Histological Methods Research Articles

Immunogenicity and Protectivity of Sputnik V Vaccine in hACE2-Transgenic Mice against Homologous and Heterologous SARS-CoV-2 Lineages Including Far-Distanced Omicron BA.5

Background: The SARS-CoV-2 virus continuously acquires mutations, leading to the emergence of new variants. Notably, the effectiveness of global vaccination efforts has significantly declined with the rise and spread of the B.1.1.529 (Omicron) variant. Methods: The study used virological, immunological and histological research methods, as well as methods of working with laboratory animals. In this study, we evaluated the Gam-COVID-Vac (Sputnik V), an adenoviral vaccine developed by the N.F. Gamaleya National Research Center for Epidemiology and Microbiology, and conducted experiments on hemizygous K18-ACE2-transgenic F1 mice. The variants studied included B.1.1.1, B.1.1.7, B.1.351, B.1.1.28/P.1, B.1.617.2, and B.1.1.529 BA.5. Results: Our findings demonstrate that the Sputnik V vaccine elicits a robust humoral and cellular immune response, effectively protecting vaccinated animals from challenges posed by various SARS-CoV-2 variants. However, we observed a notable reduction in vaccine efficacy against the B.1.1.529 (Omicron BA.5) variant. Conclusions: Our results indicate that ongoing monitoring of emerging mutations is crucial to assess vaccine efficacy against new SARS-CoV-2 variants to identify those with pandemic potential. If protective efficacy declines, it will be imperative to develop new vaccines tailored to current variants of the virus.

Quercetin ameliorates depressive-like phenotypes and memory deficits in hypoxia-induced murine model of stress

BackgroundHypoxia is believed to induce a form of stress that contributes to the exacerbation of certain disease conditions such as heart diseases. Most of these conditions revolve around the central nervous system (CNS) since the brain consumes a significant percentage of oxygen. PurposeThis study aims to determine the specific effect of hypoxia on mood and cognitive behaviours using behavioural assessment and histological methods in mice. Additionally, this study investigated the counter effect of quercetin (QCN) on the hypoxia-induced changes. MethodsA total of thirty (30) mice were equally divided into five (5) groups. Group 1 and 2 received 5 % DMSO while only the latter was subjected to the hypoxia protocol. Groups 3–5 received graded doses of quercetin (10, 20 and 40 mg/kg), respectively. Approximately 24 h after a consecutive seven-day treatment, the mice were tested for depressive-like symptoms as well as dysfunctional memory function using recognized pharmacological tests. Afterwards, histology of specific brain regions was conducted. Finally, the data were analysed by one-way ANOVA and statistical significance was set at p<0.05. ResultsIn comparison with the normal control, mice of the model control group scored significantly (p<0.05) higher in the tests that assess depression and significantly (p<0.05) lower in the tests that assessed memory function, both indicators of adverse effects of hypoxia. Also, the contributing brain regions were also negatively affected by hypoxia. However, a significant attenuation of these effects was observed in the groups that received quercetin. ConclusionIn conclusion, quercetin is a nutritional flavonoid that possesses the ability to improve behaviour and cognition in individuals regularly exposed to hypoxia or suffering from conditions that affect oxygen delivery within the body.

Investigating mechanoreceptor variability and morphometric proxies in Rhesus Macaques: Implications for primate precision touch studies.

The origin of primates has long been associated with an increased emphasis on manual grasping and touch. Precision touch, facilitated by specialized mechanoreceptors in glabrous skin, provides critical sensory feedback for grasping-related tasks and perception of ecologically-relevant stimuli. Despite its importance, studies of mechanoreceptors in primate hands are limited, in part due to challenges of sample availability and histological methods. Dermatoglyphs have been proposed as alternative proxies of mechanoreceptor density. We investigated the relationships between mechanoreceptors (Meissner and Pacinian corpuscles), dermatoglyphs, and demography in the apical finger pads of 15 juvenile to adult rhesus macaques (Macaca mulatta) from a free-ranging population at Cayo Santiago Primate Field Station (Puerto Rico). Our results indicate substantial interindividual variation in mechanoreceptor density (Meissner corpuscles: 11.9-43.3 corpuscles/mm2; Pacinian corpuscles: 0-4.5 corpuscles/mm2). While sex and digit were generally not associated with variation, there was strong evidence of a developmental effect. Specifically, apical pad length, Meissner corpuscle size, and Pacinian corpuscle depth increased while mechanoreceptor densities decreased throughout juvenescence, suggesting that primate mechanoreceptors change as fingers grow during adolescence and then stabilize at physical maturity. We also found Meissner corpuscle density was significantly associated with dermatoglyph ridge width and spacing, such that density predicted by a dermatoglyph model was strongly correlated with observed values. Dermatoglyphs thus offer a useful proxy of relative Meissner corpuscle density in primates, which opens exciting avenues of noninvasive research. Finally, our results underscore the importance of considering demographic factors and methodology in comparative studies of primate touch.

