Search Results (1,029)

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a promising biotechnology tool for genome editing. However, in living organisms, several pharmacokinetic challenges arise, including off-target side effects due to incorrect distribution, low bioavailability caused by membrane impermeability, and instability resulting from enzymatic degradation. Therefore, innovative delivery strategies must be developed to address these issues. Modified nanoparticles offer a potential solution for the non-invasive delivery of CRISPR/Cas9 ribonucleoproteins (Cas9 RNPs). Cas9 RNPs encapsulated in nanoparticles are protected from enzymatic degradation, similar to how microRNAs are shielded within exosomes. It is well-established that certain materials, including proteins, are expressed selectively in specific cell types. For example, the α-7 nicotinic receptor is expressed in endothelial and neuronal cells, while the αvβ3 integrin is expressed in cancer cells. These endogenous materials can facilitate receptor-mediated endocytosis or transcytosis. Nanoparticles encapsulating Cas9 RNPs and coated with ligands targeting such receptors may be internalized through receptor-mediated mechanisms. Once internalized, Cas9 RNPs could perform the desired gene editing in the nucleus after escaping the endosome through mechanisms such as the proton sponge effect or membrane fusion. In this review, I discuss the potential and advantages of delivering Cas9 RNP-encapsulated nanoparticles coated with ligands through receptor-mediated endocytosis or transcytosis. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV) cause serious economic losses to the swine industry worldwide. Both viruses show a tropism for macrophages, based on the use of specific entry mediators (e.g., Siglec-1 and CD163). Identifying additional mediators of viral entry is essential for advancing antiviral and vaccine development. In this context, monoclonal antibodies (mAbs) are valuable tools. This study employed a library of 166 mAbs targeting porcine alveolar macrophages (PAMs) to identify candidates capable of blocking early infection stages, including viral binding, internalization, and fusion. Immunofluorescence analysis revealed 74 mAbs with cytoplasmic staining and 70 mAbs with membrane staining. Fifteen reacted with blood monocytes as determined by flow cytometry. mAb blocking assays were performed at 4 °C and 37 °C to analyze the ability of mAbs to block PRRSV and/or ASFV infections in PAMs. The mAb 28C10 significantly blocked PRRSV (96% at 4 °C and 80% at 37 °C) and ASFV (64% at 4 °C and 81% at 37 °C) infections. The mAb 28G10B6 significantly blocked PRRSV (86% at 4 °C and 74% at 37 °C) and partially blocked ASFV (35% at 4 °C and 64% at 37 °C) infections. mAb 26B8F5-I only partially blocked PRRSV infection (65% at 4 °C and 46% at 37 °C). Western blotting and mass spectrometry identified the corresponding proteins as Siglec-1 (28C10; 250 kDa), MYH9 (28G10B6; 260 kDa), and ANXA1 (26B8F5-I; 37 kDa). Our findings are indicative that Siglec-1, MYH9, and ANXA1 play a role in PRRSV/ASFV entry into macrophages. Full article

Dominant optic atrophy (DOA) is the most commonly inherited optic neuropathy. The majority of DOA is caused by mutations in the OPA1 gene, which encodes a dynamin-related GTPase located to the mitochondrion. OPA1 has been shown to regulate mitochondrial dynamics and promote fusion. Within the mitochondrion, proteolytically processed OPA1 proteins form complexes to maintain membrane integrity and the respiratory chain complexity. Although OPA1 is broadly expressed, human OPA1 mutations predominantly affect retinal ganglion cells (RGCs) that are responsible for transmitting visual information from the retina to the brain. Due to the scarcity of human RGCs, DOA has not been studied in depth using the disease affected neurons. To enable studies of DOA using stem-cell-derived human RGCs, we performed CRISPR-Cas9 gene editing to generate OPA1 mutant pluripotent stem cell (PSC) lines with corresponding isogenic controls. CRISPR-Cas9 gene editing yielded both OPA1 homozygous and heterozygous mutant ESC lines from a parental control ESC line. In addition, CRISPR-mediated homology-directed repair (HDR) successfully corrected the OPA1 mutation in a DOA patient’s iPSCs. In comparison to the isogenic controls, the heterozygous mutant PSCs expressed the same OPA1 protein isoforms but at reduced levels; whereas the homozygous mutant PSCs showed a loss of OPA1 protein and altered mitochondrial morphology. Furthermore, OPA1 mutant PSCs exhibited reduced rates of oxygen consumption and ATP production associated with mitochondria. These isogenic PSC lines will be valuable tools for establishing OPA1-DOA disease models in vitro and developing treatments for mitochondrial deficiency associated neurodegeneration. Full article

