P65 Protein Research Articles

The P2 protein of wheat yellow mosaic virus acts as a viral suppressor of RNA silencing in Nicotiana benthamiana to facilitate virus infection.

Wheat yellow mosaic virus (WYMV) causes severe viral wheat disease in Asia. The WYMV P1 protein encoded by RNA2 has viral suppressor of RNA silencing (VSR) activity to facilitate virus infection, however, VSR activity has not been identified for P2 protein encoded by RNA2. In this study, P2 protein exhibited strong VSR activity in Nicotiana benthamiana at the four-leaf stage, and point mutants P70A and G230A lost VSR activity. Protein P2 interacted with calmodulin (CaM) protein, a gene-silencing associated protein, while point mutants P70A and G230A did not interact with it. Competitive bimolecular fluorescence complementation and competitive co-immunoprecipitation experiments showed that P2 interfered with the interaction between CaM and calmodulin-binding transcription activator 3 (CAMTA3), but the point mutants P70A and G230A could not. Mechanical inoculation of wheat with in vitro transcripts of WYMV infectious cDNA clone further confirmed that VSR-deficient mutants P70A and G230A decreased WYMV infection in wheat plants compared with the wild type. In addition, RNA silencing, temperature, ubiquitination and autophagy had significant effects on accumulation of P2 protein in N. benthamiana leaves. In conclusion, WYMV P2 plays a VSR role in N. benthamiana and promotes virus infection by interfering with calmodulin-related antiviral RNAi defense.

The Oncolytic Avian Reovirus p17 Protein Inhibits Invadopodia Formation in Murine Melanoma Cancer Cells by Suppressing the FAK/Src Pathway and the Formation of theTKs5/NCK1 Complex.

To explore whether the p17 protein of oncolytic avian reovirus (ARV) mediates cell migration and invadopodia formation, we applied several molecular biological approaches for studying the involved cellular factors and signal pathways. We found that ARV p17 activates the p53/phosphatase and tensin homolog (PTEN) pathway to suppress the focal adhesion kinase (FAK)/Src signaling and downstream signal molecules, thus inhibiting cell migration and the formation of invadopodia in murine melanoma cancer cell line (B16-F10). Importantly, p17-induced formation of invadopodia could be reversed in cells transfected with the mutant PTENC124A. p17 protein was found to significantly reduce the expression levels of tyrosine kinase substrate 5 (TKs5), Rab40b, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), and matrix metalloproteinases (MMP9), suggesting that TKs5 and Rab40b were transcriptionally downregulated by p17. Furthermore, we found that p17 suppresses the formation of the TKs5/NCK1 complex. Coexpression of TKs5 and Rab40b in B16-F10 cancer cells reversed p17-modulated suppression of the formation of invadopodia. This work provides new insights into p17-modulated suppression of invadopodia formation by activating the p53/PTEN pathway, suppressing the FAK/Src pathway, and inhibiting the formation of the TKs5/NCK1 complex.

Network pharmacology prediction, molecular docking, and molecular dynamics simulation-based strategy to explore the potential mechanism of Huashanshen dripping pill against asthma.

Asthma is a heterogeneous disease characterized by chronic airway inflammation. Huashanshen dripping pills (HSS) are commonly utilized for relieving asthma, relieving cough, and expelling phlegm. At present, the molecular mechanism against airway inflammation remains unclear. In this study, network pharmacology, molecular docking technology, and molecular dynamic simulation were used to predict the therapeutic pathways of HSS for asthma. The ovalbumin-induced mouse model was used to further validate the prediction by RT-qPCR, western blot, immunofluorescence, and related methods. The findings indicate that HSS improves lung function and relieves lung inflammation by reducing inflammatory cell infiltration around the bronchus and reducing eosinophilic counts in bronchoalveolar lavage fluid (BALF). In addition, it lowers the levels of inflammatory cytokines and the expression levels of interleukin-4, interleukin-5, and interleukin-13 mRNA. HSS also inhibits the phosphorylation and nuclear translocation of NF-κB p65 protein. All results suggested that HSS can decrease airway inflammation in asthmatic mice by inhibiting NF-κB signaling pathway. This finding will shed light on how it can be used to treat asthma.

Maize lipid droplet-associated protein 2 is recruited by a virus to enhance viral multiplication and infection through regulating cellular fatty acid metabolism.

