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Objective: Blood lipid levels play a critical role in the progression of atherosclerosis. However, even with adequate lipid reduction, significant residual cardiovascular risk remains. Therefore, it is necessary to seek novel therapeutic strategies for atherosclerosis that can not only lower lipid levels but also inhibit inflammation simultaneously. Methods: The fusion protein FD03-IL-1Ra was designed by linking the Angiopoietin-like 3 (ANGPTL3) nanobody and human interleukin-1 receptor antagonist (IL-1Ra) sequences to a mutated human immunoglobulin gamma 1 (IgG1) Fc. This construct was transfected into HEK293 cells for expression. The purity and thermal stability of the fusion protein were assessed using SDS-PAGE, SEC-HPLC, and differential scanning calorimetry. Binding affinities of the fusion protein to ANGPTL3 and IL-1 receptor were measured using Biacore T200. The biological activity of the fusion protein was validated through in vitro experiments. The therapeutic efficacy of the fusion protein was evaluated in an ApoE-/- mouse model of atherosclerosis, including serum lipid level determination, histological analysis of aorta and aortic sinus sections, and detection of inflammatory and oxidative stress markers. ImageJ software was utilized for quantitative image analysis. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc test. Results: The FD03-IL-1Ra fusion protein was successfully expressed, with no polymer formation detected, and it demonstrated good thermal and conformational stability. High affinity for both murine and human ANGPTL3 was exhibited by FD03-IL-1Ra, and it was able to antagonize hANGPTL3's inhibition of LPL activity. FD03-IL-1Ra also showed high affinity for both murine and human IL-1R, inhibiting IL-6 expression in A549 cells induced by IL-1β stimulation, as well as suppressing IL-1β-induced activity inhibition in A375.S2 cells. Our study revealed that the fusion protein effectively lowered serum lipid levels and alleviated inflammatory responses in mice. Furthermore, the fusion protein enhanced plaque stability by increasing collagen content within atherosclerotic plaques. Conclusions: These findings highlighted the potential of bifunctional interleukin-1 receptor antagonist and ANGPTL3 antibody fusion proteins for ameliorating the progression of atherosclerosis, presenting a promising novel therapeutic approach targeting both inflammation and lipid levels. [ABSTRACT FROM AUTHOR]

Cervical cancer is the fourth most common cause of cancer and the fourth most common cause of cancer deaths in women. Some reports have shown the effect of Streptococcus agalactiae proteins and capsule products against cancer cell lines. This study aimed to design and produce a fusion of recombinant protein (containing the capsules of Streptococcus agalactiaes with a linker and Antti cancer sequences) CpsA-CpsC-L-ACAN in the plasmid pET-22b (+) vector. Construct pET-22b (+) was designed by researchers, optimized with bioinformatics software, and synthesized by a Biometrics company. For the confirmation of recombinant protein, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed. Antibacterial activity was performed according to protocol CLSI2020. Based on bioinformatics analysis, the recombinant protein had good spatial structure and stability and half-life. Iterative Threading ASSEmbly Refinement (I-TASSER) results predicted a surfing topology with C-score values (−3.60). The results of Gastro-Oesophageal Reflux 4 (GOR4) analysis showed a little extended strand (30.63%), a random coil with a percentage (50.18%), and an alpha helix with a percentage (19.19%). The recombinant protein was confirmed by SDS-PAGE and western blotting with Anti-his tag. The purpose of peptide fusion design in this article is to help in the development of anti-cervical cancer medicine. Of course, this issue needs to be investigated in animal and human phases. In this article, only bioinformatics investigations and validation of the results in the laboratory have been discussed. [ABSTRACT FROM AUTHOR]

DNA Fragmentation Factor (DFF) is a heterodimer protein involved in DNA fragmentation during apoptosis, which acts as a trigger downstream of caspase-3 activation. DFF40 catalytically active homo-oligomers break down chromosomal DNA. Previous scientific investigations have revealed a link between DFF40 expression changes and various cancers. DFF40 deletion or down-regulation has been observed in some cancers. Consequently, therapeutic strategies involving the DFF40 molecule compensating led to an increased rate of cancer cell apoptosis. In this review article, we aimed to introduce cancers with low expression of this protein first. The second part of this paper focuses on studies that utilized exogenous DFF40 protein produced by recombinant DNA technology and surveyed during in vitro and in vivo tests. Finally, compensation for diminished expression of the mentioned protein via gene therapy-based techniques to make up for this apoptotic molecule's low expression is the topic of the last part of this review article. [ABSTRACT FROM AUTHOR]

Hippocampal neurons can be the first to be impaired with neurodegenerative disorders, including Alzheimer's disease (AD). Most drug candidates for causal therapy of AD cannot either enter the brain or accumulate around hippocampal neurons. Here, we genetically engineered insulin-fusion proteins, called hippocampal neuron-targeting (Ht) proteins, for targeting protein drugs to hippocampal neurons because insulin tends to accumulate in the neuronal cell layers of the hippocampus. In vitro examinations clarified that insulin and Ht proteins were internalized into the cultured hippocampal neurons through insulin receptor-mediated macropinocytosis. Cysteines were key determinants of the delivery of Ht proteins to hippocampal neurons, and insulin B chain mutant was most potent in delivering cargo proteins. In vivo accumulation of Ht proteins to hippocampal neuronal layers occurred after intracerebroventricular administration. Thus, hippocampal neuron-targeting technology can provide great help for developing protein drugs against neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Background: Nipah virus is a deadly infectious virus that was first isolated and identified from Malaysia. Since then, a number of Nipah virus outbreaks have been reported from Bangladesh and India. Transmission of the disease occurs through Pteropus genus fruit bats. The case fatality rate of this infection is very high when compared with other viral zoonoses. At present, there are no approved vaccines or drugs available to prevent or treat this infection. A number of studies are ongoing to develop an efficient vaccine candidate to combat this deadly virus. The majority of them concentrate on the structural and non-structural proteins, which are the main targets of neutralizing antibodies. Materials and Methods: Here, we analyzed the genome sequence identity of two Nipah virus strains, Indian and Malaysian, and also the amino acid identity between the two structural proteins (Attachment glycoprotein G and Fusion protein F) and one non-structural protein (W protein) of those two strains. Results and Discussion: It was found that there is a considerable amount of nucleotide sequence homology between the initial strain that originated from Malaysia and the strain that is now found in India. Furthermore, the Structural and Non-structural proteins of these two strains exhibit a high degree of similarity. Conclusion: Hence, a vaccine candidate designed using either NiV M or NiV B can be effectively used as a potent vaccine. [ABSTRACT FROM AUTHOR]

Background: Variations in the fusion (F) protein of respiratory syncytial virus (RSV) with main antigenic sites I–V and Ø may affect the development of RSV vaccines and therapies. Methods: In the study, 30 respiratory specimens positive for RSV were randomly selected from children with acute lower respiratory infections (ALRI) in Beijing every year from 2012 to 2021 for F gene sequencing. Then, 300 F gene sequences and 508 uploaded to GenBank from China were subjected to phylogenetic analysis. Results: The results indicated the nucleotide identities were 95.4–100% among 446 sequences of RSV A, and 96.3–100% among 362 of RSV B. The most common variant loci were N80K (100.00%) and R213S (97.76%) for site Ø, and V384I/T (98.43%) for site I among sequences of RSV A, and M152I (100.00%), I185V (100.00%), and L172Q/H (94.48%) for site V, and R202Q (99.45%) for site Ø among sequences of RSV B. N276S appears in 95.29% sequences of RSV A, while S276N and N262 I/S appear in 1.38% and 0.55% sequences of RSV B, respectively. No variation was found in all sequences at the binding sites of 14N4 and motavizumab. Conclusions: There were cumulative variations of the RSV F gene, especially at some binding sites of antigenic sites. [ABSTRACT FROM AUTHOR]

