Cervical cancer is a major health burden driven by persistent infection with high-risk human papillomavirus (HPV). Genomic instability biomarkers from the micronucleus cytome (MNcyt) assay may improve identification of women at risk of progression beyond cytology and HPV testing. A cross-sectional study of 184 women attending routine cervical cancer screening was conducted in Popayán, Colombia. Exfoliated cervical epithelial cells were collected for HPV DNA genotyping, liquid-based cytology, and MNcyt analysis using the Feulgen-Fast Green DNA-specific method. For each sample, 2,000 cells were scored for micronuclei, nuclear buds, binucleated cells, pyknotic cells, karyorrhectic cells, karyolytic cells, and cells with condensed chromatin. Group differences and trends were assessed across ordered HPV/cytology categories; genotype analyses used age-adjusted models and, among HPV-positive women, models adjusted for age and cytology. Women were classified as HPV-negative with negative for intraepithelial lesion or malignancy (HPV-/NILM, n = 69), HPV-positive with NILM (HPV+/NILM, n = 47), HPV-positive with atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion (HPV+/ASCUS-LSIL, n = 49), and HPV-positive with high-grade squamous intraepithelial lesion or worse (HPV+/≥HSIL, n = 19). Significant positive trends across these ordered categories were observed for micronuclei (p = 0.044), nuclear buds (p = 0.029), and binucleated cells (p = 0.011). Among HPV-positive women, genotype differences adjusted for age and cytology were observed for nuclear buds (p = 0.046), karyolytic cells (p = 0.010), and condensed chromatin (p = 0.035). Overall, micronuclei, nuclear buds, and binucleated cells showed consistent patterns of genomic instability across HPV/cytology categories, while genotype-focused analyses also identified differences in selected genotoxic and cytotoxic endpoints. These findings support the potential value of MNcyt biomarkers as a complementary approach for risk assessment in HPV-related cervical disease.

Sperm DNA integrity is a crucial determinant of fertility and early embryonic development. The Sperm Chromatin Dispersion (SCD) test provides a simple, practical, and cost-effective tool for assessing DNA fragmentation; however, the diagnostic reliability depends heavily on the staining technique used to visualize halos of dispersed chromatin. This study evaluated and optimized several commonly and widely used histochemical stains associated with a modified SCD protocol in bovine spermatozoa to improve accuracy and interpretation; hence providing new and useful data for scientists in the field. Frozen-thawed semen samples from five fertile bulls were processed using SCD procedures and stained with Toluidine Blue (TB), Giemsa (G), May Grünwald-Giemsa (MGG), Eosin Thiazine (ET), Feulgen & Rossenbeck (F&R), Hematoxylin & Eosin (H&E), Nuclear Fast Red (NFR), Eosin-Nigrosine (EN), and various modifications of the above stains. Most stains and their modifications (TB, G, ET, NFR, EN) failed to provide consistent or distinguishable halos, whilst specific modifications of MGG, F&R, and H&E produced reproducible halos. The MGG-modified protocol enabled halo visualization but introduced notable nonspecific background staining that reduced distinctiveness of halos. Specific modified protocols of F&R and H&E also visualized halos but reacted variably with agarose: F&R did not produce nonspecific background staining while H&E did. In conclusion, our results demonstrate that specific modifications of MGG, F&R, and H&E visualized halos and thus are suitable for SCD test.

Tissue samples from European sea bass (Dicentrarchus labrax) naturally infected with Aeromonas veronii biovar veronii were subjected to bacteriological, molecular, pathological, and immunohistochemical analyses. The fish were sourced from aquaculture farms in the Aegean region of Turkiye. Clinical and postmortem examinations revealed granuloma formation as the most prominent lesion, primarily affecting the skin, spleen, kidneys, and liver. Sequencing of the 16S rRNA gene from all isolates showed 96-98% identity with A. veronii biovar veronii in the BLASTN 2.13.0+ database. Histopathological analysis demonstrated that granulomas originated from clusters of epithelioid macrophages and progressed through five distinct stages, culminating in caseous necrosis. Immunohistochemical labeling localized A. veronii antigens within necrotic regions and within the cytoplasm of macrophages in granulomatous lesions. These findings suggest that macrophages serve as the primary host cells for the bacterium and play a key role in its intra- and inter-tissue dissemination.