Pathogen infection induces massive reprogramming of host primary metabolism. Lipid and fatty acid (FA) metabolism is generally disrupted by pathogens and co-opted for their proliferation. Lipid droplets (LDs) that play important roles in regulating cellular lipid metabolism are utilized by a variety of pathogens in mammalian cells. However, the function of LDs during pathogenic infection in plants remains unknown. We show here that infection by rice black streaked dwarf virus (RBSDV) affects the lipid metabolism of maize, which causes elevated accumulation of C18 polyunsaturated fatty acids (PUFAs) leading to viral proliferation and symptom development. The overexpression of one of the two novel LD-associated proteins (LDAPs) of maize (ZmLDAP1 and ZmLDAP2) induces LD clustering. The core capsid protein P8 of RBSDV interacts with ZmLDAP2 and prevents its degradation through the ubiquitin-proteasome system mediated by a UBX domain-containing protein, PUX10. In addition, silencing of ZmLDAP2 downregulates the expression of FA desaturase genes in maize, leading to a decrease in C18 PUFAs levels and suppression of RBSDV accumulation. Our findings reveal that plant virus may recruit LDAP to regulate cellular FA metabolism to promote viral multiplication and infection. These results expand the knowledge of LD functions and viral infection mechanisms in plants.

Molecular cloning and functional study of a Rel homologous gene in sea urchin Strongylocentrotus intermedius

NF-κB (Nuclear factor-kappa B) family proteins are versatile transcription factors that play crucial regulatory roles in cell development, growth, apoptosis, inflammation, and immune response. However, there is limited research on the function of these key genes in echinoderms. In this study, an NF-κB family gene (SiRel) was identified in sea urchin Strongylocentrotus intermedius. The gene has an open reading frame length of 1809 bp and encodes for 602 amino acids. Domain prediction results revealed that the N-terminal of SiRel protein encodes a conserved Rel homology domain (RHD), including the RHD-DNA binding domain and the RHD-dimerization domain. Multiple sequence comparison results showed that the protein sequences of these two domains were conserved. Phylogenetic analysis indicated that SiRel clustered with Strongylocentrotus purpuratus p65 protein and Rel protein of other echinoderms. Results from quantitative real-time PCR demonstrated detectable SiRel mRNA expression in all tested sea urchin tissues, with the highest expression level found in the gills. And SiRel mRNA expression levels were significantly induced after LPS (Lipopolysaccharide) and poly(I:C) (Polyinosinic:polycytidylic acid) stimulation. In addition, SiRel protein expression can be found in cytoplasm and nucleus of HEK293T cells. Co-immunoprecipitation results showed that SiRel could interact with sea urchin IκB (Inhibitor of NF-κB) protein. Western blotting and dual-luciferase reporter gene assay results indicated that overexpression of SiRel in HEK293T cells could impact the phosphorylation levels of JNK (c-Jun N-terminal kinase) and Erk1/2 (Extracellular signal-regulated kinases1/2) and activate interleukin-6 (IL-6), activating protein 1 (AP-1), interferon (IFN)α/β/γ, and signal transducer and activator of transcription 3 (STAT3) reporter genes in HEK293T cells. In conclusion, this study reveals that SiRel plays an important role in the innate immune response of sea urchins and enriches our understanding of comparative immunology theory.

Glucose prevents the acquisition of the capacitated state in pig spermatozoa.

Mammalian spermatozoa need to undergo a process named capacitation to be able to fertilize an oocyte. During their journey in the female tract, spermatozoa obtain energy while exposed to a changing environment containing a variety of metabolic substrates. The energy requirements for sperm capacitation are species-specific. In addition, the available energy source can hinder the process of sperm capacitation and eventually the acrosome reaction. To evaluate whether the metabolic substrates available in the in vitro sperm capacitation medium allow or interfere with the pig sperm capacitation process. The effect of different metabolic substrates on sperm capacitation process was evaluated by analyzing phosphorylation in the p32 protein; the acrosome reaction and the ATP intracellular content. The presence of glucose in the in vitro capacitating medium diminishes, in a concentration-dependent manner, parameters associated with the capacitated status: induced acrosome exocytosis, plasma membrane destabilization, and protein tyrosine phosphorylation. Conversely, sperm incubation with pyruvate or lactate, either individually or in combination, allows the attainment of the capacitated status. Unexpectedly, pig spermatozoa incubated without any extracellular energy substrates or with a non-metabolizable substrate (l-glucose) for 4h displayed similar sperm viability to the control and exhibited a capacitated phenotype. The capacitation-like phenotype observed in starved pig spermatozoa (absence of glucose, lactate, and pyruvate) was dependent on extracellular bicarbonate and calcium levels, and these spermatozoa exhibited lower intracellular ATP content compared to those not capacitated. Nevertheless, the intracellular content of calcium was not modified in comparison to the control. Our findings suggest that the metabolic substrates used to fuel pig sperm metabolism are important in achieving the capacitated status. The results of this work could be used to refine the capacitating medium employed in pig in vitro fertilization.