Cryo-electron tomography enables capture of 3D images of membrane fusion intermediate states with resolution of fusion proteins and membrane leaflet remodeling. Studies of class I, II, and III viral membrane fusion proteins are revealing general features as well as differences in stages of membrane reorganization, leading to complete fusion and pore formation. Viral fusion machinery is comprised of fusion proteins, viral membrane, and internal structural components such as matrix proteins that regulate fusion protein conformation and function. Tightly docked membrane contact zones with closely apposed proximal headgroup layers appear to be a near universal intermediate state along the fusion pathway. Computational modeling that builds upon observed structural information from cryo-electron tomography may offer a path towards a detailed mechanistic understanding of the forces and energetics that drive protein-mediated membrane fusion. Protein-mediated membrane fusion is the dynamic process where specialized protein machinery undergoes dramatic conformational changes that drive two membrane bilayers together, leading to lipid mixing and opening of a fusion pore between previously separate membrane-bound compartments. Membrane fusion is an essential stage of enveloped virus entry that results in viral genome delivery into host cells. Recent studies applying cryo-electron microscopy techniques in a time-resolved fashion provide unprecedented glimpses into the interaction of viral fusion proteins and membranes, revealing fusion intermediate states from the initiation of fusion to release of the viral genome. In combination with complementary structural, biophysical, and computation modeling approaches, these advances are shedding new light on the mechanics and dynamics of protein-mediated membrane fusion. [ABSTRACT FROM AUTHOR]

Fusing insect derived neurotoxic peptides with Galanthus nivalis agglutinin (GNA) has been shown to enhance the insecticidal activity of the neuropeptides, especially when administered orally. This study produced a recombinant scorpion insect specific neurotoxin BjαIT, GNA, and a fusion protein BjαIT/GNA using Pichia pastoris as an expression host. Recombinant rBjαIT/GNA was found to be easily degraded during expression in yeast which and produced a main protein product with a molecular weight of approximately 14 kDa. Cytotoxicity results showed that rBjαIT, rGNA, and rBjαIT/GNA had no toxicity to mammalian NIH/3T3 cells. Adding rBjαIT or rBjαIT/GNA at a concentration as low as 1 ng/mL to insect cell culture medium inhibited the proliferation of insect Sf9 cells, with rBjαIT exhibiting stronger cytotoxicity, while 20 ng/mL rGNA did not inhibit the proliferation of Sf9 cells. Silkworm larval injection results showed that rBjαIT/GNA was the most toxic of the three proteins, followed by rBjαIT, and rGNA. When rBjαIT/GNA was injected at a concentration of 0.129 nmol/g body weight 46.7% of silkworm died within 48 h. Feeding newly hatched silkworms with rBjαIT/GNA at a leaf surface concentration of 40 µg/cm2 resulted in 76.7% mortality within 24 h. However, rBjαIT/GNA showed almost no oral insecticidal activity against second instar silkworms. The results indicated that rBjαIT/GNA has stronger injection insecticidal activity and feeding insecticidal activity than rBjαIT and rGNA individually, making it more suitable for biological control. [ABSTRACT FROM AUTHOR]

The search for novel therapeutic agents to treat breast cancer has compelled the development of fusion proteins that synergize the functional benefits of different bioactive peptides. Leptulipin, derived from scorpion venom, exhibits antitumor properties. On the other hand, p28, a peptide from the bacterial protein azurin, enhances cell penetration. The current study investigated the design and computational evaluation of a Leptulipin-p28 fusion protein for breast cancer treatment. The amino acid sequences of Leptulipin and p28 were joined via a rigid linker to maintain structural and functional integrity. Secondary and tertiary structure predictions were performed using online servers of GOR-IV and I-TASSER. Physicochemical properties and solubility were analyzed using ProtParam and Protein-Sol. Validation and quality assessment of the fusion protein were confirmed through Rampage and ERRAT2. Finally, the fusion protein was docked with 2 receptors (VEGFR and Cadherin) and docked complexes were simulated on GROMACS. The Leptulipin-p28 fusion protein exhibited a stable structure exhibiting a high quality score of 92 on ERRAT and Ramachandran plot analysis highlighting 76.3% of residues in the favorable region. Docking studies with VEGFR and Cadherin receptors followed by 100 ns simulations on GROMACS showed stable complex formation. Molecular dynamics simulations confirmed the stability and robust interaction of the fusion protein-receptor complexes over time. The computational analysis indicates that the Leptulipin-p28 fusion protein holds promise as a multitarget therapeutic agent in breast cancer. The current findings warrant further investigation through in vitro and in vivo studies to validate the current outcomes. [ABSTRACT FROM AUTHOR]

The development of immune cell engagers (ICEs) can be limited by logistical and functional restrictions associated with fusion protein designs, thus limiting immune cell recruitment to solid tumors. Herein, a high affinity superantigen‐based multivalent ICE is developed for simultaneous activation and recruitment of NK and T cells for tumor treatment. Yeast library‐based directed evolution is adopted to identify superantigen variants possessing enhanced binding affinity to immunoreceptors expressed on human T cells and NK cells. High‐affinity superantigens exhibiting improved immune‐stimulatory activities are then incorporated into a superantigen‐based tri‐functional yeast‐display‐enhanced multivalent immune cell engager (STYMIE), which is functionalized with a nanobody, a Neo‐2/15 cytokine, and an Fc domain for tumor targeting, immune stimulation, and prolonged circulation, respectively. Intravenous administration of STYMIE enhances NK and T cell recruitment into solid tumors, leading to enhanced inhibition in multiple tumor models. The study offers design principles for multifunctional ICEs. [ABSTRACT FROM AUTHOR]

Introducing genetic material into hard-to-transfect mammalian cell lines and primary cells is often best achieved through retroviral infection. An ideal retroviral vector should offer a compact, selectable, and screenable marker while maximizing transgene delivery capacity. However, a previously published retroviral vector featuring an EGFP/Puromycin fusion protein failed to meet these criteria in our experiments. We encountered issues such as low infection efficiency, weak EGFP fluorescence, and selection against infected cells. To address these shortcomings, we developed a novel retroviral vector based on the Moloney murine leukemia virus. This vector includes a compact bifunctional EGFP and Puromycin resistance cassette connected by a 2A peptide. Our extensively tested vector demonstrated superior EGFP expression, efficient Puromycin selection, and no growth penalty in infected cells compared with the earlier design. These benefits were consistent across multiple mammalian cell types, underscoring the versatility of our vector. In summary, our enhanced retroviral vector offers a robust solution for efficient infection, reliable detection, and effective selection in mammalian cells. Its improved performance and compact design make it an ideal choice for a wide range of applications involving precise genetic manipulation and characterization in cell-based studies. [ABSTRACT FROM AUTHOR]

Chimeric RNAs, distinct from DNA gene fusions, have emerged as promising therapeutic targets with diverse functions in cancer treatment. However, the functional significance and therapeutic potential of most chimeric RNAs remain unclear. Here we identify a novel fusion transcript of solute carrier family 2‐member 11 (SLC2A11) and macrophage migration inhibitory factor (MIF). In this study, we investigated the upregulation of SLC2A11–MIF in The Cancer Genome Atlas cohort and a cohort of patients from Sun Yat‐Sen Memorial Hospital. Subsequently, functional investigations demonstrated that SLC2A11–MIF enhanced the proliferation, antiapoptotic effects, and metastasis of bladder cancer cells in vitro and in vivo. Mechanistically, the fusion protein encoded by SLC2A11–MIF interacted with polypyrimidine tract binding protein 1 (PTBP1) and regulated the mRNA half‐lives of Polo Like Kinase 1, Roundabout guidance receptor 1, and phosphoinositide‐3‐kinase regulatory subunit 3 in BCa cells. Moreover, PTBP1 knockdown abolished the enhanced impact of SLC2A11–MIF on biological function and mRNA stability. Furthermore, the expression of SLC2A11–MIF mRNA is regulated by CCCTC‐binding factor and stabilized through RNA N4‐acetylcytidine modification facilitated by N‐acetyltransferase 10. Overall, our findings revealed a significant fusion protein orchestrated by the SLC2A11–MIF–PTBP1 axis that governs mRNA stability during the multistep progression of bladder cancer. [ABSTRACT FROM AUTHOR]

