Search Results (66)

The complement system is crucial for immune defense, linking innate and adaptive immunity. In the classical and lectin pathways, C4 is split into C4b, triggering opsonization, lysis, and the removal of pathogens and damaged cells. Dysregulated activation of C4 and other components of the classical pathway can lead to tissue damage and heightened inflammation, whereas appropriate regulation of C4b activity serves to mitigate excessive inflammation and prevent injury. ELISA analysis demonstrated C4 activation and cleavage during the co-incubation of PRRSV with fresh porcine serum. Immunoelectron microscopy revealed that porcine red blood cells could immunologically adhere to PRRSV, and C4b was involved in this adhesion process. BLAST (NCBI BLAST+ 2.14.1) analysis revealed that porcine CR1-like CCPs 1-3, CR1-like CCPs 12-14, and CR1-like CCPs 19-21 share high similarity with the CCP 1-3 region of human CR1, which mediates C4b binding. Yeast two-hybrid assays confirmed that all three CR1-like fragments bind C4b. To elucidate the interaction mechanism, homology models of C4b and CR1-like fragments were constructed, followed by molecular docking and dynamics simulations, identifying 18 key amino acids in porcine CR1-like involved in C4b binding. Surface plasmon resonance further validated the binding affinity of CR1-like CCPs 1-3, its mutant 118I, and C4b. These results enhance our understanding of complement regulation and provide a foundation for developing therapeutic strategies targeting complement-related diseases. Full article

LigiLactobacillus saerimneri (L. sae) has shown considerable promise as a probiotic in recent years, particularly in poultry production. Comprehensive evaluation of its genetic functions, safety profile, and immunogenicity is essential prior to practical application. Our previous study demonstrated that the chicken-derived strain L. sae M-11 colonizes effectively and exhibits a favorable safety profile at adequate dosages. In this study, we further evaluated the potential of L. sae M-11 by analyzing its genetic basis for intestinal adaptation, metabolic features, safety risks, and suitability as a delivery vector. Comparative genomic analysis revealed that L. sae has evolved distinctive genetic features and functional specialization that may facilitate host adaptation. Genomic stability assessments and virulence factor screening confirmed that L. sae M-11 poses no substantial health risks. Furthermore, based on transmembrane protein predictions, the LPQTGE-motif protein was identified as a cell wall anchor in genetically engineered L. sae M-11 using immunoelectron microscopy. Notably, this delivery system selectively activated peripheral blood monocyte-derived dendritic cells (PB-MoDCs) in vitro, as evidenced by the up-regulation of maturation markers (CD83, CD80), pro-inflammatory cytokines (IL-1β, IL-6), Th1-associated IL-12, and the chemokine CXCLi1. However, it exhibited a limited antigen presentation capacity, indicated by low expression levels of CD40, MHCII, DEC205, TNF-α, and IFN-γ. The prospects and challenges associated with the application of L. sae M-11 have been discussed. Overall, these findings support the potential development of L. sae M-11 as a microbial cell factory and mucosal delivery vector. Full article

SSB proteins play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. This study investigates the transcript levels, identification, expression and purification, subcellular localization, and immune protective potential of the SSB-like proteins of Eimeria necatrix (EnSSB), exploring its role in the development of E. necatrix and its potential as a candidate antigen for a subunit vaccine against avian coccidiosis. The level of EnSSB gene transcription was highest in unsporulated oocysts (UO), followed by gametocytes (GAM) (p < 0.05). The gene consisted of an open reading frame of 1488 nucleotides encoding a protein of 495 amino acid residues with a predicted molecular weight of 53.31 kDa. EnSSB contained a SSB domain with a conserved OB (oligonucleotide/oligosaccharide binding) fold. The molecular mass of the native protein, as determined by Western blot analysis, was ~58 kDa in second-generation merozoites (MZ-2) and UO. In addition to the 58 kDa band, four other bands (~98 kDa, ~82 kDa, ~36 kDa and ~28 kDa) were detected in GAM. No bands were detected in MZ-3. Indirect immunofluorescence and immuno-electron microscopy localized EnSSB in the cytoplasm of macrogametocytes but not in wall-forming bodies and oocyst wall. Animal challenge experiments demonstrated that rEnSSB elicited robust IgY responses, increased splenic T lymphocytes and body weight gain, reduced intestinal lesion scores and oocyst shedding, and presented anticoccidial index (ACI) more than 160. These findings not only offer a foundation for understanding the role of EnSSB protein in regulating the development of E. necatrix, but also present a potential protective antigen of E. necatrix for the development of a subunit vaccine against avian coccidiosis. Full article