Quantitative and semiquantitative assessment of biomarker expression using immunohistochemistry (IHC) can be performed using several analytical approaches. Manual counting remains widely used but is inherently subjective and susceptible to observer variability. Rule-based image-analysis platforms may provide accessible alternatives, highlighting the need for direct method-comparison and agreement studies. c-Fos is a well-established marker of neuronal activation commonly quantified by counting IHC-positive cells. This study compared manual counting, ImageJ, and CellProfiler for quantifying c-Fos-positive cells in rat brainstem sections. Of 125 images initially evaluated, one was excluded as an extreme outlier, leaving 124 images for final analysis. Each image was independently analyzed by two trained observers using all three methods. ImageJ and CellProfiler were applied through their standard graphical interfaces and built-in functions using predefined user-guided threshold ranges, without additional plugins, scripts, machine-learning models, or custom code. Agreement was assessed using intraclass correlation coefficients (ICCs) and Bland-Altman analysis. No statistically significant difference in c-Fos-positive cell counts was observed among the three methods (Friedman test, P = 0.07). Median (IQR) counts were 6.75 (0.50-13.50) for manual counting, 6.25 (1.00-14.38) for ImageJ, and 6.00 (0.63-14.50) for CellProfiler. Inter-observer agreement was excellent for manual counting (ICC = 0.983), ImageJ (ICC = 0.964), and CellProfiler (ICC = 0.962). Inter-method agreement was also excellent (single-measures ICC = 0.971; 95% CI = 0.961-0.979). Bland-Altman analysis showed small mean biases of 0.33, 0.27, and -0.07 cells, with no significant proportional bias. Manual counting, ImageJ, and CellProfiler produced statistically comparable c-Fos-positive cell counts with excellent agreement. Under the predefined user-guided threshold ranges used, both software platforms closely matched manual counting; however, image-dependent threshold adjustment remains a potential source of observer variability.

Hepatocellular carcinoma (HCC) is a globally prevalent malignancy often triggered by oxidative stress and exposure to hepatotoxic agents such as N-nitrosodiethylamine (NDEA), which induces DNA adducts, inflammation, and liver dysfunction. Natural compounds like plumbagin (PL) and griseofulvin (GF) have demonstrated anticancer potential; however, their in vivo efficacy in NDEA-induced HCC models remains insufficiently explored. This study investigated the protective effects of PL and GF against NDEA-induced hepatocarcinogenesis in male Swiss albino mice. Animals were randomly assigned to seven groups, including normal control, NDEA control (200 mg/kg), and treatment groups receiving PL (2 mg/kg and 6 mg/kg, intraperitoneally), GF (225 mg/kg and 450 mg/kg, orally), or the reference drug sorafenib (30 mg/kg, orally) for 14 days. Biochemical, hematological, and oxidative stress markers were evaluated alongside histopathological analysis. NDEA exposure significantly elevated liver enzymes (ALT, AST, ALP), cholesterol, and malondialdehyde (MDA), while reducing antioxidant enzyme levels (SOD, CAT, GST, and GSH-Px), indicating marked hepatic damage. Treatment with PL and GF led to significant, dose-dependent restoration of these parameters, with notable reductions in MDA and improvements in antioxidant activity. Histopathological assessments further confirmed these findings, showing reduced fibrosis, enhanced hepatocyte regeneration, and improved liver architecture, particularly with high-dose PL, which demonstrated efficacy comparable to sorafenib. In conclusion, PL and GF exhibited substantial hepatoprotective and anticancer effects in the NDEA-induced HCC model, primarily by restoring liver function, reducing oxidative stress, and improving histological outcomes. These findings support their potential as complementary therapeutic agents in liver cancer management.

This article reports a visualization method that combines Sudan red 7B staining with UV autofluorescence in rice root secondary cell walls. Sudan red 7B stains the aliphatic domains of suberin (SPAD), as well as intracellular lipids, whereas after UV excitation of unstained sections the aromatic domains of suberin (SPPD) emitted violet autofluorescence. However, when Sudan red 7B staining preceded UV excitation, the suberized cell walls fluoresced in varying colors from red to magenta, violet or bluish white. The optical phenomenon, Inner Filter Effect (IFE), exhibited this color variation as the differential solubility of Sudan Red 7B stain-SPAD suberin domain complex acted as an optical filter that shifted the violet autofluorescence from SPPD suberin domains to red. At the time of visualization, the red fluorescence spectrally mixed with violet autofluorescence such that the suberized cell walls fluoresced in varying colors. Using this visualization procedure, the intramolecular deposition and distribution pattern for SPAD and SPPD suberin domains in the outer apoplastic barrier can be effectively ascertained.

