Glycan-Imprinted Nanoparticle as Artificial Neutralizing Antibody for Efficient HIV-1 Recognition and Inhibition.

The HIV-1 envelope is a heavily glycosylated class 1 trimeric fusion protein responsible for viral entry into CD4+ immune cells. Developing neutralizing antibodies against the specific envelope glycans is an alternative method for antiviral therapies. This work presents the first-ever development and characterization of artificial neutralizing antibodies using molecular imprinting technology to recognize and bind to the envelope protein of HIV-1. The prepared envelope glycan-imprinted nanoparticles (GINPs) can successfully prevent HIV-1 from infecting target cells by shielding the glycans on the envelope protein. In vitro experiments showed that GINPs have strong affinity toward HIV-1 (Kd = 36.7 ± 2.2 nM) and possess high anti-interference and specificity. GINPs demonstrate broad inhibition activity against both tier 1 and tier 2 HIV-1 strains with a pM-level IC50 and exhibit a significant inhibitory effect on long-term viral replication by more than 95%. The strategy provides a promising method for the inhibition and therapy of HIV-1 infection.

Design of Vif-Derived Peptide Inhibitors with Anti-HIV-1 Activity by Interrupting Vif-CBFß Interaction.

One of the major functions of the accessory protein Vif of human immunodeficiency virus type 1 (HIV-1) is to induce the degradation of APOBEC3 (A3) family proteins by recruiting a Cullin5-ElonginB/C-CBFß E3 ubiquitin ligase complex to facilitate viral replication. Therefore, the interactions between Vif and the E3 complex proteins are promising targets for the development of novel anti-HIV-1 drugs. Here, peptides are designed for the Vif-CBFß interaction based on the sequences of Vif mutants with higher affinity for CBFß screened by a yeast surface display platform. We identified two peptides, VMP-63 and VMP-108, that could reduce the infectivity of HIV-1 produced from A3G-positive cells with IC50 values of 49.4 µM and 55.1 µM, respectively. They protected intracellular A3G from Vif-mediated degradation in HEK293T cells, consequently increasing A3G encapsulation into the progeny virions. The peptides could rapidly enter cells after addition to HEK293T cells and competitively inhibit the binding of Vif to CBFß. Homology modeling analysis demonstrated the binding advantages of VMP-63 and VMP-108 with CBFß over their corresponding wild-type peptides. However, only VMP-108 effectively restricted long-term HIV-1 replication and protected A3 functions in non-permissive T lymphocytes. Our findings suggest that competitive Vif-derived peptides targeting the Vif-CBFß interaction are promising for the development of novel therapeutic strategies for acquired immune deficiency syndrome.

Prokaryote-derived phosphorylated Tau epitope vaccine is immunogenic and non-T-cell activated in the mice model.

Accumulation of phosphorylated Tau protein is a prominent pathological hallmark of Alzheimer's disease (AD). However, current vaccines targeting phosphorylation sites are primarily modified using chemical reactions, which exhibit low efficiency in terms of linking to the vaccine carrier. Despite the identification of over 2000 phosphorylation sites on approximately 20% of E. coli proteins through proteomic studies, it remains unclear whether recombinant Tau proteins expressed in bacteria undergo direct phosphorylation. Additionally, limited information is available regarding the immunogenicity and safety profiles of prokaryotic-derived pTau epitope vaccines. Our study discovered that the prokaryotic system can induce phosphorylation on four residues (T181, T205, S262, and S396) of the full-length Tau protein. Based on this finding, we developed a prokaryotic-modified phosphorylated Tau protein vaccine and immunized wild-type mice, resulting in enhanced immunogenicity and a favorable safety profile.

The XCL1-Mediated DNA Vaccine Targeting Type 1 Conventional Dendritic Cells Combined with Gemcitabine and Anti-PD1 Antibody Induces Potent Antitumor Immunity in a Mouse Lung Cancer Model.