Glomerular podocytes act as a part of the filtration barrier in the kidney. The activity of this filter is regulated by ionotropic and metabotropic glutamate receptors. Adjacent podocytes can potentially release glutamate into the intercellular space; however, little is known about how podocytes release glutamate. Here, we demonstrated vesicular glutamate transporter 3 (VGLUT3)-dependent glutamate release from podocytes. Immunofluorescence analysis revealed that rat glomerular podocytes and an immortal mouse podocyte cell line (MPC) express VGLUT1 and VGLUT3. Consistent with this finding, quantitative RT-PCR revealed the expression of VGLUT1 and VGLUT3 mRNA in undifferentiated and differentiated MPCs. In addition, the exocytotic proteins vesicle-associated membrane protein 2, synapsin 1, and synaptophysin 1 were present in punctate patterns and colocalized with VGLUT3 in MPCs. Interestingly, approximately 30% of VGLUT3 colocalized with VGLUT1. By immunoelectron microscopy, VGLUT3 was often observed around clear vesicle-like structures in differentiated MPCs. Differentiated MPCs released glutamate following depolarization with high potassium levels and after stimulation with the muscarinic agonist pilocarpine. The depletion of VGLUT3 in MPCs by RNA interference reduced depolarization-dependent glutamate release. These results strongly suggest that VGLUT3 is involved in glutamatergic signalling in podocytes and may be a new drug target for various kidney diseases. Full article

Retinoic acid (RA) is commonly used to differentiate SH-SY5Y neuroblastoma cells. This effect is sustained by a specific modulation of gene transcription, leading to marked changes in cellular proteins. In this scenario, autophagy may be pivotal in balancing protein synthesis and degradation. The present study analyzes whether some autophagy-related proteins and organelles are modified during RA-induced differentiation of SH-SY5Y cells. RA-induced effects were compared to those induced by starvation. SH-SY5Y cells were treated with a single dose of 10 µM RA or grown in starvation, for 3 days or 7 days. After treatments, cells were analyzed at light microscopy and transmission electron microscopy to assess cell morphology and immunostaining for specific markers (nestin, βIII-tubulin, NeuN) and some autophagy-related proteins (Beclin 1, LC3). We found that both RA and starvation differentiate SH-SY5Y cells. Specifically, cell differentiation was concomitant with an increase in autophagy proteins and autophagy-related organelles. However, the effects of a single dose of 10 μM RA persist for at least 7 days, while prolonged starvation produces cell degeneration and cell loss. Remarkably, the effects of RA are modulated in the presence of autophagy inhibitors or stimulators. The present data indicate that RA-induced differentiation is concomitant with an increased autophagy. Full article

The ability to reproduce depends on metabolic status. In rodents, the ventral premammillary nucleus (PMv) integrates metabolic and reproductive signals. While leptin (adiposity-related) signaling in the PMv is critical for female fertility, male reproductive functions are strongly influenced by glucose homeostasis. The anorexigenic peptide nesfatin-1 is a leptin-independent central regulator of blood glucose. Therefore, its integrative role in male rats can be assumed. To investigate this, we mapped the distribution of nesfatin-1 mRNA- and protein-producing cells in the PMv during postnatal development via in situ hybridization and immunohistochemistry, respectively. Fos-nesfatin-1, double immunostaining was used to determine the combined effect of heterosexual pheromone challenge and insulin-induced hypoglycemia on neuronal activation in adults. We found that ~75% of the pheromone-activated neurons were nesfatin-1 cells. Hypoglycemia reduced pheromone-induced cell activation, particularly in nesfatin-1 neurons. Immuno-electron microscopy revealed innervation of PMv nesfatin-1 neurons by urocortin3-immunoreactive terminals, reportedly originating from the medial amygdala. Nesfatin-1 immunopositive neurons expressed GPR10 mRNA, a receptor associated with metabolic signaling, but did not respond with accumulation of phosphorylated STAT3 immunopositivity, a marker of leptin receptor signaling, in response to intracerebroventricular leptin treatment. Our results suggest that PMv nesfatin-1 neurons are primarily responsible for integrating reproductive and metabolic signaling in male rats. Full article

