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.
Previous molecular methods for sex determination of sturgeon of the Acipenser genus have been repeatedly validated for females, but have largely relied on suboptimal solutions to compensate for the lack of a confirmed control for males. By combining sturgeon cytochrome b (cyt b) gene amplification as a reference within existing qPCR techniques, this limitation is addressed through the demonstration of both the presence and confirmed absence of the female-specific AllWSex2 amplicon. Duplexing produces distinct melting curve profiles that enable simultaneous evaluation of both Acipenser confirmation and female identification targets. Incorporating the cyt b primer pair results in clearly distinguishable peak patterns for reliable sex determination, eliminating the need for additional probe detection, and making the approach more cost-effective, faster, and efficient than traditional multiplexing techniques. The method also highlights the importance of developing and appending existing assays to include said endogenous reference controls, such as the cyt b marker used here.
RNA interference (RNAi) screening is widely used for systematic gene perturbation, but reproducible readouts depend on careful assay design and consistent performance. Here, we describe a plate-based small interfering RNA (siRNA) screening workflow that combines repeated green fluorescent protein (GFP) fluorescence measurements (0-72 h) with an orthogonal endpoint XTT metabolic readout performed in the same wells. Using a medium-scale siRNA library and technical replicate plates, we establish a quality control (QC)-guided framework for evaluating assay performance, providing a robust basis for downstream gene-level hit identification. Complementary metrics, including coefficient of variation (CV), strictly standardized mean difference (SSMD), and Spearman rank correlation, were used to assess variability, control separation, and replicate concordance across plates and time points. These analyses revealed improved assay performance at later time points, with increased control separation and reproducibility, supporting the selection of 72 h as the most robust endpoint for downstream comparisons in the present screening workflow. Together, the workflow and QC framework provide a transferable strategy for qualifying plate-based RNAi screening experiments, enabling reliable downstream hit selection and follow-up studies.
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.
This study developed a detection method for antisense oligonucleotides (ASOs) by ligase detection reaction (LDR) using T4 RNA ligase 2 (T4Rnl2). The LDR was applied to detect a nusinersen-based ASO, an 18-mer 2'-O-methoxyethyl-modifiedoligonucleotide with phosphorothioate linkages. The method enabled detection of the ASO at concentrations of 0.08-10 nM in aqueous samples. In addition, when combined with mixed-mode solid phase extraction for ASO spiked samples, the method detected 0.8-100 nM in serum and 0.04-25 nmol/g tissue in spinal tissue. The method was further applied to determine ASO levels in plasma and spinal tissue following intrathecal injection in mice. These results demonstrated that LDR using T4Rnl2 was a simple and sensitive approach for detecting ASOs in biological samples.
In laboratory mice, the H2-Ld gene confers resistance to encephalitis upon infection with Toxoplasma gondii, a foodborne parasite capable of infecting all endotherm animals tested. H2-Ld is carried by inbred strains with major histocompatibility complex (MHC) haplotypes H-2a (e.g., A/J) and H-2d (e.g., BALB/c), but not by those with haplotypes H-2b (e.g., C57BL/6) and H-2k (e.g., C3H/He). As H2-Ld is typically detected by serological typing of ex vivo spleen samples, testing is usually terminal. Here we present a polymerase chain reaction (PCR)-based method to reliably detect the H2-Ld gene in ear biopsies from live mice, thus allowing researchers to ascertain the H2-Ld genotype for breeding or other in vivo procedures.
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.
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.
Glucose-lowering treatment and antidiabetic drugs show great potential for preventing and intervening in diabetes mellitus (DM)-associated cognitive dysfunction or advanced dementia. This study delves into the role and mechanism of low-carbohydrate diet (LCD) combined with Xiaokeping (XKP) on these conditions. Type 2 diabetes (T2D) rat models were constructed via high-fat diet and streptozotocin (STZ) injection, and treated with LCD and XKP alone or in combination for 8 weeks. Body weight and indicators of blood glucose and lipids were determined. Additionally, memory and spatial learning abilities were assessed. Tissue staining of the hippocampus was used to observe pathological changes. Oxidative stress, levels of inflammatory factors, and phosphatidylinositol 3-kinase (PI3K)/Akt serine/threonine kinase (Akt) were analyzed. The results demonstrated that LCD and/or XKP treatment recovered body weight, blood glucose, and lipid balance, while enhancing spatial memory and attenuating lesion in the hippocampus of diabetic rats. LCD and/or XKP treatment increased superoxide dismutase, decreased malondialdehyde, interleukin-1β/6, and tumor necrosis factor alpha level in the hippocampus of diabetic rats, which suggests a reduction in oxidative stress and neuroinflammation. Moreover, LCD and/or XKP treatment activated the PI3K/Akt pathway. LCD combined with XKP displayed superior efficacy compared to monotherapy. Collectively, LCD combined with XKP mixture improves cognitive decline of T2D rats by suppressing oxidative stress and neuroinflammation in the hippocampus, through a mechanism involving the activation of the PI3K/Akt pathway.