With the advent of cancer immunotherapy, there is a growing interest in vaccine development as a means to activate the cellular immune system against cancer. Despite the promise of DNA vaccines in this regard, their effectiveness is hindered by poor immunogenicity, leading to modest therapeutic outcomes across various cancers. The role of Type 1 conventional dendritic cells (cDC1), capable of cross-presenting vaccine antigens to activate CD8+T cells, emerges as crucial for the antitumor function of DNA vaccines. To address the limitations of DNA vaccines, a promising approach involves targeting antigens to cDC1 through the fusion of XCL1, a ligand specific to the receptor XCR1 on the surface of cDC1. Here, female C57BL/6 mice were selected for tumor inoculation and immunotherapy. Additionally, recognizing the complexity of cancer, this study explored the use of combination therapies, particularly the combination of cDC1-targeted DNA vaccine with the chemotherapy drug Gemcitabine (Gem) and the anti-PD1 antibody in a mouse lung cancer model. The study's findings indicate that fusion antigens with XCL1 effectively enhance both the immunogenicity and antitumor effects of DNA vaccines. Moreover, the combination of the cDC1-targeted DNA vaccine with Gemcitabine and anti-PD1 antibody in the mouse lung cancer model demonstrates an improved antitumor effect, leading to the prolonged survival of mice. In conclusion, this research provides important support for the clinical investigation of cDC1-targeting DNA vaccines in combination with other therapies.

Metformin improved a heterologous prime-boost of dual-targeting cancer vaccines to inhibit tumor growth in a melanoma mouse model.

Therapeutic cancer vaccines, which induce anti-tumor immunity by targeting specific antigens, constitute a promising approach to cancer therapy. Our previous work proposed an optimized heterologous immunization strategy using cancer gene vaccines co-targeting MUC1 and survivin. Administration of a DNA vaccine three times within a week followed by a single recombinant MVA (rMVA) boost was able to efficiently induce anti-tumor immunity and inhibit tumor growth in tumor-bearing mouse models However, the complex immunosuppressive tumor microenvironment always limits infiltration by vaccine-induced T cells. Modifying the immunosuppressive microenvironment of tumors would be a breakthrough in enhancing the therapeutic effects of a cancer vaccine. Recent studies have reported that metformin, a type 2 diabetes drug, may ameliorate the tumor microenvironment, thereby enhancing anti-tumor immunity. Here, we tested whether the combinational therapeutic strategy of cancer vaccines administered with a heterologous prime-boost strategy with metformin enhanced anti-tumor effects in a melanoma mouse model. The results showed that metformin promoted the transition of M2-tumor-associated macrophages (M2-TAM) to M1-TAM, induced more tumor-infiltrating proliferative CD4 and CD8 T cells, and decreased exhausted T cells. This combinational treatment induced anti-tumor immunity from cancer vaccines, ameliorating the tumor microenvironment, showing improved tumor inhibition, and prolonging survival in tumor-bearing mice compared with either a cancer vaccine or metformin alone.

The Recent Progress of Oxygen Reduction Electrocatalysts Used at Fuel Cell Level.

Proton exchange membrane fuel cells (PEMFCs) are gaining significant interest as an attractive substitute for traditional fuel cells, with higher energy density, lower environmental pollution, and better operation efficiency. However, the cathode reaction, i.e., the oxygen reduction reaction (ORR), is widely proved to be inefficient, and therefore an obstacle to the widespread development of PEMFCs. The requirement for affordable highly-efficient ORR catalysts is extremely urgent to be met, especially at fuel cell level. Unfortunately, most previous reports focus on the ORR performance at rotating disk electrodes (RDE) level instead of membrane electrode assembly (MEA) level, making it harder to evaluate ORR catalysts operating under real vehicle conditions. Obviously, it is extremely necessary to develop an in-depth understanding of the structure-activity relationship of highly-efficient ORR catalysts applied at MEA level. In this work, an overview of the latest advances in ORR catalysts is provided with an emphasis on their performance at MEA level, hoping to cover the novel and systemic insights for innovative and efficient ORR catalyst design and applications in PEMFCs.

A postmeiotically bifurcated roadmap of honeybee spermatogenesis marked by phylogenetically restricted genes.

