Search Results (58)

Ovarian hormones are essential regulators of vascular homeostasis, yet their deficiency’s effects on the meningeal microvasculature remain incompletely understood. We used high-resolution imaging to assess the cranial dura mater (CDM) blood and lymphatic microvasculature in ovariectomized (OVX) and control (intact or sham-operated) mice, followed by morphometric analysis of microvessel architecture. Immunofluorescent staining and Western blotting were employed to evaluate markers of vascular remodeling and profibrotic signaling. Blood microvascular quantification revealed a significant reduction in total microvessel length two weeks post-OVX, primarily due to arteriolar, but not venular, shortening. At the same time, the lengths of individual segments of both arterioles and venules were also significantly decreased, indicating microvascular fragmentation. Despite these changes, total vessel surface area remained preserved, suggesting compensatory dilation, particularly in arterioles. OVX also increased overall vessel tortuosity, again selectively affecting arterioles. Region-specific analysis of lymphatic networks associated with the coronal suture (CS) showed significantly increased surface area of podoplanin-positive lymphatic vessels. Elevated α-smooth muscle actin (α-SMA) expression in vascular and stromal compartments in OVX animals, along with increased transforming growth factor beta (TGF-β) levels, indicated early profibrotic changes. These findings highlight the selective vulnerability of arterial and lymphatic microvascular structures to hormonal deficiency post-OVX and suggest an association between hormonal status, microvascular remodeling, and profibrotic alterations in the CDM. Full article

Indocalamus tessellatus (Munro) P. C. Keng is a bamboo species with significant economic and ecological value, and exhibits considerable resistance to abiotic stresses. However, systematic evaluation of reference genes for gene expression analysis in this species is lacking. Analysis of multi-tissue transcriptomes yielded 3801 relatively stable genes; from these, we selected eleven new candidates along with nine widely adopted reference genes. We then evaluated these candidates under four conditions: drought (15% PEG-6000), salt (200 mM NaCl), waterlogging (root submergence in water), and a multi-tissue panel (leaf, leaf sheath, culm, shoot, and root). Under stress, early and sustained time points were sampled to capture dynamic transcriptional responses. Expression stability was assessed using geNorm, NormFinder, BestKeeper, and ΔCt, and results were integrated with RefFinder to generate comprehensive stability rankings for each condition. The most stable reference genes were condition-dependent: MD10B and PP2A under drought, eIF1A and Ite23725 under salt stress, PP2A and eIF4A under waterlogging, and 60S and UBP1 across different tissues. Notably, commonly used genes such as UBI and Actin7 were less stable. Peroxidase (POD) was used as a validation marker because it is a known stress-responsive gene, providing a sensitive readout of normalization accuracy. Validation confirmed that selecting suitable reference genes is essential for dependable expression quantification. These findings provide a robust set of reference genes for qRT-PCR studies in I. tessellatus, supporting future molecular and functional research in bamboo. Full article

Background/Objectives: Under physiological conditions, vascular smooth muscle cells (VSMCs) are in a quiescent contractile state, but under pathological conditions, such as atherosclerosis, they change their phenotype to synthetic, characterized by increased proliferation, migration, and production of an extracellular matrix. Furthermore, VSMCs can undergo calcification, switching to an osteoblast-like phenotype, contributing to plaque instability. Methods: In this study, we analyzed the phenotypic changes in VSMCs during the transition from a physiological to a pathological state, a key process in the progression of atherosclerosis, using confocal and transmission electron microscopy, real-time PCR, and intracellular calcium quantification. Results: Confocal and transmission electron microscopy revealed a prominent remodeling of the actin cytoskeleton, increasing autophagic vacuoles in synthetic VSMCs and the deposition of calcium microcrystals in calcified cells. Immunofluorescence analysis revealed differential expression of α-SMA (contractile marker) and galectin-3 (synthetic marker), confirming the phenotypic changes. Real-time PCR further validated these changes, showing upregulation of RUNX-2, a marker of osteogenic transition, in calcified VSMCs. Conclusions: This study highlights the dynamic plasticity of VSMCs and their role in atherosclerosis progression. Understanding the characteristics of these phenotypic transitions can help develop targeted therapies to mitigate vascular calcification and plaque instability, potentially countering cardiovascular disease. Full article