This protocol describes a methodology combining TurboID - a recently developed proximity biotinylation technique - with conventional epitope-tag based co-immunoprecipitation (Co-IP) to analyze protein-protein interactions (PPIs) in cell culture systems. This integrated approach allows the targeted examination of both transient and stable protein interactors, enhancing our understanding of protein dynamics. TurboID captures transient interactions often missed by Co-IP, which captures high affinity, stable interactors. Combination of both techniques enables direct comparison of interaction strengths, providing insights into the dynamic nature of protein interactions within cells. The rapid biotinylation capability of TurboID reduces background noise and false positives while Co-IP enriches stable interactors, together improving data quality and interpretation of the interactomic landscape. Demonstrating the efficacy of this methodology, proteins relevant to the pathology of Spinal Muscular Atrophy were utilized to explore variations at the interactome level. The use of identical starting cell lysates for both TurboID and Co-IP minimized variability and ensured datasets were comparable, allowing for consistency and enhancing the reliability of findings regarding the nature and strength of protein interactions. This novel framework effectively combines both innovative and classical techniques while maintaining consistency in sample handling, advancing our understanding of the intricate networks that govern cellular processes. Full data-set available at https://www.ebi.ac.uk/pride/ reference ID PXD066858.
METHOD SUMMARY:Paraformaldehyde-fixed mouse liver samples are sectioned on a vibratome at 100 µm. The free-floating sections are permeabilized, followed by exposure to one of two heat-induced antigen retrieval methods selected based on the antigen of interest. Antigen retrieval is carried out in a mechanical stabilization apparatus developed here to prevent free-floating sections from deforming. The immunostaining is then carried out with noted modifications to previously established protocols. MULTIDISCIPLINARY ABSTRACT:Spatial organ biology widely relies on slicing tissues into one cell-thick sections. These studies have advanced our understanding of organ structure and function; yet, thin sections have been insufficient to investigate three-dimensional organization in complex organs. A more recent approach utilizes thicker sections, enabling visualization of volumetric tissue architecture. Currently, few protocols exist to strengthen immunofluorescence signal on thick tissue sections while maintaining tissue integrity, particularly for difficult antigens. Here, we developed a simple, affordable method that mechanically stabilizes thick tissue sections and preserves tissue integrity during heat-mediated antigen retrieval used to enhance antigen accessibility. This method widens the utility of thick tissue sections and facilitates holistic analysis of complex tissues, opening new possibilities for spatial organ research.
Cell line stability of mammalian cell lines is an important concern for manufacturing of biopharmaceuticals. Characterization of the master cell bank (MCB) for monoclonal antibody A (Mab A) via multiplex fluorescence in situ hybridization (M-FISH) analysis identified two distinct karyotypic cell populations. As a result, a regulatory agency requested additional analysis to support a single clonal lineage of the MCB and demonstrate control of the two cell populations in subsequently derived cell banks. Next Generation Sequencing (NGS) revealed 11 transgene integration site sequences. A multiplex (measure two amplicons) Droplet Digital PCR (ddPCR) method was developed and characterized those 11 integration site copy numbers in the MCB. Analysis of 12 subclones from the MCB displayed that among 7 subclones there was loss of at least one of five specific integration sites. The characterization of these five unstable sites in the end production cells (EPCs) of four manufacturing batches are consistent with the MCB. The conservation of five other integration sites among all clones supported that these cells were derived from the same population and that loss of the unstable integration sites contributed to the two distinct karyotypic populations.
Long interspersed nuclear element-1 (LINE-1 or L1) is the only autonomously active retrotransposon in the human genome and produces both sense and antisense transcripts from its 5' untranslated region (5'UTR) of L1Hs, a human-specific L1 subfamily. Among these, ORF0 is an antisense transcript-derived protein implicated in retrotransposition activity, yet its mRNA expression has been difficult to quantify because strand discrimination is required for accurate detection. Here, we developed a strand-specific quantitative polymerase chain reaction (qPCR) method incorporating multiplex gene-specific tagged primers in the reverse transcription (RT) step, which enables simultaneous quantification of antisense ORF0 and sense 5'UTR and ORF2 transcripts. Validation in 5-aza-2'-deoxycytidine-treated neuroblastoma cells confirmed dose-dependent increases in expression of ORF0 and sense transcripts, demonstrating the strand specificity and functionality of this method. Application to human postmortem prefrontal cortex and cerebellum samples revealed stable expression of ORF0 and sense transcripts. This assay provides a robust and scalable tool for the precise quantification of strand-specific L1 transcription, complementing locus-specific analyses, and offering a platform for future studies on the role of ORF0 and L1 antisense transcription.
