19 cells followed by Raji tumor cells led to marked prolongation of survival. The best case scenario after sequential treatment was a survival time of more than 200 days; the average survival time of mice in the non-sequential treatment group was 80 days. We treated mice with autologous CD19-modified T cells after initial treatment with CAR-T-19 cells. The overall survival and recurrence-free survival times of mice receiving sequential treatment were significantly longer. The percentages of CAR+ T cells in peripheral blood increased. Sequential therapy with autologous CAR-T-19 and aT19 cells provides a new strategy for generating memory CAR-T cells, which may lead ultimately to increased clinical efficacy.

Development of in vitro and in vivo neutralization assays based on the pseudotyped H7N9 virus.

H7N9 viral infections pose a great threat to both animal and human health. This avian virus cannot be handled in level 2 biocontainment laboratories, substantially hindering evaluation of prophylactic vaccines and therapeutic agents. Here, we report a high-titer pseudoviral system with a bioluminescent reporter gene, enabling us to visually and quantitatively conduct analyses of virus replications in both tissue cultures and animals. For evaluation of immunogenicity of H7N9 vaccines, we developed an in vitro assay for neutralizing antibody measurement based on the pseudoviral system; results generated by the in vitro assay were found to be strongly correlated with those by either hemagglutination inhibition (HI) or micro-neutralization (MN) assay. Furthermore, we injected the viruses into Balb/c mice and observed dynamic distributions of the viruses in the animals, which provides an ideal imaging model for quantitative analyses of prophylactic and therapeutic monoclonal antibodies. Taken together, the pseudoviral systems reported here could be of great value for both in vitro and in vivo evaluations of vaccines and antiviral agents without the need of wild type H7N9 virus.

Valuable antibody detection method for classifying hepatitis E virus genotypes.

Nucleotide-based methods are conventionally used to classify the hepatitis E virus (HEV) genotypes. A serological enzyme immunoassay (EIA) using open reading frame 3 (ORF3) C-terminal peptides was developed to conveniently and accurately classify and evaluate the genotypes of HEV. The sera of mice immunized with HEV genotype 1, 3, and 4 reacted highly specifically to the peptides of the corresponding genotypes. Most (84.2%) clinical sera infected with HEV genotype 4 were positive for anti-HEV antibodies when tested with the ORF3 peptides of genotype 4, but were negative for genotypes 1 and 3. Monkey and clinical serial sera infected with HEV reacted strongly to the homologous genotype ORF3 peptides. The indirect EIAs were more sensitive, with stronger reactivity, than commercial anti-HEV immunoglobulin G assays when serial sera from monkeys infected with HEV genotype 1 or 4 were tested. All our results indicate that the serological typing EIA assays described in this study are more effective and convenient for the classification of HEV genotypes than molecular approaches, and can be used to screen large numbers of serum samples and differentiate genotypes for the diagnosis of HEV infections.

Neutralizing antibodies against adenovirus type 2 in normal and HIV-1-infected subjects: Implications for use of Ad2 vectors in vaccines.

Pre-existing neutralizing antibodies (NAbs) directed against vaccine vectors have attracted considerable research attention. Therefore, our aim was to establish a high-throughput economical neutralization assay to investigate the epidemiology of adenovirus type 2 (Ad2)-specific immunity in China and developed countries, including in a Chinese Human immunodeficiency virus (HIV)-1-infected population, and to guide the application of Ad2-vectored vaccines. We established a FluoroSpot-based anti-Ad2-virus neutralization assay using a recombinant replication-deficient Ad2 that expresses enhanced green fluorescent protein and standardized the critical parameters, including the choice of cell line, cell concentration, viral infective dose, and incubation time. The sera of 561 healthy individuals from China and developed countries and from 230 HIV-1-infected Chinese individuals were screened with this assay for Nabs against Ad2. The prevalence of anti-Ad2 NAbs was high in both China (92.2%) and developed countries (86.9%). Of the Ad2-seropositive individuals, 64.6% in China and 77.4% in developed countries had high NAb titers (> 810). The frequency of anti-Ad2 NAbs was higher in Anhui (97.5%) than in Beijing (88.7%). Their prevalence differed significantly according to age in Beijing, but not in Anhui Province, but by sex in neither province. Ad2 seroprevalence was as high among HIV-1-infected individuals (88.7%) as among healthy individuals (92.2%) in China. In conclusion, a simple, intuitive, high-throughput, economical fluorescence-based neutralization assay was developed to determine anti-Ad2 NAbs titers. Ad2 exposure was high in both healthy and HIV-1-infected populations in China, so vectors based on Ad2 may be inappropriate for human vaccines.