PURPOSE: Evaluate the safety and efficacy of efbemalenograstim alfa for reducing the risk of febrile neutropenia in breast cancer patients undergoing myelosuppressive chemotherapy. METHODS: A phase III, randomized, double-blind, placebo-controlled study was conducted. A total of 122 subjects received up to 4 cycles of TA chemotherapy (75 mg/m2 docetaxel + 60 mg/m2 doxorubicin). Patients were randomized in a 2:1 ratio to subcutaneously inject a single 20 mg of efbemalenograstim alfa or placebo on day 2 of cycle 1, and all subjects received efbemalenograstim alfa on day 2 of cycles 2, 3, and 4. Duration of severe (grade 4) neutropenia (DSN), depth of neutrophil nadir, incidence of febrile neutropenia (FN), time to neutrophil recovery, and safety information were recorded. RESULTS: For the primary endpoint, the mean DSN in cycle 1 was 1.3 days and 3.9 days for efbemalenograstim alfa and placebo respectively (95% CI, 2.3, 3.4). As the lower bound of the 95% CI was > 0, superiority of efbemalenograstim alfa over placebo can be declared. In addition, the incidence of FN in Cycle 1 was lower in efbemalenograstim alfa group than in placebo group (4.8% vs. 25.6%; p = 0.0016). Patients in the efbemalenograstim alfa group required less intravenous antibiotics (3.6% vs. 17.9%; p = 0.0119). Most adverse events were consistent with those expected for breast cancer patient receiving TA chemotherapy. CONCLUSION: Efbemalenograstim alfa is effective and safe for significantly decreasing the duration of severe neutropenia and the incidence of febrile neutropenia in breast cancer patients who are receiving TA chemotherapy. TRIAL REGISTRATION: NCT02872103, August 19, 2016.

Background: Variations in the fusion (F) protein of respiratory syncytial virus (RSV) with main antigenic sites I–V and Ø may affect the development of RSV vaccines and therapies. Methods: In the study, 30 respiratory specimens positive for RSV were randomly selected from children with acute lower respiratory infections (ALRI) in Beijing every year from 2012 to 2021 for F gene sequencing. Then, 300 F gene sequences and 508 uploaded to GenBank from China were subjected to phylogenetic analysis. Results: The results indicated the nucleotide identities were 95.4–100% among 446 sequences of RSV A, and 96.3–100% among 362 of RSV B. The most common variant loci were N80K (100.00%) and R213S (97.76%) for site Ø, and V384I/T (98.43%) for site I among sequences of RSV A, and M152I (100.00%), I185V (100.00%), and L172Q/H (94.48%) for site V, and R202Q (99.45%) for site Ø among sequences of RSV B. N276S appears in 95.29% sequences of RSV A, while S276N and N262 I/S appear in 1.38% and 0.55% sequences of RSV B, respectively. No variation was found in all sequences at the binding sites of 14N4 and motavizumab. Conclusions: There were cumulative variations of the RSV F gene, especially at some binding sites of antigenic sites.

Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins. We delineate a novel assay protocol in miniaturized format to quantify available biotin binding sites based on the affinity of the anionic dye 4′-hydroxyazobenzene-2-carboxylic acid for biotin binding sites within avidin. We apply this assay as a quality control assay to evaluate the number of available biotin binding sites in different fusion protein production batches. This method offers a streamlined alternative to fluorescence-based assays commonly employed to assess biotin binding, is less time-consuming than other methods and is applicable to diverse fusion proteins.

Recombinant human growth hormone (rhGH) and GH receptor antagonists (GHAs) are used clinically to treat a range of disorders associated with GH deficiency or hypersecretion, respectively. However, these biotherapeutics can be difficult and expensive to manufacture with multiple challenges from recombinant protein generation through to the development of long-acting formulations required to improve the circulating half-life of the drug. In this review, we summarize methodologies and approaches used for making and purifying recombinant GH and GHA proteins, and strategies to improve pharmacokinetic and pharmacodynamic properties, including PEGylation and fusion proteins. Therapeutics that are in clinical use or are currently under development are also discussed.

Abstract The primary challenge to improving existing cancer treatment is to develop drugs that specifically target tumor cell. NGR peptide is tumor homing peptide that selectively target cancer cells while interleukin 15 is a pleiotropic cytokine with anticancer properties. This study computationally engineered a IL15-NGR fusion peptide by linking the homing peptide NGR with the targeting peptide IL-15. After evaluating and validating the chimeric peptide, we docked it to the IL-15Rα/IL-15Rβ/γc heterodimer receptor, examining the interactions and binding energy and lastly, molecular dynamics simulations were performed. The secondary and tertiary structures, along with physicochemical properties of the designed IL-15-NGR chimeric protein, were predicted using GOR IV, trRosetta and ProtParam online servers respectively. The quality and 3D structure validation were confirmed via ProSA-web and SAVES 6.0 analysis which predicted an ERRAT score of 96.72%, with 97.6% of residues in the Ramachandran plot, validating its structure. Finally, Docking, MD simulations and interaction analysis were performed using ClusPro 2.0 and GROMACS and PDBsum, which exhibited significant hydrogen bonding and salt bridges, confirming the formation of a stable docked complex. These results were further corroborated by simulation analysis, which demonstrated a stable and dynamic behavior of the docked complex in a biological environment. The predicted high expression value of fusion protein was 0.844 in E.coli using SOLUPROT tool. These findings suggest efficient expression of the IL15-NGR fusion protein if its gene is inserted into E. coli and indicates its potential as a safe and effective anticancer treatment, paving the way for targeted therapeutic interventions. Graphical abstract

The primary challenge to improving existing cancer treatment is to develop drugs that specifically target tumor cell. NGR peptide is tumor homing peptide that selectively target cancer cells while interleukin 15 is a pleiotropic cytokine with anticancer properties. This study computationally engineered a IL15-NGR fusion peptide by linking the homing peptide NGR with the targeting peptide IL-15. After evaluating and validating the chimeric peptide, we docked it to the IL-15Rα/IL-15Rβ/γc heterodimer receptor, examining the interactions and binding energy and lastly, molecular dynamics simulations were performed. The secondary and tertiary structures, along with physicochemical properties of the designed IL-15-NGR chimeric protein, were predicted using GOR IV, trRosetta and ProtParam online servers respectively. The quality and 3D structure validation were confirmed via ProSA-web and SAVES 6.0 analysis which predicted an ERRAT score of 96.72%, with 97.6% of residues in the Ramachandran plot, validating its structure. Finally, Docking, MD simulations and interaction analysis were performed using ClusPro 2.0 and GROMACS and PDBsum, which exhibited significant hydrogen bonding and salt bridges, confirming the formation of a stable docked complex. These results were further corroborated by simulation analysis, which demonstrated a stable and dynamic behavior of the docked complex in a biological environment. The predicted high expression value of fusion protein was 0.844 in E.coli using SOLUPROT tool. These findings suggest efficient expression of the IL15-NGR fusion protein if its gene is inserted into E. coli and indicates its potential as a safe and effective anticancer treatment, paving the way for targeted therapeutic interventions. [ABSTRACT FROM AUTHOR]

HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated peptide, HxTx-Hv1h/CPP-1838, created by fusing HxTx-Hv1h with CPP-1838. Additionally, we aimed to establish a robust recombinant expression system for HxTx-Hv1h/CPP-1838. We successfully achieved the secretory production of HxTx-Hv1h, its fusion with Galanthus nivalis agglutinin (GNA) forming HxTx-Hv1h/GNA and HxTx-Hv1h/CPP-1838 in yeast. Purified HxTx-Hv1h exhibited contact toxicity against Megoura crassicauda, with a 48 h median lethal concentration (LC50) of 860.5 μg/mL. Fusion with GNA or CPP-1838 significantly enhanced its aphidicidal potency, reducing the LC50 to 683.5 μg/mL and 465.2 μg/mL, respectively. The aphidicidal efficacy was further improved with the addition of surfactant, decreasing the LC50 of HxTx-Hv1h/CPP-1838 to 66.7 μg/mL—over four times lower compared to HxTx-Hv1h alone. Furthermore, we engineered HxTx-Hv1h/CPP-1838 multi-copy expression vectors utilizing the BglBrick assembly method and achieved high-level recombinant production in laboratory-scale fermentation. This study is the first to document a CPP fusion strategy that enhances the transdermal aphidicidal activity of a natural toxin like HxTx-Hv1h and opens up the possibility of exploring the recombinant production of HxTx-Hv1h/CPP-1838 for potential applications. [ABSTRACT FROM AUTHOR]

The coronavirus disease 2019 (COVID-19) pandemic, which caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), lead to a crisis with devastating disasters to global public economy and health. Several studies suggest that the SARS-CoV-2 nucleocapsid protein (N protein) is one of uppermost structural constituents of SARS-CoV-2 and is relatively conserved which could become a specific diagnostic marker. In this study, eight single domain antibodies recognized the N protein specifically which were named pN01–pN08 were screened using human phage display library. According to multiple sequence alignment and molecular docking analyses, the interaction mechanism between antibody and N protein was predicted. ELISA results indicated pN01–pN08 with high affinity to protein N. To improve their efficacy, two fusion proteins were prepared and their affinity was tested. These finding showed that fusion proteins had higher affinity than single domain antibodies and will be used as diagnosis for the pandemic of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Heparinases, including heparinases I–III (HepI, HepII, and HepIII, respectively), are important tools for producing low‐molecular‐weight heparin, an improved anticoagulant. The poor thermostability of heparinases significantly hinders their industrial and laboratory applications. To improve the thermostability of heparinases, we applied a rigid linker (EAAAK)5 (R) and a flexible linker (GGGGS)5 (F) to fuse maltose‐binding protein (MBP) and HepI, HepII, and HepIII from Pedobacter heparinus, replacing the original linker from the plasmid pMAL‐c2X. Compared with their parental fusion protein, MBP‐fused HepIs, HepIIs, and HepIIIs with linkers (EAAAK)5 or (GGGGS)5 all displayed enhanced thermostability (half‐lives at 30°C: 242%–464%). MBP‐fused HepIs and HepIIs exhibited higher specific activity (127%–324%), whereas MBP‐fused HepIIIs displayed activity similar to that of their parental fusion protein. Kinetics analysis revealed that MBP‐fused HepIIs showed a significantly decreased affinity toward heparin with increased Km values (397%–480%) after the linker replacement, whereas the substrate affinity did not change significantly for MBP‐fused HepIs and HepIIIs. Furthermore, it preliminarily appeared that the depolymerization mechanism of these fusion proteins may not change after linker replacement. These findings suggest the superior enzymatic properties of MBP‐fused heparinases with suitable linker designs and their potential for the bioproduction of low‐molecular‐weight heparin. [ABSTRACT FROM AUTHOR]

In previous studies, we developed a novel fusion protein named "melittin‐MIL‐2" which exhibited more anti‐tumor activity. However, it remains unclear whether melittin‐MIL‐2 possesses antitumor immune effect on lung adenocarcinoma. In this study, the immune effect and mechanism of melittin‐MIL‐2 inhibiting the growth and invasion of lung adenocarcinoma will be investigated, in order to provide novel perspectives for the immunotherapy of lung cancer. The results indicated that melittin‐MIL‐2 promoted T cell proliferation, enhanced NK cell cytotoxicity, and boosted IFN‐γ secretion in PBMCs. After melittin‐MIL‐2 stimulation, perforin expression and LAK/NK‐like killing activities of human PBMCs and NK cells were significantly enhanced. Melittin‐MIL‐2 is capable of hampering the development and proliferation of lung adenocarcinoma cell A549. ICAM‐1 and Fas expression in A549 cells exposed to melittin‐MIL‐2 rose significantly. The expression levels of TLR8 and VEGF in A549 cells decreased significantly after melittin‐MIL‐2 stimulation. In vivo, melittin‐MIL‐2 substantially impeded the growth of lung adenocarcinoma and formed an immune‐stimulating microenvironment locally in tumor tissues. In conclusion, the novel fusion protein melittin‐MIL‐2 exhibits strong anti‐tumor immune effect in lung adenocarcinoma cell A549 via activating the LFA‐1/ICAM‐1 and Fas/FasL pathways to enhance cytolytic activity, upregulating the secretion of IFN‐γ and perforin, and boosting LAK/NK‐like killing activities. Immuno‐effector cells and their secreted cytokines can form immune stimulation microenvironment locally in lung adenocarcinoma Lewis mice tissue. [ABSTRACT FROM AUTHOR]

Although functional studies on carbohydrate-binding module (CBM) have been carried out extensively, the role of tandem CBMs in the enzyme containing multiple catalytic domains (CDs) is unclear. Here, we identified a multidomain enzyme (Lc25986) with a novel modular structure from lignocellulolytic bacterial consortium. It consists of a mannanase domain, two CBM65 domains (LcCBM65-1/LcCBM65-2), and an esterase domain. To investigate CBM function and domain interactions, full-length Lc25986 and its variants were constructed and used for enzymatic activity, binding, and bioinformatic analyses. The results showed that LcCBM65-1 and LcCBM65-2 both bind mannan and xyloglucan but not cellulose or β-1,3-1,4-glucan, which differs from the ligand specificity of reported CBM65s. Compared to LcCBM65-2, LcCBM65-1 showed a stronger ligand affinity and a preference for acetylation sites. Both CBM65s stimulated the enzymatic activities of their respective neighboring CDs against acetylated mannan, but did not contribute to the activities of the distal CDs. The time course of mannan hydrolysis indicated that the full-length Lc25986 was more effective in the complete degradation of mixed acetyl/non-acetyl substrates than the mixture of single-CD mutants. When acting on complex substrates, LcCBM65-1 not only improved the enzymatic activity of the mannanase domain, but also directed the esterase domain to the acetylated polysaccharides. LcCBM65-2 adopted a low affinity to reduce interference with the catalysis of the mannanase domain. These results demonstrate the importance of CBMs for the synergism between the two CDs of a multidomain enzyme and suggest that they contribute to the adequate degradation of complex substrates such as plant cell walls. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) remains the primary cause of lower respiratory tract infections, particularly in infants and the elderly. In this study, we employed reverse genetics to generate a chimeric influenza virus expressing neuraminidase‐3F protein conjugate with three repeats of the RSV F protein protective epitope inserted into the NA gene of A/California/7/2009 ca (CA/AA ca), resulting in rFlu/RSV/NA‐3F (hereafter, rFRN3). The expression of NA‐3F protein was confirmed by Western blotting. The morphology and temperature‐sensitive phenotype of rFRN3 were similar to CA/AA ca. Its immunogenicity and protective efficiency were evaluated in BALB/c mice and cotton rats. Intranasal administration of rFRN3 elicited robust humoral, cellular, and to some extent, mucosal immune responses. Compared to controls, rFRN3 protected animals from RSV infection, attenuated lung injury, and reduced viral titers in the nose and lungs post‐RSV challenge. These results demonstrate that rFRN3 can trigger RSV‐specific immune responses and thus exhibits potent protective efficacy. The "dual vaccine" approach of a cold‐adapted influenza vector RSV vaccine will improve the prophylaxis of influenza and RSV infection. rFRN3 thus warrants further clinical investigations as a candidate RSV vaccine. [ABSTRACT FROM AUTHOR]

