Background: Highly pathogenic avian influenza virus (H5N1) is a highly contagious and deadly pathogen among birds, but it can also be transmitted to humans. The surface glycoprotein, hemagglutinin (HA), is the most important antigenic determinant against which immune responses are elicited during infection or following vaccination. The virus neutralization test (VNT) is considered the gold standard method for detecting and measuring antibody levels; however, it requires the use of live virus. An alternative approach for neutralization assays involves the use of pseudoviruses, which are non-replicative particles expressing HA. Objectives: This study aimed to evaluate the suitability of the constructed pseudovirus for assessing virus-neutralizing antibodies. Methods: A pseudovirus harboring H5 HA was constructed using retroviral vectors. Following transfection of HEK293T cells with pcDNA-HA, pLOX, and psPAX plasmids, pseudovirus production and titration were evaluated by measuring P24 protein, and the amount of green fluorescent protein (GFP) expression was detected by flow cytometry. The functionality and specificity of the H5 pseudovirus were assessed by VNT. Results: Chicken sera containing H5 antibodies effectively neutralized the pseudovirus in a titer-dependent manner. The results of the neutralization test and the hemagglutination inhibition (HI) test were consistent. The cross-reactivity analysis of H5 pseudovirus and human influenza viruses with chicken and human sera highlighted the specificity of the H5 pseudoviruses. Conclusions: The VNTs based on pseudoviruses expressing H5 on their surface are recognized as reliable and safe alternatives for detecting neutralizing antibodies to avian influenza viruses in routine biosafety level 2 (BSL-2) laboratories.

A novel in-frame calreticulin mutation induces activation of JAK-STAT signaling and thrombocytosis

CD24-targeted cystine and glucose oxidase cascade catalytic nanosystem triggers disulfidptosis in neuroblastoma

Deciphering letermovir's mode of action and resistance mutation effects.

CXCL17 activates three MAS-related G protein-coupled receptors independently of its conserved C-terminal fragment.

IκBα controls the canonical activation of NFκB. IκBα gain-of-function due to NFKBIA variants affecting the N-terminus of IκBα-especially residues 32 and 36-manifests with combined immunodeficiency. The role of NFKBIA variants affecting other IκBα domains has not been described. Variants in NFKBIA were identified using whole-exome sequencing. IκBα expression has been quantified by flow cytometry and Western blotting. Activation-induced IκBα degradation, NFκB1 activation, and nuclear translocation as well as inflammasome activity were evaluated. The p.Gln228* variant in NFKBIA, identified in a family with a history of arthritis and psoriasis, did not affect induced degradation of IκBα. Its expression in HEK293T cells confirmed its truncating effect and revealed reduced NFκB activation. Similar to transfected HEK293T cells, peripheral blood mononuclear cells from patients harbouring the p.Gln228* variant displayed substantially reduced nuclear levels of NFκB1 p50. The latter findings suggest that the p.Gln228* variant causes a functional insufficiency of NFκB1. Similar to patients with NFκB1 haploinsufficiency, high serum levels of interleukin (IL)-18, as well as enhanced induced apoptosis-associated speck-like protein containing a caspase recruitment domain speck formation and IL-1β secretion in vitro, suggest enhanced inflammasome activation. NFKBIA variants not localising to the signal reception domain can affect IκBα function and consequently restrict the canonical activation of NFκB. In contrast to the phenotype of N-terminal variants, which is dominated by combined immunodeficiency, the here-reported C-terminal deletion of IκBα was identified in patients with autoinflammatory arthritis and psoriasis.

Human CXCL17 binds and activates fish GPR25 orthologs.