Comprehensive transcriptomic analysis identifies three distinct subtypes of pituitary adenomas: insights into tumor behavior, prognosis, and stem cell characteristics

BackgroundPituitary adenomas (PAs) are the second most common intracranial tumor. While current diagnostic practices rely primarily on histological testing, they often fail to capture the molecular complexities of pituitary adenomas, underscoring the need for a molecular-based classification to refine therapeutic strategies and prognostic assessments. This study aims to provide a molecularly unbiased classification of pituitary adenomas and explore their unique gene expression patterns and clinical features.MethodsWe performed unsupervised hierarchical clustering of the gene expression profiles of 117 PA samples to identify three distinct molecular subtypes. Subsequently, we analyzed the compiled transcriptomic profiles of each individual subtype for pathway enrichment. We also validated the new classification with a validation set containing 158 PAs and 24 pituitary adenoma stem cells (PASCs).ResultsConsensus clustering of transcriptomic data from 117 pituitary adenoma (PA) samples identified three distinct molecular subtypes, each showing unique gene expression patterns and associated biological processes: Group I is enriched in signaling pathways, such as the cAMP signaling pathway and the calcium signaling pathway. Group II is primarily related to metabolic processes, including nitrogen metabolism and arginine biosynthesis in cancer. Group III predominantly shows enrichment in immune responses and potential malignant transformation of the disease, especially through cancer-related pathways such as the JAK–STAT signaling pathway and the PI3K–Akt signaling pathway. The immune profiling revealed distinct patterns for each subtype: Group I had higher dendritic cells and fewer CD8+ T cells, Group II had more monocytes and macrophages, and Group III had elevated levels of T cells. Additionally, there were differences in clinical characteristics and prognosis among the subtypes, with Group III having a worse prognosis, despite the smaller tumor size compared to other groups. Notably, differences in PASCs correlated with the molecular subtypes, with Group III stem cells being enriched in tumorigenesis pathways, PI3K–Akt signaling pathway and Ras signaling pathway.ConclusionOur study introduces a novel molecular classification for pituitary adenomas, independent of traditional histological methods. Each subtype features distinct genetic, molecular, and immunological profiles. We have isolated pituitary adenoma stem-like cells (PASCs), pairing them with tumor tissues for detailed transcriptomic analysis. These PASCs exhibit diverse molecular traits consistent with the new classification.

New CRISPR/Cas9-based Fgfr2C361Y/+ mouse model of Crouzon syndrome exhibits skull and behavioral abnormalities.

Crouzon syndrome (CS), a syndromic craniosynostosis, is a craniofacial developmental deformity caused by mutations in fibroblast growth factor receptor 2 (FGFR2). Previous CS mouse models constructed using traditional gene editing techniques faced issues such as low targeting efficiency, extended lineage cycles, and inconsistent and unstable phenotypes. In this study, a CRISPR/Cas9-mediated strategy was employed to induce a functional augmentation of the Fgfr2 point mutation in mice. Various techniques, including bone staining, micro-CT, histological methods, and behavioral experiments, were employed to systematically examine and corroborate phenotypic disparities between mutant mice (Fgfr2C361Y/+) and their wild-type littermates. Confirmed via PCR-Sanger sequencing, we successfully induced the p.Cys361Tyr missense mutation in the Fgfr2 IIIc isoform of the extracellular domain (corresponding to the p.Cys342Tyr mutation in humans) based on Fgfr2-215 transcript (ENSMUST00000122054.8). Fgfr2C361Y/+ mice exhibited characteristics consistent with the phenotypic features associated with CS, including skull-vault craniosynostosis, skull deformity, shallow orbits accompanied by exophthalmos, midface hypoplasia with malocclusion, and shortened skull base, notably without any apparent limb defects. Furthermore, mutant mice displayed behavioral abnormalities encompassing deficits in learning and memory, social interaction, and motor dysfunction, without anxiety-related disorders. Histopathological examination of the hippocampal region revealed structural abnormalities, suggesting possible brain development impairment secondary to craniosynostosis. In conclusion, we constructed a novel gene-edited Fgfr2C361Y/+ mice strain based on CRISPR/Cas9, which displayed skull and behavioral abnormalities, serving as a new model for studying genetic molecular mechanisms and exploring treatments for CS. KEY MESSAGES: CRISPR/Cas9 crafted a Crouzon model by enhancing Fgfr2-C361Y in mice. Fgfr2C361Y/+ mice replicate CS phenotypes-craniosynostosis and midface anomalies. Mutant mice show diverse behavioral abnormalities, impacting learning and memory. Fgfr2C361Y/+ mice offer a novel model for cranial suture studies and therapeutic exploration.