Botulinum toxin (BoNT), the most potent substance known to humans, likely evolved not to kill but to serve other biological purposes. While its use in cosmetic applications is well known, its medical utility has become increasingly significant due to the intricacies of its structure and function. The toxin’s structural complexity enables it to target specific cellular processes with remarkable precision, making it an invaluable tool in both basic and applied biomedical research. BoNT’s potency stems from its unique structural features, which include domains responsible for receptor recognition, membrane binding, internalization, and enzymatic cleavage. This division of labor within the toxin’s structure allows it to specifically recognize and interact with synaptic proteins, leading to precise cleavage at targeted sites within neurons. The toxin’s mechanism of action involves a multi-step process: recognition, binding, and catalysis, ultimately blocking neurotransmitter release by cleaving proteins like SNAP-25, VAMP, and syntaxin. This disruption in synaptic vesicle fusion causes paralysis, typically in peripheral neurons. However, emerging evidence suggests that BoNT also affects the central nervous system (CNS), influencing presynaptic functions and distant neuronal systems. The evolutionary history of BoNT reveals that its neurotoxic properties likely provided a selective advantage in certain ecological contexts. Interestingly, the very features that make BoNT a potent toxin also enable its therapeutic applications, offering precision in treating neurological disorders like dystonia, spasticity, and chronic pain. In this review, we highlight the toxin’s structural, functional, and evolutionary aspects, explore its clinical uses, and identify key research gaps, such as BoNT’s central effects and its long-term cellular impact. A clear understanding of these aspects could facilitate the representation of BoNT as a unique scientific paradigm for studying neuronal processes and developing targeted therapeutic strategies. Full article

The study of pathogenic viruses has always posed significant biosafety challenges. In particular, the study of highly pathogenic viruses requires methods with low biological risk but relatively high sensitivity and convenience in detection. In recent years, pseudoviruses, which consist of a backbone of one virus and envelope proteins of another virus, have become one of the most widely used tools for exploring the mechanisms of viruses binding to cells, membrane fusion and viral entry, as well as for screening the libraries of antiviral substances, evaluating the potential of neutralizing monoclonal antibodies, developing neutralization tests, and therapeutic platforms. During the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pseudotyped virus-based assays played a pivotal role in advancing our understanding of virus–cell interactions and the role of its proteins in disease pathogenesis. Such tools facilitated the search for potential therapeutic agents and accelerated epidemiological studies on post-infection and post-vaccination humoral immunity. This review focuses on the use of pseudoviruses as a model for large-scale applications to study enveloped viruses. Full article

SARS-CoV-2 viral entry requires membrane fusion, which is facilitated by the fusion peptides within its spike protein. These predominantly hydrophobic peptides insert into target membranes; however, their precise mechanistic role in membrane fusion remains incompletely understood. Here, we investigate how FP1 (SFIEDLLFNKVTLADAGFIK), the N-terminal fusion peptide, modulates membrane stability and barrier function across various model membrane systems. Through a complementary suite of biophysical techniques—including electrophysiology, fluorescence spectroscopy, and atomic force microscopy—we demonstrate that FP1 significantly promotes pore formation and alters the membrane’s mechanical properties. Our findings reveal that FP1 reduces the energy barrier for membrane defect formation and stimulates the appearance of stable conducting pores, with effects modulated by membrane composition and mechanical stress. The observed membrane-destabilizing activity suggests that, beyond its anchoring function, FP1 may facilitate viral fusion by locally disrupting membrane integrity. These results provide mechanistic insights into SARS-CoV-2 membrane fusion mechanisms and highlight the complex interplay between fusion peptides and target membranes during viral entry. Full article