HtpB, the chaperonin of the bacterial pathogen L. pneumophila, is found in extracellular locations, even the cytoplasm of host cells. Although chaperonins have an essential cytoplasmic function in protein folding, HtpB exits the cytoplasm to perform extracellular virulence-related functions that support L. pneumophila’s lifestyle. The mechanism by which HtpB reaches extracellular locations is not currently understood. To address this experimental gap, immunoelectron microscopy, trypsin-accessibility assays, and cell fractionation were used to localize HtpB in various L. pneumophila secretion mutants. Dot/Icm type IV secretion mutants displayed less surface-exposed HtpB and more periplasmic HtpB than parent strains. The analysis of periplasmic extracts and outer membrane vesicles of these mutants, where HtpB co-localized with bona fide periplasmic proteins, confirmed the elevated levels of periplasmic HtpB. Genetic complementation of the mutants recovered parent strain levels of surface-exposed and periplasmic HtpB. The export of GSK-tagged HtpB into the cytoplasm of infected cells was also Dot/Icm-dependent. The translocating role of the Dot/Icm system was not specific for HtpB because GroEL, the chaperonin of Escherichia coli, was found at the cell surface and accumulated in the periplasm of Dot mutants when expressed in L. pneumophila. These findings establish that a functional Dot/Icm system is required for HtpB to reach extracellular locations, but the mechanism by which cytoplasmic HtpB reaches the periplasm remains partially unidentified. Full article

Monoclonal gammopathy of renal significance (MGRS) refers to a group of renal disorders caused by a monoclonal immunoglobulin (MIg), secreted by a non-malignant B-cell clone. Unlike overt multiple myeloma or B-cell proliferation, MGRS does not meet those diagnostic criteria. However, it is associated with significant morbidity, due to severe renal, and sometimes systemic, lesions induced by the MIg. Early recognition is crucial, as chemotherapy to suppress MIg secretion often improves outcomes. The spectrum of renal diseases in MGRS is broad, including both well-known conditions like AL amyloidosis and newly described lesions. Kidney biopsy is essential to determine the specific lesion associated with MGRS and assess its severity. Diagnosis involves integrating morphologic alterations using techniques such as light microscopy, immunofluorescence (IF), electron microscopy, and, in some cases, IF staining for Ig isotypes, immunoelectron microscopy, and proteomic analysis. Additionally, a complete hematologic evaluation, including serum and urine protein electrophoresis, immunofixation, and a serum-free light-chain assay, is necessary. Full article

Adenomyosis (AM) is a gynecological disease characterized by the invasion of endometrial glands and stroma within the myometrium. The etiology and pathogenesis of AM remain inadequately understood. Pale cells were identified as a novel cell type characterized by the absence of desmosomal contacts and light-colored cytoplasm. These cells were observed to migrate individually through ultra-micro ruptures in the basal membrane of the endometrial glands, translocating into the stroma and then further into the myometrium. Our study aimed to explore the possible stem cell properties of these pale cells. Forty hysterectomy specimens were analyzed using immunohistochemistry and immunofluorescence to assess negative E-cadherin expression and the positive expression of stem cell markers SSEA-1, MSI-1, and SOX-2. Immunohistochemical analysis revealed the presence of pale cells and occasionally rounded, enlarged E-cadherin-negative cells predominantly in the basal endometrial epithelium. The stem cell marker SSEA-1 was significantly elevated in the basalis epithelium, as well as in the ectopic epithelium. SSEA-1 positive cells were also identified in the stroma and myometrium. Sporadic colocalization of SSEA-1+/E-cadherin– cells was confirmed through immunofluorescence. The positive staining of pale cells for SSEA-1 and MSI-1 was also confirmed at the ultrastructural level by immunoelectron microscopy. These findings indicate that pale cells may possess stem cell characteristics, particularly a positive SSEA-1 profile, warranting further in vitro investigation into their role in the pathogenesis of adenomyosis. Full article