Ginkgetin attenuates bone loss in OVX mice by inhibiting the NF-κB/IκBα signaling pathway.

Osteoporosis is a disease associated with bone resorption, characterized primarily by the excessive activation of osteoclasts. Ginkgetin is a compound purified from natural ginkgo leaves which has various biological properties, including anti-inflammation, antioxidant, and anti-tumor effects. This study investigated the bone-protective effects of ginkgetin in ovariectomized (OVX) mice and explored their potential signaling pathway in inhibiting osteoclastogenesis in a mouse model of osteoporosis. Biochemical assays were performed to assess the levels of Ca, ALP, and P in the blood. Micro CT scanning was used to evaluate the impact of ginkgetin on bone loss in mice. RT-PCR was employed to detect the expression of osteoclast-related genes (ctsk, c-fos, trap) in their femoral tissue. Hematoxylin and eosin (H&E) staining was utilized to assess the histopathological changes in femoral tissue due to ginkgetin. The TRAP staining was used to evaluate the impact of ginkgetin osteoclast generation in vivo. Western blot analysis was conducted to investigate the effect of ginkgetin on the expression of p-NF-κB p65 and IκBα proteins in mice. Our findings indicate that ginkgetin may increase the serum levels of ALP and P, while decreasing the serum level of Ca in OVX mice. H&E staining and micro CT scanning results suggest that ginkgetin can inhibit bone loss in OVX mice. The TRAP staining results showed ginkgetin suppresses the generation of osteoclasts in OVX mice. RT-PCR results demonstrate that ginkgetin downregulate the expression of osteoclast-related genes (ctsk, c-fos, trap) in the femoral tissue of mice, and this effect is dose-dependent. Western blot analysis results reveal that ginkgetin can inhibit the expression of p-NF-κB p65 and IκBα proteins in mice. Ginkgetin can impact osteoclast formation and activation in OVX mice by inhibiting the NF-κB/IκBα signaling pathway, thereby attenuating bone loss in mice.

Effects of dietary Chitosan oligosaccharides supplementation on Th17/Treg balance and gut microbiota of early weaned pigeon squabs

Our previous study found that early weaning is associated with decreased growth performance, intestinal barrier impairment, and an imbalance in Th17/Treg in pigeon squabs. Chitosan oligosaccharides (COS) has been substantiated to regulate gut microbiota and restore Th17/Treg equilibrium in mammals, thereby ameliorating growth performance. However, the potential effects of COS in altricial birds remain unclear. Three hundred healthy 7-day-old American king pigeon squabs were selected with similar body weights and randomly divided into 5 groups. The 5 treatment groups were as follows: the control group (CON), fed with artificial pigeon milk; 4 supplementation groups, fed with artificial pigeon milk +100 (COS1), 150 (COS2), 200 (COS3), and 250 (COS4) mg/kg COS, respectively. Results showed that dietary supplementation of COS significantly enhanced the growth performance of weaned squabs. Compared to the CON group, the COS groups exhibited increased villus length and villus area in the jejunum and ileum, accompanied by improvements in morphological structure and mucosal permeability. COS was found to reduce the levels of Th17-associated cytokines and increase the levels of Treg-associated cytokines. COS downregulated the expression of retinoic acid receptor-related orphan receptor C (RORC), a key transcription factor of Th17 cells, while upregulated the expression of Forkhead box protein P3 (FOXP3), a key transcription factor of Treg cells. Dietary COS supplementation increased gut bacterial diversity, altered the relative abundance of several bacteria taxa and enhanced the concentration of short-chain fatty acids (SCFA). Correlation analysis demonstrated a close association between gut microbiota, SCFAs, and indicators related to the Th17/Treg balance. Moreover, we found that SCFAs correlated more strongly with Th17/Treg-related indexes than gut microbiota. These results demonstrated that COS could relieve early weaning stress in pigeon squabs and the optimal dosage of dietary COS supplementation was suggested to be 200 mg/kg. In addition, COS had a protective effect on maintaining intestinal immune balance by modulating microbiota and Th17/Treg related signaling pathways, in which SCFAs might play a crucial role as messengers.