Diagnosis and management of multiple primary lung cancer.

Multiple primary lung cancer (MPLC), can be categorized as synchronous multiple primary lung cancer (sMPLC) and metachronous multiple primary lung cancer (mMPLC), which are becoming increasingly common in clinical practice. A precise differential diagnosis between MPLC and intrapulmonary metastases (IPM) is essential for determining the appropriate management strategy. MPLC is primarily diagnosed through histology, imaging, and molecular methods. Imaging serves as an essential foundation for preoperative diagnosis, while histology is a critical tool for establishing a definitive diagnosis. As molecular biology advances, the diagnosis of MPLC has stepped into the era of molecular precision. Surgery is the preferred treatment approach, with stereotactic radiotherapy and ablation being viable options for unresectable lesions. Targeted therapy and immunotherapy can be considered for specific patients. A multidisciplinary team approach to evaluation and the application of combination therapy can benefit more patients. Looking ahead, the development of more authoritative guidelines will be instrumental in streamlining the diagnosis and management of MPLC.

The Possible Protective Effect of Taurine on Bisphenol Induced Structural Changes on the Cerebral Cortex of Rats: Histological and Immunohistochemical Study.

Bisphenol A (BPA) is a chemical compound that has been used in many industries, such as paints and dental sealants. Taurine is a semi-essential amino acid with antioxidant, anti-inflammatory, and anti-apoptotic actions. This study aimed to evaluate the possible protective effect of taurine on BPA-induced structural changes in the cerebral cortex of rats using histological and immunohistochemical methods. 35 Wistar rats (180-200 gm) were divided into control: 10 rats; Group I: 5 rats received corn oil (0.5 mL/day); Group II (Bisphenol low dose; BPAL): 5 rats received a low dose of BPA (25 mg/kg/three times/week); Group III (Bisphenol high dose; BPAH): 5 rats received a high dose of BPA (100 mg/kg/three times/week; Group IV: (BPAL + taurine): 5 rats received taurine 100 mg/kg/day and BPAL (25 mg/kg/three times/week); Group V: (BPAH + taurine): 5 rats received taurine 100 mg/kg/day and BPH (100 mg/kg/ three times/week). BPAL& BPAH groups showed significant dose-dependent histological changes of the neuropil, pyramidal, and neuroglial cells at H&E stained sections, significantly increased GFAP, caspase- 3 immunohistochemical reaction with cells positive for Ki67 with many mitotic figures. BPAL + taurine and BPAH + taurine groups showed amelioration of the previously mentioned results. Taurine ameliorated the structural changes induced by BPA in the cerebral cortex of rats.

Novel Fluorescence-Based Methods to Determine Infarct and Scar Size in Murine Models of Reperfused Myocardial Infarction.