Rearranged during transfection (RET) rearrangement oncoprotein-mediated Ras/MAPK signaling cascade is constitutively activated in cancers. Here, we demonstrate a unique signal niche. The niche is a ternary complex based on the chimeric RET liquid-liquid phase separation. The complex comprises the rearranged kinase (RET fusion); the adaptor (GRB2), and the effector (SHC1). Together, they orchestrate the Ras/MAPK signal cascade, which is dependent on tyrosine kinase. CCDC6-RET fusion undergoes LLPS requiring its kinase domain and its fusion partner. The CCDC6-RET fusion LLPS promotes the autophosphorylation of RET fusion, with enhanced kinase activity, which is necessary for the formation of the signaling niche. Within the signal niche, the interactions among the constituent components are reinforced, and the signal transduction efficiency is amplified. The specific RET fusion-related signal niche elucidates the mechanism of the constitutive activation of the Ras/MAPK signaling pathway. Beyond just focusing on RET fusion itself, exploration of the ternary complex potentially unveils a promising avenue for devising therapeutic strategies aimed at treating RET fusion-driven diseases. [ABSTRACT FROM AUTHOR]

Interleukin-35 (IL-35) is an important immunosuppressive cytokine that has been shown to play a role in the immune response of various diseases. In this study, we cloned the coding sequence of human IL-35 gene, constructed single subunit expression vectors pXC17.4-p35 and pcDNA3.1 (+)-EBI3, and co-transfected CHO-K1 cells to express IL-35 in vitro. No binding was found between subunits of p35 and EBI3. Knobs-into-Holes (KIH) can solve the problem of heavy chain mismatch of heterologous antibodies. Therefore, expression vectors pXC17. 4-p35-Fch and pcDNA3.1(+)-EBI3-Fck were constructed by fusing KIH structures on the basis of the original sequences to express the recombinant fusion protein of KIH-IL-35. The expression vectors of two subunits were exchanged at the same time to verify the influence of different vectors on the expression level of KIH-IL-35. The analysis of various protein detection methods showed that the correct expression rate of KIH-IL-35 structure was significantly improved. Affinity purification of KIH-IL-35 was performed after large amount of expression, and the binding activity of KIH-IL-35 to glycoprotein 130 (gp130) was detected by ELISA. The results showed that the binding of KIH-IL-35 to gp130 was concentration dependent. The indirect activity of KIH-IL-35 and M1 cells was detected by cell activity assay. Further results showed that the inhibition rate of M1 cells increased in a dose-dependent manner with the concentration of KIH-IL-35. In addition, a method for determining IL-35 activity by activated human peripheral blood mononuclear cells was successfully established. Activated PBMCs increased in a dose-dependent manner with KIH-IL-35 concentration. In summary, this study utilized the KIH-IL-35 model to enhance the expression of recombinant human IL-35 and validated its high activity in vitro, providing new ideas for the study of IL-35 and the recombinant expression of similar heterodimeric cytokines. [ABSTRACT FROM AUTHOR]

Following an interseasonal rise in mainly pediatric respiratory syncytial virus (RSV) cases in Germany in 2021, an exceptionally high number of adult cases was observed in the subsequent respiratory season of 2022/2023. The aim of this study was to compare the clinical presentation of RSV infections in the pre- and post-SARS-CoV-2 pandemic periods. Additionally, the local epidemiology of the RSV fusion protein was analyzed at a molecular genetic and amino acid level. RSV detections in adults peaked in calendar week 1 of 2023, 8 weeks earlier than the earliest peak observed in the three pre-pandemic seasons. Although the median age of the adult patients was not different (66.5 vs. 65 years), subtle differences between both periods regarding comorbidities and the clinical presentation of RSV cases were noted. High rates of comorbidities prevailed; however, significantly lower numbers of patients with a history of lung transplantation (p = 0.009), chronic kidney disease (p = 0.013), and immunosuppression (p = 0.038) were observed in the 2022/2023 season. In contrast, significantly more lower respiratory tract infections (p < 0.001), in particular in the form of pneumonia (p = 0.015) and exacerbations of obstructive lung diseases (p = 0.008), were detected. An ICU admission was noted for 23.7% of all patients throughout the study period. Sequence analysis of the fusion protein gene revealed a close phylogenetic relatedness, regardless of the season of origin. However, especially for RSV-B, an accumulation of amino acid point substitutions was noted, including in antigenic site Ø. The SARS-CoV-2 pandemic had a tremendous impact on the seasonality of RSV, and the introduction of new vaccination and immunization strategies against RSV warrants further epidemiologic studies of this important pathogen. [ABSTRACT FROM AUTHOR]

The significant growth of the global protein drug market, including fusion proteins, emphasizes the crucial role of optimizing amino acid sequences to enhance the productivity and bioefficacy. Among these fusion proteins, RBP-IIIA-IB, comprising retinol-binding protein in conjunction with the albumin domains, IIIA and IB, has displayed efficacy in alleviating liver fibrosis by inhibiting the activation of hepatic stellate cells (HSCs). This study aimed to address the issue of the low productivity in RBP-IIIA-IB. To induce structural changes, the linking sequence, EVDD, between domain IIIA and IB in RBP-IIIA-IB was modified to DGPG, AAAA, and GGPA. Among these, RBP-IIIA-AAAA-IB demonstrated an increase in yield (>4-fold) and a heightened inhibition of HSC activation. Furthermore, we identified amino acid residues that could form disulfide bonds when substituted with cysteine. Through the mutation of N453S-V480S in RBP-IIIA-AAAA-IB, the productivity further increased by over 9-fold, accompanied by an increase in anti-fibrotic activity. Overall, there was a more than 30-fold increase in the fusion protein's yield. These findings demonstrate the effectiveness of modifying linker sequences and introducing extra disulfide bonds to improve both the production yield and biological efficacy of fusion proteins. [ABSTRACT FROM AUTHOR]

Objective: To study the transport activity of bacterial rhodopsins, proteoliposomes with the same orientation of the protein molecules should be obtained. Methods: The genes of fusion proteins containing Exiguobacterium sibiricum proteorhodopsin (ESR) and various N-terminal soluble domains have been constructed. Results and Discussion: Effective synthesis in Escherichia coli cells was observed only in the case of fusion with chaperone Caf1M and maltose-binding protein MBP expressed as precursors with their own signal sequences. The study of the isolated MBP-ESR protein in micelles and proteoliposomes demonstrated formation and decay of the main photocycle intermediates at pH > 8. The photoelectric response of the fusion proteins Caf-ESR and MBP-ESR is comparable in amplitude to the wild-type ESR response, indicating their homogeneous orientation in the proteoliposome membrane. Conclusions: The obtained constructs can be used to create bacterial expression systems for various retinal proteins, ensuring their uniform incorporation into proteoliposomes. [ABSTRACT FROM AUTHOR]

Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galβ1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates. IMPORTANCE All cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease. [ABSTRACT FROM AUTHOR]