Exosomes originating from neural stem cells undergoing necroptosis participate in cellular communication by inducing TSC2 upregulation of recipient cells following spinal cord injury.

We previously demonstrated that inhibiting neural stem cells necroptosis enhances functional recovery after spinal cord injury. While exosomes are recognized as playing a pivotal role in neural stem cells exocrine function, their precise function in spinal cord injury remains unclear. To investigate the role of exosomes generated following neural stem cells necroptosis after spinal cord injury, we conducted single-cell RNA sequencing and validated that neural stem cells originate from ependymal cells and undergo necroptosis in response to spinal cord injury. Subsequently, we established an in vitro necroptosis model using neural stem cells isolated from embryonic mice aged 16-17 days and extracted exosomes. The results showed that necroptosis did not significantly impact the fundamental characteristics or number of exosomes. Transcriptome sequencing of exosomes in necroptosis group identified 108 differentially expressed messenger RNAs, 104 long non-coding RNAs, 720 circular RNAs, and 14 microRNAs compared with the control group. Construction of a competing endogenous RNA network identified the following hub genes: tuberous sclerosis 2 (Tsc2), solute carrier family 16 member 3 (Slc16a3), and forkhead box protein P1 (Foxpl). Notably, a significant elevation in TSC2 expression was observed in spinal cord tissues following spinal cord injury. TSC2-positive cells were localized around SRY-box transcription factor 2-positive cells within the injury zone. Furthermore, in vitro analysis revealed increased TSC2 expression in exosomal receptor cells compared with other cells. Further assessment of cellular communication following spinal cord injury showed that Tsc2 was involved in ependymal cellular communication at 1 and 3 days post-injury through the epidermal growth factor and midkine signaling pathways. In addition, Slc16a3 participated in cellular communication in ependymal cells at 7 days post-injury via the vascular endothelial growth factor and macrophage migration inhibitory factor signaling pathways. Collectively, these findings confirm that exosomes derived from neural stem cells undergoing necroptosis play an important role in cellular communication after spinal cord injury and induce TSC2 upregulation in recipient cells.

Sinomenine hydrochloride improves DSS-induced colitis in mice through inhibition of the TLR2/NF-κB signaling pathway

BackgroundSinomenine hydrochloride (SH) has anti-inflammatory and immunosuppressive effects, and its effectiveness in inflammatory diseases, such as rheumatoid arthritis, has been demonstrated. However, whether SH has a therapeutic effect on dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in mice and its mechanism of action have not been clarified. This study aimed to investigate the therapeutic effects and mechanism of action of SH on UC. MethodsTwenty-four mice were randomly divided into control, model, SH low-dose (SH-L, 20mg/kg), and SH high-dose (SH-H, 60mg/kg) groups with six mice in each group. Disease activity index (DAI), colonic mucosal damage index, and colonic histopathology scores were calculated. The expression levels of related proteins, genes, and downstream inflammatory factors in the Toll-like receptor 2/NF-κB (TLR2/NF-κB) signaling pathway were quantified. ResultsSH inhibited weight loss, decreased DAI and histopathological scores, decreased the expression levels of TLR2, MyD88, P-P65, P65 proteins, and TLR2 genes, and also suppressed the expression of inflammatory factors TNF-α, IL-1 β, and IL-6 in the peripheral blood of mice. ConclusionThe therapeutic effect of SH on DSS-induced UC in mice may be related to the inhibition of the TLR2/NF-κB signaling pathway.