While antiretroviral therapy (ART) has transformed HIV-1 into a manageable chronic illness, its long-term affordability and accessibility remain major challenges in resource-limited settings. Additionally, adverse side effects can compromise treatment adherence and effectiveness. These limitations highlight the need for novel, affordable therapeutic alternatives. In this study, we evaluated the anti-HIV-1 activity of Product Nkabinde (PN), a traditional herbal formulation comprising four plant extracts, and Gnidia sericocephala (G. sericocephala), to assess their potential as alternative or complementary therapies. HIV-1 subtype B and subtype C viral stocks were produced by transfecting HEK293T cells with envelope plasmids and an env-deficient HIV-1 backbone vector using polyethylenimine. TZM-bl cells were treated with PN and G. sericocephala extracts, alone or combined with antiretrovirals (AZT, raltegravir, maraviroc, amprenavir), then infected with the viruses. Viral infectivity was measured using the luciferase assay, and results were validated in peripheral blood mononuclear cells (PBMCs) using HIV-1 p24 ELISA. The PN extract exhibited a dose-dependent antiviral effect, with the optimal concentration achieving 93% and 96% inhibition of HIV-1 subtype B and C, respectively, in TZM-bl cells, comparable to AZT. In HIV-1 infected PBMCs, treatment with AZT, PN, or G. sericocephala resulted in a sustained reduction of p24 antigen levels over 11 days compared to untreated controls. While NL4.3 showed partial inhibition (p24 levels >20,000pg/mL), strains CM070P.1, YU2, and CM019P.1.2 exhibited consistently low p24 production levels (<20,000pg/mL), indicating strain-dependent antiviral activity. PN, combined with maraviroc inhibited YU2 replication by 81.3% (p = 0.0361), while combinations with raltegravir and AZT suppressed subtype C strains CM070P.1 and CM019P.1.2 by 98.7% (p = 0.0083) and 99% (p = 0.0428), respectively, compared to either PN or the antiretroviral alone. Gnidia sericocephala combined with AZT inhibited NL4.3 by 80.3% (p = 0.0105), and its combinations with maraviroc, raltegravir, and amprenavir suppressed CM070P.1 replication by 87% (p = 0.0093), 86% (p = 0.0168), and 90% (p = 0.0006), respectively, relative to either test agent alone. Fractional inhibitory concentration index (FICI) analysis indicated no synergistic or antagonistic interactions. Thus, this current data suggests that PN and G. sericocephala possess anti-HIV-1 activity.

BackgroundThe converging biology between enveloped viruses and extracellular vesicles (EVs) has raised interest in the application of engineered EVs as antiviral therapeutics. Following the recent COVID-19 pandemic, EVs engineered with either the ACE2-receptor or Spike-protein have been proposed as strategy to either decoy SARS-CoV-2, or to compete with its cell entry. For generic use as a platform for future pandemic preparedness, a systematic and quantitative comparison of both strategies is required to assess their limitations and benefits across different variants of concern.MethodsHere we generated EVs decorated with either the ACE2-receptor or the Spike-protein of (Wuhan)-SARS-CoV-2 and used single vesicle imaging for in-depth quantitative characterisation. These vesicles were then systematically tested for anti-viral activity across SARS-CoV-2 variants of concern using both, pseudotype and live virus cellular infection models including primary human bronchial and nasal explants.ResultsSpike-protein EVs or ACE2-EVs recovered from transiently transfected HEK293T cells comprised only a small fraction of the EV secretome (5% or 20%, respectively) and were primarily derived from the plasma membrane rather than multivesicular bodies. Redirecting intracellular trafficking of the Spike protein by mutating its transmembrane or subcellular localisation domains did not increase the yields of Spike-EVs. Both types of vesicles inhibited SARS-CoV-2 (D614G) in a dose dependent manner with kinetics and immunohistochemistry consistent with an inhibition at the initial cell entry stage. ACE2-EVs were more potent than Spike-EVs and at least 500–1000 times more potent than soluble antibodies in a pseudotype model. Surprisingly, ACE2-EVs switched from an inhibitory to an enhancer activity for the Omicron BA.1 variant whereas Spike-EVs retained their activity across all variants of concern.ConclusionsWhile our data show that both types of engineered EVs potently inhibit SARS-CoV, the decoy versus competition strategy may result in diverging outcomes when considering viral evolution into new variants of concern. While Spike-EVs retain their competition for receptor binding even against higher affinity viral Spike mutations, the formation of complexes between ACE2-EVs and the virus may not only result in inhibition by decoy. As EVs are actively internalised by cells themselves, they may shuttle the virus into cells, resulting in a productive alternative cell entry route for variants such as Omicron, that diverge from strict plasma membrane protease cleavage to the use of endosomal proteases for release of their genome.

Abstract A zinc-coordinated ATP stimulus-responsive nanostructure was developed for CRISPR gene delivery. The system was based on low-molecular-weight polyethylenimine (PEI1.8k) attached to phenylboronic acid (PBA) and a pyridine zinc chelate. The nanostructure could form a polyplex with p-CRISPR DNA. The aim was for the PBA would bind to the ribose moiety of ATP and become hydrophilic, thus releasing the plasmid cargo. The in vitro findings demonstrated that the combination of a Zn-coordinated pyridine ligand, an ATP-sensitive PBA moiety, and a cationic backbone could act synergistically to condense DNA, enhance cellular internalization, disrupt endosomes effectively, and resist serum protein binding. This delivery platform enabled efficient CRISPR plasmid transfection in HEK 293T cells, even at low plasmid concentrations and under serum conditions ranging from 5% to 30%, outperforming the commonly used PEI25k. We suggest that this novel method has the potential to be an appropriate non-viral vector for CRISPR/Cas9 gene editing in vivo and that it may potentially have therapeutic significance.