Catalytic DNA molecules (DNAzymes) offer a programmable means to manipulate RNA without genomic modification. Although DNAzymes have been thoroughly characterized in culture and in various animals, their utility in the prevalent model system, Caenorhabditis elegans, has not been explored. Here, we examine the feasibility of catalytic DNA-mediated RNA targeting in C. elegans. Cholesterol-conjugated 8-17 DNAzymes were efficiently delivered to intestinal cells following ingestion, with no visibile toxic effects on growth or health, reducing targeted reporter fluorescence and endogenous gene function. The observed dependence on cholesterol tagging was consistent with a requirement for cellular uptake. Our findings provide a proof-of-principle demonstration of catalytic DNA activity in C. elegans, and highlight considerations for design and optimization. This exploratory study lays the foundation for expanding catalytic nucleic acid technologies in nematodes, supporting future development of programmable RNA tools in a powerful model system.
Precision-cut tissue slices (PCTS), defined as thin, uniformly cut sections of fresh organs that preserve native tissue architecture and cell-cell interactions, serve as valuable ex vivo models for studying tissue physiology, toxicology, and metabolism of drug substances. However, existing PCTS preparation protocols typically focus on single-organ preparation, leaving the feasibility of sequential multi-organ slicing remains uncertain. This study evaluated the viability of rat liver and kidney slices during a 72-hour incubation period, considering factors such as sex, organ collection method, coring technique, and slicing duration. Viability was assessed based on intracellular Adenosine Triphosphate (ATP) as an indicator of metabolic activity. No sex-related differences in ATP levels were observed in 18-20-month-old rats. ATP content was also comparable between central and peripheral liver regions. In contrast, kidney slices composed of the medulla demonstrated significantly higher ATP levels than those predominantly composed of cortex, reflecting regional differences in microanatomy and metabolic adaptability. Furthermore, slices obtained from anesthetized rats showed better ATP recovery during incubation than those from euthanized rats, highlighting the importance of adequate oxygen supply maintained through blood circulation during organ collection. The sequential preparation of liver and kidney slices within 4 hours after organ collection was feasible, as slicing performed within 0-2 hours and 2-4 hours did not significantly affect viability. These findings provide practical insights for optimizing PCTS preparation supporting reliable slice viability, and enabling efficient multi-organ processing when tissue slicer capacity is limited.
Circadian rhythms are biological processes that occur in approximately 24-hour cycles in organisms. Disruption of circadian rhythms (CD) is thought to have adverse effects on many organs, including the eyes. The aim of this study was to evaluate the effects of circadian disruption on the cornea and conjunctiva, as well as the impact of two forms of omega-3 supplementation on the ocular surface, using a rat model of circadian disruption. This experimental study included 32 female Wistar albino rats, which were divided into four groups. The control group (Group 1) was maintained under normal feeding and sleeping conditions without any disruption. Circadian disruption was induced in the other three groups, which were administered saline (Group 2), fish oil (Group 3), or flaxseed oil (Group 4) via oral gavage. On the 31st day, all rats were euthanized, and corneal, conjunctival, and palpebral tissues were collected from both eyes through enucleation. Histological examination and immunohistochemical analysis of Caspase-3 (Cas-3), tumor necrosis factor-alpha (TNF-α), and PERIOD-2 (PER-2) were performed on the corneal and conjunctival tissues. Group 2 exhibited significantly thicker corneas compared to Group 1 (P < 0.001). Additionally, hyperemia, inflammatory cell infiltration in the conjunctiva, higher expression levels of Cas-3 and TNF-α, and decreased PER-2 expression were observed in the corneal and conjunctival tissues of Group 2. These pathological changes were minimal or absent in Groups 3 and 4. Notably, Group 3 showed better amelioration of these alterations compared to Group 4. Disruption of circadian rhythms can have a negative impact on the cornea and conjunctiva. Omega-3 supplementation demonstrated a significant protective effect against ocular tissue damage induced by circadian disruption. Fish oil was more effective than flaxseed oil in reducing corneal thickening, inflammation, and apoptotic marker expression, highlighting its potential as a therapeutic intervention for circadian rhythm-related ocular pathologies.
The aim of this study was to determine the effects of three linear cyanobacterial oligopeptides: microginin-FR1 (MG-FR1), anabaenopeptin-B (ANA-B), aeruginosin 98A (AER-A), aeruginosin 98B (AER-B) and one cyclic oligopeptide anabaenopeptin-B (ANA-B) on RTgill-W1 fluorescent biomarkers of cell line derived from Rainbow trout gill cells. The following cellular parameters were determined after 48-h exposure to different concentrations (205 nM, 512 nM and 1004 nM) of the oligopeptides: Hoechst and Propidium Iodide (PI) fluorescence, intracellular glutathione level and cytoskeletal F-actin structure. The study showed that AER-B and MG-FR1 were the most potent inhibitors of Hoechst fluorescence, AER-B additionally increased PI fluorescence level. MG-FR1 and ANA-B substantially reduced glutathione and F-actin fluorescence level in RTgill-W1 cells. The obtained results suggest that both linear and cyanobacterial oligopeptides should be considered as factors inducing detrimental alterations in RTgill-W1 cells, suggesting that in natural conditions exposure of fish to cyanobacterial metabolites may result in cytotoxic effects in fish gills.