Determination of infarct and scar size following myocardial infarction (MI) is commonly used to evaluate the efficacy of potential cardioprotective treatments in animal models. However, histological methods to determine morphological features in the infarcted heart have barely improved since implementation while still consuming large parts of the tissue and offering little options for parallel analyses. We aim to develop a new fluorescence technology for determining infarct area and area at risk that is comparable to 2,3,5-triphenyltetrazolium chloride (TTC) staining but allows for multiple analyses on the same heart tissue. For early and late time points following MI, we compared classical histochemical approaches with fluorescence staining methods. Reperfused MI was induced in male mice, the hearts were extracted 24 h, 7-, 21-, or 28-days later and fluorescently stained by combining Hoechst and phalloidin. This approach allowed for clear visualization of the infarct area, the area at ischemic risk and the remote area not affected by MI. The combined fluorescence staining correlated with the classic TTC/Evans Blue staining 24 h after MI (r = 0.8334). In later phases (>7 d) post-MI, wheat germ agglutinin (WGA) is equally accurate as classical Sirius Red (r = 0.9752), Masson's (r = 0.9920) and Gomori's Trichrome (r = 0.8082) staining for determination of scar size. Additionally, feasibility to co-localize fluorescence-stained immune cells in specific regions of the infarcted myocardium was demonstrated with this protocol. In conclusion, this new procedure for determination of post-MI infarct size is not inferior to classical TTC staining, yet provides substantial benefits, including the option for unbiased software-assisted analysis while sparing ample residual tissue for additional analyses. Overall, this enhances the data quality and reduces the required animal numbers consistent with the 3R concept of animal experimentation.

The Wound-Healing Effect of a Novel Fibroblasts-Impregnated Hydroxyethylcellulose Gel in a Rat Full-Thickness Burn Model: A Preclinical Study.

Background/Objectives: The objective of this study was to assess the efficacy of a cell-containing wound dressing based on fibroblasts in hydroxyethylcellulose (HEC) gel for the local treatment of deep partial-thickness and/or full-thickness skin burns in an animal model. Methods: The rats (male Wistar, n = 100) were subjected to a full-thickness thermal burn (16 cm2). Radical necrectomy was performed one day after the burn. Three days later, the rats were randomly assigned to one of four groups: group 1 (no treatment), group 2 (chloramphenicol and methyluracil ointment, a routine clinical treatment), group 3 (a gel without cells, mock treatment), and group 4 (a dermal fibroblast-impregnated HEC gel). The treatment lasted for five days. The wound-healing process was evaluated by planimetric, cytologic, histologic, and immunohistochemical methods. Results: The differences in the rate of wound healing and the characteristics of wound cytology were identified. In the group 4, a regenerative type of cytogram was revealed, characterized by a significantly increased number of fibroblastic cells in comparison to samples from non-treated and mock-treated animals. Biopsy samples of burn wounds from animals in the group 4l demonstrated the presence of mature granulation tissue and a large number of microvessels. The repair process was stimulated, as evidenced by the increased thickness of newly formed granulation tissue and epidermis in the wound zone, elevated cellularity, and enhanced re-epithelialization activity. The number of Ki-67-positive proliferating cells was significantly higher in group 4 than in the control groups). A small number of non-proliferating donor fibroblasts was observed in the wound area 3 days after the end of treatment. Conclusions: The cell product is an effective agent for promoting wound healing during the regenerative phase. The experiments demonstrated that a gel populated by dermal fibroblasts can stimulate reparative regeneration processes in deep partial- and full-thickness burn wounds.

Clinical, Morphological and Multiplex Analysis of a Case of COVID-19-Associated Placental and Fetal Organ Damage at 16 Weeks Gestation

Abstract. COVID-19-associated damage to the placenta and fetal organs is possible among infected pregnant women. This phenomenon is associated with the presence of the virus in the placental tissue itself and transplacental transmission of SARS-CoV-2 from mother to fetus and occurs in case of severe course of the disease in the mother. However, reliable and clinically significant morphological changes in COVID-19-associated placental damage are still a subject of controversy and speculation. The aim of this article is to present a clinical case analysis and evaluation of the morphological features and results of multiplex analysis of COVID-19- associated fetal and placental tissue damage. Materials and methods. Placental and fetal tissue samples were examined using routine histological methods and immunohistochemical studies of the expression of antibodies to CD15 (MMA), CD3 (MRQ-39), CD68 (KP-1), CD138 (DB-A38). The spectroscopic study was performed using the InSpectr M spectrometer with an excitation wavelength of 532 nm. Results The study revealed an inflammatory infiltrate in the fetal membranes, in the umbilical vein wall, in the stroma of the villi, basal plate and intervillous space, forming thrombi of the vessels of the villi, chorionic plate, intervillous space, trophoblast necrosis, abundant deposition of perivillous fibrin. Spectroscopically, signs of changes in the quantitative and qualitative composition of the amino acids proline, phenylalanine, tyrosine and tryptophan, as well as the appearance of non-standard amino acids (hydroxyproline) and protein isoforms, signs of instability of the acid-base balance were revealed. Conclusion. Among the identified changes, both those characteristic of COVID-19-associated placental damage and non-specific ones associated with the body's inflammatory/cytokine response to COVID-19 and persistent placental infection are determined. The results of the multiplex analysis identified a promising direction in understanding the biological basis and essence of this pathology.

