Search Results (3,395)

Phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, is associated with adaptation to environmental stress. However, its potential role in diapause is not known. Here, two cytoplasmic genes encoding PEPCK (SmPEPCK1-1 and SmPEPCK1-2) in Sitodiplosis mosellana, a significant wheat pest undergoing obligatory larval diapause during the third instar, were cloned, and their expression patterns during diapause and thermal stress were assessed, together with the effects of SmPEPCK1-2 knockdown on larval development. Both SmPEPCK1-1 and SmPEPCK1-2 were evolutionarily conserved and contained canonical functional domains and motifs. Their expression was induced by diapause, and was found to be tissue-specific, with the highest levels observed in the fat bodies of diapausing larvae. Furthermore, exposure to heat stress in oversummering larvae or cold stress in overwintering larvae enhanced the expression of both genes within specific temperature ranges (35–40 °C and −10–0 °C, respectively). RNA interference-mediated knockdown of SmPEPCK1-2 did not affect cocoon-breaking rates and timing but significantly prolonged the duration of larval development from cocoon-breaking to pupation. These findings indicate that both SmPEPCK genes are closely involved in tolerance to diapause-related stress, with SmPEPCK1-2 also contributing to the regulation of larval development. Full article

The increasing prevalence of multidrug-resistant (MDR) Campylobacter species poses a serious threat to food safety and public health, highlighting the urgent need for natural antimicrobial alternatives to conventional antibiotics. This study investigated the antibacterial potential and mechanism of action of seven essential oils (EOs), Cymbopogon citratus, Mentha pulegium, Artemisia absinthium, Myrtus communis, Thymus algeriensis, Thymus capitatus, and Eucalyptus globulus, against multidrug-resistant Campylobacter jejuni and Campylobacter coli. The antimicrobial activity was first assessed by the agar disk diffusion and broth microdilution methods to determine inhibition zones, minimum inhibitory concentrations (MICs), and minimum bactericidal concentrations (MBCs). The most active EOs were further evaluated through time–kill kinetics, cell lysis, salt tolerance, and membrane integrity assays to elucidate their bactericidal mechanisms. Results showed that E. globulus, T. algeriensis, and M. communis exhibited the strongest inhibitory effects, particularly against C. jejuni, with MIC values ranging from 3.125% to 6.25%, while C. coli was more resistant. Time–kill and lysis experiments demonstrated rapid bacterial reduction and significant decreases in optical density, indicating cell disruption. Additionally, EO treatments reduced salt tolerance and induced leakage of cytoplasmic materials, confirming membrane damage. Overall, these findings suggest that selected essential oils exert potent antimicrobial effects through membrane disruption and osmotic imbalance, offering promising natural strategies to control MDR Campylobacter in food systems. The application of such bioactive compounds could contribute significantly to improving food quality, extending shelf life, and enhancing food safety. Full article

Following the downregulation of testicular endocrine and germinative function by slow-release gonadotropin-releasing hormone (GnRH)-agonist implants, testicular functions are quickly restored after implant removal. As an intact blood–testis barrier (BTB) is crucial for normal spermatogenesis and its integrity is FSH- and androgen-dependent, alterations in the BTB gene and protein expressions during downregulation and subsequent restart seem inevitable. We investigated occludin (OCLN), claudin (CLDN) 3, 5, 11, and connexin (CX) 43 mRNA-, and CLDN11 and CX43 protein expressions during GnRH implant-induced downregulation (W0) and restart of spermatogenesis after implant removal (week, W, 3–12). Untreated juvenile (JG) and adult dogs (CG) served as controls. Sertoli cells were significantly affected by treatment (reduced nuclear area, OCLN, and CLDN5 expressions). All investigated genes (except CLDN3) differed significantly during restart (W0–12) compared with CG (p < 0.05). CLDN11 and CX43 immunopositive staining was absent or diffuse cytoplasmic at downregulation and relocated at W9, indicating disruption and subsequent restorage of BTB. As W0 and JG differed considerably, our results suggest that the model cannot mimic puberty. In conclusion, GnRH implant-induced long-term gonadotropin suppression disrupts testicular CX43 and CLDN11 distribution and changes gap and tight junction mRNA expression. Treatment effects are reversible suggesting re-establishment of the BTB. Full article