Microsecretory adenocarcinoma of the hard palate: a case report and literature review

Microsecretory adenocarcinoma (MSA) is a new type of salivary gland neoplasm identified in the 2022 World Health Organization Classification of Head and Neck Tumour (Skalova et al., Head Neck Pathol 16:40-53, 2022) and is characterized by a unique set of histomorphologic and immunohistochemical features and a recurrent MEF2C::SS18 fusion. MSA was initially misdiagnosed as another salivary gland tumour due to its similar morphology; until recently, only fewer than 50 cases were reported. We present a case of MSA of the hard palate with diverse architectural growth patterns, bland cytological features, abundant basophilic intraluminal secretions and fibromyxoid stroma. The tumour cells were positive for the SOX10, S100, and p63 protein and negative for the p40 protein according to immunohistochemistry. SS18 gene rearrangement was demonstrated via break-apart fluorescence in situ hybridization. We also provided a comprehensive literature review and integrated the clinicopathological features, immunophenotype, and molecular alterations of the disease. A comprehensive understanding of MSA enables us to accurately distinguish and categorize MSA from other salivary gland tumours with analogous morphologies.

Hinesol attenuates DSS-induced ulcerative colitis through the suppression of Src-mediated NF-κB and chemokine signaling pathway.

As a common inflammatory bowel disease, ulcerative colitis (UC) is featured with inflammation, oxidative damage, and the impairment of intestinal mucosal barrier, which bring threat to patients' quality of live. Hinesol, derived from Atractylodes lancea, is a unique sesquiterpenoid. Our study proposed to survey the effects and mechanism of hinesol in UC. UC mouse model was constructed using dextran sulfate sodium (DSS). Lipopolysaccharide (LPS) was applied for RAW264.7 cells stimulation to construct cell inflammatory model. The changes of disease activity index (DAI), body weight, colon length, and intestinal pathology in mice were analyzed to estimate the severity of colitis. Enzyme-linked immunosorbent assay was applied to check the changes of interleukin (IL)-1β, IL-18, IL-6, and tumor necrosis factor (TNF)-α. The levels of myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione peroxidase (GSH-px), catalase (CAT), and malondialdehyde (MDA) were estimated by corresponding reagent kit. The changes of phosphorylated (p)-NF-κB P65, and p-IκBα, ZO-1, Occludin, Claudin-1, Src, XCL1, CCL2, and CXCL16 protein were examined using western blot. Flow cytometry and cell counting kit-8 assay were utilized for assessment of cell apoptosis and viability. We found that DSS reduced mice body weight, increased DAI, shorten colon length, and led to severe enteric mucosal injury, while hinesol improved the above symptoms induced by DSS. In DSS mice, hinesol raised the levels of ZO-1, Occludin, Claudin-1, SOD, GSH-px, and CAT and decreased the levels of TNF-α, IL-18, IL-1β, IL-6, MPO, and MDA. Additionally, in DSS mice and LPS-stimulated RAW264.7 cells, hinesol inhibited the high expression of Src, XCL1, CCL2, CXCL16, p-NF-κB P65, and p-IκBα. The molecular docking showed that there was a good interaction between hinesol and Src. Moreover, in LPS-stimulated RAW 264.7 cells, Src overexpression partially reversed the inhibition of hinesol on cell apoptosis, pro-inflammatory factors, and oxidative stress. In conclusion, hinesol alleviated DSS-induced colitis, which might have a bearing on the inhibition of Src-mediated NF-κB and chemokine signaling pathway.

Sevoflurane impedes neuropathic pain by maintaining endoplasmic reticulum stress and oxidative stress homeostasis through inhibiting the activation of the PLCγ/CaMKII/IP3R signaling pathway.

To investigate the effect of sevoflurane on neuropathic pain induced by chronic constriction injury (CCI) of sciatic nerve in mice, and to elucidate its mechanism by animal experiments. Thirty-two C57BL/6 mice were randomly divided into four groups: Sham group, Model group, Control group and Sevoflurane group. First, a mouse model of neuropathic pain was established. Then, the mice in each group were killed on Day 14 after operation to harvest the enlarged lumbosacral spinal cord. In contrast with the Model group, the Sevoflurane group displayed a significantly increased paw withdrawal mechanical threshold (PWMT) and significantly prolonged paw withdrawal thermal latency (PWTL) from Day 5 after operation. The morphological changes of lumbosacral spinal cord were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy. Pathological results showed that sevoflurane reduced nuclear pyknosis in lumbosacral spinal cord tissue, with a large number of mitochondrial crista disappearance and mitochondrial swelling. The results of Western blotting showed that sevoflurane significantly decreased the protein expressions of phosphorylated phospholipase Cγ (p-PLCγ), phosphorylated calcium/calmodulin-dependent protein kinase II (p-CaMKII) and phosphorylated inositol 1,4,5-triphosphate receptor (p-IP3R), and reduced the protein expressions of endoplasmic reticulum (ER) stress proteins glucose-regulated protein 78 (GRP78) and GRP94, oxidative stress-related proteins P22 and P47 and inflammatory factors nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), interleukin-1 β (IL-1β), and tumor necrosis factor-α (TNF-α). Sevoflurane inhibits neuropathic pain by maintaining ER stress and oxidative stress homeostasis through inhibiting the activation of the PLCγ/CaMKII/IP3R signaling pathway.