Haploid males of hymenopteran species produce gametes through an abortive meiosis I followed by meiosis II that can either be symmetric or asymmetric in different species. Thus, one spermatocyte could give rise to two spermatids with either equal or unequal amounts of cytoplasm. It is currently unknown what molecular features accompany these postmeiotic sperm cells especially in species with asymmetric meiosis II such as bees. Here we present testis single-cell RNA sequencing datasets from the honeybee (Apis mellifera) drones of 3 and 14 days after emergence (3d and 14d). We show that, while 3d testes exhibit active, ongoing spermatogenesis, 14d testes only have late-stage spermatids. We identify a postmeiotic bifurcation in the transcriptional roadmap during spermatogenesis, with cells progressing toward the annotated spermatids (SPT) and small spermatids (sSPT), respectively. Despite an overall similarity in their transcriptomic profiles, sSPTs express the fewest genes and the least RNA content among all the sperm cell types. Intriguingly, sSPTs exhibit a relatively high expression level for Hymenoptera-restricted genes and a high mutation load, suggesting that the special meiosis II during spermatogenesis in the honeybee is accompanied by phylogenetically young gene activities.

YTHDF2-mediated regulations bifurcate BHPF-induced programmed cell deaths.

N6-methyladenosine (m6A) is a critical regulator in the fate of RNA, but whether and how m6A executes its functions in different tissues remains largely obscure. Here we report downregulation of a crucial m6A reader, YTHDF2, leading to tissue-specific programmed cell deaths (PCDs) upon fluorene-9-bisphenol (BHPF) exposure. Currently, Bisphenol A (BPA) substitutes are widely used in plastic manufacturing. Interrogating eight common BPA substitutes, we detected BHPF in 14% serum samples of pregnant participants. In a zebrafish model, BHPF caused tissue-specific PCDs triggering cardiac and vascular defects. Mechanistically, BHPF-mediated downregulation of YTHDF2 reduced YTHDF2-facilitated translation of m6A-gch1 for cardiomyocyte ferroptosis, and decreased YTHDF2-mediated m6A-sting1 decay for caudal vein plexus (CVP) apoptosis. The two distinct YTHDF2-mediated m6A regulations and context-dependent co-expression patterns of gch1/ythdf2 and tnfrsf1a/ythdf2 contributed to YTHDF2-mediated tissue-specific PCDs, uncovering a new layer of PCD regulation. Since BHPF/YTHDF2-medaited PCD defects were also observed in mammals, BHPF exposure represents a potential health threat.

Cell Membrane-Coated Oncolytic Adenovirus for Targeted Treatment of Glioblastoma.

An oncolytic virus is a promising strategy for glioblastoma (GBM) therapy. However, there are still some challenges such as the blood-brain barrier (BBB) and preexisting immunity for targeted treatment of GBM with an oncolytic virus. In this study, two kinds of cell membrane-coated oncolytic adenoviruses (NCM-Ad and GCM-Ad) were prepared using neural stem cells (NSCs) and GBM cells as sources of membranes, respectively, and were shown to improve the targeted infectivity on GBM cells and avoid the immune clearance of preexisting neutralizing antibodies in vitro and in vivo. Specifically, NCM-Ad showed a strong ability to cross the BBB and target tumor cells in vivo. To improve the cytotoxicity to GBM, a capsid dual-modified oncolytic adenovirus (A4/k37) was also encapsulated, and NCM-A4/k37 showed outstanding tumor targeting and inhibition capacity in an orthotopic xenograft tumor model of GBM upon intravenous administration. This study provides a promising oncolytic virus-based targeted therapeutic strategy for glioma.

Dose-related immunomodulatory effects of recombinant TRAIL in the tumor immune microenvironment.

BACKGROUND: In addition to specifically inducing tumor cell apoptosis, recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has also been reported to influence the cancer immune microenvironment; however, its underlying effects and mechanisms remain unclear. Investigating the immunomodulatory effects and mechanisms of recombinant TRAIL in the tumor microenvironment (TME) may provide an important perspective and facilitate the exploration of novel TRAIL strategies for tumor therapy. METHODS: Immunocompetent mice with different tumors were treated with three doses of recombinant TRAIL, and then the tumors were collected for immunological detection and mechanistic investigation. Methodological approaches include flow cytometry analysis and single-cell sequencing. RESULTS: In an immunocompetent mouse model, recombinant soluble mouse TRAIL (smTRAIL) had dose-related immunomodulatory effects. The optimal dose of smTRAIL (2 mg/kg) activated innate immune cells and CD8+ T cells, whereas higher doses of smTRAIL (8 mg/kg) promoted the formation of a tumor-promoting immune microenvironment to counteract the apoptotic effects on tumor cells. The higher doses of smTRAIL treatment promoted M2-like macrophage recruitment and polarization and increased the production of protumor inflammatory cytokines, such as IL-10, which deepened the suppression of natural killer (NK) cells and CD8+ T cells in the tumor microenvironment. By constructing an HU-HSC-NPG.GM3 humanized immune system mouse model, we further verified the immunomodulatory effects induced by recombinant soluble human TRAIL (shTRAIL) and found that combinational administration of shTRAIL and trabectedin, a macrophage-targeting drug, could remodel the tumor immune microenvironment, further enhance antitumor immunity, and strikingly improve antitumor effects. CONCLUSION: Our results highlight the immunomodulatory role of recombinant TRAIL and suggest promising therapeutic strategies for clinical application.

