Search Results (8)

Euonymus bungeanus is a highly valued ornamental tree/shrub species widely utilized in landscaping and afforestation in Northeast Asia, yet molecular studies on this species remain limited due to the lack of validated reference genes for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). In this study, 16 candidate reference genes were selected based on classical plant reference genes and our previous transcriptome data. Their expression stability was comprehensively evaluated using 64 samples collected from diverse tissues and plants subjected to various abiotic stress/hormone treatments across multiple time points. Across all samples analyzed, PBG1 (20S proteasome beta subunit G1) exhibited the highest overall expression stability, followed by VAPD (vacuolar ATP synthase subunit D) and EIF4A (eukaryotic translation initiation factor 4A). For tissue-specific analysis, TSR2 (pre-rRNA-processing protein), VAPD, and PBG1 demonstrated the greatest stability. Under specific stress conditions, PBG1 and EIF4A were identified as the most stable genes under low- and high-temperature conditions. PP2A (protein phosphatase 2A) and TUB6 (beta-6 tubulin) were optimal for drought stress, while TSR2, SRP (nuclear speckle splicing regulatory-like protein), and PBG1 exhibited superior stability under salt stress. These findings establish a validated panel of reference genes enabling accurate and reliable gene expression normalization in E. bungeanus, thereby facilitating future functional genomics studies in this economically and ecologically important species. Full article

Background/Objectives: Pseudomonas aeruginosa is one of the leading causes of ventilator-associated pneumonia (VAP) and ventilator-associated tracheobronchitis (VAT), with a worldwide spread of difficult-to-treat high-risk clones. This study aimed to investigate the virulence potential and to characterize phenotypic and genotypic antimicrobial resistance (AMR) in P. aeruginosa causing VAP/VAT in the Intensive Care Unit (ICU), University Hospital of Split, Croatia. Methods: The study included P. aeruginosa isolates obtained from ICU patients who met the criteria for VAP or VAT, between January 2023 and January 2024. Isolates were identified using MALDI-TOF MS and tested for antimicrobial susceptibility (AST). A subset of phenotypically multidrug-resistant (MDR) isolates was further analyzed using whole-genome sequencing (WGS) and multilocus sequence typing. Results: A high rate of resistance was detected to ceftazidime (23.4%), imipenem (39.6%), and meropenem (43.8%). WGS confirmed the presence of multiple AMR genes, including the bla_VIM-2 gene, whose genetic environment highlights a complex MDR locus integrating multiple AMR determinants and mobile genetic elements. All tested isolates possessed genes for class C (bla_PDC34, bla_PDC374 or bla_PDC16) and class D (bla_OXA-2, bla_OXA-10 or bla_OXA-50) β-lactamases, fosA, aph(3′)-IIb and crpP genes. Additionally, WGS analysis revealed the presence of numerous virulence genes including those for adherence (Type IV pili and Fap protein production), motility (such as flgF), biofilm formation (e.g., algE and mucE), quorum sensing (lasI, lasR, rhlI and rhlR), exotoxin (toxA and plcH) and exoenzyme activity (exoU, exoT, exoS, exoY, pcrV, hcp1 and lasA). The isolates belonged to four different sequence types: ST235, ST446, the high-risk ST253 and the widely distributed ST395. Phylogenomic comparison demonstrated that the isolates from this study do not originate from a single clonal source, but instead represent multiple globally distributed high-risk P. aeruginosa lineages introduced into the clinical setting. Conclusions: Due to the emergence of high-risk clones with broad AMR and strong virulence potential, ineffectiveness of standard empirical therapy may be anticipated, highlighting the urgent need for new therapeutic approaches (including those targeting major virulence factors). Full article

