An engineered influenza virus to deliver antigens for lung cancer vaccination.

The development of cancer neoantigen vaccines that prime the anti-tumor immune responses has been hindered in part by challenges in delivery of neoantigens to the tumor. Here, using the model antigen ovalbumin (OVA) in a melanoma model, we demonstrate a chimeric antigenic peptide influenza virus (CAP-Flu) system for delivery of antigenic peptides bound to influenza A virus (IAV) to the lung. We conjugated attenuated IAVs with the innate immunostimulatory agent CpG and, after intranasal administration to the mouse lung, observed increased immune cell infiltration to the tumor. OVA was then covalently displayed on IAV-CPG using click chemistry. Vaccination with this construct yielded robust antigen uptake by dendritic cells, a specific immune cell response and a significant increase in tumor-infiltrating lymphocytes compared to peptides alone. Lastly, we engineered the IAV to express anti-PD1-L1 nanobodies that further enhanced regression of lung metastases and prolonged mouse survival after rechallenge. Engineered IAVs can be equipped with any tumor neoantigen of interest to generate lung cancer vaccines.

Transcriptome analysis revealed that short-term stress in Blattella germanica to ß-cypermethrin can reshape the phenotype of resistance adaptation.

Previous studies on insect resistance have primarily focused on resistance monitoring and the molecular mechanisms involved, while overlooking the process of phenotype formation induced by insecticide stress. In this study, we compared the expression profiles of a beta-cypermethrin (ß-CYP) resistant strain (R) and a susceptible strain (S) of Blattella germanica after ß-CYP induction using transcriptome sequencing. In the short-term stress experiment, we identified a total of 792 and 622 differentially expressed genes (DEGs) in the S and R strains. Additionally, 893 DEGs were identified in the long-term adaptation experiment. To validate the RNA-Seq data, we performed qRT-PCR on eleven selected DEGs, and the results were consistent with the transcriptome sequencing data. These DEGs exhibited down-regulation in the short-term stress group and up-regulation in the long-term adaptation group. Among the validated DEGs, CUO8 and Cyp4g19 were identified and subjected to knockdown using RNA interference. Subsequent insecticide bioassays revealed that the mortality rate of cockroaches treated with ß-CYP increased by 69.3% and 66.7% after silencing the CUO8 and Cyp4g19 genes (P<0.05). Furthermore, the silencing of CUO8 resulted in a significant thinning of the cuticle by 59.3% and 53.4% (P<0.05), as observed through transmission electron microscopy and eosin staining, in the S and R strains, respectively. Overall, our findings demonstrate that the phenotypic plasticity in response to short-term stress can reshape the adaptive mechanisms of genetic variation during prolonged exposure to insecticides. And the identified resistance-related genes, CUO8 and Cyp4g19, could serve as potential targets for controlling these pest populations.

Motif-to-Core Nucleation in a Decahedral Evolution Pattern.

The atomic precision of ultrasmall metal nanoclusters has opened the door to elucidating the structural evolution principles of metal nanomaterials at the molecular level. Here, we report a novel set of super-atomic Ag clusters, including [Ag19(TBBT)16(DPPP)4]+ (Ag19), [Ag22(DMAT)8(DPPM)4Cl8]2+ (Ag22), Ag26(SPh3,5-CF3)15(DPPF)4Cl5 (Ag26), and [Ag30(DMAT)12(DPPP)4Cl8]2+ (Ag30). The core structures of these clusters correspond to one decahedral Ag7, perpendicular bi-decahedrons, three-dimensional penta-decahedrons, and hexa-decahedrons, respectively. The Ag atoms in AgS2 blocks show a strong correlation with the decahedral cores: the five equatorial Ag atoms in the decahedral Ag7 core of Ag19 all adopt the AgS2 coordination, while the Ag atoms in AgS2 blocks of Ag22, Ag26, and Ag30 unexceptionally constitute additional decahedral structures with the core Ag atoms. Specifically, two and four core Ag atoms of Ag26 and Ag30 clusters occupy positions that highly resemble that of Ag (in AgS2 motifs) of Ag22. The strong structural correlation demonstrates the motif-to-core evolution of the surface Ag (on AgS2) to build extra-decahedral blocks. Density functional theory calculations indicate that the 2e, 4e, 6e, and 8e clusters (from Ag19 to Ag30) adopt 1S2, 1S21P2, 1S21P4, and 1S21P6 electron configurations, all of which feature excellent super-atomic characters.