One of the main causes of death worldwide is cancer, a disorder in which a solid or liquid mass of cells known as a tumor develops when one or more cells lose the capacity to regulate their development. This study aims to assess the potential of chitosan as an anticancer agent in place of standard therapy regimens that have some degree of unselective cytotoxicity. PCR was performed for the RNA extraction of Caspase-3 and β-actin genes, and Cq values and quantification curves for each gene were recorded. Additionally, SRB and FITC apoptosis investigations were used to assess the effectiveness of chitosan powder’s anticancer activity against breast cancer cells. Full article

Background: Fibroblast activation protein (FAP) is a cell surface glycoprotein expressed by myofibroblastic cells in areas of active tissue remodeling, such as wound healing, fibrosis, and certain chronic inflammatory lesions. As FAP is uniquely present in chronic inflammatory lesions and has an important role in extracellular matrix (ECM) turnover, it appears to have all the characteristics necessary for involvement in atherosclerosis and atherosclerotic plaque rupture and has become a potential target in the treatment of myocardial infarction. Methods: To further understand the role of FAP, its expression in atherosclerotic plaques was examined in a genetically modified mouse model of accelerated atherosclerosis (Apobec1 −/− Ldlr −/− double-knockout mice). The immunohistochemical Fap staining of atherosclerotic plaques in a mouse model of atherosclerosis was correlated with quantification of Fap mRNA obtained from the atherosclerotic plaques of the aortic arch. Fap distribution was characterized in mouse atherosclerotic plaques relative to other markers of activated smooth muscle cells, such as alpha smooth muscle actin and myosin heavy chain (Acta2 and Myh2), ECM turnover (Ki-67, procollagen III and Mmp-9), and inflammation in atherosclerosis (Cd-44, Il-12 and Tgf beta) using immunohistochemistry (IH) and RT-PCR analysis. Results: The mouse model of accelerated atherosclerosis showed an increasing presence of Fap with the progression of atherosclerosis and a high expression level in advanced atherosclerotic lesions compared with other markers of ECM turnover and inflammation in atherosclerosis. Conclusions: FAP exhibits a distinct pattern of expression in a mouse model of atherosclerosis as compared to other markers of activated vascular smooth muscle cells, ECM degeneration, and inflammatory cytokines. Full article

Idiopathic pulmonary fibrosis (IPF) is the most common type of fibrosis in lungs, characterized as a chronic and progressive interstitial lung disease involving pathological findings of fibrosis with a median survival of 3 years. Despite the knowledge accumulated regarding IPF from basic and clinical research, an effective medical therapy for the condition remains to be established. Thus, it is necessary for further research, including stem cell therapy, which will provide new insights into and expectations for IPF treatment. Recently, it has been reported that one of the new therapeutic candidates for IPF is adipose-derived mesenchymal stem cells (ADSCs), which have several benefits, such as easy accessibility and minimal morbidity compared to bone marrow-derived mesenchymal stem cells. Therefore, we investigated the possibility of ADSCs as a therapeutic candidate for IPF. Using human lung fibroblasts (LFs) from IPF patients, we demonstrated that human IPF LFs cocultured with ADSCs led to reduced fibrosis-related genes. Further analysis revealed that ADSCs prevented the activation of the ERK signaling pathway in IPF LFs via the upregulation of protein tyrosine phosphatase receptor-type R (PTPRR), which negatively regulates the ERK signaling pathway. Moreover, we demonstrated that intravascular administration of ADSCs improved the pathogenesis of bleomycin-induced pulmonary fibrosis with reduced collagen deposition in histology and hydroxyproline quantification and collagen markers such as the gene expression of types I and III collagen and α-smooth muscle actin (α-SMA) in a murine model. ADSC transfer was also investigated in a humanized mouse model of lung fibrosis induced via the infusion of human IPF LFs, because the bleomycin installation model does not fully recapitulate the pathogenesis of IPF. Using the humanized mouse model, we found that intravascular administration of ADSCs also improved fibrotic changes in the lungs. These findings suggest that ADSCs are a promising therapeutic candidate for IPF. Full article