Background: The apicomplexan parasite Toxoplasma gondii causes serious diseases in animals and humans. The in vitro efficacy of the antimicrobial peptide mixture tyrothricin, composed of tyrocidines and gramicidins, against T. gondii tachyzoites was investigated. Methods: Effects against T. gondii were determined by monitoring inhibition of tachyzoite proliferation and electron microscopy, host cell and splenocyte toxicity was measured by Alamar blue assay, and early embryo toxicity was assessed using zebrafish embryos. Differential affinity chromatography coupled to mass spectrometry and proteomics (DAC-MS-proteomics) was employed to identify potential molecular targets in T. gondii cell-free extracts. Results: Tyrothricin inhibited T. gondii proliferation at IC₅₀s < 100 nM, with tyrocidine A being the active and gramicidin A the inactive component. Tyrothricin also impaired fibroblast, T cell and zebrafish embryo viability at 1 µM. Electron microscopy carried out after 6 h of treatment revealed cytoplasmic vacuolization and structural alterations in the parasite mitochondrion, but these changes appeared only transiently, and tachyzoites recovered after 96 h. Tyrothricin also induced a reduction in the mitochondrial membrane potential. DAC-MS-proteomics identified 521 proteins binding only to tyrocidine A. No specific binding to gramicidin A was noted, and four proteins were common to both peptides. Among the proteins binding specifically to tyrocidine A were several SRS surface antigens and secretory proteins, mitochondrial inner and outer membrane proteins associated with the electron transfer chain and porin, and several calcium-binding proteins putatively involved in signaling. Discussion: These results suggest that tyrocidine A potentially affected multiple pathways important for parasite survival and development. Full article

The presence of aberrant double-stranded DNA (dsDNA) in the cytoplasm of cells is sensed by unique pattern recognition receptors (PRRs) to trigger innate immune response. The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) signaling pathway is activated by the presence of non-self or mislocalized self-dsDNA from nucleus or mitochondria released in response to DNA damage or cellular stress in the cytoplasm. Activation of cGAS leads to the synthesis of the second messenger cyclic GMP–AMP (cGAMP), which binds and activates STING, triggering downstream signaling cascades that result in the production of type I interferons (IFNs) and proinflammatory cytokines. Here, we show that diverse immunostimulatory dsDNA ligands and chemotherapy agents like Doxorubicin and Taxol trigger the integrated stress response (ISR) by activating endoplasmic reticulum (ER) stress kinase, protein kinase RNA-like ER kinase (PERK), in addition to the canonical IFN pathways. PERK-mediated phosphorylation and inactivation of the alpha subunit of eukaryotic translation initiation factor-2 (eIF2α) result in the formation of stress granules (SGs). SG formation by dsDNA was significantly reduced in PERK knockout cells or by inhibiting PERK activity. Transcriptional induction of IFNβ and cytokines, ISR signaling, and SG formation by dsDNA was dampened in cells lacking PERK activity, STING, or key stress-granule nucleating protein, Ras-GAP SH3 domain-binding protein 1 (G3BP1), demonstrating an important role of the signal transduction pathway mediated by STING and SG assembly. Lastly, STING regulates reactive oxygen species (ROS) production in response to DNA damage, highlighting the crosstalk between DNA sensing and oxidative stress pathways. Together, our data identify STING–PERK–G3BP1 signaling axis that couples cytosolic DNA sensing to stress response pathways in maintaining cellular homeostasis. Full article

Disruption of the blood–milk barrier and inhibition of enzymatic activity caused by abnormal external stimuli, accompanied by the occurrence of autophagy, are among the major factors contributing to the onset of clinical mastitis (CM) in dairy cows. However, the molecular mechanisms through which external stimuli and autophagy regulate CM in dairy cows are not fully understood. This study examined mammary gland (MG) tissue samples collected from healthy dairy cows and those with CM caused by Staphylococcus aureus (n = 3 per group) to observe histological changes and autophagic phenomena, identify candidate biomolecular targets involved in external stimuli in dairy cows affected by mastitis through proteomic and bioinformatic analyses, and analyze their expression and distribution patterns in MG tissues. Pathological examination revealed that the MG tissues of the CM group exhibited significant alveoli collapse and inflammatory cell infiltration, accompanied by autolysosome and phagolysosome activation, and elevated expression of lysosomal and autophagic markers. Bioinformatic analysis identified five biological processes (BPs) and 144 differentially expressed proteins (DEPs) associated with external stimuli, among which beclin 1 (BECN1) was involved in all five BPs. Pathway enrichment analysis revealed that BECN1 participated in six autophagy-related signaling pathways. BECN1 was localized in the cytoplasm of mammary epithelial cells, and both mRNA and protein levels of BECN1 were significantly upregulated in the CM group compared with those in the controls (p < 0.01). These findings suggest that BECN1 expression is closely associated with CM in dairy cows and correlates with autophagy-related responses to external stimuli, and its elevated expression is positively correlated with Staphylococcus aureus–induced CM severity. Our results offer preliminary observations relevant to the molecular mechanisms by which BECN1, the autophagy-regulating biomolecule BECN1 influences the development of CM. Full article