[Expression and purification of dormancy survival regulator Rv2628 of Mycobacterium tuberculosis and preparation of rabbit anti-Rv2628 polyclonal antibody].

Objective To express and purify the dormancy survival regulator Rv2628 of Mycobacterium tuberculosis, so as to prepare and identify its rabbit polyclonal antibody. Methods Using the genome of Mycobacterium tuberculosis H37Rv strain as a template, the Rv2628 gene was amplified to construct a recombinant expression plasmid which was transformed into an Escherichia coli protein expression strain and induced expression by IPTG. The target protein was purified using Ni-NTA chromatography column, and the rabbit polyclonal antibody was obtained by immunizing New Zealand white rabbits with purified Rv2628 protein. The specificity of polyclonal antibodies was verified by Western blot analysis and indirect ELISA, respectively. Results The PET-30A-RV2628 recombinant carrier was successfully constructed. After induction by IPTG, the RV2628 protein was mainly expressed in the form of inclusion. The high-purity Rv2628 protein was obtained by Ni-NTA column purification. Rabbit anti-Rv2628 polyclonal antibody was obtained after immunizing the rabbit. The antibody had good antigen binding properties and the antibody titer reached 1:1 093 500. Conclusion The high-purity Rv2628 protein and high-titer rabbit anti-Rv2628 polyclonal antibody were successfully prepared.

Synthesis of Novel Pentacyclic Triterpenoid Derivatives that Induce Apoptosis in Cancer Cells through a ROS-dependent, Mitochondrial-Mediated Pathway.

Betulinic acid (BA) and oleanolic acid (OA) are plant-derived conjugates found in various medicinal plants that have emerged as potential antitumor agents. Herein, a series of novel BA and OA derivatives were synthesized by conjugation with per-O-methylated-ß-cyclodextrin (PM-ß-CD), and their anticancer properties against a panel of three human cancer cell lines were evaluated. Two OA-PM-ß-CD conjugates (48 and 50) were observed to be the most potent conjugates against the three cell lines (MCF-7, BGC-823, and HL-60), with a 15- to 20-fold decrease in the IC50 values (IC50: 6.06-8.47 µM) compared with their parental conjugate (OA). Annexin V-FITC/propidium iodide staining and Western blot analysis revealed that both conjugates induced apoptosis in HL-60 cells. Additionally, in the representative conjugate 48-treated HL-60 cells, a decrease in mitochondrial membrane potential and subsequent release of cytochrome c into the cytosol were observed, indicating the activation of the intrinsic apoptosis pathway. Furthermore, 48 dramatically induced the generation of reactive oxygen species (ROS) in HL-60 cells, and the corresponding effect could be reversed using the ROS scavenger N-acetylcysteine. Collectively, these results suggest that the novel pentacyclic triterpenoid derivatives trigger the intrinsic apoptotic pathways via the ROS-mediated activation of caspase-3 signaling, inducing cell death in human cancer cells.

Research on the Physics-Intelligence Hybrid Theory Based Dynamic Scenario Library Generation for Automated Vehicles.