ABSTRACT Human parainfluenza virus 3 (HPIV3) infection is driven by the coordinated action of viral surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F). Receptor-engaged HN activates F to insert into the target cell membrane and drive virion-cell membrane fusion. For F to mediate entry, its precursor (F0) must first be cleaved by host proteases. F0 cleavage has been thought to be executed during viral glycoprotein transit through the trans-Golgi network by the ubiquitously expressed furin because F0 proteins of laboratory-adapted viruses contain a furin recognition dibasic cleavage motif RXKR around residue 108. Here, we show that the F proteins of field strains have a different cleavage motif from laboratory-adapted strains and are cleaved by unidentified proteases expressed in only a narrow subset of cell types. We demonstrate that extracellular serine protease inhibitors block HPIV3 F0 cleavage for field strains, suggesting F0 cleavage occurs at the cell surface facilitated by transmembrane proteases. Candidate proteases that may process HPIV3 F in vivo were identified by a genome-wide CRISPRa screen in HEK293/dCas9-VP64 + MPH cells. The lung-expressed extracellular serine proteases TMPRSS2 and TMPRSS13 are both sufficient to cleave HPIV3 F and enable infectious virus release by otherwise non-permissive cells. Our findings support an alternative mechanism of F activation in vivo, reliant on extracellular membrane-bound serine proteases expressed in a narrow subset of cells. The proportion of HPIV3 F proteins cleaved and infectious virus release is determined by host cell expression of requisite proteases, allowing just-in-time activation of F and positioning F cleavage as another key regulator of HPIV3 spread.IMPORTANCEEnveloped viruses cause a wide range of diseases in humans. At the first step of infection, these viruses must fuse their envelope with a cell membrane to initiate infection. This fusion is mediated by viral proteins that require a critical activating cleavage event. It was previously thought that for parainfluenza virus 3, an important cause of respiratory disease and a representative of a group of important pathogens, this cleavage event was mediated by furin in the cell secretory pathways prior to formation of the virions. We show that this is only true for laboratory strain viruses, and that clinical viruses that infect humans utilize extracellular proteases that are only made by a small subset of cells. These results highlight the importance of studying authentic clinical viruses that infect human tissues for understanding natural infection.

Abstract The development of immune cell engagers (ICEs) can be limited by logistical and functional restrictions associated with fusion protein designs, thus limiting immune cell recruitment to solid tumors. Herein, a high affinity superantigen‐based multivalent ICE is developed for simultaneous activation and recruitment of NK and T cells for tumor treatment. Yeast library‐based directed evolution is adopted to identify superantigen variants possessing enhanced binding affinity to immunoreceptors expressed on human T cells and NK cells. High‐affinity superantigens exhibiting improved immune‐stimulatory activities are then incorporated into a superantigen‐based tri‐functional yeast‐display‐enhanced multivalent immune cell engager (STYMIE), which is functionalized with a nanobody, a Neo‐2/15 cytokine, and an Fc domain for tumor targeting, immune stimulation, and prolonged circulation, respectively. Intravenous administration of STYMIE enhances NK and T cell recruitment into solid tumors, leading to enhanced inhibition in multiple tumor models. The study offers design principles for multifunctional ICEs.

Abstract Chimeric RNAs, distinct from DNA gene fusions, have emerged as promising therapeutic targets with diverse functions in cancer treatment. However, the functional significance and therapeutic potential of most chimeric RNAs remain unclear. Here we identify a novel fusion transcript of solute carrier family 2‐member 11 (SLC2A11) and macrophage migration inhibitory factor (MIF). In this study, we investigated the upregulation of SLC2A11–MIF in The Cancer Genome Atlas cohort and a cohort of patients from Sun Yat‐Sen Memorial Hospital. Subsequently, functional investigations demonstrated that SLC2A11–MIF enhanced the proliferation, antiapoptotic effects, and metastasis of bladder cancer cells in vitro and in vivo. Mechanistically, the fusion protein encoded by SLC2A11–MIF interacted with polypyrimidine tract binding protein 1 (PTBP1) and regulated the mRNA half‐lives of Polo Like Kinase 1, Roundabout guidance receptor 1, and phosphoinositide‐3‐kinase regulatory subunit 3 in BCa cells. Moreover, PTBP1 knockdown abolished the enhanced impact of SLC2A11–MIF on biological function and mRNA stability. Furthermore, the expression of SLC2A11–MIF mRNA is regulated by CCCTC‐binding factor and stabilized through RNA N4‐acetylcytidine modification facilitated by N‐acetyltransferase 10. Overall, our findings revealed a significant fusion protein orchestrated by the SLC2A11–MIF–PTBP1 axis that governs mRNA stability during the multistep progression of bladder cancer.

ABSTRACT T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) is an inhibitory receptor expressed by T and natural killer cells. Here, we used TIGIT knockout (KO) mice to demonstrate that mouse TIGIT directly interacts with Candida albicans. Reduced fungal growth and colonization were observed when TIGIT-KO splenocytes were co-cultured with C. albicans compared to the wild type (WT). In a systemic candidiasis model, TIGIT-KO mice exhibited improved survival and reduced body weight loss compared to WT mice. Organ-specific fungal burden assessment revealed significantly lower fungal loads in the kidneys, spleen, and lungs of TIGIT-KO mice. Finally, we show that the agglutinin-like sequence proteins ALS6, ALS7, and ALS9 of C. albicans are ligands for TIGIT and that the absence of these proteins abolishes the TIGIT effect in vivo. Our results identify the significance of TIGIT in modulating host defense against C. albicans and highlight the potential therapeutic implications for C. albicans infections.IMPORTANCEOur results identify the significance of T cell immunoreceptor with immunoglobulin and ITIM domain in modulating host defense against Candida albicans and highlight the potential therapeutic implications for C. albicans infections.

ABSTRACT Infection with respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract disease in young children and older people. Despite intensive efforts over the past few decades, no direct-acting small-molecule agents against RSV are available. Most small-molecule candidates targeting the RSV fusion (F) protein pose a considerable risk of inducing drug-resistant mutations. Here, we explored the in vitro and in vivo virological properties of the K394R variant, a cross-resistant mutant capable of evading multiple RSV fusion inhibitors. Our results demonstrated that the K394R variant is highly fusogenic in vitro and more pathogenic than the parental strain in vivo. The small molecule (2E,2′E)-N,N′-((1R,2S,3S)−3-hydroxycyclohexane-1,2-diyl)bis(3-(2-bromo-4-fluorophenyl) acrylamide) (CL-A3-7), a structurally optimized compound derived from a natural caffeoylquinic acid derivative, substantially reduced in vitro and in vivo infections of both wild-type RSV and the K394R variant. Mechanistically, CL-A3-7 significantly inhibited virus–cell fusion during RSV entry by blocking the interaction between the viral F protein and the cellular insulin-like growth factor 1 receptor (IGF1R). Collectively, these results indicate severe disease risks caused by the K394R variant and reveal a new anti-RSV mechanism to overcome K394R-associated resistance.IMPORTANCERespiratory syncytial virus (RSV) infection is a major public health concern, and many small-molecule candidates targeting the viral fusion (F) protein are associated with a considerable risk of inducing drug-resistant mutations. This study investigated virological features of the K394R variant, a mutant strain conferring resistance to multiple RSV fusion inhibitors. Our results demonstrated that the K394R variant is highly fusogenic in cell cultures and more pathogenic than the parental strain in mice. The small-molecule inhibitor CL-A3-7 substantially reduced in vitro and in vivo infections of both wild-type RSV and the K394R variant by blocking the interaction of viral F protein with its cellular receptor, showing a new mechanism of action for small-molecules to inhibit RSV infection and overcome K394R-associated resistance.

Abstract Rhabdomyosarcoma (RMS), such as other childhood tumors, has witnessed treatment advancements in recent years. However, high-risk patients continue to face poor survival rates, often attributed to the presence of the PAX3/7-FOXO1 fusion proteins, which has been associated with metastasis and treatment resistance. Despite efforts to directly target these chimeric proteins, clinical success remains elusive. In this study, the main aim was to address this challenge by investigating regulators of FOXO1. Specifically, we focused on TRIB3, a potential regulator of the fusion protein in RMS. Our findings revealed a prominent TRIB3 expression in RMS tumors, highlighting its correlation with the presence of fusion protein. By conducting TRIB3 genetic inhibition experiments, we observed an impairment on cell proliferation. Notably, the knockdown of TRIB3 led to a decrease in PAX3-FOXO1 and its target genes at protein level, accompanied by a reduction in the activity of the Akt signaling pathway. Additionally, inducible silencing of TRIB3 significantly delayed tumor growth and improved overall survival in vivo. Based on our analysis, we propose that TRIB3 holds therapeutic potential for treating the most aggressive subtype of RMS. The findings herein reported contribute to our understanding of the underlying molecular mechanisms driving RMS progression and provide novel insights into the potential use of TRIB3 as a therapeutic intervention for high-risk RMS patients.