Valine-glutamine (VQ) motif-containing proteins play important roles in diverse plant developmental processes and signal transduction in response to biotic and abiotic stresses. However, no systematic investigation has been conducted on VQ genes in watermelon. In this study, we identified 31 watermelon VQ genes, which were classified into six subfamilies (I–VI). All of the deduced proteins contained a conserved FxxxVQxL/F/VTG motif. Eleven ClVQs were involved in segment duplication, which was the main factor in the expansion of the VQ family in watermelon. Numerous stress- and hormone-responsive cis-elements were detected in the putative promoter region of the ClVQ genes. Green fluorescent protein fusion proteins for ten selected ClVQs were localized in the nucleus, but three ClVQs also showed signals in cell membranes and the cell wall, thus confirming their predicted divergent functionality. Quantitative real-time PCR (qRT-PCR) analysis indicated that the majority of ClVQ genes were specifically or preferentially expressed in certain tissues or organs, especially in the male flower. Analyses of RNA-sequencing data under osmotic, cold, and drought stresses and Cucumber green mottle mosaic virus (CGMMV) infection revealed that the majority of ClVQ genes, especially those from subfamily IV, were responsive to these stresses. The results provide useful information for the functional characterization of watermelon ClVQ genes to unravel their biological roles. Full article

Mitochondrial homeostasis is crucial for maintaining cellular energy production and preventing oxidative stress, which is essential for overall cellular function and longevity. Mitochondrial damage and dysfunction often occur concomitantly in myocardial ischemia–reperfusion injury (MIRI). Notoginsenoside R1 (NGR1), a unique saponin from the traditional Chinese medicine Panax notoginseng, has been shown to alleviate MIRI in previous studies, though its precise mechanism remains unclear. This study aimed to elucidate the mechanisms of NGR1 in maintaining mitochondrial homeostasis in hypoxia/reoxygenation (H/R) H9c2 cells. The results showed that NGR1 pretreatment effectively increased cell survival rates post-H/R, reduced lactate dehydrogenase (LDH) leakage, and mitigated cell damage. Further investigation into mitochondria revealed that NGR1 alleviated mitochondrial structural damage, improved mitochondrial membrane permeability transition pore (mPTP) persistence, and prevented mitochondrial membrane potential (Δψm) depolarization. Additionally, NGR1 pretreatment enhanced ATP levels, increased the activity of mitochondrial respiratory chain complexes I–V after H/R, and reduced excessive mitochondrial reactive oxygen species (mitoROS) production, thereby protecting mitochondrial function. Further analysis indicated that NGR1 upregulated the expression of mitochondrial biogenesis-related proteins (PGC-1α, Nrf1, Nrf2) and mitochondrial fusion proteins (Opa1, Mfn1, Mfn2), while downregulating mitochondrial fission proteins (Fis1, Drp1) and reducing mitochondrial autophagy (mitophagy) levels, as well as the expression of mitophagy-related proteins (Pink1, Parkin, BNIP3) post-H/R. Therefore, this study showed that NGR1 can maintain mitochondrial homeostasis by regulating mitophagy, mitochondrial fission–fusion dynamics, and mitochondrial biogenesis, thereby alleviating H9c2 cell H/R injury and protecting cardiomyocytes. Full article

In this study, a novel rapid immunochromatographic (IC) test for African swine fever virus (ASFV) antibodies is presented. An immunochromatographic test (IC) is a detection technique that combines membrane chromatography with immunolabeling. This approach saves time for antibody preparation, resulting in a shorter production cycle. p54 is an important structural protein of African swine fever, and an ideal protein for serotype diagnosis. Gold nanoparticles are attached to the ASFV p54-Fc fusion protein, and the ASFV-specific antigen p54 and Staphylococcus aureus protein A (SPA) are labeled on a nitrocellulose membrane, at positions T and C, respectively. We developed a SPA double sandwich IC test strip, and assessed its feasibility using ASFV p54 and p54-Fc fusion proteins as antigens. ASFV p54 and p54-Fc fusion proteins were expressed and purified. A sandwich cross-flow detection method for p54, which is the primary structural protein of ASFV, was established, using colloidal gold conjugation. Our method can detect ASFV antibodies in field serum samples in about 15 min using a portable colloidal gold detector, demonstrating high specificity and sensitivity (1:320), and the coincidence rate was 98% using a commercial ELISA kit. The dilution of the serum sample can be determined by substituting the absorbance (T-line) interpreted by portable devices into the calibration curve function formula of an African swine fever virus standard serum. In summary, our method is rapid, cost-effective, precise, and highly selective. Additionally, it introduces a new approach for constructing IC test strips using SPA protein without antibody preparation, making it a reliable on-site antibody test for ASFV. Full article