Chronic kidney disease (CKD) accelerates vascular dysfunction and cardiovascular disease, partly through the accumulation of the uraemic toxin indoxyl sulphate (IS). Thrombospondin-1 (TSP1) and its receptor CD47 have been implicated in vascular pathology, but their role in CKD-associated vascular remodelling is unknown. We investigated the contribution of TSP1–CD47 signalling to vascular smooth muscle cell (VSMC) dysfunction in CKD. Human aortic VSMCs (hVSMCs) were exposed to IS, TSP1, or plasma from patients with CKD. CKD was induced in wild-type (WT) and CD47-deficient (CD47KO) mice using 5/6 nephrectomy. Vascular changes were assessed by histology, immunohistochemistry, and molecular analyses. IS, TSP1, and CKD plasma increased TSP1 expression in hVSMCs, reduced proliferation, elevated β-galactosidase activity, and activated phosphorylated ERK1/2 and cytoplasmic aryl hydrocarbon receptor. These effects were attenuated by CD47 blockade. CKD plasma further enhanced IS- and TSP1-induced senescence. In vivo, 5/6 nephrectomy induced aortic wall thickening in WT but not in CD47KO mice. Aortic pERK1/2 was reduced in CD47KO mice despite persistent TSP1 upregulation. IS and TSP1 promote VSMC senescence through CD47-dependent ERK1/2 and AhR signalling. CD47 deletion protects against CKD-induced vascular remodelling, suggesting that CD47 blockade may represent a novel therapeutic strategy to mitigate vascular complications in CKD. Full article

One group of human proteins found in the cytoplasm, but not in the nucleus, is characterized by the presence of short (6–9 aa), specific amino acid sequences thought to be involved in retaining proteins in the cytoplasm (cytoplasmic retention sequences). While strong evidence supports the ability of some peptides to act in this way, the number of such supported cases is small. We have taken the view that the situation would be improved by enhancing the methods available to identify cytoplasmic retention (CR) sequences. Here, we describe an appropriate bioinformatic method to identify CR peptides using information about their location at the ends of cytoplasmic proteins. The method was then used to link seven different human cytoplasmic proteins with sequences suggested to have cytoplasmic retention activity. Further bioinformatic analysis was carried out with isoforms of the cytoplasmic proteins identified. Amino acid sequence information showed that while the proposed CR amino acid sequences can be the same or distinct in different protein isoforms, they are always located at the same site in the protein. For instance, while the proposed retention sequence of CCDC57 isoform X18 is MLARLVSNS, in isoform 7 it is SEPALNEL, yet the two sequences are each located between amino acids 5 and 13 in the CCDC57 sequence. The results support the view that the protein isoform is involved in determining the location of the CR sequence in a protein, while the amino acid sequence itself affects other variables such as the sub-region of the cytoplasm the protein needs to occupy. Overall, the study yielded identification of 15 candidate CR peptides in which 10 of the 15 have unrelated amino acid sequences. Full article

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), poses a severe threat to global wheat production. The adaptor protein complex AP-1 plays a crucial role in vesicular trafficking, yet its function in rust fungi remains poorly understood. In this study, a gene encoding an AP-1 σ subunit, designated PsAP1, was identified in Pst. The expression of PsAP1 was highly induced during the early infection stage. Heterologous expression of PsAP1 in a Fusarium graminearum mutant partially restored its pathogenic defects. Subcellular localization analysis revealed that PsAP1 localizes to the plasma membrane, cytoplasm, and nucleus. Silencing PsAP1 in wheat using Barley stripe mosaic virus-mediated host-induced gene silencing (BSMV-HIGS) significantly attenuated Pst pathogenicity, reducing hyphal growth by 6.7% (colony diameter), sporulation by 61.6% (lesion length), and pathogen biomass by 66%, along with enhanced accumulation of host reactive oxygen species. Transcriptomic analysis further demonstrated that silencing PsAP1 disrupted multiple pathways, including MAPK signaling, glutathione metabolism, and carbohydrate metabolism. These findings indicate that PsAP1 facilitates Pst infection by modulating vesicular trafficking, suppressing host immunity, and reprogramming host metabolism. This study provides novel insights into the pathogenic mechanisms of rust fungi and suggests a potential target for disease control. Full article