Periodic Mesoporous Organosilica as a Nanoadjuvant for Subunit Vaccines Elicits Potent Antigen-Specific Germinal Center Responses by Activating Naive B Cells.

Infection diseases such as AIDS and COVID-19 remain challenging in regard to protective vaccine design, while adjuvants are critical for subunit vaccines to induce strong, broad, and durable immune responses against variable pathogens. Here, we demonstrate that periodic mesoporous organosilica (PMO) acts as a multifunctional nanoadjuvant by adsorbing recombinant protein antigens. It can effectively deliver antigens to lymph nodes (LNs), prolong antigen exposure, and rapidly elicit germinal center (GC) responses by directly activating naive B cells via the C-type lectin receptor signaling pathway. In mice, both the gp120 trimer (HIV-1 antigen) and the receptor-binding domain (SARS-CoV-2 antigen) with the PMO nanoadjuvant elicit potent and durable antibodies that neutralize heterologous virus strains. LN immune cells analysis shows that PMO helps to effectively activate the T-follicular helper cells, GC B cells, and memory B cells and eventually develop broad and durable humoral responses. Moreover, the PMO nanoadjuvant elicits a strong cellular immune response and shapes this immune response by eliciting high levels of effector T helper cell cytokines. This study identifies a promising nanoadjuvant for subunit vaccines against multiple pathogens.

Effect of particles size of TiC on oxidation resistance of in-situ TiC/Ni composite.

The oxidation resistance of TiC/Ni composites is crucial for its application in high-temperature oxidation environment. The in-situ TiC/Ni composites are fabricated by reactive sintering method, and the influence of TiC particle size on oxidation resistance of composite is studied. The particle size of TiC increases from 1.54 µm to 2.40 µm as the sintering holding time prolongs from 2 h to 6 h, due to the dissolution-reprecipitation mechanism. The oxidation kinetics of in-situ TiC/Ni composite with different TiC particle size oxidized at 800 °C for 100 h obeys parabolic kinetics. The oxidation mass gain of composite increases from 7.471 mg•cm-2 to 8.454 mg•cm-2, and the oxide scale on composites becomes thicker, as the particle size of TiC increases from 1.54 µm to 2.40 µm. The reduction of TiC particle size facilitates the formation of a dense and continuous oxide scale on composite, helpful to restrict the diffusion of O, Ti and Ni atoms during oxidation. Therefore, the reduction of TiC particle size is contributed to the optimization of oxidation resistance of in-situ TiC/Ni composites.

Prevalence and association of diabetic nephropathy in newly diagnosed Chinese patients with diabetes in the Hebei province: A single-center case-control study.