The testing and evaluation system has been the key technology and security with its necessity in the development and deployment of maturing automated vehicles. In this research, the physics-intelligence hybrid theory-based dynamic scenario library generation method is proposed to improve system performance, in particular, the testing efficiency and accuracy for automated vehicles. A general framework of the dynamic scenario library generation is established. Then, the parameterized scenario based on the dimension optimization method is specified to obtain the effective scenario element set. Long-tail functions for performance testing of specific ODD are constructed as optimization boundaries and critical scenario searching methods are proposed based on the node optimization and sample expansion methods for the low-dimensional scenario library generation and the reinforcement learning for the high-dimensional one, respectively. The scenario library generation method is evaluated with the naturalistic driving data (NDD) of the intelligent electric vehicle in the field test. Results show better efficient and accuracy performances compared with the ideal testing library and the NDD, respectively, in both low- and high-dimensional scenarios.

A Label-Free Photoelectrochemical Biosensor Based on CRISPR/Cas12a System Responsive Deoxyribonucleic Acid Hydrogel and "Click" Chemistry.

A novel label-free photoelectrochemical (PEC) biosensor is presented in this work. As a barrier, the DNA hydrogel could block the coupling between g-C3N4 and CdS quantum dots (QDs). Therefore, extremely low photocurrent signals were obtained. The presence of target microRNA-21 can initiate the rolling circle amplification (RCA) reaction, which in turn produces many repeated sequences to activate the CRISPR/Cas12a system. The trans-cleavage activity of the CRISPR/Cas12a system led to the degradation of DNA hydrogels efficiently. As a result, the g-C3N4/CdS QDs heterojunction was formed through "click" chemistry. Through the amplification of the RCA and CRISPR/Cas12a system, the sensitivity of the PEC biosensor was improved significantly with the detection limit of 3.2 aM. The proposed sensor also showed excellent selectivity and could be used to detect actual samples. In addition, the modular design could facilitate the detection of different objects. Thus, the proposed CRISPR/Cas12a system responsive DNA hydrogel provides a simple, sensitive, and flexible way for label-free PEC analysis.

CRISPR/Cas12a system responsive DNA hydrogel for label-free detection of non-glucose targets with a portable personal glucose meter.

In this work, personal glucose meter (PGM) as a portable electrochemical device was utilized for sensitive detection of non-glucose targets: N-gene and PCB77, respectively. DNA hydrogel, which can respond to CRISPR/Cas system, was prepared for label-free encapsulating invertase. In the presence of targets, the repeated sequence for the activation of Cas12a was obtained due to the performance of RCA. Unlike "one-to-one" recognition, activated Cas12a can efficiently cleave multiple single-stranded linker DNAs on DNA hydrogels, thus releasing many invertase that can be used for PGM detection. With the amplification of RCA and CRISPR/Cas system, high detection sensitivity can be obtained even using portable PGM. The detection limits for N-gene and PCB77 were 2.6 fM and 3.2 × 10-5 µg/L, respectively, with high specificity and good practical application performance. The developed biosensor can be used for online monitoring with the merit of low cost, easy operation and can be used for various targets analysis.

Self-adhesive PMIA membranes with chitosan porous beads immobilized pullulanase for efficient biological aging of beer.

A novel pulluanase@chitosan porous beads/Poly (m-phthaloyl-m-phenylenediamine) (PULL@CPB/PMIA) membrane with good separation and biocatalysis properties was prepared by a self-adhesive method by introducing an immobilized enzyme (PULL@CPB) onto the PMIA membrane. The combination of PULL@CPB and PMIA could increase the one-step refining of protoplasmic beer as well as the ability of biocatalysis to lower the alcohol-to-ester ratio. The experimental results showed that the addition of PULL@CPB and the increase in the ratio of EtOH/water in the coagulation bath both increased the load of pullulanase on the membrane surface, while excessive addition decreased the activity of pullulanase. Under the amount of PULL@CPB is 0.5 g·L-1 and the ratio of EtOH/water is 60%, the relative activity of pullulanase in PULL@CPB modified PMIA membrane was the highest, which was 91.7% of the initial activity. The interception rates of protein and macromolecular ß-glucan were 92.2% and 87.3%, respectively, under the condition of beer flux (162.3 L·m-2·h-1). At the same time, the PULL@CPB/PMIA membrane has strong antibacterial performance and thus plays a role in extending the shelf life of beer. Compared with free pullulanase, the thermal stability, pH stability, organic solvent stability, and storing stability of immobilized pullulanase were significantly improved. The effects of PULL@CPB dosage and operating temperature on biocatalysis efficiency were discussed. The immobilized pullulanase activity on the membrane remained at 70.8% after 10 continuous uses. Therefore, the PMIA membrane is an excellent carrier of pullulanase, so its bioactive membrane has a wide range of prospects in food, medicine, and other fields.