Motility is a fundamental property of microglial processes. Their dynamic movement is highly sensitive to alterations in the surrounding microenvironment and serves as an early indicator of pathological conditions in the central nervous system (CNS). Thus, comprehensive and accurate quantification of microglial process dynamics is critically needed. To address this, we developed a novel approach based on 3D alignment of original two-photon time-lapse images to correct misalignments caused by microglial migration or animal motion. This alignment facilitates subsequent colocalization analysis, allowing quantification of complete microglial structural dynamics at single-pixel resolution. Compared to conventional methods, our technique offers enhanced comprehensiveness and precision, enabling simultaneous measurement of process extension or retraction, microglial loss or regeneration, and clear visualization of temporal structural changes. Finally, we quantified the dynamic motility of microglia in abilateral common carotid artery occlusion/reperfusion (BCCAO/R) model using this method. We anticipate that this method will significantly improve the quantitative accuracy of dynamic motility assessments for microglia and other cell types in vivo.

Liver fibrosis is characterized by scar formation as a response to injury. Although many drugs have demonstrated antifibrotic efficacy in vitro and in animal models, effectively translating these into effective clinical therapies remains a challenge. Nanotechnology offers potential therapeutic approaches for treating liver fibrosis. This study aimed to assess and compare the antifibrotic effects of losartan and losartan nanoparticles (LP-NPs) on CCl4-induced liver fibrosis in rats.Thirty-two adult male Sprague-Dawley rats were divided into four groups: a control group, a CCl4-induced fibrosis group, a losartan-treated group, and an LP-NPs-treated group. Liver fibrosis was induced by CCl4 injections over six weeks. Losartan or LP-NPs were orally administered for four weeks. At the conclusion of the study, rats were euthanized, and blood samples were collected for liver enzyme assessment. Liver specimens were subjected to histopathological examination (H&E, Sirius red staining), immunohistochemistry (α-SMA and TIMP1), and quantitative real-time PCR (qRT-PCR) for gene expression analysis of α-SMA and TIMP1. Morphometrical studies quantified collagen areas in Sirius red-stained sections and optical density of α-SMA and TIMP1 in immunohistochemically stained sections.The results showed that CCl4 induced significantly elevated liver enzymes, severe distortion of hepatic architecture, marked steatosis and fibrosis. Additionally, there was an increase in collagen content, accompanied by strong positive immunostaining and upregulated gene expression of α-SMA and TIMP1. Both losartan and LP-NPs treatments significantly improved these parameters compared to the CCl4-only group. Notably, LP-NPs demonstrated significantly greater improvements in liver enzyme levels, collagen percentage area, and the gene expression of α-SMA and TIMP1 compared to the standard losartan group. However, no significant difference was observed between the two treatments in α-SMA optical density.In conclusion, this investigation demonstrates that nanoformulation significantly enhanced losartan's antifibrotic efficacy. LP-NPs improved liver function and histological architecture, reduced collagen content after CCl4 administration, and demonstrated greater therapeutic efficacy than conventional losartan.

In vitro cell culture models are fundamental to biomedical research, yet the choice between primary cell cultures and immortalized cell lines profoundly influences experimental outcomes. Immortalized lines offer convenience and unlimited proliferative capacity but frequently acquire genetic, epigenetic, and phenotypic alterations that compromise tissue-specific functions and overall biological fidelity. In contrast, primary cell cultures preserve native morphology, signaling pathways, and functional characteristics of their tissue of origin, providing a more physiologically relevant model. While previous reviews have outlined the general advantages of primary cells, few critically synthesize comparative data across cell types or evaluate strategies to mitigate inherent limitations such as finite lifespan and donor variability. This review critically examines the comparison between primary and immortalized cells across different cell types in terms of preserved tissue-specific characteristics, genomic and epigenetic fidelity, predictive value in drug testing and disease modeling, and emerging roles in personalized medicine. We further discuss innovative approaches, including conditional reprogramming, optimized donor selection, pooled-donor strategies, and standardized culture protocols, that enhance reproducibility and functional stability. By integrating comparative evidence with methodological advances, this review provides a critical framework for selecting appropriate in vitro models, highlighting the translational significance of primary cells in drug discovery, precision medicine, and biomedical research.