Background and Aims: Hepatoblastoma (HBL) and fibrolamellar hepatocellular carcinoma (FLC) are the most common liver malignancies in children and young adults. FLC oncogenesis is associated with the generation of the fusion kinase, DNAJB1-PKAc (J-PKAc). J-PKAc has been found in 90% of FLC patients’ tumors but not in other liver cancers. Since previous studies of J-PKAc were performed with adolescent patients, we asked if young children may express J-PKAc and if there are consequences of such expression. Methods: The biobank of the pediatric HBL/HCN-NOS specimens was examined by QRT-PCR, Western blots, RNA-Seq, and immunostaining with fusion-specific antibodies. Results: J-PKAc is expressed in 70% of the HBL/HCN-NOS patients. RNA-Seq analysis revealed that HBL tumors that do not have cells expressing J-PKAc show elevated expression of the membrane attack complex (MAC), which eliminates cells expressing J-PKAc. The fusion-positive HBL/HCN-NOS samples have several signaling pathways that are different from fusion-negative HBLs. Upregulated pathways included genes involved in the G1 to S transition and in liver cancer. Downregulated pathways included over 60 tumor suppressors, the CYP family, and the SLC family. The repression of these genes involves J-PKAc-β-catenin-TCF4-mediated elevation of the HDAC1-Sp5 pathway. The identified upregulated and downregulated pathways are direct targets of the fusion kinase. The J-PKAc kinase is also detected in livers of 1-year-old children with biliary atresia (BA). Conclusions: J-PKAc is expressed in both HBL tumor and BA liver samples, contributing to the development of HBL and creating a transcriptome profiling consistent with the potential development of liver cancer in young patients. Full article

This study investigated the modification of nanosilver (Ag) by Co-Pc (phthal–cyanine) and the synergistic effect of Ag-Co/CNT (carbon nanotube) for the long-term stability of AEMFCs (anion exchange membrane fuel cells). This study also aimed to use non-precious metal catalysts on both the cathode and anode to reduce the catalyst costs. Through a simple and efficient chemical synthesis method, a composite catalyst consisting of Co-Pc-modified Ag/CNT was successfully prepared and characterized for its structure and composition. Co-Pc and Ag were chosen for their high durability and catalytic activity in fuel cells, combined with a multi-wall carbon nanotube (MWCNT) as a carrier for the cathode catalyst, and the anode catalyst used Pd-CeO₂/C. The performance of the cell module was tested based on a commercial anion exchange membrane (X37-50RT). The experiment focused on different synthesis times and ratios of catalyst and ionomer, observing the enhancement in Co on the active sites of Ag/CNT. Finally, the cell performance was tested for the optimal loading amount. It was observed that when the loading of the nanosilver–cobalt/carbon nanotube (Ag-Co/CNT) is 1 mg/cm², the highest power density is 434.1 mW/cm². Through 100 cycles of testing, only an 18% decrease was observed, while the decrease in open circuit voltage was approximately 4.6%. Compared to nanosilver (Ag/CNT), the Co-Pc-modified nano-Ag with the degradation rate has significantly slowed down, and its catalytic activity has also improved significantly. The enhanced stability of this synergistic effect is mainly attributed to the introduction of cobalt metal, which prevents excessive fusion of nano-Ag particles and surface oxidation, effectively maintaining durability in catalytic activity. Full article

Lactococcus lactis is a potential bacterial cell factory to develop delivery systems for vaccines and therapeutic proteins. Much progress has been made in applications using engineered L. lactis against, e.g., inflammatory bowel disease and cervical cancer, but the improvement of secretion and cell anchoring efficacy is still desirable. A double-labeling method based on biarsenical hairpin binding and nickel–polyhistidine affinity was used for visualization of protein trafficking and the quantification of targeted proteins on the cell surface and in the cytoplasm. To investigate the importance of mature domain targeting signals (MTSs), we generated truncated constructs encoding 126, 66, and 26 amino acid residues from the N-terminus of the basic membrane protein A (BmpA) and fused those with the gene for the human papillomavirus serotype 16 (HPV16) E7 oncoprotein. Overexpression of fusion proteins was observed to come at the cost of cell proliferation. L. lactis cells produced and displayed the shortest fusion protein only with difficulty, suggesting that the entire absence of a homologous sequence containing MTSs significantly impedes the export and surface anchoring of fusion proteins. With 40 amino acids following the signal peptide and containing one MTS, effective translocation was possible. Mutations of MTSs towards increased hydrophobicity resulted in increased secreted and surface-displayed fusion protein, suggesting the potential to design rationally improved constructs. Full article