Women become susceptible to hypertension as they transition to menopause (i.e., perimenopause); however, the underlying mechanisms are unclear. Animal studies using an accelerated ovarian failure (AOF) model of peri-menopause (peri-AOF) demonstrate that peri-AOF hypertension is associated with increased postsynaptic NMDA receptor plasticity in the paraventricular hypothalamic nucleus (PVN), a brain area critical for blood pressure regulation. However, recent evidence indicates that presynaptic NMDA receptors also play a role in neural plasticity. Here, using immuno-electron microscopy, we examine the influence of peri-AOF hypertension on the subcellular distribution of the essential NMDA GluN1 receptor subunit in PVN axon terminals in peri-AOF and in male mice. Hypertension was produced by 14-day slow-pressor angiotensin II (AngII) infusion. The involvement of estrogen signaling was investigated by co-administering an estrogen receptor beta (ERß) agonist. Although AngII induced hypertension in both peri-AOF and male mice, peri-AOF females showed higher cytoplasmic GluN1 levels. In peri-AOF females, activation of ERß blocked hypertension and increased plasmalemmal GluN1 in axon terminals. In contrast, stimulation of ERß did not inhibit hypertension or influence presynaptic GluN1 localization in males. These results indicate that sex-dependent recruitment of presynaptic NMDA receptors in the PVN is influenced by ERß signaling in mice during early ovarian failure. Full article

Diabetes mellitus affects 537 million adults around the world. Adropin is expressed in different cell types. Our aim was to investigate the cellular localization in the endocrine pancreas and its effect on modulating pancreatic endocrine hormone release in streptozotocin (STZ)-induced diabetic rats. Adropin expression in the pancreas was investigated in normal and diabetic rats using immunohistochemistry and immunoelectron microscopy. Serum levels of insulin, glucagon pancreatic polypeptide (PP), and somatostatin were measured using a Luminex^® χMAP (Magpix^®) analyzer. Pancreatic endocrine hormone levels in INS-1 832/3 rat insulinoma cells, as well as pancreatic tissue fragments of normal and diabetic rats treated with different concentrations of adropin (10⁻⁶, 10⁻⁹, and 10⁻¹² M), were measured using ELISA. Adropin was colocalized with cells producing either insulin, glucagon, or PP. Adropin treatment reduced the number of glucagon-secreting alpha cells and suppressed glucagon release from the pancreas. The serum levels of GLP-1 and amylin were significantly increased after treatment with adropin. Our study indicates a potential role of adropin in modulating glucagon secretion in animal models of diabetes mellitus. Full article

The zoonotic transmission of hepatitis E virus (HEV) genotypes 3 (HEV-3) and 4 (HEV-4), and rabbit HEV (HEV-3ra) has been documented. Vaccination against HEV infection depends on the capsid (open reading frame 2, ORF2) protein, which is highly immunogenic and elicits effective virus-neutralizing antibodies. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles (VLPs). However, research on the production of ORF2 proteins from these HEV genotypes in E. coli to form VLPs has been modest. In this study, we constructed 21 recombinant plasmids expressing various N-terminally and C-terminally truncated HEV ORF2 proteins for HEV-3, HEV-3ra, and HEV-4 in E. coli. We successfully obtained nine HEV-3, two HEV-3ra, and ten HEV-4 ORF2 proteins, which were primarily localized in inclusion bodies. These proteins were solubilized in 4 M urea, filtered, and subjected to gel filtration. Results revealed that six HEV-3, one HEV-3ra, and two HEV-4 truncated proteins could assemble into VLPs. The purified VLPs displayed molecular weights ranging from 27.1 to 63.4 kDa and demonstrated high purity (74.7–95.3%), as assessed by bioanalyzer, with yields of 13.9–89.6 mg per 100 mL of TB medium. Immunoelectron microscopy confirmed the origin of these VLPs from HEV ORF2. Antigenicity testing indicated that these VLPs possess characteristic HEV antigenicity. Evaluation of immunogenicity in Balb/cAJcl mice revealed robust anti-HEV IgG responses, highlighting the potential of these VLPs as immunogens. These findings suggest that the generated HEV VLPs of different genotypes could serve as valuable tools for HEV research and vaccine development. Full article

