Background Dengue virus (DENV) is a major global health concern with frequent hepatic involvement that contributes to disease severity. In vitro models using human hepatocyte–derived cell lines are essential for elucidating viral pathogenesis and supporting antiviral research. The HC‐04 cell line, derived from human hepatocytes, has not been extensively characterized as a model for DENV infection. Methods HC‐04 cells were infected with DENV‐2 (strain 16681) at a multiplicity of infection (MOI) of 10. Morphological changes were assessed by inverted microscopy. In addition, ultrastructural alterations, cell necrosis, and viral replication were studied by transmission electron microscopy and immunogold electron microscopy for Bax, GRP78, DENV Nonstructural Protein 3 (NS3) and envelope (E) protein, ethidium bromide/acridine orange (EB/AO) staining, and plaque assay, respectively. C6/36 mosquito cells were used as a comparative control for the plaque assay. Additional assessments of morphological changes, viral replication, and necrosis were performed at MOIs of 1 and 5. Results Infected HC‐04 cells exhibited cytoplasmic granulation starting from Day 2 postinfection (p.i.), followed by irregular alignment and detachment from Day 4 p.i. Ultrastructural abnormalities on Day 5 p.i. included cytoplasmic vacuolization, lipid droplet accumulation, swollen mitochondria with disrupted cristae, and marked expansion of the rough endoplasmic reticulum. EB/AO staining demonstrated significantly higher necrosis in infected cells than controls. Plaque assays showed viable virus, but the titers were lower than in C6/36 cells ( p = 0.0162), and intracellular DENV NS3 and E protein were not detected. Conclusions DENV‐2 infection induced marked cytopathic and ultrastructural alterations and necrosis in HC‐04 cells. However, it cannot be confirmed whether the cells are permissive to DENV.
Background The relationship between Streptococcus pyogenes (Group A Streptococcus) and COVID‐19 has been of interest because of possible coinfections and their clinical implications. This discussion examines the interaction between these pathogens and focuses on rates of coinfection, effects, and diagnostic considerations. Materials and Methods This study on 119 COVID‐19 patients involved sampling pharyngeal and nasal secretions for streptococcal culture and PCR testing. RNA extraction, cDNA synthesis, and RT‐qPCR targeted the N gene of SARS‐CoV‐2. Results This study on 119 COVID‐19 patients revealed a prevalence of 52.94%, with higher rates among women and older individuals. Streptococcal infection prevalence was 34.45%, significantly elevated in COVID‐19 patients compared with noninfected individuals ( p < 0.001). Notably, streptococcal infection rates were higher in men, older age groups, and those exhibiting symptoms like shortness of breath, cough, fever, weakness, and lethargy ( p < 0.001). Conclusion The co‐occurrence of S . pyogenes infections with COVID‐19 complicates diagnosis, necessitating comprehensive laboratory testing. Understanding their epidemiology informs public health strategies for surveillance, prevention, and control, highlighting the complexities in infectious disease management.
Plasmodium infects hepatocytes prior to establishing a pathogenic blood stage infection. During liver stage development, parasites modulate their host cells to survive, grow, and multiply, resulting in the formation of merozoites that are released into the blood stream. Compared with the number of parasite proteins that are exported into the erythrocyte, evidence exists for the export of only a very few parasite proteins into the host hepatocyte. Here, we screened multiple Plasmodium berghei candidate proteins for liver stage expression and export into the hepatocyte. To ascertain expression and export, proteins were tagged with either mCherry or 3xcMyc. This screening resulted in the identification of a novel liver stage exported protein, that is, LSEP. Although LSEP fused to mCherry failed to translocate into the hepatocyte, cMyc-tagged LSEP was exported into the hepatocyte cytosol, indicating that the tag structure or size influences export into the hepatocyte. In addition, the known liver stage-specific and exported protein LISP2 was tagged with mCherry/cMyc in multiple locations (N-terminal, internal, and C-terminal). Notably, only LISP2 tagged with cMyc at its N-terminus translocated into the hepatocyte, showing that both the choice of the tag and the location of the tag are important for identifying proteins that are exported into the hepatocyte cytosol. Identifying novel exported proteins may, in turn, lead to the identification of potential targets for possible therapeutics.
Porphyromonas gulae has been established as a pathogen in animal periodontal disease and expresses several virulence factors, including fimbriae, lipopolysaccharide, and proteases. P. gulae fimbriae are classified into three genotypes (A, B, and C) based on the fimbrillin (fimA) gene encoding a fimbrial subunit protein. We previously reported that P. gulae organisms with type C fimbriae were clearly more virulent as compared to those with other fimbria types, though the basic function of this is not fully understood. Here, we investigated the function of fimbriae using fimA-deficient mutants. In the present study, bacterial fimbriae were related to biofilm tenacity, whereas biofilm formation was not. Also, P. gulae fimbriae are involved in bacterial adhesion and entry into gingival epithelial cells. The role of P. gulae fimbriae in bacterial pathogenicity was then determined using fimA-deficient mutants with a silkworm infection model. Additionally, P. gulae infection can induce inflammatory responses in gingival epithelial cells and also inhibit cell migration and proliferation. In contrast, a P. gulae fimA-deficient mutant did not cause several of those cellular responses. Together, the results show that fimbriae contribute to the pathogenicity of P. gulae by affecting bacterial functions and host cell interactions.
Candida albicans (C. albicans) is an opportunistic yeast that forms biofilms, which confer increased resistance to antifungal agents, making infections very difficult to treat. One of the mechanisms regulating this virulence is quorum sensing (QS), which can be defined as a cellular communication mechanism based on the production of autoinducer, namely, farnesol. We previously reported that the essential oils of Cistus ladanifer, Matricaria chamomilla, and Pistacia lentiscus inhibit C. albicans. In this context, a virtual screening of 48 molecules derived from these plants, as well as their ADMET property predictions, was undertaken to identify the most promising ligands for inhibiting QS receptors (CYC and RAS1) compared to farnesol. The latter exhibited a binding affinity of -7.0 kcal/mol for both CYC and RAS1. Two phytomolecules, namely, chamazulene and gamma-muurolene, exhibited stronger binding affinities of -7.6 and -8.1 kcal/mol, respectively, for CYC, while toward the RAS1 receptor, they showed identical binding affinities of -7.1 kcal/mol. According to the binding free energy calculations performed using Prime MM-GBSA, the complexes formed by gamma-muurolene exhibited high stability, maintained by coulombic, lipophilic, and Van der Waals interactions, demonstrating that gamma-muurolene is a promising inhibitor of QS. Molecular dynamic simulations confirmed the conformational stability of the CYC-gamma-muurolene and RAS1-gamma-muurolene complexes, characterized by low RMSD values, suggesting favorable dynamic behavior. The results as a whole provide a solid foundation for further experimental validation.