Background: Altered gene expression in cancers holds great potential to improve the diagnostics and differentiation of primary and metastatic liver cancers. In this study, the expression of the protein-coding genes ring finger protein 135 (RNF135), ephrin-B2 (EFNB2), ring finger protein 125 (RNF125), homeobox-C 4 (HOXC4), actin-binding LIM protein 1 (ABLIM1) and oncostatin M receptor (OSMR) and the long non-coding RNAs (lncRNA) prospero homeobox 1 antisense RNA 1 (PROX1-AS1) and leukemia inhibitory factor receptor antisense RNA 1 (LIFR-AS1) was investigated in hepatocellular carcinoma, cholangiocarcinoma, colorectal liver metastases and pancreatic ductal adenocarcinoma liver metastases. Methods: This study included 149 formalin-fixed, paraffin-embedded samples from 80 patients. After RNA isolation, quantification, reverse transcription and preamplification, real-time qPCR was performed. The gene expression between different groups was calculated relative to the expression of the reference genes using the ∆∆Cq method and statistically analyzed. The expression of the genes was additionally analyzed using the AmiCA and UCSC Xena platforms. Results: In primary cancers, our results showed differential expression between primary tumors and healthy tissues for all the genes and lncRNA examined. Moreover, we found downregulation of RNF135 in hepatocellular carcinoma, downregulation of OSMR in colorectal liver metastases and upregulation of HOXC4 in cholangiocarcinoma compared to primary liver cancers and metastatic cancers. The major finding is the upregulation of ABLIM1 in cholangiocarcinoma compared to hepatocellular carcinoma, colorectal liver metastases, pancreatic ductal adenocarcinoma liver metastases and healthy liver tissue. We propose ABLIM1 as a potential biomarker that differentiates cholangiocarcinoma from other cancers and healthy liver tissue. Conclusions: This study emphasizes the importance of understanding the differences in gene expression between healthy tissues and primary and metastatic cancers and highlights the potential use of altered gene expression as a diagnostic biomarker in these malignancies. Full article

Background/Objectives: Pterygium is a nonneoplastic elastotic degeneration characterized by subepithelial growth. It manifests as an ocular lesion originating from the bulbar conjunctiva, extending to the corneal surface, and reaching the visual axis in some cases. Although the exact cause is unknown, prolonged exposure to ultraviolet radiation is considered the most significant contributing factor. Chronic irritation and actinic damage are likely responsible for the typical fibrovascular reactions observed in pterygium. Additionally, growth factors, cytokines, and matrix metalloproteinases play roles in the pathogenesis of pterygium. This study compared recurrent and primary pterygium cases at the molecular level to gain new insights into the etiology of pterygium. Methods: Total protein was extracted from surgical samples of patients with primary and recurrent pterygium, and the levels of transforming growth factor beta 1 (TGF-β1), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), IL-8, and IL-10 were analyzed using the enzyme-linked immunosorbent assay technique. Target gene expression levels were analyzed using the ΔΔCt method after cDNA synthesis from isolated RNA, with normalization to GAPDH and quantification performed with SYBR Green PCR Master Mix. Results: Among the studied cytokines, IL-10 levels were higher in primary pterygium than in recurrent pterygium (722.0 ± 600.9/421.4 ± 266.8) (p = 0.0054). Other cytokines (IL-6, IL-8, IL-1β, and TGF-β1) were detected at similar levels in both primary and recurrent pterygium (p = 0.2986). Additionally, the TGF-β1 gene expression was found to be significantly upregulated in recurrent pterygium tissue compared to primary pterygium tissue (p = 0.034). Conclusions: This increase suggests that TGF-β1 may contribute to the recurrence mechanisms of pterygium through processes such as fibroblast activation and tissue remodeling. The higher levels of IL-10 in primary pterygium compared to recurrent pterygium indicate an enhanced early protective response aimed at limiting pterygium progression and controlling the inflammatory process. Full article

Naturally fermented dairy products are an important component of the human diet. They are a valuable source of nutrients as well as vitamins and minerals. Their importance as a source of probiotic bacterial strains should not be overlooked. A number of studies highlight the positive effects of species of the probiotic lactic acid bacteria on the intestinal microbiome and the overall homeostasis of the body, as well as a complementary treatment for some diseases. However, data on the effects on the intestinal epithelial cells of postmetabolites released by probiotic bacteria are incomplete. This is likely due to the fact that these effects are species- and strain-specific. In the present study, we investigated the effects of postmetabolites produced by a pre-selected candidate probiotic strain Limosilactobacillus fermentum on HT-29 intestinal epithelial cells. Our data showed a pronounced proliferative effect, evaluated by flow cytometry, quantification of the cell population and determination of the mitotic index. This was accompanied by the stabilization of the cell monolayer, measured by an increase in TEER (transepithelial electric resistance) and the reorganization of actin filaments. The data obtained are a clear indication of the positive effects that the products secreted by L. fermentum strain 53 have on intestinal epithelial cells. Full article