Histologic stains which identify specific tissue and cellular components are a staple of all diagnostic pathology laboratories. Histotechnology scientists are constantly attempting to find methods and reagents which are specific, simple to use, and cost effective. Turmeric (Curcuma longa) is a yellow spice readily available in grocery stores and is inexpensive. Previously, turmeric has been investigated as a natural histological stain, with most studies describing predominantly cytoplasmic staining and suggesting turmeric as a replacement for eosin. However, the present study supports the use of turmeric as a counterstain as staining localization is strongly dependent on the solvent, co-solute and procedure used for extraction and for staining. In contrast to several prior reports using alcoholic turmeric extracts, alcohol-based preparations in this study produced little to no appreciable tissue staining despite exhibiting intense coloration in solution. Instead, an aqueous extract with addition of aluminum was the most effective. A 0.5% turmeric solution prepared in aqueous 5% aluminum sulfate produced a bright yellow stain in histologic sections. While yellow chromatin against a colorless background provided limited contrast as a routine nuclear stain substitute, pairing turmeric with selected companion stains yielded favorable results. As a counterstain for alcian blue, Prussian blue, von Kossa, and rhodanine methods, turmeric provided excellent visual contrast. Turmeric is also markedly less expensive than nuclear fast red, supporting its potential as an economical alternative for selected histological applications.

Coccidiosis is a significant enteric disease impacting economically important livestock, caused by a multidrug-resistant parasite. This research was performed to assess the immunohistochemical and molecular insights of Coriandrum sativum leaves extract (CSLE) against Eimeria papillata infection in murine models. CSLE subjected to phytochemical analysis. Thirty male Swiss albino mice were included: Group 1: Control; Group 2: Supplemented group receiving CSLE at 300 mg/kg B.W. (CSLE); Group 3: Infected with 1000 sporulated E. papillata oocysts; Group 4: Infected-treated with CSLE at 300 mg/kg B.W. (Infected + CSLE). Group 5: Infected-treated with amprolium at 120 mg/kg B.W. (Infected+Amp). Following 60 min of infection, groups 4 and 5 received daily treatment for five successive days with CSLE and Amp, respectively. Measurements of oocyst suppression and jejunal glutathione peroxidase (GPx) activity were conducted. Concentrations of Caspase-3 (Casp3) as well as jejunal nuclear factor kappa B (NFκB) and Casp-3 cells were measured immunohistochemically. Gene expression of apoptotic genes was assessed. The CSLE exhibited a substantial concentration of total phenolics and flavonoids, in addition to notable antioxidant activity (48.04 ± 2.98% DPPH scavenging). CSLE treatment exhibited significant oocyst suppression and minimized parasitic stages in the jejunum. CSLE reinstated jejunal GPx activity and mitigated the infection-induced elevation in Casp3 protein levels and its gene expression. Furthermore, CSLE modified apoptotic gene expressions, significantly downregulating BCL-2-associated protein x (BAX) and upregulating B-cell leukemia/lymphoma 2 (BCL2) following CSLE therapy. These data collectively underscore the significant anticoccidial, antioxidant, and antiapoptotic capabilities of CSLE, indicating its potential as a natural therapeutic treatment for E. papillata infection.

Despite the commercial success of protein- and peptide-based therapeutics, their clinical application remains limited due to rapid clearance and inactivation via metabolic pathways. In this study, we PEGylated Aspergillus flavus-derived uricase (Uox) using methoxy polyethylene glycol (5 kDa) activated with Succinimidyl Succinamide (mPEG-SS) at a molar ratio of 1:10 (Uox:mPEG-SS) to enhance its pharmacological properties. This study aimed to evaluate and compare the kinetic behavior, thermodynamic stability, and immunogenic potential of native and PEGylated uricase. mPEG-SS was synthesized through a two-step reaction and characterized by FT-IR,1 H-NMR spectroscopy, and TLC. Successful conjugation to Uox (mPEG-SS@Uox) was confirmed using DLS and SDS-PAGE. Kinetic and thermodynamic analyses revealed a 16.44% increase in thermal stability and a 23.37% increase in half-life at 45°C upon PEGylation, with only a modest 27.3% reduction in catalytic efficiency, likely due to steric hindrance. Immunogenicity assays using CFSE-labeled PBMC proliferation and cytokine profiling via ELISA indicated that PEGylated Uox induced significantly lower immune activation compared to the native enzyme (p < 0.0045), with no statistical difference from the control group (p = 0.2426). These findings demonstrate that PEGylation with 5 kDa mPEG-SS substantially improves uricase's stability and diminishes its in vitro immunogenic potential (as evidenced by reduced PBMC activation), making it a promising candidate for further preclinical development in the treatment of hyperuricemia and gout.