Upconverting nanoparticles based nanodevice for DNAzymes amplified miRNAs detection and artificially controlled chemo-gene therapy.

Despite the great promise of cancer theranostic platforms, accurate diagnosis and effective treatment are still highly challenging. In this work, nanodevice for intracellular miRNAs detection and artificially controlled drug releasement was developed based on upconverting nanoparticles (UCNPs). For analysis aspect, DNAzymes amplified miRNA-21 detection was carried out, giving excellent sensitivity with detection limits of 1.8 × 10-11 M. Moreover, intracellular fluorescence imaging permitted in situ diagnoses of miRNA-21 expression in living cells. Once the test identifies tumor markers, treatment can be performed. Here, artificially controlled chemo-gene synergetic therapy nanodevice was obtained by integrating UCNPs with photocleavable linkers (PC-linkers). In vitro and in vivo experiments verified the potential application of prepared nanodevice in cancer theranostics.

Discovery of Pentacyclic Triterpenoid PROTACs as a Class of Effective Hemagglutinin Protein Degraders.

Influenza hemagglutinin that drives viral entry into cells via the membrane fusion process is an up-and-coming antiviral drug target. Herein, we described for the first time the design, synthesis, and biological characteristics of a new class of pentacyclic triterpenoid-based proteolysis targeting chimeras (PROTACs) to enhance the degradation of hemagglutinin target. Among these PROTACs, V3 showed the best degradation effect on the hemagglutinin with a median degradation concentration of 1.44 µM in a ubiquitin and proteasome-dependent manner and broad-spectrum anti-influenza A virus activity but not affected the entry of influenza virus. Moreover, intravenous injection of V3 protected mice against influenza A virus-induced toxic effects. Further diazirine-containing photo-crosslinking mass spectrometric analysis of hemagglutinin complexes indicated crosslinking to Asn15, Thr31, and Asn27, a novel target of hemagglutinin. Taken together, our data revealed that oleanolic acid-based PROTACs could degrade hemagglutinin protein, providing a new direction toward the discovery of potential anti-influenza drugs.

Influenza Virus Precision Diagnosis and Continuous Purification Enabled by Neuraminidase-Resistant Glycopolymer-Coated Microbeads.

Rapid diagnosis and vaccine development are critical to prevent the threat posed by viruses. However, rapid tests, such as colloidal gold assays, yield false-negative results due to the low quantities of viruses; moreover, conventional virus purification, including ultracentrifugation and nanofiltration, is multistep and time-consuming, which limits laboratory research and commercial development of viral vaccines. A rapid virus enrichment and purification technique will improve clinical diagnosis sensitivity and simplify vaccine production. Hence, we developed the surface-glycosylated microbeads (glycobeads) featuring chemically synthetic glycoclusters and reversible linkers to selectively capture the influenza virus. The surface plasmon resonance (SPR) evaluation indicated broad spectrum affinity of S-linked glycosides to various influenza viruses. The magnetic glycobeads were integrated into clinical rapid diagnosis, leading to a 30-fold lower limit of detection. Additionally, the captured viruses can be released under physiological conditions, delivering purified viruses with >50% recovery and without decreasing their native infectivity. Notably, this glycobead platform will facilitate the sensitive detection and continuous one-step purification of the target virus that contributes to future vaccine production.

Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells.

The preferential activation of regulatory T (Treg) cells by interleukin-2 (IL-2), which selectively binds to the trimeric IL-2 receptor (IL-2R) on Treg cells, makes this cytokine a promising therapeutic for the treatment of autoimmune diseases. However, IL-2 has a narrow therapeutic window and a short half-life. Here, we show that the pharmacokinetics and half-life of IL-2 can be substantially improved by orthogonally conjugating the cytokine to poly(ethylene glycol) (PEG) moieties via a copper-free click reaction through the incorporation of azide-bearing amino acids at defined sites. Subcutaneous injection of a PEGylated IL-2 that optimally induced sustained Treg-cell activation and expansion over a wide range of doses through highly selective binding to trimeric IL-2R led to enhanced therapeutic efficacy in mouse models of lupus, collagen-induced arthritis and graft-versus-host disease without compromising the immune defences of the host against viral infection. Site-specific PEGylation could be used more generally to engineer cytokines with improved therapeutic performance for the treatment of autoimmune diseases.

Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation*.

There is an urgent need to develop antiviral drugs and alleviate the current COVID-19 pandemic. Herein we report the design and construction of chimeric oligonucleotides comprising a 2'-OMe-modified antisense oligonucleotide and a 5'-phosphorylated 2'-5' poly(A)4 (4A2-5 ) to degrade envelope and spike RNAs of SARS-CoV-2. The oligonucleotide was used for searching and recognizing target viral RNA sequence, and the conjugated 4A2-5 was used for guided RNase L activation to sequence-specifically degrade viral RNAs. Since RNase L can potently cleave single-stranded RNA during innate antiviral response, degradation efficiencies with these chimeras were twice as much as those with only antisense oligonucleotides for both SARS-CoV-2 RNA targets. In pseudovirus infection models, chimera-S4 achieved potent and broad-spectrum inhibition of SARS-CoV-2 and its N501Y and/or ΔH69/ΔV70 mutants, indicating a promising antiviral agent based on the nucleic acid-hydrolysis targeting chimera (NATAC) strategy.

Photoswitchable CAR-T Cell Function In Vitro and In Vivo via a Cleavable Mediator.

Chimeric antigen receptor (CAR)-T-based therapeutics are a breakthrough in cancer treatment; however, they are hampered by constitutive activation, which leads to worrisome side effects. Engineering CAR-T cells to be as tightly controllable as possible remains a topic of ongoing investigation. Here, we report a photoswitchable approach that uses a mediator for the at-will regulation of CAR-T cells. This mediator carries dual folate and fluorescein isothiocyanate moieties tethered by an ortho-nitrobenzyl ester photocleavable linker. CAR-T cells were shown to be highly cytotoxic to targeted cells only in the presence of the mediator and acted in a dose-dependent manner. The toxicity of CAR-T cells can be rapidly terminated by cleavage of the mediator, and the effects of CAR-T cells can be activated again by resupplementation with the mediator without compromising tumor therapy. The approach described here provides a direction for enhancing the controllability of CAR-T cells and can likely be applied in other immunotherapies.

Rhodamine B-based fluorescent probes for molecular mechanism study of the anti-influenza activity of pentacyclic triterpenes.

