Folic Acid Functionalized AQ4N/Gd@PDA Nanoplatform with Real-Time Monitoring of Hypoxia Relief and Enhanced Synergistic Chemo/Photothermal Therapy in Glioma.

Purpose: Hypoxia is often associated with glioma chemoresistance, and alleviating hypoxia is also crucial for improving treatment efficacy. However, although there are already some methods that can improve efficacy by alleviating hypoxia, real-time monitoring that can truly achieve hypoxia relief and efficacy feedback still needs to be explored. Methods: AQ4N/Gd@PDA-FA nanoparticles (AGPF NPs) were synthesized using a one-pot method and were characterized. The effects of AGPF NPs on cell viability, cellular uptake, and apoptosis were investigated using the U87 cell line. Moreover, the effectiveness of AGPF NPs in alleviating hypoxia was explored in tumor-bearing mice through photoacoustic imaging. In addition, the diagnosis and treatment effect of AGPF NPs were evaluated by magnetic resonance imaging (MRI) and bioluminescent imaging (BLI) on orthotopic glioma mice respectively. Results: In vitro experiments showed that AGPF NPs had good dispersion, stability, and controlled release. AGPF NPs were internalized by cells through endocytosis, and could significantly reduce the survival rate of U87 cells and increase apoptosis under irradiation. In addition, we monitored blood oxygen saturation at the tumor site in real-time through photoacoustic imaging (PAI), and the results showed that synergistic mild-photothermal therapy/chemotherapy effectively alleviated tumor hypoxia. Finally, in vivo anti-tumor experiments have shown that synergistic therapy can effectively alleviate hypoxia and inhibit the growth of orthotopic gliomas. Conclusion: This work not only provides an effective means for real-time monitoring of the dynamic feedback between tumor hypoxia relief and therapeutic efficacy, but also offers a potential approach for the clinical treatment of gliomas.

High-Throughput Screening of Functional Neo-Antigens and Their Specific T-Cell Receptors via the Jurkat Reporter System Combined with Droplet Microfluidics.

T-cell receptor (TCR)-engineered T cells can precisely recognize a broad repertoire of targets derived from both intracellular and surface proteins of tumor cells. TCR-T adoptive cell therapy has shown safety and promising efficacy in solid tumor immunotherapy. However, antigen-specific functional TCR screening is time-consuming and expensive, which limits its application clinically. Here, we developed a novel integrated antigen-TCR screening platform based on droplet microfluidic technology, enabling high-throughput peptide-major histocompatibility complex (pMHC)-to-TCR paired screening with a high sensitivity and low background signal. We introduced DNA barcoding technology to label peptide antigen candidate-loaded antigen-presenting cells and Jurkat reporter cells to check the specificity of pMHC-TCR candidates. Coupled with the next-generation sequencing pipeline, interpretation of the DNA barcodes and the gene expression level of the Jurkat T-cell activation pathway provided a clear peptide-MHC-TCR recognition relationship. Our proof-of-principle study demonstrates that the platform could achieve pMHC-TCR paired high-throughput screening, which is expected to be used in the cross-reactivity and off-target high-throughput paired testing of candidate pMHC-TCRs in clinical applications.

A Novel Proteogenomic Integration Strategy Expands the Breadth of Neo-Epitope Sources.

Tumor-specific antigens can activate T cell-based antitumor immune responses and are ideal targets for cancer immunotherapy. However, their identification is still challenging. Although mass spectrometry can directly identify human leukocyte antigen (HLA) binding peptides in tumor cells, it focuses on tumor-specific antigens derived from annotated protein-coding regions constituting only 1.5% of the genome. We developed a novel proteogenomic integration strategy to expand the breadth of tumor-specific epitopes derived from all genomic regions. Using the colorectal cancer cell line HCT116 as a model, we accurately identified 10,737 HLA-presented peptides, 1293 of which were non-canonical peptides that traditional database searches could not identify. Moreover, we found eight tumor neo-epitopes derived from somatic mutations, four of which were not previously reported. Our findings suggest that this new proteogenomic approach holds great promise for increasing the number of tumor-specific antigen candidates, potentially enlarging the tumor target pool and improving cancer immunotherapy.

IntroSpect: Motif-Guided Immunopeptidome Database Building Tool to Improve the Sensitivity of HLA I Binding Peptide Identification by Mass Spectrometry.

Although database search tools originally developed for shotgun proteome have been widely used in immunopeptidomic mass spectrometry identifications, they have been reported to achieve undesirably low sensitivities or high false positive rates as a result of the hugely inflated search space caused by the lack of specific enzymic digestions in immunopeptidome. To overcome such a problem, we developed a motif-guided immunopeptidome database building tool named IntroSpect, which is designed to first learn the peptide motifs from high confidence hits in the initial search, and then build a targeted database for refined search. Evaluated on 18 representative HLA class I datasets, IntroSpect can improve the sensitivity by an average of 76%, compared to conventional searches with unspecific digestions, while maintaining a very high level of accuracy (~96%), as confirmed by synthetic validation experiments. A distinct advantage of IntroSpect is that it does not depend on any external HLA data, so that it performs equally well on both well-studied and poorly-studied HLA types, unlike the previously developed method SpectMHC. We have also designed IntroSpect to keep a global FDR that can be conveniently controlled, similar to a conventional database search. Finally, we demonstrate the practical value of IntroSpect by discovering neoepitopes from MS data directly, an important application in cancer immunotherapies. IntroSpect is freely available to download and use.