The Greater Caribbean manatee, recently reclassified as endangered, is a keystone species in coastal and freshwater ecosystems. Its long-term conservation requires integrative strategies that combine in situ and ex situ approaches. Although skin-derived cell cultures have been reported for other sirenian species, none have yet been established and characterized for the Greater Caribbean manatee. Thus, the aim of this study was to establish and characterize skin-derived fibroblast cultures from the Greater Caribbean manatee. Skin biopsies obtained from seven animals were used to establish primary cultures. The cells were expanded in vitro, identified as fibroblasts by vimentin immunostaining, and characterized by ultrastructural features, karyotype stability, and the absence of microbial contamination under antibiotic-free culture conditions. Fibroblasts at passages 2-10 were characterized for morphology, viability, metabolic activity, population doubling time, oxidative stress levels, mitochondrial membrane potential, and apoptotic profile. Explant-derived cultures adhered within 24 h, reached sub-confluence after a mean of 8.4 days, and a full confluence by 17.9 days, sustaining active proliferation for up to 84.6 days across ten passages. Cells displayed typical fibroblast-like morphology, high viability (≥ 84%), and stable metabolic activity and proliferation rates (P > 0.05) throughout the culture period. Reactive oxygen species levels increased significantly at passages 4, 8, and 10 relative to passage 2 (P < 0.05), accompanied by a mild reduction in mitochondrial membrane potential, while overall cellular functionality was preserved. Apoptotic and necrotic events exhibited only transient fluctuations without evidence of progressive cellular damage. Cytogenetic analyses revealed a stable karyotype of 2n = 48, with normal XX/XY constitution and no detectable chromosomal abnormalities. Cultures remained free of microbial contamination for 30 days under antibiotic-free conditions. Therefore, this study establishes the first established fibroblast culture from the Greater Caribbean manatee, offering a novel and reliable platform for functional, genomic, cytogenetic, and toxicological studies in conservation biotechnology.

This study aimed to investigate the changes in HSP27 and HSP70 immunoexpression after hyperthermia in the lung and heart of rats on days 5, 10, 20, 25, and 30 of postnatal development. The body temperature of rat pups in the hyperthermia groups was increased to 40°C under the infrared lamp. The recovery time after hyperthermia was kept at 5 hours. HSP27 was present in cardiomyocytes and bronchiole and alveolar epithelial cells of control rats. Compared to the control groups after hyperthermia, HSP27 expression was only significantly different on day PND10 for cardiomyocyte cells (P < 0.05), but not on any day of postnatal development for bronchiole and alveolar epithelial cells (P > 0.05). In control group rats, HSP70 was almost absent in cardiomyocytes and alveolar epithelial cells but was evident in bronchiole epithelial cells. A significant increase in HSP70 expression after hyperthermia was observed throughout the postnatal days in cardiomyocytes and alveolar epithelial cells, but only on day PND20 in bronchiole epithelial cells (P < 0.05). In conclusion, during postnatal development, HSP27 is essential for the development of cardiomyocytes and bronchiole and alveolar epithelial cells, and HSP70 is essential for the development of bronchiole epithelial cells. On the other hand, HSP70 may participate in cytoprotective and repair mechanisms against hyperthermia-induced stress in cardiomyocytes and alveolar epithelial cells during postnatal development.

Canine mammary gland tumors (CMGTs) are among the most common neoplasms in female dogs, and serve as valuable models for human breast cancer in terms of histopathology and biological behavior. This study aimed to investigate the relationship between mast cell density (MCD) (total, peritumoral, and intratumoral) and microvascular density (MVD), and to determine their association with clinicopathological features in 40 canine mammary carcinomas (CMCs). The mast cells (MCs) assessment was conducted by utilizing standard Giemsa staining. To evaluate angiogenesis, the immunohistochemical expression of CD31 was assessed. Results revealed that peritumoral MCD was significantly higher than intratumoral MCD (P < 0.001). Higher histological grades (Grade III) exhibited increased MVD (P = 0.09) and total/intratumoral MCD (P = 0.031 and P = 0.020, respectively). Necrosis was associated with elevated MVD (P = 0.04) and intratumoral MCD (P = 0.027). However, no correlation was found between MVD and MCD, nor were significant differences observed among histological subtypes and lymphovascular invasion. These findings indicate that mast cells might play a role in tumor progression, potentially by promoting angiogenesis. Additionally, their distribution could serve as an extra histopathological marker that, when combined with traditional grading, may enhance prognostic accuracy for CMCs. Further studies with larger cohorts are needed to highlight the complex role of mast cells in the tumor microenvironment.