Search Results (100)

Nephrotic syndrome (NS) is characterized by proteinuria, hypoalbuminemia, edema, and hyperlipidemia. Apart from the traditional causes of NS, such as minimal change disease, focal segmental glomerulosclerosis, diabetes, infections, malignancies, autoimmune conditions, and nephrotoxic agents, there are also rare causes of NS, whose knowledge is of the utmost importance. The aim of this article was to highlight the less well-known causes that have a significant impact on diagnosis and treatment. Genetic syndromes such as Schimke immuno-osseous dysplasia, familial lecithin-cholesterol acyltransferase deficiency with two clinical variants (fish-eye Disease and the p.Leu364Pro mutation), lead to NS through mechanisms involving podocyte and lipid metabolism dysfunction. Congenital disorders of glycosylation and Nail–Patella Syndrome emphasize the role of deranged protein processing and transcriptional regulation in glomerular injury. The link of NS with type 1 diabetes, though rare, suggests an etiology on the basis of common HLA loci and immune dysregulation. Histopathological analysis, particularly electron microscopy, shows mainly podocyte damage, mesangial sclerosis, and alteration of the basement membrane, which aids in differentiating rare forms. Prompt recognition of these novel etiologies by genetic analysis, renal biopsy, and an interdisciplinary panel is essential to avoid delays in diagnosis and tailored treatment. Full article

Extracellular vesicles (EVs) have emerged as promising acellular tools for modulating immune responses for tissue engineering applications. This study explores the potential of human fibroblast-derived EVs delivered within a three-dimensional (3D) injectable scaffold composed of polycaprolactone (PCL) nanofibers and collagen (PNCOL) to reprogram macrophage behavior and support scaffold integrity under inflammatory conditions. EVs were successfully isolated from human fibroblasts using ultracentrifugation and characterized for purity, size distribution and surface markers (CD63 and CD9). Macrophage-laden PNCOL scaffolds were prepared under three conditions: macrophage-only (MP), fibroblast co-encapsulated (F-MP), and EV-encapsulated (EV-MP) groups. Structural integrity was assessed via scanning electron microscopy and Masson’s trichrome staining, while immunomodulatory effects were evaluated through metabolic assays, gene expression profiling, and immunohistochemistry for macrophage polarization markers (CD80, CD206). When co-encapsulated with pro-inflammatory (M1) macrophages in PNCOL scaffolds, fibroblast-derived EVs preserved scaffold structure and significantly enhanced macrophage metabolic activity compared to the control (MP) and other experimental group (F-MP). The gene expression and immunohistochemistry data demonstrated substantial upregulation of anti-inflammatory markers (TGF-β, CD163, and CCL18) and surface protein CD206, indicating a phenotypic shift toward M2-like macrophages for EV-encapsulated scaffolds relative to the other groups. The findings of this study demonstrate that fibroblast-derived EVs integrated into injectable PCL–collagen scaffolds offer a viable, cell-free approach to modulate inflammation, preserve scaffold structure, and support regenerative healing. This strategy holds significant promise for advancing immuno-instructive platforms in regenerative medicine, particularly in settings where conventional cell therapies face limitations in survival, cost, or safety. 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

Utilizing high-resolution microscopy in conjunction with a new antibody highly specific for rat alpha4 Na,K-ATPase, we describe changes in alpha4 expression during spermatogenesis and in sperm incubated under capacitating and noncapacitating conditions. Immunohistochemical analyses showed alpha4 expression at low levels in spermatogonia and in pachytene spermatocytes. Alpha4 then becomes highly expressed on round spermatids and the midpiece of elongated spermatozoa within the seminiferous tubules. In noncapacitating conditions, alpha4 was confined mainly to the flagellum of mature sperm; however, under capacitating conditions, sperm acquired intense alpha4 staining along the acrosomal region of the sperm head. To visualize the precise localization of alpha4 in the sperm head, we performed an ultrastructural analysis using immuno-scanning electron microscopy. Under capacitating conditions, sperm exhibited alpha4 staining along the dorsal surface of the sperm head associated with the acrosome. In addition, after 4 h of incubation in motility buffer, we observed an increase in alpha4 protein in sperm that could be blocked with chloramphenicol, a mitochondrial-type ribosome inhibitor. These findings demonstrate that both the localization and expression level of alpha4 Na,K-ATPase are dynamic aspects of sperm maturation and suggest that sperm motility and capacitation may be supported by these changes to the location and amount of this protein. 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

Goose parvovirus (GPV) is an etiological agent of Derzsy’s disease, afflicting geese and Muscovy ducks worldwide. Its high mortality rate among goslings and ducklings causes large losses to the waterfowl industry. Toward molecular and structural characterization, virus-like particles (VLPs) of GPV were produced, and the capsid structure was determined by cryogenic electron microscopy (cryo-EM) at a resolution of 2.4 Å. The capsid exhibited structural features conserved among parvoviruses, including surface two-fold depressions, three-fold protrusions, and five-fold channels. A structural comparison of the GPV viral protein (VP) structure with other adeno-associated viruses (AAVs), including human AAV2, AAV5, and quail AAV (QAAV), revealed unique conformations of several surface-accessible variable regions (VRs). Furthermore, the GPV capsid was found to be thermally stable at physiological pH, but less so under lower pH conditions. As a member of the genus Dependoparvovirus, GPV could also be bound by cross-reactive anti-AAV capsid antibodies that bind to the five-fold region of the viruses, as shown by native immuno-dot blot analysis. Finally, the GPV VP structure was compared to those of other bird dependoparvoviruses, which revealed that VR-III may be important for GPV and Muscovy duck parvovirus (MDPV) infection. 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

Outer membrane vesicles (OMVs) are extracellular structures, ranging in size from 10 to 300 nm, produced by Gram-negative bacteria. They can be incorporated into the outer membrane of a recipient’s cells, which may enable the transfer of substances with lytic properties. Due to the scarce information regarding the OMVs produced by Proteus mirabilis, the aim of this study was to test the blebbing abilities of the clinical P. mirabilis O77 and O78 strains and to determine the blebs’ interactions with bacterial cells, including their possible bactericidal activities. The production of OMVs was visualised by Transmission electron microscopy (TEM). The presence of OMVs in the obtained samples as well as the phenomenon of OMV fusion to recipient cells were confirmed by Enzyme-Linked ImmunoSorbent Assay (ELISA) and Western blotting assays. The bacteriolytic activity of the OMVs was examined against P. mirabilis clinical strains and reference Staphylococcus aureus and Escherichia coli strains. It was shown that each of the two tested P. mirabilis strains could produce OMVs which were able to fuse into the cells of the other strain. The lytic properties of the O78 OMVs against another P. mirabilis O78 strain were also demonstrated. This promising result may help in the future to better understand the mechanisms of the pathogenesis and to treat the infections caused by P. mirabilis. 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