Dysfunction of the membrane transporter P5B-ATPase 13A2 (ATP13A2) has been linked to neurodegenerative disorders, while its overexpression has been associated with colorectal cancer. ATP13A2 localizes to lysosomes and late endosomes, where it exports polyamines such as spermine into the cytosol. We previously showed that ATP13A2 expression alters lipid homeostasis and reduces the levels of bis(monoacylglycero)phosphate (BMP), an anionic phospholipid essential for lipid digestion in acidic compartments, suggesting that ATP13A2-mediated spermine export may affect lysosomal lipid catabolism. α/β-hydrolase domain-containing 6 (ABHD6), the enzyme responsible for BMP catabolism, was detected by immunofluorescence and immunoblot analysis in SH-SY5Y cells overexpressing human ATP13A2 and treated with spermine. The activities of the lipid-degrading hydrolases acid ceramidase (ACase) and glucocerebrosidase (GCase) were measured using specific fluorogenic substrates. ATP13A2-expressing cells showed higher ABHD6 expression, and spermine treatment promoted its translocation to the cytoplasm. Spermine induced a transient increase in ACase activity, followed by a stronger inhibition in ATP13A2-expressing cells. Moreover, GCase activity was elevated in these cells but also showed greater spermine-induced inhibition. Altogether, these results suggest that ATP13A2-mediated spermine export modulates the lipid digestion capacity of the endolysosomal system and support a functional interplay between polyamine and lipid metabolism in these organelles. Full article

Foot-and-mouth disease virus (FMDV) infection elicits sustained, high-level interleukin-10 (IL-10) secretion in cattle and pigs, which correlates with lymphopenia and immunosuppression. We previously showed that macrophages are the principal source of IL-10 during FMDV infection in mice, but the viral trigger and host pathways remained unknown. In the present study, we examined whether the FMDV structural protein VP3 regulates IL-10 expression. To this end, a eukaryotic VP3 expression vector was transfected into porcine alveolar macrophages (3D4/21 cells), and IL-10 expression together with related signaling pathways was interrogated by qRT-PCR, ELISA, Western blot, co-immunoprecipitation (Co-IP), confocal microscopy, and luciferase reporter assays. The results showed that VP3 significantly increased IL-10 mRNA and protein levels (p < 0.001) in a time-dependent manner. Mechanistically, VP3 promoted phosphorylation of PI3K, AKT, and mTOR; this effect was abolished by the PI3K inhibitor LY294002, which also abrogated VP3-induced IL-10 secretion (p < 0.05). Furthermore, VP3 upregulated mRNA expression of STAT3, ATF1, and CREB (p < 0.05) and enhanced IL-10 promoter activity. The STAT3 inhibitor Stattic reduced IL-10 secretion by 22% (p < 0.05). Co-IP and confocal microscopy confirmed direct binding of VP3 to PI3K in the cytoplasm. In conclusion, FMDV VP3 induces IL-10 overexpression by directly activating the PI3K/AKT-mTOR signaling pathway, thereby elucidating a key mechanism of FMDV-induced immunosuppression. Full article

Heat shock protein (HSP) gene families are known to be expressed preferentially over normal cellular proteins when poultry cells are exposed to environmental stressors. Bordetella avium infection leads to bordetellosis, which is characterized by severe inflammation of the tracheal epithelium, representing a severe stressor for the infected turkey poult. The purpose of this experiment was to examine the expression of HSP60 and HSP70 in the tracheae of B. avium-challenged poults. Tracheae were dissected from five poults per treatment at 7, 11, 14, and 21 days of age, and snap-frozen rings were examined immunohistochemically for the presence of HSP60 and HSP70 using HSP60 and HSP70 monoclonal antibodies (MABs). Infected poults expressed HSP60 predominantly on the apical surface of ciliated epithelial cells and weakly near the nucleus, and HSP70 was expressed only in the cytoplasm. Early in the development of bordetellosis, infected poults expressed more HSP60 and HSP70 than control poults of the same age, but at 14 days after infection, infected birds showed significantly decreased HSP60 proteins, which was associated with loss of tracheal epithelium. With loss of tracheal epithelium, the expression of HSP70 decreased at 11 days in the infected poults, and by day 21, very low levels of the proteins were observed in the infected poults compared to control poults with intact tracheal epithelium. It was concluded that decreased expression of HSP60 was indicative of HSP60 proteins becoming antigenic targets for the host’s immune system since immunologically targeted B. avium cells express GroEL (highly conserved HSP60 family equivalent) and DnaK (highly conserved HSP70 equivalent). The potential targeting of host and pathogen HSP60 by the innate immune system could feasibly facilitate loss of tracheal epithelium during the development of bordetellosis. Full article