The cytochrome P4501A1 (CYP1A1) inhibitor bergamottin enhances host tolerance to multidrug-resistant Vibrio vulnificus infection

PurposeVibrio vulnificus (V. Vulnificus) infection is characterized by rapid onset, aggressive progression, and challenging treatment. Bacterial resistance poses a significant challenge for clinical anti-infection treatment and is thus the subject of research. Enhancing host infection tolerance represents a novel infection prevention strategy to improve patient survival. Our team initially identified cytochrome P4501A1 (CYP1A1) as an important target owing to its negative modulation of the body's infection tolerance. This study explored the superior effects of the CYP1A1 inhibitor bergamottin compared to antibiotic combination therapy on the survival of mice infected with multidrug-resistant V. Vulnificus and the protection of their vital organs. MethodsAn increasing concentration gradient method was used to induce multidrug-resistant V. Vulnificus development. We established a lethal infection model in C57BL/6J male mice and evaluated the effect of bergamottin on mouse survival. A mild infection model was established in C57BL/6J male mice, and the serum levels of creatinine, urea nitrogen, aspartate aminotransferase, and alanine aminotransferase were determined using enzyme-linked immunosorbent assay to evaluate the effect of bergamottin on liver and kidney function. The morphological changes induced in the presence of bergamottin in mouse organs were evaluated by hematoxylin and eosin staining of liver and kidney tissues. The bacterial growth curve and organ load determination were used to evaluate whether bergamottin has a direct antibacterial effect on multidrug-resistant V. Vulnificus. Quantification of inflammatory factors in serum by enzyme-linked immunosorbent assay and the expression levels of inflammatory factors in liver and kidney tissues by real-time quantitative polymerase chain reaction were performed to evaluate the effect of bergamottin on inflammatory factor levels. Western blot analysis of IκBα, phosphorylated IκBα, p65, and phosphorylated p65 protein expression in liver and kidney tissues and in human hepatocellular carcinomas-2 and human kidney-2 cell lines was used to evaluate the effect of bergamottin on the nuclear factor kappa-B signaling pathway. One-way ANOVA and Kaplan-Meier analysis were used for statistical analysis. ResultsIn mice infected with multidrug-resistant V. Vulnificus, bergamottin prolonged survival (p = 0.014), reduced the serum creatinine (p = 0.002), urea nitrogen (p = 0.030), aspartate aminotransferase (p = 0.029), and alanine aminotransferase (p = 0.003) levels, and protected the cellular morphology of liver and kidney tissues. Bergamottin inhibited interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α expression in serum (IL-1β: p = 0.010, IL-6: p = 0.029, TNF-α: p = 0.025) and inhibited the protein expression of the inflammatory factors IL-1β, IL-6, TNF-α in liver (IL-1β: p = 0.010, IL-6: p = 0.011, TNF-α: p = 0.037) and kidney (IL-1β: p = 0.016, IL-6: p = 0.011, TNF-α: p = 0.008) tissues. Bergamottin did not affect the proliferation of multidrug-resistant V. Vulnificus or the bacterial load in the mouse peritoneal lavage fluid (p = 0.225), liver (p = 0.186), or kidney (p = 0.637). ConclusionBergamottin enhances the tolerance of mice to multidrug-resistant V. Vulnificus infection. This study can serve as a reference and guide the development of novel clinical treatment strategies for V. Vulnificus.