Neurodegenerative diseases such as Alzheimer’s disease (AD) pose substantial challenges to patients and health-care systems, particularly in countries with aging populations. Immunotherapies, including the marketed antibodies lecanemab (Leqembi®) and donanemab (KisunlaTM), offer promise but face hurdles due to limited delivery across the blood–brain barrier (BBB). This limitation necessitates high doses, resulting in increased costs and a higher risk of side effects. This study explores transferrin receptor (TfR)-binding camelid single-domain antibodies (VHHs) for facilitated brain delivery. We developed and evaluated fusion proteins (FPs) combining VHHs with human IgG Fc domains or single-chain variable fragments (scFvs) of the anti-amyloid-beta (Aβ) antibody 3D6. In vitro assessments showed varying affinities of the FPs for TfR. In vivo evaluations indicated that specific VHH-Fc and VHH-scFv fusions reached significant brain concentrations, emphasizing the importance of optimal TfR binding affinities. The VHH-scFv fusions were further investigated in mouse models with Aβ pathology, showing higher retention compared to wild-type mice without Aβ pathology. Our findings suggest that these novel VHH-based FPs hold potential for therapeutic and diagnostic applications in AD, providing a strategy to overcome BBB limitations and enhance brain targeting of antibody-based treatments. Furthermore, our results suggest that a given bispecific TfR-binding fusion format has a window of “optimal” affinity where parenchymal delivery is adequate, while blood pharmacokinetics aligns with the desired application of the fusion protein.

ABSTRACTHuman metapneumovirus (HMPV) is one of the main pathogens causing severe respiratory infections in children, as a common cause of immunodeficiency-related deaths in children and elderly individuals, the prevalence of HMPV has been showing an increasing trend during the last years. However, no vaccines or effective treatment plans are available currently. In this present, based on candidate proteins highly associated with viral virulence and has promising protective potential, we screened for immunodominant cytotoxic T cells, helper T cells, and Linear B-cell epitopes from the most promising candidate Fusion protein, together with G, SH, M, and M2. All epitopes were predicted to have strong antigenicity by Vaxijen and pose no potential toxicity, allergenicity, or hormonology to human proteins by Toxinpred, Allerpred, and Blast analysis, meanwhile, high conservancy is demanded to cover different subtypes. adjuvants β-defensin II and Pam2Cys was attached with EAAAK linkers to improve vaccine’s efficiency. Then, calculation of physicochemical properties proved the protein vaccine as a product can stably exist in the human body. Besides, we assessed the docking between the vaccine and immune receptors to evaluate its ability to stimulate immune responses, and the dynamic simulation further confirmed that the vaccine can tightly bind with immune receptors, which approved that the construction has the potential to induce strong humoral and cellular immune response. Finally, the vaccine was constructed into a multi-epitope mRNA vaccine, the immune simulations suggest that this is a vaccine candidate for controlling HMPV infection.

• VLP associated RSV-F and hMPV-F immunogens properly display pre-fusion epitopes. • VLP associated immunogens induce better neutralizing responses than soluble ones. • The VLP platform benefits manifest primarily during prime immunization. Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccines have been long overdue. Structure-based vaccine design created a new momentum in the last decade, and the first RSV vaccines have finally been approved in older adults and pregnant individuals. These vaccines are based on recombinant stabilized pre-fusion F glycoproteins administered as soluble proteins. Multimeric antigenic display could markedly improve immunogenicity and should be evaluated in the next generations of vaccines. Here we tested a new virus like particles-based vaccine platform which utilizes the direct fusion of an immunogen of interest to the structural human immunodeficient virus (HIV) protein Gag to increase its surface density and immunogenicity. We compared, in mice, the immunogenicity of RSV-F or hMPV-F based immunogens delivered either as soluble proteins or displayed on the surface of our VLPs. VLP associated F-proteins showed better immunogenicity and induced superior neutralizing responses. Moreover, when combining both VLP associated and soluble immunogens in a heterologous regimen, VLP-associated immunogens provided added benefits when administered as the prime immunization. [ABSTRACT FROM AUTHOR]

Introduction: Respiratory syncytial virus (RSV) fusion (F) protein is essential for facilitating virus entry into host cells, providing a hopeful path for combating viral diseases. However, F protein inhibitors can rapidly select for viral resistance. Thus, discovering new inhibitors of F-protein is necessary to enrich the RSV drug development pipeline. Methods: In this study, we screen 25 bioactive compounds from Chinese herbal medicines that exhibit a strong binding to the RSV-F protein using surface plasmon resonance. Results: After screening, we found emodin could strongly bind to RSV-F protein, and could effectively curb RSV infection. Further investigations certificated that emodin specifically disrupts the attachment and internalization phases of RSV infection by targeting the RSV-F protein. In vivo studies with mice infected with RSV demonstrated that emodin effectively reduces lung pathology. This therapeutic effect is attributed to emodin's capacity to diminish proinflammatory cytokine production and reduce viral load in the lungs. Discussion: In conclusion, our findings provide initial insights into the mechanism by which emodin counters RSV infection via engagement with the RSV-F protein, establishing it as a viable contender for the development of novel therapeutic agents aimed at RSV. [ABSTRACT FROM AUTHOR]

The PAX8/PPARγ rearrangement, producing the PAX8–PPARγ fusion protein (PPFP), is thought to play an essential role in the oncogenesis of thyroid follicular tumors. To identify PPFP-targeted drug candidates and establish an early standard of care for thyroid tumors, we performed ensemble-docking-based compound screening. Specifically, we investigated the pocket structure that should be adopted to search for a promising ligand compound for the PPFP; the position of the ligand-binding pocket on the PPARγ side of the PPFP is similar to that of PPARγ; however, the shape is slightly different between them due to environmental factors. We developed a method for selecting a PPFP structure with a relevant pocket and high prediction accuracy for ligand binding. This method was validated using PPARγ, whose structure and activity values are known for many compounds. Then, we performed docking calculations to the PPFP for 97 drug or drug-like compounds registered in the DrugBank database with a thiazolidine backbone, which is one of the characteristics of ligands that bind well to PPARγ. Furthermore, the binding affinities of promising ligand candidates were estimated more reliably using the molecular mechanics Poisson–Boltzmann surface area method. Thus, we propose promising drug candidates for the PPFP with a thiazolidine backbone. [ABSTRACT FROM AUTHOR]

[Objective]This study is aimed to explore the binding ability of different sources of CBM to beech-xylanan, and to fuse exogenous CBM with high binding ability to the C-terminal and N-terminal of Streptomyces L10904 xylanase(XYN), to explore the effects of exogenous CBM on the enzymatic properties of xylanase.[Method]First, through the substrate adsorption method, the concentration of CBM in the solution before and after adsorption was detected by Coomassie Brilliant blue G250 method, and the substrate binding rate of CBM was calculated. CBM1 and CBM4 with better xylan binding ability were screened. In order to explore the effect of the fusion location of CBM with high substrate binding ability on the enzymatic properties of xylanase, CBM1 and CBM4 were fused with the C-terminal and N-terminal of XYN by flexible binding peptide, and four recombinant enzymes were obtained by expression in Escherichia coli BL21(DE3). They were named CBM1-XYN, XYN-CBM1, CBM4-XYN, and XYN-CBM4.[Result]The binding rates of CBM1 and CBM4 to xylan were 89% and 95%, respectively. The specific activities of XYN, CBM1-XYN, XYN-CBM1, CBM4-XYN and XYN-CBM4 were 32 274.81, 49 342.21, 602.48, 230.42 and 2 362.24 U/mg, respectively, measured at 60℃ and pH 7.0. The specific activities of CBM1-XYN were 1.5 times higher than the specific activities of XYN. The analysis of enzymatic properties showed that CBM1 improved the temperature stability and pH stability of XYN, XYN and CBM1-XYN were incubated at 60℃ for 1 h, and the residual enzyme activity of CBM1-XYN and XYN was 81% and 28%, respectively. In the pH range of 3-11, CBM1-XYN maintained more than 90% enzyme activity after incubation at 4℃ for 12 h.[Conclusion]Streptomyces derived xylanase was heterogeneically expressed in E. coli BL21(DE3), and CBM1 and CBM4 with high substrate binding rates were screened. CBM was successfully fused to XYN by protein fusion technology, and CBM1-XYN with improved enzymatic properties was obtained, which improved the temperature stability, pH tolerance and specific enzyme activity of xylanase. [ABSTRACT FROM AUTHOR]