Corrigendum: Evaluating SARS-CoV-2 Spike Protein Transfection in HEK-293T Cells for VLP Applications

BackgroundThe treatment of non-small cell lung cancer (NSCLC) patients is correlated with the efficacy of immune checkpoint blockade therapy (ICB) targeting programmed cell death ligand 1 (PD-L1) or its cognate receptor (PD-1) on cancer cells or infiltrating immune cells. Analysis of PD-L1/PD-1 expression in tumor tissue represents a crucial step before PD-L1/PD-1 blocker usage.MethodsWe used directed evolution of protein variants derived from a 13 kDa Myomedin loop-type combinatorial library with 12 randomized amino acid residues to select high-affinity binders of human PD-L1 (hPD-L1). After the ribosome display, individual clones were screened by ELISA. Detailed analysis of binding affinity and kinetics was performed using LigandTracer. The specificity of Myomedins was assessed using fluorescent microscopy on HEK293T-transfected cells and cultured cancer cells in vitro, formalin-fixed paraffin-embedded (FFPE) sections of human tonsils, and FFPE tumor samples of NSCLC patients.ResultsSeven identified PD-L1 binders, called MLE, showed positive staining for hPD-L1 on transfected HEK293T cells and cultured MCF-7 cells. MLE031, MLE105, MLE249, and MLE309 exhibited high affinity to both human and mouse PD-L1-transfected HEK293T cells measured with LigandTracer. The diagnostic potential of MLE variants was tested on human tonsillitis tissue and compared with diagnostic anti-PD-L1 antibody DAKO 28-8 and PD-L1 IHC 22C3 pharmDx antibody. MLE249 and MLE309 exhibited an excellent overlap with diagnostic DAKO 28-8 (Pearson´s coefficient (r) = 0.836 and 0.731, respectively) on human tonsils on which MLE309 exhibited also excellent overlap with diagnostic 22C3 antibody (r = 0.876). Using three NSCLC tissues, MLE249 staining overlaps with 28-8 antibody (r = 0.455–0.883), and MLE309 exhibited overlap with 22C3 antibody (r = 0.534–0.619). Three MLE proteins fused with Fc fragments of rabbit IgG, MLE249-rFc, MLE309-rFc and MLE031-rFc, exhibited very good overlap with anti-PD-L1 antibody 28-8 on tonsil tissue (r = 0.691, 0.610, and 0.667, respectively). Finally, MLE249-rFc, MLE309-rFc and MLE031-rFc exhibited higher sensitivity in comparison to IHC 22C3 antibody using routine immunohistochemistry staining system Ventana, which is one of gold standards for PD-L1 diagnosis.ConclusionsWe demonstrated the development of MLE Myomedins specifically recognizing hPD-L1 that may serve as a refinement tool for clinical PD-L1 detection.

Human T cell leukemia virus type 1 (HTLV 1) is an oncogenic retrovirus responsible for the development of adult T cell leukemia (ATL). The minus strand of HTLV-1 provirus encodes an oncoprotein named HTLV-1 bZIP factor (HBZ), which plays a pivotal role in viral replication and T cell proliferation. Of particular interest is the spliced HBZ isoform (sHBZ), which is predominantly expressed in ATL cells and localizes within the nucleolus, conferring immortalizing properties to T cells. Our previous study has shown that sHBZ colocalizes and associates with Nucleophosmin/B23, a nucleolar phosphoprotein with multiple functions. In this study, through an optimized nucleolar isolation method, we first confirmed sHBZ's nucleolar localization via Western blotting in transfected HEK293T cells, chronically HTLV-1-infected T cell lines, and freshly infected HeLa cells. We further demonstrated that the sHBZ/B23 association predominantly occurs in the nucleolus by co-immunoprecipitation of cell fractions. Our study highlights the nucleolar localization of sHBZ and its possibly essential interaction with this nucleolar-residing protein, leading to cell immortalization.