Background and Clinical Significance: We present an 85-year-old male case of primary hepatic SCC manifesting as multiple liver nodules with atypical imaging findings. Case Presentation: The patient was negative for hepatitis B surface antigen and hepatitis C virus antibody. Serum tumor markers were all within normal limits. Contrast-enhanced ultrasonography with perflubutane demonstrated hypervascular nodules in the early vascular phase, early washout in the portal phase, and a defect in the postvascular phase (10 mm in S5 and 25 mm in S6). Histopathological examination revealed irregularly shaped tumor cells with large hyperchromatic nuclei and basophilic cytoplasm, surrounded by dense fibrous stroma forming cords, solid nests, and sheet-like structures. Immunohistochemical analysis showed positivity for AE1/AE3, p40, CK5/6, c-kit, and NCAM. Conclusions: The lesions were diagnosed as primary hepatic squamous cell carcinoma and suggested the possible involvement of hepatic progenitor cells, supporting the hypothesis of de novo carcinogenesis. Full article

Background/Objectives: The ineffectiveness of current treatments for glioblastoma underscores the urgent need for effective alternatives. This study aimed to investigate the effectiveness of sodium dichloroacetate (NaDCA) and a sodium valproate NaDCA combination (NaVPA–NaDCA) on formed patients’ primary cell tumors on the chick embryo chorioallantoic membrane (CAM). Methods: Glioblastoma tissue samples were obtained from three patients during tumor surgery. WHO grade IV, IDH wild-type, and a strong positive cytoplasmic GFAP reaction in tumor cells characterized the investigated glioblastoma cases. The tumor cells GBM2-2F, GBM2-3F, and GBM-4M from the patients were examined. Histological examination of tumor invasion into CAM, angiogenesis, and immunohistochemical expression of GFAP-, PCNA-, p53-, EZH2- and vimentin-positive cells were examined. Results: No difference in GFAP expression was observed between the patient’s GBM tumor tissue and the tumor formed on CAM from the same patient’s tumor cells. There were no significant differences in invasion or in the frequency of GFAP- and p53-positive cells among the study control groups. The expression of PCNA-, EZH2-, and vimentin-positive cells in control tumors varied significantly. Treatment significantly reduced the incidence of tumor invasion in GBM2-2F and GBM2-4M and did not affect GBM2-3F tumors; treatment also significantly reduced GFAP expression in GBM2-3F and GBM2-4M and did not affect GBM2-2F tumors. The treatment with NaVPA–NaDCA significantly reduced the expression of PCNA, p53, EZH2 and vimentin in the tested tumors. Conclusions: Data demonstrated an antitumor effect of NaVPA–NaDCA in an in vivo model of a patient’s primary glioblastoma cells. Full article

Background/Objectives: Oral potentially malignant disorders (OPMDs), including oral leukoplakia (OLK), oral lichen planus (OLP), and actinic cheilitis (AC), have varying risks of progression to oral squamous cell carcinoma (OSCC). Biomarkers such as aldehyde dehydrogenase 1 (ALDH1) and mammary serine protease inhibitor (Maspin) have shown potential for diagnostic and prognostic use in oral cancer. The present study aimed to evaluate the immunoexpression of aldehyde dehydrogenase 1, a cancer stem cell marker associated with aggressiveness, and the mammary serine protease inhibitor, a potential tumor suppressor, in OPMD and OSCC tissues. Methods: A retrospective analysis was performed on 145 biopsy specimens collected from January 2015 to January 2023, including normal epithelium, OPMDs (OLK, OLP, AC), and OSCC. ALDH1 and Maspin expression levels were evaluated using immunohistochemistry, considering both the percentage of positive cells and staining intensity. Statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS, version 29.0; IBM Corp., Chicago, IL, USA). Results: Normal oral epithelium showed no expression of ALDH1, whereas 40.6% of OPMDs and 44.4% of OSCC samples exhibited high cytoplasmic ALDH1 expression. Nuclear ALDH1 expression was elevated in 29.7% of OPMDs and 38.9% of OSCCs (p < 0.001). Nuclear Maspin expression was high in 95.2% of normal tissues, in 67.2% of OPMDs and in 55.6% of OSCCs (p < 0.001). Maspin showed strong nuclear and cytoplasmic expression in normal tissue, but its expression decreased in OPMDs and OSCCs, with statistically significant reductions in both compartments (p < 0.001). Conclusions: The results indicate that ALDH1 upregulation and Maspin downregulation are hallmark events in oral carcinogenesis. Their combined evaluation provides a powerful tool for assessing dysplastic severity and malignant transformation risk in OPMDs. Future studies on larger cohorts are needed to confirm the prognostic utility of this dual-marker model. Full article