Intranasal lipopolysaccharide administration prevents chronic stress-induced depression- and anxiety-like behaviors in mice.

We recently reported that intraperitoneal injection of a low dose of lipopolysaccharide (LPS) prevents chronic stress-induced depression-like behaviors in mice. In this study, we reported that a single intranasal LPS administration (10 µg/mouse) one day prior to stress exposure produced prophylactic effects on chronic social defeat stress (CSDS)-induced depression-like behaviors, which was indicated by the reduction in social interaction time in the social interaction test and the decrease in immobility time in the tail suspension test and forced swimming test. The single intranasal LPS administration prior to stress exposure was also found to prevent CSDS-induced anxiety-like behaviors, including prevention of CSDS-induced decrease in the time spent in open arms in the elevated plus maze test, decrease in the time spent in lit side in the light-dark test, and decrease in the time spent in central regions in the open field test, along with no changes in locomotor activity. Further analysis showed that the single intranasal LPS administration one day prior to stress exposure prevented CSDS-induced increase in levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß mRNA in the hippocampus and prefrontal cortex. Inhibition of innate immune stimulation by minocycline pretreatment not only abrogated the preventive effect of intranasal LPS administration on CSDS-induced depression- and anxiety-like behaviors, but also abrogated the preventive effect of intranasal LPS administration on CSDS-induced neuroinflammatory responses in the hippocampus and prefrontal cortex. These results demonstrate that intranasal administration of innate immune stimulants could be a potential approach for the prevention of depression and anxiety.

Pharmacological Actions of Myricetin in the Nervous System: A Comprehensive Review of Preclinical Studies in Animals and Cell Models.

Myricetin is a natural flavonoid extracted from a variety of plants, such as medicinal herbs, vegetables, berries, and tea leaves. A growing body of evidence has reported that myricetin supplementation display therapeutic activities in a lot of nervous system disorders, such as cerebral ischemia, Alzheimer's disease, Parkinson's disease, epilepsy, and glioblastoma. Myricetin supplementation can also protect against pathological changes and behavioral impairment induced by multiple sclerosis and chronic stress. On the basis of these pharmacological actions, myricetin could be developed as a potential drug for the prevention and/or treatment of nervous system disorders. Mechanistic studies have shown that inhibition of oxidative stress, cellular apoptosis, and neuroinflammatory response are common mechanisms for the neuroprotective actions of myricetin. Other mechanisms, including the activation of the nuclear factor E2-related factor 2 (Nrf2), extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (Akt), cyclic adenosine monophosphate-response element binding protein (CREB), and brain-derived neurotrophic factor (BDNF) signaling, inhibition of intracellular Ca2+ increase, inhibition of c-Jun N-terminal kinase (JNK)-p38 activation, and suppression of mutant protein aggregation, may also mediate the neuroprotective effects of myricetin. Furthermore, myricetin treatment has been shown to promote the activation of the inhibitory neurons in the hypothalamic paraventricular nucleus, which subsequently produces anti-epilepsy effects. In this review, we make a comprehensive understanding about the pharmacological effects of myricetin in the nervous system, aiming to push the development of myricetin as a novel drug for the treatment of nervous system disorders.

Lycopene ameliorates PTSD-like behaviors in mice and rebalances the neuroinflammatory response and oxidative stress in the brain.

Posttraumatic stress disorder (PTSD) is a mental disorder that can translate into severe economic problems. Lycopene is an aliphatic hydrocarbon carotenoid extracted from plants, including papayas, tomatoes, and water melons. Previous studies have shown that lycopene can produce antidepressant-like effects in rodent models of depression. However, little is known about its anti-PTSD-like effect. This was addressed in the present study by using the single prolonged stress (SPS) protocol to induce PTSD-like behavioral deficits in mice. Our results showed that 12 days of lycopene treatment at the dose of 10 and 20 mg/kg, but not at 5 mg/kg, ameliorated the PTSD-like phenotype induced by SPS, including the increase in freezing time in contextual fear paradigm, the decrease in time and entries in open arms in elevated plus maze test, and the decrease in distance and time in the central area of the open field test, without affecting the mouse locomotor activity. Mechanistic studies revealed that lycopene treatment (20 mg/kg, 12 days) could suppress the SPS-induced increase in levels of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), and nitrite in the hippocampus and prefrontal cortex in mice, as well as the increased markers that indicate high levels of oxido-nitrosative stress in the hippocampus and prefrontal cortex in SPS mice. Lycopene treatment (20 mg/kg, 12 days) also suppressed the SPS-induced decrease in brain derived neurotrophic factor (BDNF) levels in the hippocampus and prefrontal cortex in mice. Overall, the anti-PTSD-like effect of lycopene may be associated with its anti-neuroinflammation and anti-oxidative stress activities.