Ovarian aging significantly impacts female fertility, with mitochondrial dysfunction emerging as a key factor. This study investigated the effects of recombinant follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on mitochondrial function and metabolism in aging female reproductive cells. Human granulosa cells (HGL5) were treated with FSH/LH or not. Mitochondrial function was assessed through various assays, including mitochondrial mass, membrane potential, ROS levels, and ATP production. Mitochondrial dynamics and morphology were analyzed using MitoTracker staining. Cellular respiration was measured using a Seahorse Bioenergetics Analyzer. Metabolic reprogramming was evaluated through gene expression analysis and metabolite profiling. In vivo effects were studied using aging mouse oocytes. FSH/LH treatment significantly improved mitochondrial function in aging granulosa cells, increasing mitochondrial mass and membrane potential while reducing ROS levels. Mitochondrial dynamics showed a shift towards fusion and elongation. Cellular respiration, ATP production, and spare respiratory capacity were enhanced. FSH/LH-induced favorable alterations in cellular metabolism, favoring oxidative phosphorylation. In aging mouse oocytes, FSH/LH treatment improved in vitro maturation and mitochondrial health. In conclusion, FSH/LH supplementation ameliorates age-related mitochondrial dysfunction and improves cellular metabolism in aging female reproductive cells. Full article

Mitochondrial function is essential for synaptic function. ATAD1, an AAA+ protease involved in mitochondrial quality control, governs fission–fusion dynamics within the organelle. However, the distribution and functional role of ATAD1 in neurons remain poorly understood. In this study, we demonstrate that ATAD1 is primarily localized to mitochondria in dendrites and, to a lesser extent, in spines in cultured hippocampal neurons. We found that ATAD1 deficiency disrupts the mitochondrial fission–fusion balance, resulting in mitochondrial fragmentation. This deficiency also impairs dendritic branching, hinders dendritic spine maturation, and reduces glutamatergic synaptic transmission in hippocampal neuron. To further investigate the underlying mechanism, we employed an ATP hydrolysis-deficient mutant of ATAD1 to rescue the neuronal deficits associated with ATAD1 loss. We discovered that the synaptic deficits are independent of the mitochondrial morphology changes but rely on its ATP hydrolysis. Furthermore, we show that ATAD1 loss leads to impaired mitochondrial function, including decreased ATP production, impaired membrane potential, and elevated oxidative stress. In conclusion, our results provide evidence that ATAD1 is crucial for maintaining mitochondrial function and regulating neurodevelopment and synaptic function. Full article

Within mammalian cells, diverse endocytic mechanisms, including phagocytosis, pinocytosis, and receptor-mediated endocytosis, serve as gateways exploited by many bacterial pathogens and toxins. Among these, caveolae-mediated endocytosis is characterized by lipid-rich caveolae and dimeric caveolin proteins. Caveolae are specialized microdomains on cell surfaces that impact cell signaling. Caveolin proteins facilitate the creation of caveolae and have three members in vertebrates: caveolin-1, caveolin-2, and caveolin-3. Many bacterial pathogens hijack caveolin machinery to invade host cells. For example, the Gram-positive facultative model intracellular bacterial pathogen Listeria monocytogenes exploits caveolin-mediated endocytosis for efficient cellular entry, translocation across the intestinal barrier, and cell–cell spread. Caveolin facilitates the internalization of group A streptococci by promoting the formation of invaginations in the plasma membrane and avoiding fusion with lysosomes, thereby aiding intracellular survival. Caveolin plays a crucial role in internalizing and modulation of host immune responses by Gram-negative bacterial pathogens, such as Escherichia coli K1, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium. Here, we summarize how bacterial pathogens manipulate the host’s caveolin system to facilitate bacterial entry and movement within and between host cells, to support intracellular survival, to evade immune responses, and to trigger inflammation. This knowledge enhances the intervention of new therapeutic targets against caveolin in microbial invasion and immune evasion processes. Full article