Flavonoid profiling of Plumula nelumbinis and evaluation of their anti‐inflammatory effects on lipopolysaccharide‐induced RAW 264.7 macrophages

AbstractThe lotus seed embryo, known as “Plumula nelumbinis,” holds significance in Chinese culture as a traditional medicinal and edible herb rich in beneficial bioactive compounds like flavonoids, alkaloids, and polyphenols. Using ultrahigh‐performance liquid chromatography‐quadrupole‐exactive Orbitrap mass spectrometry, we meticulously analyzed the total flavonoids in Plumula nelumbinis (PNF) sourced from Hangzhou's West Lake. Our investigation preliminarily identified 29 flavonoid compounds, including 21 flavonoid O‐glycosides, five flavonoid C‐glycosides, and three aglycones, through comparisons with standards and literature references. Subsequent cellular anti‐inflammatory experiments using lipopolysaccharide (LPS)‐induced RAW264.7 cells demonstrated that treatment with 300 μg/mL of lotus seed flavonoids significantly reduced inflammatory factors' production, such as nitric oxide, prostaglandin E2, tumor necrosis factor, interleukin‐6, and interleukin‐1β. Additionally, PNF effectively lowered intracellular reactive oxygen species levels, mitigated LPS‐induced cell apoptosis, and nuclear translocation of nuclear factor‐κB (NF‐κB) p65 protein, collectively suppressing the NF‐κB p65 inflammatory pathway and demonstrating potent anti‐inflammatory properties. Crucially, the observed gene expression patterns of inflammatory factors aligned with their respective protein secretion levels. This study provides a comprehensive exploration of lotus seed flavonoids' anti‐inflammatory potential, highlighting their significance in potential therapeutic applications and laying the groundwork for future advancements in related functional foods.

Antiproliferative Effect of 7-Ketositosterol in Breast and Liver Cancer Cells: Possible Impact on Ceramide, Extracellular Signal-Regulated Kinases, and Nuclear Factor Kappa B Signaling Pathways.

Background: This study aimed to examine the effect of 7-Ketositosterol (7-KSS), on sphingomyelin/ceramide metabolites and apoptosis in human breast MCF-7 and human liver HepG2 cancer cells. Methods: Anti-proliferative effects of 7-KSS treatment were assessed at different concentrations and periods. Cell viability was assessed through MTT analysis, whereas the levels of sphingosine-1-phosphate (S1P), sphingomyelins (SMs), and ceramides (CERs) were measured using LC-MS/MS. Phosphorylated 44/42 ERK1/2 and NF-κB p65 (Ser536) protein levels were measured by Western blot analysis and immunofluorescence staining. Apoptosis was evaluated by TUNEL staining and flow cytometric assessment of annexin-V and propidium iodide (PI) labeling. Results: Treatment with 7-KSS significantly decreased cell survival and S1P, p-44/42 ERK1/2, and p-NF-κB p65 protein levels in cancer cells compared to controls. A substantial rise was detected in intracellular amounts of C16-C24 CERs and apoptosis in cancer cells incubated with 7-KSS. Conclusions: 7-KSS stimulated ceramide accumulation and apoptosis while decreasing cell proliferation via downregulating S1P, p-44/42 ERK1/2, and p-NF-κB p65 protein levels.

An ultrasensitive strip sensor for rapid detection of African swine fever virus

African swine fever (ASF) is a highly contagious disease caused by the African swine fever virus (ASFV) infecting pigs, which has caused huge economic losses in countries around the world. Currently, there is no effective vaccine, and the prevention and control of ASF is mainly through rapid detection, so it is particularly important to carry identify and develop rapid detection methods for ASFV. In this study, recombinant plasmid PET-28a(+)-p30 was constructed, and the recombinant protein was obtained by inducing expression and Ni+ resin affinity column purification. Mice were immunized with recombinant p30 protein, and after three immunizations, ten strains of hybridoma cells that stably secreted monoclonal antibodies (mAbs) against p30 protein were obtained by cell fusion and subcloning. A colloidal gold immunochromatography assay (GICA) based on double antibody sandwich technology was established to screen the paired antibodies, and the trapping and detecting antibodies were mAb-11F11 and mAb-7A8, respectively, with a detection limit of 1 ng/mL, which laid an important material foundation for the early detection of ASF in the future.

Frequency of Euroglyphus maynei sensitization in respiratory allergies: a real-life study with bioinformatic analysis and geographical exploration of allergen prevalence.