Transcriptomes and proteomes can be normalized with a handful of RNAs or proteins (or their peptides), such as GAPDH, β-actin, RPBMS, and/or GAP43. Even with hundreds of standards, normalization cannot be achieved across different molecular mass ranges for small molecules, such as lipids and metabolites, due to the non-linearity of mass by charge ratio for even the smallest part of the spectrum. We define the amount (or range of amounts) of metabolites and/or lipids per a defined amount of a protein, consistently identified in all samples of a multiple-model organism comparison, as the normative level of that metabolite or lipid. The defined protein amount (or range) is a normalized value for one cohort of complete samples for which intrasample relative protein quantification is available. For example, the amount of citrate (a metabolite) per µg of aconitate hydratase (normalized protein amount) identified in the proteome is the normative level of citrate with aconitase. We define normativity as the amount of metabolites (or amount range) detected when compared to normalized protein levels. We use axon regeneration as an example to illustrate the need for advanced approaches to the normalization of proteins. Comparison across different pharmacologically induced axon regeneration mouse models entails the comparison of axon regeneration, studied at different time points in several models designed using different agents. For the normalization of the proteins across different pharmacologically induced models, we perform peptide doping (fixed amounts of known peptides) in each sample to normalize the proteome across the samples. We develop Regen V peptides, divided into Regen III (SEB, LLO, CFP) and II (HH4B, A1315), for pre- and post-extraction comparisons, performed with the addition of defined, digested peptides (bovine serum albumin tryptic digest) for protein abundance normalization beyond commercial labeled relative quantification (for example, 18-plex tandem mass tags). We also illustrate the concept of normativity by using this normalization technique on regenerative metabolome/lipidome profiles. As normalized protein amounts are different in different biological states (control versus axon regeneration), normative metabolite or lipid amounts are expected to be different for specific biological states. These concepts and standardization approaches are important for the integration of different datasets across different models of axon regeneration. Full article

Electrochemical biosensors are valued for their sensitivity and selectivity in detecting biological molecules. Having the advantage of generating signals that can be directly or indirectly proportional to the concentration of the target analyte, these biosensors can achieve specificity by utilizing a specific biorecognition surface designed to recognize the target molecule. Electrochemical biosensors have garnered substantial attention, as they can be used to fabricate compact, cost-effective devices, making them promising candidates for point-of-care testing (POCT) devices. This study introduces a label-free electrochemical biosensor employing a gold screen-printed electrode (SPE) to detect lysophosphatidic acid (LPA), a potential early ovarian cancer biomarker. We employed the gelsolin–actin system, previously introduced by our group, in combination with fluorescence spectrometry, as a biorecognition element to detect LPA. By immobilizing a gelsolin–actin complex on an SPE, we were able to quantify changes in current intensity using cyclic voltammetry and differential pulse voltammetry, which was directly proportional to the LPA concentration in the solution. Our results demonstrate the high sensitivity of the developed biosensor for detecting LPA in goat serum, with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.9 µM and 2.76 µM, respectively, highlighting its potential as a promising tool for early-stage diagnosis of ovarian cancer. Full article

Background and Objectives: Wound healing encompasses a multitude of factors and entails the establishment of interactions among components of the basement membrane. The quantification of particle concentrations can serve as valuable biomarkers for assessing biomechanical muscle properties. The objective of this study was to examine the immunoexpression and immunoconcentration of myometrial collagen type VI, elastin, alpha-smooth muscle actin, and smooth muscle myosin heavy chain, as well as the expression of platelets and clusters of differentiation 31 in the uterine scar following a cesarean section (CS). Materials and Methods: A total of 177 biopsies were procured from a cohort of pregnant women who were healthy, specifically during the surgical procedure of CS. The participants were categorized into seven distinct groups. Group 1 consisted of primiparas, with a total of 52 individuals. The subsequent groups were organized based on the duration of time that had elapsed since their previous CS. The analysis focused on the immunoexpression and immunoconcentration of the particles listed. Results: No significant variations were observed in the myometrial immunoconcentration of collagen type VI, elastin, smooth muscle myosin, and endothelial cell cluster of differentiation 31 among the analyzed groups. The concentration of alpha-smooth muscle actin in the myometrium was found to be significantly higher in patients who underwent CS within a period of less than 2 years since their previous CS, compared to those with a longer interval between procedures. Conclusions: Our findings indicate that the immunoconcentration of uterine myometrial scar collagen type VI, elastin, smooth muscle myosin heavy chain, alpha-smooth muscle actin, and endothelial cell marker cluster of differentiation 31 remains consistent regardless of the duration elapsed since the previous CS. The findings indicate that there are no significant alterations in the biomechanical properties of the uterine muscle beyond a period of 13 months following a CS. Full article