Chronic pancreatitis (CP) is a progressive and irreversible fibroinflammatory disease that markedly increases susceptibility to pancreatic cancer and remains without effective targeted therapies. Among the genetic contributors to CP, the carboxypeptidase A1 p.Ser282Pro (CPA1S282P) variant has been proposed to promote disease through misfolding-induced endoplasmic reticulum stress (ERS), although the broader pathogenic landscape remains incompletely defined. This study generated a rabbit model mimicking the human CPA1S282P mutation using the SpRY-ABE-8.17 system. Homozygous CPA1S282P rabbits exhibited characteristic human CP phenotypes following alcohol induction, including visceral pain, elevated serum lipase and amylase, inflammatory cell infiltration, and extensive pancreatic fibrosis. Biochemical analyses confirmed that the p.S282P mutation induced CPA1 misfolding and elevated the expression of ERS markers GRP78 and CHOP in both transfected HEK293T cells and homozygous mutant rabbits. Notably, the CPA1S282P mutation markedly disrupted intra-pancreatic lipid homeostasis, contributing to the development of CP in mutant rabbits. This study successfully established the first rabbit model of CP that accurately recapitulates CP caused by a defined human point mutation. Additionally, this study provides insights into a previously unrecognized link between CPA1 and intra-pancreatic lipid metabolism, offering a foundation for identifying novel therapeutic targets for human CP.

A major challenge in the delivery of genetic material is finding safe and effective vectors to deliver genetic material into target cells. Although virus-based vectors have proven to be effective in delivering genes to the cell nucleus, they often turn out to be harmful and interact with host cells. On the other hand, naturally available or chemically synthesized nonviral vectors are relatively safer but have low delivery efficiency. Research regarding the use of lipopeptide-based compounds as non-viral vectors for the delivery of genetic material is still limited. This study aims to synthesize NLS-based peptide, HKHK-PKKKRKV (P) and its lipopeptide, C16-HKHK-PKKKRKV (LP), and to evaluate their capability to transport genetic material into the nucleus in vitro. Synthesis of peptide and its lipopeptide was performed using the solid-phase method on 2-chlorotrityl chloride resin with Fmoc chemistry. HATU/HOAt was used as coupling reagents for amide bond formation between 2 amino acids, and DIC/Oxyma was used for fatty acid conjugation to the peptidyl resin. The yields of peptide and lipopeptide obtained were 33 and 26.4 %, respectively. Their structures were confirmed by ESI-TOF-MS, 1H-NMR, and 13C-NMR. The physicochemical analysis of peptide and lipopeptide showed that they were capable of condensing and protecting DNA from enzyme degradation at a ratio of 1:8 to DNA. The peptide and lipopeptide were found to be less efficient for transfection in HEK-293T cells because they required a high concentration (16,000 ng/μL). Nonetheless, peptide and lipopeptide concentrations of 250 - 16,000 ng/μL showed no toxicity to the cells. HIGHLIGHTS Successful synthesis of NLS-based peptide, HKHK-PKKKRKV, and its amphiphilic lipopeptide C16-HKHK-PKKKRKV using solid-phase method on 2-chlrotrityl resin with Fmoc chemistry. Implementation of efficient synthesis lipopeptide techniques, which overcame difficult aggregation and hydrophobic interaction issues throughout the assembly process. Demonstration of the lipopeptide’s capability as a versatile non-viral vector for gene delivery with adaptable properties for nuclear localization, cellular entrance, and nucleic acid binding. GRAPHICAL ABSTRACT

This study characterized a male infertile patient suffering from azoospermia and a female infertile patient with embryonic arrest from one consanguineous family. We aimed to elucidate the novel pathogenic mechanism and provide support for the genetic diagnosis of infertility. Mutations were detected by whole-exome sequencing (WES) and confirmed by Sanger sequencing. Effect of the mutations on mRNA level was investigated by sequencing the cDNA amplification product. Functional characterization of the mutations was investigated through transfection in HEK293T cells in vitro, and the subcellular localization and protein levels were evaluated by immunofluorescence and western blot. Preimplantation genetic testing was performed to analyze chromosomal anomalies of the arrested embryos. The novel homozygous whole exon deletion of exon 19 inMEI1 was detected by WES. Amplification and sequencing of the cDNA verified the deletion of whole exon of MEI1. Quantitative expression revealed that the deletion result in almost no detectable expression of MEI1 Exon 19 in patients, and their mother is a heterozygous carrier of this whole exon deletion. Western blotting revealed that the whole exon deletion in MEI1 result in production of truncated MEI1 protein, corresponding to the predicted premature termination at Exon 20 of MEI1. Highly chromosomal anomalies were revealed in arrested embryos with MEI1 mutation. Overall, this study reported the first exon rearrangement of MEI1, thus broadening the mutational pattern and spectrum of MEI1-associated infertility, and also revealed the aneuploidies of embryos with MEI1 mutation as a potential reason for declined developmental potency and embryonic arrest.