The Helicobacter pylori vapD gene is transcribed and expressed when the bacteria are within the gastric cell. In this current study, we investigated how vapD knockout affects the survival of H. pylori inside human gastric adenocarcinoma cells. We constructed an H. pylori 26695 vapD (Hp ΔvapD) mutant strain. H. pylori 26695 wt and Hp ΔvapD strains were grown in synthetic media and were co-cultured with AGS cells. From the start, the growth curve, total protein concentration and colony-forming units (CFUs) of each strain were measured. From each co-culture, CFUs and total RNA were obtained, and transcript levels of GAPDH, vapD, vacA, ureA, and 16s Hp were measured by qRT-PCR. Hp ΔvapD did not affect the growth rate of the strain in synthetic media, showing that the vapD gene is not necessary when the bacteria grow outside eukaryote cells. However, in the intracellular environment, the number of CFUs recovered from the Hp ΔvapD strain from AGS cells decreased after 36 h. Transcription levels of the vacA gene from the Hp ΔvapD strain were 10,000-fold lower than those of H. pylori wt, to the point of being undetectable. The results suggest that the vapD gene contributed to maintaining H. pylori inside gastric cells. Full article

The emergence of drug resistance and the limited number of approved antitubercular drugs prompted identification and development of new antitubercular compounds to cure Tuberculosis (TB). In this work, an attempt was made to identify potential natural compounds that target mycobacterial proteins. Three plant extracts (A. aspera, C. gigantea and C. procera) were investigated. The ethyl acetate fraction of the aerial part of A. aspera and the flower ash of C. gigantea were found to be effective against M. tuberculosis H₃₇Rv. Furthermore, the GC-MS analysis of the plant fractions confirmed the presence of active compounds in the extracts. The Mycobacterium target proteins, i.e., available PDB dataset proteins and proteins classified in virulence, detoxification, and adaptation, were investigated. A total of ten target proteins were shortlisted for further study, identified as follows: BpoC, RipA, MazF4, RipD, TB15.3, VapC15, VapC20, VapC21, TB31.7, and MazF9. Molecular docking studies showed that β-amyrin interacted with most of these proteins and its highest binding affinity was observed with Mycobacterium Rv1636 (TB15.3) protein. The stability of the protein-ligand complex was assessed by molecular dynamic simulation, which confirmed that β-amyrin most firmly interacted with Rv1636 protein. Rv1636 is a universal stress protein, which regulates Mycobacterium growth in different stress conditions and, thus, targeting Rv1636 makes M. tuberculosis vulnerable to host-derived stress conditions. Full article

Radopholus similis is a migratory endoparasitic nematode that is extremely harmful to host plants. Venom allergen-like proteins (VAPs) are members of the cysteine-rich secretory protein family that are widely present in plants and animals. In this study, we cloned a VAP gene from R. similis, designated as RsVAP. RsVAP contains an open reading frame of 1089 bp encoding 362 amino acids. RsVAP is specifically expressed in the esophageal gland, and the expression levels of RsVAP are significantly higher in juveniles than in other life stages of R. similis. This expression pattern of RsVAP was consistent with the biological characteristics of juveniles of R. similis, which have the ability of infection and are the main infection stages of R. similis. The pathogenicity and reproduction rate of R. similis in tomato was significantly attenuated after RsVAP was silenced. In tobacco leaves transiently expressing RsVAP, the pathogen-associated molecular pattern-triggered immunity (PTI) induced by a bacterial flagellin fragment (flg22) was inhibited, while the cell death induced by two sets of immune elicitors (BAX and Gpa2/RBP-1) was repressed. The RsVAP-interacting, ras-related protein RABA1d (LeRabA1d) was identified in tomato hosts by yeast two-hybrid and co-immunoprecipitation assays. RsVAP may interact with LeRabA1d to affect the host defense response, which in turn facilitates nematode infection. This study provides the first evidence for the inhibition of plant defense response by a VAP from migratory plant-parasitic nematodes, and, for the first time, the target protein of R. similis in its host was identified. Full article