Diabetes is a chronic disease and has huge pressure on patients and the medical system, especially for patients with diabetic complications, for example, diabetic nephropathy. Diabetic nephropathy is a diabetic complication associated with damage to the kidney. To improve the quality of life of patients with diabetes, it is necessary to understand the factors that are associated with diabetic nephropathy. The objective of the study was to find the prevalence of diabetic nephropathy in newly diagnosed patients with diabetes and to develop the association between clinicopathological parameters and diabetic nephropathy. In a case-control study, demographics, anthropometric, and clinicopathological parameters of a total of 305 newly diagnosed patients with diabetes (the fasting blood glucose ≥ 7.0 mM/L and/or glycosylated hemoglobin ≥ 6.5 mM/L) in Hebei province were included in the analysis. If the urine albumin to creatinine ratio was ≥ 30 (microalbuminuria) then patients were considered diabetic nephropathy. Among enrolled patients, 206 (68%) were males and 99 (32%) were females and they were 46 to 71 years old. Demographic variables and health-related behaviors were the same among patients with diabetes either with nephropathy (case group, n = 135) or patients without nephropathy (control group, n = 170, P > .05 for all). The prevalence of diabetic nephropathy was 44%. Female to male ratio was 1:1.7 in the case group. Patients with diabetic nephropathy had higher body weight (P < .0001), waist circumference (P = .0006), and body mass index (P = .0002) than those of patients without nephropathy. Abnormal urinary globulin (P = .041, odd ratio (OR): 1.1231) was associated with diabetic nephropathy. Aspartate transaminase (P = .0651, OR: 0.8541), alkaline phosphatase (P = .0661, OR: 0.8122), hypertension (P = .0821, OR: 0.8214), and blood urea nitrogen (P = .0842, OR: 0.9411) were not significantly associated with diabetic neuropathy. However, they are near the statistical cutoff value. The prevalence of diabetic nephropathy in newly diagnosed diabetic patients of Hebei province is higher than those of the other provinces. Urinary globulin excretion had a weak association with the presence of nephropathy defined by urinary albumin excretion in patients with diabetes. The presence of other diabetic complications is also an essential parameter for diabetic nephropathy. Males are more susceptible to diabetic nephropathy than females if diabetic (Evidence Level: V; Technical Efficacy: Stage 3).

Molecular Mechanism of Adenovirus Late Protein L4-100K Chaperones the Trimerization of Hexon.

Assembly of the adenovirus capsid protein hexon depends on the assistance of the molecular chaperone L4-100K. However, the chaperone mechanisms remain unclear. In this study, we found that L4-100K was involved in the hexon translation process and could prevent hexon degradation by the proteasome in cotransfected human cells. Two nonadjacent domains, 84-133 and 656-697, at the N-terminal and C-terminal regions of human adenovirus type 5 L4-100K, respectively, were found to be crucial and cooperatively responsible for hexon trimer expression and assembly. These two chaperone-related domains were conserved in the sequence of L4-100K and in the function of hexon assembly across different adenovirus serotypes. Different degrees of cross-activity of hexon trimerization with different serotypes were detected in subgroups B, C, and D, which were proven to be controlled by the interaction between the C-terminal chaperone-related domain of L4-100K and hypervariable regions (HVR) of hexon. Additionally, HVR-chimeric hexon mutants were successfully assembled with the assistance of the 1-697 mutant. Structural analysis of 656-697 by nuclear magnetic resonance and structural prediction of L4-100K using Robetta showed that the two conserved domains are mainly composed of α-helices and are located on the surface of the highly folded core region. Our research provides a more complete understanding of hexon assembly and guidance for the development of hexon-chimeric adenovirus vectors that will be safer, smarter, and more efficient. IMPORTANCE Adenovirus vectors have been widely used in clinical trials of vaccines and gene therapy, although some deficiencies remain. Chimeric modification of the hexon was expected to improve the potency of preexisting immune evasion and targeting, but in many cases, viral packaging is prevented by the inability of the chimeric hexon to assemble correctly. So far, few studies have examined the mechanisms of hexon trimer assembly. Here, we show how the chaperone protein L4-100K contributes to the assembly of the adenovirus capsid protein hexon, and these data will provide a guide for novel adenovirus vector design and development, as we desired.

Epitope-based minigene vaccine targeting fibroblast activation protein α induces specific immune responses and anti-tumor effects in 4 T1 murine breast cancer model.

Fibroblast activation protein (FAPα) is a tumor stromal antigen expressed by cancer-associated fibroblasts (CAFs) in more than 90 % of malignant epithelial carcinomas. FAPα-based immunotherapy has been reported and showed that FAPα-specific immune response can remold immune microenvironment and contribute to tumor regression. Many FAPα-based vaccines have been investigated in preclinical trials, which can elicit strong and durable cytolytic T lymphocytes (CTL) with good safety. However, epitope-based FAPα vaccines are rarely reported. To break tolerance against self-antigens, analogue epitopes with modified peptides at the anchor residues are typically used to improve epitope immunogenicity. To investigate the feasibility of a FAPα epitope-based vaccine for cancer immunotherapy in vivo, we conducted a preclinical study to identify a homologous CTL epitope of human and mouse FAPα and obtained its analogue epitope in BALB/c mice, and explored the anti-tumor activity of their minigene vaccines in 4 T1 tumor-bearing mice. By using in silico epitope prediction tools and immunogenicity assays, immunodominant epitope FAP.291 (YYFSWLTWV) and its analogue epitope FAP.291I9 (YYFSWLTWI) were identified. The FAP.291-based epitope minigene vaccine successfully stimulated CTLs targeting CAFs and exhibited anti-tumor activity in a 4 T1 murine breast cancer model. Furthermore, although the analogue epitope FAP.291I9 enhanced FAP.291-specific immune responses, improvement of anti-tumor immunity effects was not observed. Check of immunosuppressive factors revealed that the high levels of IL-10, IL-13, myeloid-derived suppressor cells and iNOS induced by FAP.291I9 increased, which considered the main cause of the failure of the analogue epitope-based vaccine. Thus, we demonstrated for the first time that the FAP.291 minigene vaccine could induce mouse CTLs and also function as a tumor regression antigen, providing the basis for future studies of FAPα epitope-based vaccines. This study may also be valuable for further improvement of the immunogenicity of analogue epitope vaccines.