Dermatophagoides pteronyssinus and Dermatophagoides farinae belong to the family Pyroglyphidae (subfamily: "Dermatophagoidinae") and have the respective allergenic proteins of Der p1, Der p2, and Der p23 and Der f1 and Der f2. Euroglyphus maynei, belongs to the family Pyroglyphidae (subfamily: "Pyroglyphinae") and its main allergenic protein is Eur m1, a source of sensitization. Sensitization to D. pteronyssinus and D. farinae is assessed through skin tests, while sensitization to E. maynei is assessed less frequently. This experimental work aims to analyze the prevalence of sensitization to E. maynei in patients with respiratory allergies treated at M. Albanesi Allergy and Immunology Unit in Bari, Italy, and the sequence homology of major allergenic proteins of E. maynei with D. farinae and D. pteronyssinus was analyzed. In this real-life study, 65 patients were enrolled. In particular, patients with respiratory allergy were subjected to skin prick tests for common respiratory allergens, including Euroglyphus maynei. The sequence homology analysis was performed between the major allergenic proteins of E. maynei and those of D. pteronyssinus and D. farinae. Sensitization to E. maynei accounts for 41.5% of patients. All patients with E. maynei sensitization had concomitant sensitization to D. farinae and D. pteronyssinus. The analysis of sequence homology of Der p1 and Der f1 proteins with the sequence of Eur m1 protein demonstrated an identity of 84.4% and 86%, respectively. Nearly 50% of house dust mites-sensitized patients have a concomitant sensitization to E. maynei. The cross-sensitization could be due to Der f1, Der p1, and Eur m1 similarity.

Combination of high anti-SKI and low anti-TMED5 antibody levels is preferable prognostic factor in esophageal carcinoma.

Given that esophageal cancer is highly malignant, the discovery of novel prognostic markers is eagerly awaited. We performed serological identification of antigens by recombinant cDNA expression cloning (SEREX) and identified SKI proto-oncogene protein and transmembrane p24 trafficking protein 5 (TMED5) as antigens recognized by serum IgG antibodies in patients with esophageal carcinoma. SKI and TMED5 proteins were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens. The serum anti-SKI antibody (s-SKI-Ab) and anti-TMED5 antibody (s-TMED5-Ab) levels were significantly higher in 192 patients with esophageal carcinoma than in 96 healthy donors. The presence of s-SKI-Abs and s-TMED5-Abs in the patients' sera was confirmed by western blotting. Immunohistochemical staining showed that the TMED5 protein was highly expressed in the cytoplasm and nuclear compartments of the esophageal squamous cell carcinoma tissues, whereas the SKI protein was localized predominantly in the nuclei. Regarding the overall survival in 91 patients who underwent radical surgery, the s-SKI-Ab-positive and s-TMED5-Ab-negative statuses were significantly associated with a favorable prognosis. Additionally, the combination of s-SKI-Ab-positive and s-TMED5-Ab-negative cases showed an even clearer difference in overall survival as compared with that of s-SKI-Ab-negative and s-TMED5-Ab-positive cases. The s-SKI-Ab and s-TMED5-Ab biomarkers are useful for diagnosing esophageal carcinoma and distinguishing between favorable and poor prognoses.

Complement regulatory protein CD46 promotes bladder cancer metastasis through activation of MMP9.

CD46, a transmembrane protein known for protecting cells from complement‑mediated damage, is frequently dysregulated in various types of cancer. Its overexpression in bladder cancers safeguards the cancer cells against both complement and antibody‑mediated cytotoxicity. The present study explored a new role of CD46 in facilitating cancer cell invasion and metastasis, examining its regulatory effect on matrix metalloproteases (MMPs) and their effect on the metastatic capability of bladder cancer cells. Specifically, CD46 alteration positively influenced MMP9 expression, but not MMP2, in several bladder cancer cell lines. Furthermore, CD46 overexpression triggered phosphorylation of p38 MAPK and protein kinase B (AKT), leading to enhanced activator protein 1 (AP‑1) activity via c‑Jun upregulation. The inhibition of p38 or AKT pathways attenuated the CD46‑induced MMP9 and AP‑1 upregulation, indicating that the promotion of MMP9 by CD46 involved activating both p38 MAPK and AKT. Functionally, the upregulation of MMP9 by CD46 translated to increased migratory and invasive capabilities of bladder cancer cells, as well as enhanced in vivo metastasis. Overall, the present study revealed a novel role for CD46 as a metastasis promoter through MMP9 activation in bladder cancers and highlighted the regulatory mechanism of CD46‑mediated MMP9 promotion via p38 MAPK and AKT activation.