Rhabdomyosarcoma (RMS), such as other childhood tumors, has witnessed treatment advancements in recent years. However, high-risk patients continue to face poor survival rates, often attributed to the presence of the PAX3/7-FOXO1 fusion proteins, which has been associated with metastasis and treatment resistance. Despite efforts to directly target these chimeric proteins, clinical success remains elusive. In this study, the main aim was to address this challenge by investigating regulators of FOXO1. Specifically, we focused on TRIB3, a potential regulator of the fusion protein in RMS. Our findings revealed a prominent TRIB3 expression in RMS tumors, highlighting its correlation with the presence of fusion protein. By conducting TRIB3 genetic inhibition experiments, we observed an impairment on cell proliferation. Notably, the knockdown of TRIB3 led to a decrease in PAX3-FOXO1 and its target genes at protein level, accompanied by a reduction in the activity of the Akt signaling pathway. Additionally, inducible silencing of TRIB3 significantly delayed tumor growth and improved overall survival in vivo. Based on our analysis, we propose that TRIB3 holds therapeutic potential for treating the most aggressive subtype of RMS. The findings herein reported contribute to our understanding of the underlying molecular mechanisms driving RMS progression and provide novel insights into the potential use of TRIB3 as a therapeutic intervention for high-risk RMS patients. [ABSTRACT FROM AUTHOR]

Background & Objective: Superantigens are bacterial toxins that induce a massive immune response in the host. Superantigen staphylococcal enterotoxin B (SEB) can form a ternary complex with its receptors, MHC class II (MHCII) and TCR, and can be used in tumor-targeting therapy, particularly when cooperating with a specific vector. In this study, SEB was fused to interleukin-13 (IL13), which forms a complex with IL13 receptor α2 (IL13Rα2) overexpressed in glioblastoma multiforme (GBM) cells for therapeutic goals. Methods We designed four fusion proteins based on the arrangement of SEB (N- or Cterminal domain) and provided a flexible inter-domain linker (no or yes), resulting in the formation of SEB-IL13, SEB-L-IL13, IL13-SEB, and IL13-L-SEB, respectively. These fusion proteins were then evaluated for their various physicochemical properties and structural characteristics. Bioinformatics tools were employed to predict, refine, and validate the three-dimensional structure of the fusion proteins. In addition, the fusion proteins were docked with IL13Rα2, MHCII, and TCR receptors through the HADDOCK 2.4 server. The candidate fusion protein was subjected to molecular dynamics simulation. Results: There were differences among the designed fusion proteins. The model with the N-terminal domain of IL13 and containing an inter-domain linker (IL13-LSEB) was stable and had a long half-life. The docking analysis revealed that the IL13-L-SEB fusion protein had a higher binding affinity to the IL13Rα2, MHCII, and TCR receptors. Finally, using molecular dynamics simulation through iMODS, acceptable results were obtained for the IL13-L-SEB docked complexes. Conclusion: The results suggest IL13-L-SEB is a promising novel fusion protein for cancer therapeutic application. [ABSTRACT FROM AUTHOR]

Natural G-protein-coupled receptors (GPCRs) rarely have an additional transmembrane (TM) helix, such as an artificial TM-linker that can unite two class A GPCRs in tandem as a single-polypeptide chain (sc). Here, we report that three groups of TM-linkers exist in the intervening regions of natural GPCR fusions from vertebrates: (1) the original consensus (i.e., consensus 1) and consensus 2~4 (related to GPCR itself or its receptor-interacting proteins); (2) the consensus but GPCR-unrelated ones, 1~7; and (3) the inability to apply 1/2 that show no similarity to any other proteins. In silico analyses indicated that all natural GPCR fusions from Amphibia lack a TM-linker, and reptiles have no GPCR fusions; moreover, in either the GPCR-GPCR fusion or fusion protein of (GPCR monomer) and non-GPCR proteins from vertebrates, excluding tetrapods, i.e., so-called fishes, TM-linkers differ from previously reported mammalian and are avian sequences and are classified as Groups 2 and 3. Thus, previously reported TM-linkers were arranged: Consensus 1 is [T(I/A/P)(A/S)–(L/N)(I/W/L)(I/A/V)GL(L/G)(A/T)(S/L/G)(I/L)] first identified in invertebrate sea anemone Exaiptasia diaphana (LOC110241027) and (330-SPSFLCI–L–SLL-340) identified in a tropical bird Opisthocomus hoazin protein LOC104327099 (XP_009930279.1); GPCR-related consensus 2~4 are, respectively, (371-prlilyavfc fgtatg-386) in the desert woodrat Neotoma lepida A6R68_19462 (OBS78147.1), (363-lsipfcll yiaallgnfi llfvi-385) in Gavia stellate (red-throated loon) LOC104264164 (XP_009819412.1), and (479-ti vvvymivcvi glvgnflvmy viir-504) in a snailfish GPCR (TNN80062.1); In Mammals Neotoma lepida, Aves Erythrura gouldiae, and fishes protein (respectively, OBS83645.1, RLW13346.1 and KPP79779.1), the TM-linkers are Group 2. Here, we categorized, for the first time, natural TM-linkers as rare evolutionary events among all vertebrates. [ABSTRACT FROM AUTHOR]

Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dosedependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression. IMPORTANCE The role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV), a member of the Flaviviridae family and a flavivirus, is known to induce acute encephalitis. Vimentin protein has been identified as a potential receptor for JEV, engaging in interactions with the viral membrane protein. The Fc fragment, an integral constituent of immunoglobulins, plays a crucial role in antigen recognition by dendritic cells (DCs) or phagocytes, leading to subsequent antigen presentation, cytotoxicity, or phagocytosis. In this study, we fused the receptor of JEV vimentin with the Fc fragment of IgG and expressed the resulting vimentin-Fc fusion protein in Escherichia coli. Pulldown experiments demonstrated the binding ability of the vimentin-Fc fusion protein to JEV virion in vitro. Additionally, we conducted inhibition assays at the cellular level, revealing the ability of vimentin-Fc protein suppressing JEV replication, it may be a promising passive immunotherapy agent for JEV. These findings pave the way for potential therapeutic strategies against JEV. [ABSTRACT FROM AUTHOR]

The coronavirus disease 2019 (COVID-19) pandemic, which caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), lead to a crisis with devastating disasters to global public economy and health. Several studies suggest that the SARS-CoV-2 nucleocapsid protein (N protein) is one of uppermost structural constituents of SARS-CoV-2 and is relatively conserved which could become a specific diagnostic marker. In this study, eight single domain antibodies recognized the N protein specifically which were named pN01–pN08 were screened using human phage display library. According to multiple sequence alignment and molecular docking analyses, the interaction mechanism between antibody and N protein was predicted. ELISA results indicated pN01–pN08 with high affinity to protein N. To improve their efficacy, two fusion proteins were prepared and their affinity was tested. These finding showed that fusion proteins had higher affinity than single domain antibodies and will be used as diagnosis for the pandemic of SARS-CoV-2.