Apoptosis-linked gene 2 (ALG-2, also known as PDCD6) is a member of the penta-EF-hand (PEF) family of Ca²⁺-binding proteins. The murine gene encoding ALG-2 was originally reported to be an essential gene for apoptosis. However, the role of ALG-2 in cell death pathways has remained elusive. In the present study, we found that cell death-inducing p53 target protein 1 (CDIP1), a pro-apoptotic protein, interacts with ALG-2 in a Ca²⁺-dependent manner. Co-immunoprecipitation analysis of GFP-fused CDIP1 (GFP-CDIP1) revealed that GFP-CDIP1 associates with tumor susceptibility gene 101 (TSG101), a known target of ALG-2 and a subunit of endosomal sorting complex required for transport-I (ESCRT-I). ESCRT-I is a heterotetrameric complex composed of TSG101, VPS28, VPS37 and MVB12/UBAP1. Of diverse ESCRT-I species originating from four VPS37 isoforms (A, B, C, and D), CDIP1 preferentially associates with ESCRT-I containing VPS37B or VPS37C in part through the adaptor function of ALG-2. Overexpression of GFP-CDIP1 in HEK293 cells caused caspase-3/7-mediated cell death. In addition, the cell death was enhanced by co-expression of ALG-2 and ESCRT-I, indicating that ALG-2 likely promotes CDIP1-induced cell death by promoting the association between CDIP1 and ESCRT-I. We also found that CDIP1 binds to vesicle-associated membrane protein-associated protein (VAP)A and VAPB through the two phenylalanines in an acidic tract (FFAT)-like motif in the C-terminal region of CDIP1, mutations of which resulted in reduction of CDIP1-induced cell death. Therefore, our findings suggest that different expression levels of ALG-2, ESCRT-I subunits, VAPA and VAPB may have an impact on sensitivity of anticancer drugs associated with CDIP1 expression. Full article

Salinity is one of the major abiotic stress factors that limit plant growth and crop yield worldwide. To understand the molecular mechanisms and screen the key proteins in response of sugar beet (Beta vulgaris L.) to salt, in the present study, the proteomics of roots and shoots in three-week-old sugar beet plants exposed to 50 mM NaCl for 72 h was investigated by isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technology. The results showed that 105 and 30 differentially expressed proteins (DEPs) were identified in roots and shoots of salt-treated plants compared with untreated plants, respectively. There were 46 proteins up-regulated and 59 proteins down-regulated in roots; and 13 up-regulated proteins and 17 down-regulated proteins found in shoots, respectively. These DEPs were mainly involved in carbohydrate metabolism, energy metabolism, lipid metabolism, biosynthesis of secondary metabolites, transcription, translation, protein folding, sorting, and degradation as well as transport. It is worth emphasizing that some novel salt-responsive proteins were identified, such as PFK5, MDH, KAT2, ACAD10, CYP51, F3H, TAL, SRPR, ZOG, V-H⁺-ATPase, V-H⁺-PPase, PIPs, TIPs, and tubulin α-2/β-1 chain. qRT-PCR analysis showed that six of the selected proteins, including BvPIP1-4, BvVP and BvVAP in root and BvTAL, BvURO-D1, and BvZOG in shoot, displayed good correlation between the expression levels of protein and mRNA. These novel proteins provide a good starting point for further research into their functions using genetic or other approaches. These findings should significantly improve the understanding of the molecular mechanisms involved in salt tolerance of sugar beet plants. Full article

In previous research, a 32 kDa protein in lymphocystis disease virus (LCDV) was identified as viral attachment protein (VAP) that specifically interacted with the 27.8 kDa cellular receptor from flounder Paralichthys olivaceus gill (FG) cells, and the recombinant VAP (rVAP) was expressed in Escherichia coli strain BL21 (DE3). In this study, monoclonal antibodies (MAbs) against 32 kDa VAP are produced by immunization of BALB/c mice with the rVAP. Seven hybridoma secreting MAbs were screened by enzyme-linked immunosorbent assay, five of which designated as 1C6, 1C8, 3B5, 3D11 and 3H10 are cloned by the limiting dilution method, depending on the strongly positive results of ELISA. Western blotting analysis shows that the five MAbs can specifically react with the 32 kDa protein of LCDV and the purified 50 kDa rVAP, and the subtype of the MAbs is identified as IgG. Immunofluorescence results demonstrate that the specific fluorescence signals for LCDV appear in the cytoplasm of FG cells at 24 h post LCDV infection. Neutralization assay results indicate that pre-incubations of LCDV with the five MAbs can significantly decrease the LCDV copy numbers and delay the development of the cytopathic effect in FG cells, revealing that the five MAbs can neutralize the LCDV particles and block viral infection in vitro. The neutralizing MAbs against 32 kDa VAP would be useful for the study on the LCDV–host interaction and might be promising inhibitors of LCDV infection in fish. Full article