Cardiovascular tissue engineering is providing many solutions to cardiovascular diseases. The complex disease demands necessitating tissue-engineered constructs with enhanced functionality. In this study, we are presenting the production of a dexamethasone (DEX)-loaded electrospun tubular polymeric poly(l-lactide) (PLA) or poly(d,l-lactide-co-glycolide) (PLGA) construct which contains iPSC-CMs (induced pluripotent stem cell cardiomyocytes), HUVSMCs (human umbilical vein smooth muscle cells), and HUVECs (human umbilical vein endothelial cells) embedded in fibrin gel. The electrospun tube diameter was calculated, as well as the DEX release for 50 days for 2 different DEX concentrations. Furthermore, we investigated the influence of the polymer composition and concentration on the function of the fibrin gels by imaging and quantification of CD31, alpha-smooth muscle actin (αSMA), collagen I (col I), sarcomeric alpha actinin (SAA), and Connexin 43 (Cx43). We evaluated the cytotoxicity and cell proliferation of HUVECs and HUVSMCs cultivated in PLA and PLGA polymeric sheets. The immunohistochemistry results showed efficient iPSC-CM marker expression, while the HUVEC toxicity was higher than the respective HUVSMC value. In total, our study emphasizes the combination of fibrin gel and electrospinning in a functionalized construct, which includes three cell types and provides useful insights of the DEX release and cytotoxicity in a tissue engineering perspective. Full article

Thoracic aortic aneurysm is found in patients with ACTA2 pathogenic variants. ACTA2 missense variants are associated with impaired aortic smooth muscle cell (SMC) contraction. This study tested the hypothesis that the Acta2^R149C/+ variant alters actin isoform expression and decreases integrin recruitment, thus, reducing aortic contractility. Stress relaxation measurements in thoracic aortic rings showed two functional regimes with a reduction of stress relaxation in the aorta from Acta2^R149C/+ mice at low tension, but not at high tension values. Contractile responses to phenylephrine and potassium chloride were 50% lower in Acta2^R149C/+ mice than in wild-type (WT) mice. Additionally, SMC were immunofluorescently labeled for specific proteins and imaged by confocal or total internal reflection fluorescence microscopy. The quantification of protein fluorescence of Acta2^R149C/+ SMC showed a downregulation in smooth muscle α-actin (SMα-actin) and a compensatory upregulation of smooth muscle γ-actin (SMγ-actin) compared to WT cells. These results suggest that downregulation of SMα-actin leads to reduced SMC contractility, while upregulation of SMγ-actin may lead to increased SMC stiffness. Decreased α5β1 and α2β1 integrin recruitment at cell-matrix adhesions further reduce the ability of mutant cells to participate in cell-matrix crosstalk. Collectively, the results suggest that mutant Acta2^R149C/+ aortic SMC have reduced contractility and interaction with the matrix, which are potential long-term contributing factors to thoracic aortic aneurysms. Full article

The quantification of viruses is necessary for both research and clinical applications. The methods available for RNA virus quantification possess several drawbacks, including sensitivity to inhibitors and the necessity of a standard curve generation. The main purpose of this study was to develop and validate a method for the quantification of recombinant, replication-deficient Semliki Forest virus (SFV) vectors using droplet digital PCR (ddPCR). This technique demonstrated stability and reproducibility using various sets of primers that targeted inserted transgenes, as well as the nsP1 and nsP4 genes of the SFV genome. Furthermore, the genome titers in the mixture of two types of replication-deficient recombinant virus particles were successfully measured after optimizing the annealing/extension temperature and virus:virus ratios. To measure the infectious units, we developed a single-cell ddPCR, adding the whole infected cells to the droplet PCR mixture. Cell distribution in the droplets was investigated, and β-actin primers were used to normalize the quantification. As a result, the number of infected cells and the virus infectious units were quantified. Potentially, the proposed single-cell ddPCR approach could be used to quantify infected cells for clinical applications. Full article