AKT3, a key component of the PI3K-AKT-MTOR pathway, is highly expressed in the brain, and its activating variants cause megalencephaly and cortical malformations. In this study, we functionally verified a novel missense AKT3 variant (p.Q78R) identified in a patient with extreme megalencephaly and intractable epilepsy. We transiently transfected HEK-293T cells with the AKTWT or AKT3Q78R and observed a significant increase of phospho-S6, a marker of mTOR complex 1 (mTORC1) activity, in AKT3Q78R transfected cells. Furthermore, considering its application in epilepsy treatment research, we identified a small interfering RNA (siRNA) capable of reducing the mRNA levels of AKTQ78R without affecting the expression levels of AKT3WT. Finally, the siRNA we identified specifically suppressed the AKT3Q78R-mediated mTORC1 activity, suggesting that this allele-specific siRNA approach holds promise for ameliorating the pathological condition.

Platelet inhibition by hypochlorous acid involves cAMP signalling.

PurposeThe translocator protein 18 kDa (TSPO) is a widely used marker for imaging neuroinflammation via Positron Emission Tomography (PET). However, the vast majority of reported TSPO PET tracers display low binding affinity to a common isoform of human TSPO (rs6971; A147T), making them unsuitable for universal use in the general population. In this study, we have developed and preclinically validated two novel tracers designed to image TSPO in patients of all genotypes.MethodsNovel analogues of known TSPO ligands were synthesised, evaluated for TSPO binding affinity in vitro (membranes prepared from transfected HEK-293T cells expressing wild-type (WT) or A147T TSPO) and radiolabelled with carbon-11 or fluorine-18. They were evaluated in situ (autoradiography on genotyped human brain tissue) and in vivo (rat, both WT and clinically relevant experimental autoimmune encephalomyelitis (EAE) neuroinflammation model) as potential polymorphism-insensitive TSPO PET tracers.ResultsTwo new TSPO ligands, DPA-813 and DPA-814, displayed equivalent single-digit nanomolar binding affinities in vitro towards both human TSPO isoforms. [11C]DPA-813 and [18F]DPA-814 were synthesised in moderate radiochemical yields, high radiochemical purity, and high molar activity. Autoradiography on human MS tissues showed high specific binding for both tracers, irrespective of the TSPO isoform. The tracers demonstrated high plasma stability after 45 min and no brain metabolism with > 99% intact tracer. Biodistribution in WT animals indicated good brain uptake for both tracers (0.28 and 0.41%ID/g for [18F]DPA-814 and [11C]DPA-813, respectively). PET imaging in the clinically relevant EAE neuroinflammation model in rats showed significantly higher uptake of [11C]DPA-813 and [18F]DPA-814 in the spinal cord of the EAE rats compared to the controls.ConclusionWe have developed two novel PET tracers that display indiscriminately high binding affinity to both common isoforms of human TSPO, show favourable metabolic stability and brain penetration in rats, and significantly higher uptake in the spinal cord of a neuroinflammatory rat model of multiple sclerosis. Going forward, first-in-human clinical validation will mark a critical juncture in the development of these tracers, which could offer substantial improvements over existing imaging tools for detecting neuroinflammation, irrespective of genetic variations.

BackgroundThe aberrant expression of α defensin 5 (DEFA5) protein in colonic inflammatory bowel diseases (IBDs) underlies the distinct pathogenesis of Crohn’s colitis (CC). It can serve as a biomarker for differentiating CC from Ulcerative colitis (UC), particularly in Indeterminate colitis (IC) cases into UC and CC. We evaluated the specificity of commercially available anti-DEFA5 antibodies, emphasizing the need to further validate their appropriateness for a given application and highlighting the necessity for novel antibodies.MethodsWe established two mice monoclonal DEFA5 antibody clones, 1A8 and 4F5, by immunizing mice with purified recombinant protein. We validated the specificity, sensitivity, and cross-reactivity of these antibodies in recognizing both endogenous and recombinant DEFA5 protein, especially for use in Immunohistochemistry (IHC), Western blot (WB), Immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA).ResultsClones 1A8 and 4F5 effectively recognized the endogenous DEFA5 in active human colon tissue from patients with diverticulitis (DV), UC, CC, and IC disease samples, as well as in transiently transfected HEK293T cells expressing DEFA5 with minimal non-confounding cross reactivity.ConclusionsThe 1A8 and 4F5 clones are useful for a wide variety of immunoassays, including WB, IHC, IP/WB, and ELISA. Their specificity enhances their potential as valuable tools for research applications in IBD colitis.