Oenothera biennis improves pregnancy outcomes by suppressing inflammation and fibrosis in an intra-uterine adhesion rat model

Intrauterine adhesion (IUA), also referred to as Asherman’s syndrome, is characterized by fibrosis, inflammation, and can cause amenorrhea and infertility due to abnormal endometrial healing. Histological and Molecular methods were used to evaluate the efficacy of EPO, which is traditionally known for its anti-inflammatory and fibrinolytic properties, in preventing the formation of IUA. Oral administration of EPO reduced the formation of adhesion bands and promoted endometrial regeneration. EPO administration decreased extracellular matrix accumulation, evidenced by the down-regulation of tissue COL1A1 and COL3A1 expression. The anti-inflammatory effect of EPO was confirmed by a reduction in oxidants and down-regulation of pro-inflammatory cytokines including TNF-α, IL-6, IFN-γ, and IL-1β. Furthermore, EPO improved embryonic development parameters, including size and weight of embryo, as well as increased embryo count and live embryo percentage in the rat IUA model. EPO also positively enhanced implantation markers, particularly enlargement and mass gain in the placenta of the treated group, consequently improving pregnancy outcomes such as the number of babies, percent of live babies, baby weight and gestation time. Histopathological investigation provides evidence that oral administration of EPO showed no toxicity on the main three organs including liver, kidney and heart. These results showed that EPO can be considered as a safe and natural product with potent anti-inflammatory and fibrinolytic properties without any observed side effects for the treatment of IUA.

Regenerative Variability of Human Juvenile Chondrocyte Sheets From Different Cell Donors in an Athymic Rat Knee Chondral Defect Model.

This study aimed to establish a combined histological assessment system of neo-cartilage outcomes and to evaluate variations in an established rat defect model treated with human juvenile cartilage-derived chondrocyte (JCC) sheets fabricated from various donors. JCCs were isolated from the polydactylous digits of eight patients. Passage 2 (P2) JCC sheets from all donors were transplanted into nude rat chondral defects for 4 weeks (27 nude rats in total). Defect-only group served as control. Histological samples were stained for safranin O, collagen 1 (COL1), and collagen 2 (COL2). (1) All samples were scored, and correlation coefficients for each score were calculated. (2) Donors were divided into "more effective" and "less effective" groups based on these scores. Then, differences between each group in each category of modified O'Driscoll scoring were evaluated. (1) Modified O'Driscoll scores were negatively correlated with %COL1 area, and positively correlated with %COL2 area and COL2/1 ratio. (2) Four of 8 donors exhibited significantly higher modified O'Driscoll scores and %COL2 areas. JCC donors were divided into two groups by average score values. Significant differences between the two groups were observed in modified O'Driscoll categories of "Nature of predominant tissue," "Reconstruction of subchondral bone," and "Safranin O staining." The combined histological evaluation method is useful for detailed in vivo efficacy assessments of cartilage defect regeneration models. Variations in histological scores among juvenile cartilage-derived chondrocyte donors were correlated to the quality of regenerated cartilage hyaline structure and subchondral bone remodeling observed in the nude rat defect model.

Physiological research of young Russian sturgeons in modern ecological conditions of the Caspian sea

In the Volga-Caspian fisheries basin, artificial reproduction of sturgeon species is the primary source of replenishing their natural populations. The aim of this study is to conduct physiological research on juvenile Russian sturgeon (Acipenser gueldenstaedtii) in the western part of the Northern Caspian Sea, to establish the features of fish homeostasis. The physiological condition of the specimens was studied using a set of physiological and biochemical parameters. Hematological, biochemical, and morpho-histological parameters were evaluated. In the blood of the fish, the contents of hemoglobin, total protein, glucose, inorganic phosphorus, calcium, creatinine, bilirubin, total lipids, cholesterol, and triglycerides were determined. In the muscle tissue of juvenile Russian sturgeon, the physiological and biochemical indicators studied included the content of the main reserve substances of the body — total lipids and water-soluble protein. Histological methods were used to assess the morpho-functional state of the gills. The variability of physiological and biochemical parameters in juvenile Russian sturgeon was calculated using descriptive statistics based on parametric and non-parametric criteria in the blood and muscles. According to the results of correlation statistical analysis, significant changes were observed in the tissues of the sturgeon juveniles, aimed at providing the body with sources of energy and nutrients used for the functioning of body tissues and serving as reserve stocks, as well as maintaining regulatory mechanisms of metabolism and its changes depending on fluctuating environmental conditions. The morphological examination of the gills revealed vascular disorders, hyperplasia, swelling, and hypertrophy of the respiratory epithelium cells, corresponding to borderline disturbances from mild to moderate damage, which was the organ’s response to external environmental influences.