Viral hepatitis is a systemic infectious diseases caused by various hepatitis viruses, primarily leading to liver damage. It is widely prevalent worldwide, with hepatitis viruses categorized into five types: hepatitis A, B, C, D, and E, based on their etiology. Currently, the detection of hepatitis viruses relies on methods such as enzyme-linked immunosorbent assay (ELISA), immunoelectron microscopy to observe and identify viral particles, and in situ hybridization to detect viral DNA in tissues. However, these methods have limitations, including low sensitivity, high error rates in results, and potential false negative reactions due to occult serum infection conditions. To address these challenges, we have designed an AuNPs-DNA walker method that uses gold nanoparticles (AuNPs) and complementary DNA strands for detecting viral DNA fragments through a colorimetric assay and fluorescence detection. The DNA walker, attached to gold nanoparticles, comprises a long walking strand with a probe sequence bound and stem-loop structural strands featuring a modified fluorescent molecule at the 3′ end, which contains the DNAzyme structural domain. Upon the addition of virus fragments, the target sequence binds to the probe chains. Subsequently, the long walking strand is released and continuously hybridizes with the stem-loop structural strand. The DNAzyme undergoes hydrolytical cleavage by Mg²⁺, breaking the stem-loop structural strand into linear single strands. As a result of these structural changes, the negative charge density in the solution decreases, weakening spatial repulsion and rapidly reducing the stability of the DNA walker. This leads to aggregation upon the addition of a high-salt solution, accompanied by a color change. Virus typing can be performed through fluorescence detection. The innovative method can detect DNA/RNA fragments with high specificity for the target sequence, reaching concentrations as low as 1 nM. Overall, our approach offers a more convenient and reliable method for the detection of hepatitis viruses. Full article

Recent advances in cancer treatment like personalized chemotherapy and immunotherapy are aimed at tumors that meet certain specifications. In this review, we describe a new approach to general cancer treatment, termed peptide-induced poptosis, in which specific peptides, e.g., PNC-27 and its shorter analogue, PNC-28, that contain the segment of the p53 transactivating 12–26 domain that bind to HDM-2 in its 1–109 domain, bind to HDM-2 in the membranes of cancer cells, resulting in transmembrane pore formation and the rapid extrusion of cancer cell contents, i.e., tumor cell necrosis. These peptides cause tumor cell necrosis of a wide variety of solid tissue and hematopoietic tumors but have no effect on the viability and growth of normal cells since they express at most low levels of membrane-bound HDM-2. They have been found to successfully treat a highly metastatic pancreatic tumor as well as stem-cell-enriched human acute myelogenous leukemias in nude mice, with no evidence of off-target effects. These peptides also are cytotoxic to chemotherapy-resistant cancers and to primary tumors. We performed high-resolution scanning immuno-electron microscopy and visualized the pores in cancer cells induced by PNC-27. This peptide forms 1:1 complexes with HDM-2 in a temperature-independent step, followed by dimerization of these complexes to form transmembrane channels in a highly temperature-dependent step parallel to the mode of action of other membranolytic but less specific agents like streptolysin. These peptides therefore may be effective as general anti-cancer agents. Full article

Proteins need to be located in appropriate spatiotemporal contexts to carry out their diverse biological functions. Mislocalized proteins may lead to a broad range of diseases, such as cancer and Alzheimer’s disease. Knowing where a target protein resides within a cell will give insights into tailored drug design for a disease. As the gold validation standard, the conventional wet lab uses fluorescent microscopy imaging, immunoelectron microscopy, and fluorescent biomarker tags for protein subcellular location identification. However, the booming era of proteomics and high-throughput sequencing generates tons of newly discovered proteins, making protein subcellular localization by wet-lab experiments a mission impossible. To tackle this concern, in the past decades, artificial intelligence (AI) and machine learning (ML), especially deep learning methods, have made significant progress in this research area. In this article, we review the latest advances in AI-based method development in three typical types of approaches, including sequence-based, knowledge-based, and image-based methods. We also elaborately discuss existing challenges and future directions in AI-based method development in this research field. Full article