Production of Soluble Murine TRAILs in Escherichia coli with Zn2+ Supplementation.

BACKGROUND: Accumulating evidence has demonstrated the immunomodulatory effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in rheumatoid arthritis and the tumor microenvironment, besides its known capacity of specifically inducing the apoptosis of cancer cells. Mice are common available animal models for studying the roles of TRAIL. However, mice express only a single TRAIL receptor (mTRAILR) with an intracellular death domain, in contrast to the two TRAIL receptors (TRAILR1 and TRAILR2) in humans. Moreover, human TRAIL binds weakly to mTRAILR, whereas mouse TRAIL has a high affinity for human TRAIL-Rs. Therefore, we considered that murine TRAIL would be more suitable than human TRAIL for exploring the immunoregulatory effect of TRAIL in immunocompetent mice or when using mouse cells as the target. To our knowledge, the detailed method for the production of recombinant murine TRAIL has not been reported. OBJECTIVE: In this study, we aimed to design and express two soluble forms of murine TRAIL and verify the properties of the protein. METHODS: Recombinant murine TRAILs were expressed in Escherichia coli BL21 (DE3, and Nichelating affinity chromatography was used for protein purification. SDS-PAGE, GDS-PAGE and HPLC were applied to analyze the protein structure. The cytotoxicity of our purified murine TRAILs was evaluated in the TRAIL-sensitive human breast cancer ZR-75-30 cells and murine breast cancer 4T1 cells. Finally, validation of the tumor-killing ability of the murine protein in vivo. RESULTS: Two soluble forms of murine TRAILs (mT_N99 and mT_N188) were purified and demonstrated with high purity and trimeric structure. In addition, Zn2+ supplement was essential to produce soluble murine TRAILs in E.coli BL21 (DE3). The two purified soluble mTRAILs showed similar cytotoxicity to cancer cells, moreover, mT_N99 also showed a good anti-tumor effect in vivo and is more suitable for the treatment of murine tumor models. CONCLUSION: A production approach for recombinant murine TRAIL was determined, which covered the design of shortened forms, expression, purification and characterization.

CAFs/tumor cells co-targeting DNA vaccine in combination with low-dose gemcitabine for the treatment of Panc02 murine pancreatic cancer.

In this study, we investigate the synergistic effect of gemcitabine (Gem) and a novel DNA vaccine in the treatment of pancreatic cancer in mice and explore the anti-tumor mechanism of this combination therapy. Fibroblast activation protein α-expressing cancer-associated fibroblasts (FAPα+ CAFs), a dominant component of the tumor microenvironment (TME), have been shown to modulate the extracellular matrix (ECM) to promote the growth, invasion, and metastasis of pancreatic cancer (PC). Therefore, FAPα+ CAFs may be an ideal target for the treatment of PC. However, treatments that solely target FAPα+ CAFs do not directly affect tumor cells. We recently constructed a novel chimeric DNA vaccine (OsFS) against human FAPα and survivin, which simultaneously targets FAPα+ CAFs and tumor cells. In Panc02 tumor-bearing mice, OsFS vaccination not only reduced the proportion of immunosuppressive cells but also promoted the recruitment of tumor-infiltrating lymphocytes, which remodeled the TME to support anti-tumor immune responses. Furthermore, after depletion of regulatory T cells (Tregs) by metronomic low-dose Gem therapy, the anti-tumor effects of OsFS were enhanced. Taken together, our results indicate that the combination of the FAPα/survivin co-targeting DNA vaccine and low-dose Gem may be an effective therapy for PC.