The antiviral activity of pentacyclic triterpenes has attracted increasing attention. However, the detailed antiviral mechanism remains fully unclear. In the present study, four C28 or C30 modified pentacyclic triterpene probes via conjugating with rhodamine B were designed and synthesized, and their anti-influenza virus activity was evaluated. The results indicated that two compounds 14 and 23 showed significant antiviral activity to influenza A/WSN/33 (H1N1) virus in Madin-Darby canine kidney (MDCK) cells with IC50 values of 8.36 and 8.24 µM, respectively. The mechanism of action studies of representative probe 23 indicated that it could inhibit the membrane fusion by binding with influenza virus hemagglutinin (HA), and the apparent dissociation constant (KD) value for probe 23-HA interaction was successfully evaluated (1.78 × 10-5 M) using surface plasmon resonance spectroscopy. In addition, the subcellular localization of probe 23 in MDCK cells was determined by confocal microscopy and flow cytometry, and the results suggested that fluorescent probe 23 was rapidly taken up in MDCK cells and accumulated in cytoplasm, but no antiviral activity was observed after its entry into cells. The present study further confirmed our previous finding that pentacyclic triterpenes could tightly bind to the viral envelope HA protein, thus blocking the virus entry into host cells.

Emergency decision-making model of environmental emergencies based on case-based reasoning method.

Environmental emergencies are characterized by high uncertainty, complex evolution, and potential for serious damage, thus posing enormous pressure and difficulties to the emergency responses of enterprises and governments. Improving the efficiency and quality of emergency decision-making constitutes the primary focus of today's research in this field. This study systematically analyzes the scenario evolution mechanism of environmental emergencies with a multi-dimensional scenario space method, and key scenario factors are identified from disaster-inducing factors, disaster-bearing factors, disaster-pregnant environments, and emergency actions. Based on these, an emergency decision-making model for environmental emergencies (EEEDM) is constructed based on case-based reasoning (CBR). First, different matching algorithms are designed for accurate numerical data, fuzzy semantic data, and symbolic data. The similarity between the target scenario and the historical scenario is calculated, and the historical scenario similarity set is built according to the given threshold value. Finally, the emergency action plan of the scenario is modified with its utility value evaluated. A solution that applies to the target scenario is then obtained. Additionally, the decision-making model proposed in this paper is validated by an example of environmental emergencies. The results show that this model is scientific and reasonable, and it can better realize the multi-dimensional expression and fast matching of the scenarios and meet the decision requirements of "scenario-response". In practice, the model is capable of providing support for relevant departments' emergency decision-making.

SHYCD induces APE1/Ref-1 subcellular localization to regulate the p53-apoptosis signaling pathway in the prevention and treatment of acute on chronic liver failure.

Background & Aims: San huang yin chi decoction(SHYCD) is derived from the yin chen hao decoction, a well-known and canonical Chinese medicine formula from the "Treatise on Febrile Diseases". Over the past decade, SHYCD has been used to treat and prevent the liver cirrhosis and liver failure. In the present study, we investigated the effects of SHYCD for acute on chronic liver failure(ACLF) and explored its potential mechanism. an ACLF rat model, which induced by carbon tetrachloride (CCl4) combined with D-galactosamine (D-GalN) and lipopolysaccharide(LPS), was used and confirmed by B-ultrasound analysis. Rats were randomly divided into control group, model group, SHYCD-H group, SHYCD-M group, SHYCD-L group, AGNHW group. Compared with the ACLF model group, High, medium, and low doses of SHYCD reduced ALT, AST, TBIL, NH3, IL-1ß, IL-6, and TNFα expression levels in the serum, Shorten PT and INR time,and increased Fbg content in the whole blood, increased survival rate of the rats, improved liver pathological changes. APE1 / Ref-1 was mainly expressed in the nucleus, but the nucleus and cytoplasm were co-expressed after hepatocyte injury. SHYCD significantly downregulated APE1/Ref-1 expression in the cytoplasm. Increased APE1/Ref-1, Bcl-2, reduced p53, caspase-3, Bax, and Cyt-c in the total protein. Base on the results, we conclused that High, medium, and low doses of SHYCD could be applied in prevention and treatment of ACLF, and dose-dependent. The possible mechanism is to promote the APE1 / Ref-1 from the cytoplasm to the nuclear transfer, regulation of p53 apoptosis signal pathway prevention and treatment of ACLF.