An Apple Fruit Fermentation (AFF) Treatment Improves the Composition of the Rhizosphere Microbial Community and Growth of Strawberry (Fragaria × ananassa Duch 'Benihoppe') Seedlings.

Plant growth can be promoted by the application of apple fruit fermentation (AFF), despite unclear of the underlying mechanisms, the effects involved in AFF on rhizosphere microorganisms have been hypothesized. We investigated the consequences of applying AFF alone or in combination with Bacillus licheniformis to strawberry tissue culture seedlings in vitro, the analyses of Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rDNA were performed to determine AFF effects on rhizosphere. Moreover, the growth index and antioxidant enzyme activities were determined 30 days after treatments. We identified five dominant bacteria in AFF: Coprinus atramentarius, Bacillus megaterium, Bacillus licheniformis, Weissella and B. subtilis. The greatest number of bacterial species were observed in the rhizosphere of control matrix (water treated), and the lowest diversity appeared in the rhizosphere soil treated with 108 cfu/mL B. licheniformis alone. Combining AFF plus B. licheniformis in one treatment resulted in the largest leaf area, plant height, root length, plant weight, and the markedly higher activities of antioxidant enzymes. We conclude that a combination of AFF plus B. licheniformis treatment to matrix can increase antioxidant enzymes activities in strawberry seedlings, optimize the status of rhizosphere microbial, and promote plant growth.

N463 Glycosylation Site on V5 Loop of a Mutant gp120 Regulates the Sensitivity of HIV-1 to Neutralizing Monoclonal Antibodies VRC01/03.

BACKGROUND: HIV-1 gp120/gp41 is heavily modified by n-linked carbohydrates that play important roles either in correct folding or in shielding vulnerable viral protein surfaces from antibody recognition. METHODS: In our previous work, 25 potential N-linked glycosylation sites (PNGS) of a CRF07_BC isolate of HIV-1 were individually mutated, and the resulting effects on infectivity and antibody-mediated neutralization were evaluated. To further understand the functional role of these PNGS, we generated double and multiple mutants from selected individual PNGS mutants. The effects were then evaluated by examining infectivity and sensitivity to antibody-mediated neutralization by neutralizing monoclonal antibodies (nMAbs) and serum antibodies from HIV-1 positive donors. RESULTS: Infectivity results showed that, among the 12 combined PNGS mutants, only 197M.1 (N197D/N301Q) lost infectivity completely, whereas all others (except for 197M.6) showed reduced viral infectivity. In terms of neutralization sensitivity to known nMAbs, we found that adding N463Q mutation to all the gp120 mutants containing N197D significantly increased neutralization sensitivity to VRC01 and VRC03, suggesting N197 and N463 have a strong synergistic effect in regulating the neutralizing sensitivity of HIV-1 to the anti-CD4bs nMAbs VRC01/VRC03. Structural analysis based on the available structures of gp120 alone and in complex with CD4 and various nMAbs elucidates a molecular rationale for this experimental observation. CONCLUSIONS: The data indicate that N463 plays an important role in regulating the CD4bs MAbs VRC01/VRC03 sensitivity in the genetic background of N197D mutation of gp120, which should provide valuable information for a better understanding of the interplay between HIV-1 and VRC01/03.