Dominant Negative Mutants of Human Immunodeficiency Virus Type 1 Viral Infectivity Factor (Vif) Disrupt Core-Binding Factor Beta-Vif Interaction.

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFß) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFß to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFß affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFß target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFß to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFß interaction may be promising in the development of a new AIDS therapeutic strategy.

Hyperphosphorylated tau mediates neuronal death by inducing necroptosis and inflammation in Alzheimer's disease.

BACKGROUND: Progressive neuronal death is the key pathological feature of Alzheimer's disease (AD). However, the molecular mechanisms underlying the neuronal death in AD patients have not been fully elucidated. Necroptosis reportedly activates and induces neuronal death in patients with Alzheimer's disease (AD); however, the main mediators and mechanisms underlying necroptosis induction in AD remain elusive. METHODS: The function of hyperphosphorylated tau (pTau) in inducing necroptosis in neuronal cell was examined using Western blotting, RT-PCR and flow cytometry. Tau-induced inflammation was identified via RNA sequencing and transwell assay. Pharmacological methods and CRISPR-Cas9 technology were used to verify the role of necrosome proteins in pTau-stimulated neuronal death and inflammation. TauP301S model mice were treated with Nec-1 s to evaluate the role of necroptosis in tau pathology. RESULTS: Hyperphosphorylated tau could induce necroptosis in neuronal cells by promoting the formation of the RIPK1/RIPK3/MLKL necrosome. In addition, pTau significantly stimulated cell-autonomous overexpression of cytokines and chemokines via the intracellular nuclear factor kappa B (NF-κB) signaling pathway. Importantly, the RIPK1/RIPK3/MLKL axis was essential for the pTau-mediated NF-κB activation and cytokine storm. Furthermore, necroptosis stimulation, NF-κB activation, and cytokine induction have been detected in TauP301S mice and blocking necroptosis markedly ameliorated behavioral defects and excessive neuroinflammation in AD mice. CONCLUSIONS: Our study, for the first time, revealed that pTau contributes to neuronal death by inducing necroptosis and inflammation, mediated by activating the RIPK1/RIPK3/MLKL and NF-κB pathways, thereby delineating the hierarchical molecular network of neuronal necroptosis induction in AD.

ZEB2/TWIST1/PRMT5/NuRD Multicomplex Contributes to the Epigenetic Regulation of EMT and Metastasis in Colorectal Carcinoma.

(1) Background: The EMT plays a crucial role in tumor metastasis, which is the major cause for colorectal carcinoma-related mortality. However, the underlying regulators and mechanisms of EMT in CRC metastasis are still poorly understood; (2) Methods: The transcriptional regulators of EMT in CRC and their functions were examined using RT2212PCR, Western blotting, and luciferase reporter assay. The components of ZEB2/TWIST1 complex and their mutual interactions were identified via affinity purification, mass spectrometry, co-immunoprecipitation, and pull-down experiments. The functional mechanisms of ZEB2/TWIST1/PRMT5/NuRD axis were determined by chromatin immunoprecipitation and luciferase reporter assay. The contribution of ZEB2/TWIST1/PRMT5/NuRD complex in the CRC metastasis was investigated using wound healing, transwell assay, and in vivo xenograft mouse model; (3) Results: We found that ZEB2 and TWIST1 were both significantly upregulated in CRC tissues and EMT of CRC cells. ZEB2 could recruit TWIST1 to the E-cadherin promoter and synergistically repressed its transcription. In addition, ZEB2 physically interacted with TWIST1, PRMT5, and the nucleosome remodeling and deacetylase (NuRD) complex to form a novel repressive multicomplex, leading to epigenetic silencing of E-cadherin in CRC cells. Notably, the combined inhibition of ZEB2 and TWIST1 and epigenetic inhibition markedly reduced CRC metastasis in mice; (4) Conclusions: We revealed for the first time that ZEB2 could recruit TWIST1, PRMT5, and NuRD to form a repressive multicomplex and epigenetically suppresses the transcription of E-cadherin, thereby inducing the EMT process and metastasis in CRC. Our results also confirmed the therapeutic potential of epigenetic inhibitors in CRC.