The novel llama-human chimeric antibody has potent effect in lowering LDL-c levels in hPCSK9 transgenic rats.

BACKGROUND: The advent of proprotein convertase subtilisin/kexin type 9 (PCSK9)-inhibiting drugs have provided an effective, but extremely expensive treatment for the management of low density lipoprotein (LDL). Our aim was to explore a cost-effective application of camelid anti-PCSK9 single domain antibodies (sdAbs), which are high variable regions of the camelid heavy chain antibodies (VHHs), as a human PCSK9 (hPCSK9) inhibitor. One female llama was immunized with hPCSK9. Screening of high affinity anti-PCSK9 VHHs was carried out based on surface plasmon resonance (SPR) technology. We reported a lysate kinetic analysis method improving the screening efficiency. To increase the serum half-life and targeting properties, the constant region fragment of the human immunoglobulin gamma sub-type 4 (IgG4 Fc) was incorporated to form a novel llama-human chimeric molecule (VHH-hFc). RESULTS: The PCSK9 inhibiting effects of the VHH proteins were analyzed in two human liver hepatocellular cells (HepG2 and Huh7) and in the hPCSK9 transgenic Sprague-Dawley (SD) rat model. The hPCSK9 antagonistic potency of the bivalent VHH-hFc exceeded the monovalent VHH (P < 0.001) in hepatocarcinoma cells. Furthermore, the llama-human chimeric VHH-Fc protein had a similar reduction (~ 40%) of the LDL-c and total cholesterol when compared to the approved evolocumab in transgenic SD rat model, but with low cost. More surprisingly, the chimeric heavy chain antibodies could be persevered for 3 months at room temperature with little loss of the affinity. CONCLUSIONS: Due to the high yield and low cost of Pichia pastoris, lipid-lowering effect and strong stability, the llama-human chimeric antibody (VHH-Fc) offers a potent therapeutic candidate for the control of the serum lipid level.

Regulatory effects of Echinococcus multilocularis extracellular vesicles on RAW264.7 macrophages.

Extracellular vesicles (EVs) play a role in intercellular communications via exchanging biological molecules, being involved in host-parasite interplay. Little is to date known about E. multilocularis EVs and their biological activities. Here spherical EVs secreted by E. multilocularis metacestodes were shown to range predominately from 34nm to 95nm in diameter. A total of 433 proteins were identified in the EVs, and the proteins involved in binding (42%) and catalytic activity (41%) were most frequently represented. Moreover, the proteins associated with EV biogenesis and trafficking, including annexin, 14-3-3, tetraspanin and heat shock protein 70kDa, were highly enriched. It was shown that the EVs remarkably suppressed NO produced by activated RAW macrophages via downregulation of inducible nitric oxide synthase expression (p <0.01). Suppression of pro-inflammatory cytokines, especially IL-1α and IL-1ß, was also observed post treatment with the EVs. Conversely, increased expression of the majority (10/11) of key components involved in the LPS/TLR4 pathway was induced by the EVs. These results demonstrate a regulatory effect of E. multilocularis EVs on macrophages, suggesting a role in parasite-host interactions.

[Screening and Applications of qPCR Primers for apomucin Gene of Echinococcus multilocularis].

Objective: To screen for the optimal qPCR primers for Echinococcus multilocularis apomucin gene (Em-apo) and analyze Em-apo expression. Methods: Primers were designed based on 4 Em-apo sequences from GeneDB. Primer specificity and PCR efficiency were determined, based on which the optimal primer pairs were selected. Alterations of Em-apo expression in 1 000 E. multilocularis protoscoleces treated with albendazole（5 µg/ml） and insulin（100 ng/ml） were separately assessed using the selected primers. DMSO used in albendazole dilution and in PBS insulin dilution were used as the control. Results: Specific primers for Em-apo-1, Em-apo-2/3, Em-apo-4 and actin were selected. qPCR melting curves revealed a single peak for each primer pair and an amplification efficiency from 95% to 101%. The qPCR showed increased expression of Em-apo-1（1.51±0.27）, Em-apo-2/3 （1.39±0.30） and Em-apo-4（1.14±0.18） after albendazole treatment in comparison to the DMSO control（1.00）（P>0.05 among the three genes）; and an unaltered Em-apo-1 expression, slightly decreased Em-apo-4 expression, and significantly decreased Em-apo-2/3 expression（0.73±0.09） after insulin treatment in comparison to the PBS control (P>0.05 among the three genes）. Conclusion: The selected specific primers for Em-apo genes can be used to analyze the gene expression by qPCR. Treatment with albendazole and insulin show certain effects on the expression of Em-apo genes in E. multilocularis protoscoleces.