Features of beta cell differentiation during the development of type 2 diabetes mellitus

Type 2 diabetes mellitus is characterized by a mild inflammatory reaction in the pancreas, which affects the structure and function of the pancreatic islets: the number of β-cells decreases and the number of α-cells increases. The work examined the features of β-cell differentiation in the development of experimental type 2 diabetes mellitus and while reducing the inflammatory process. Biochemical, histological methods, enzyme-linked immunosorbent assay, immunohistochemical methods were used using primary antibodies to insulin, glucagon, proliferation marker Ki-67 and secondary antibodies labeled with fluorescent dyes. Streptozotocin and nicotinamide were used to model type 2 diabetes mellitus, and the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione was used to reduce the inflammatory response. Previous studies have shown that it changes the macrophage phenotype from proinflammatory M1 to anti-inflammatory M2. In type 2 diabetes mellitus, against the background of a decrease in the number of macrophages with the CD163 marker and the concentration of the cytokine TGF-β1, which have an anti-inflammatory effect, in the pancreatic islets, a decrease in the number of β-cells and their functional activity was observed, while the content of α-cells synthesizing glucagon increased. After administration of the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione, the opposite picture was observed in the pancreatic islets: against the background of an increase in the number of CD163+ macrophages and the content of TGF-β1, the number of β cells increased and the number of α cells decreased-cells. The increase in the number of insulin-synthesizing cells was not accompanied by their mitotic activity. It is likely that a decrease in the number of CD163+ macrophages and the level of the antiinflammatory cytokine TGF-β1 in the islets are factors contributing to changes in the cell microenvironment and, as a consequence, the differentiation of β-cells into α-cells. On the contrary, an increase in the number of CD163+ macrophages and TGF-β1 against the background of administration of the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione presumably promotes reverse differentiation of α-cells into β-cells and restoration of insulin synthesis pancreas. Targeted effects on the microenvironment of cells in the pancreatic islet in type 2 diabetes mellitus may be a new approach to treating the disease.

Preventing Site-Specific Calpain Proteolysis of Junctophilin-2 Protects Against Stress-Induced Excitation-Contraction Uncoupling and Heart Failure Development.

Excitation-contraction (E-C) coupling processes become disrupted in heart failure (HF), resulting in abnormal Ca2+ homeostasis, maladaptive structural and transcriptional remodeling, and cardiac dysfunction. Junctophilin-2 (JP2) is an essential component of the E-C coupling apparatus but becomes site-specifically cleaved by calpain, leading to disruption of E-C coupling, plasmalemmal transverse tubule degeneration, abnormal Ca2+ homeostasis, and HF. However, it is not clear whether preventing site-specific calpain cleavage of JP2 is sufficient to protect the heart against stress-induced pathological cardiac remodeling in vivo. Calpain-resistant JP2 knock-in mice (JP2CR) were generated by deleting the primary JP2 calpain cleavage site. Stress-dependent JP2 cleavage was assessed through in vitro cleavage assays and in isolated cardiomyocytes treated with 1 μmol/L isoproterenol by immunofluorescence. Cardiac outcomes were assessed in wild-type and JP2CR mice 5 weeks after transverse aortic constriction compared with sham surgery using echocardiography, histology, and RNA-sequencing methods. E-C coupling efficiency was measured by in situ confocal microscopy. E-C coupling proteins were evaluated by calpain assays and Western blotting. The effectiveness of adeno-associated virus gene therapy with JP2CR, JP2, or green fluorescent protein to slow HF progression was evaluated in mice with established cardiac dysfunction. JP2 proteolysis by calpain and in response to transverse aortic constriction and isoproterenol was blocked in JP2CR cardiomyocytes. JP2CR hearts are more resistant to pressure-overload stress, having significantly improved Ca2+ homeostasis and transverse tubule organization with significantly attenuated cardiac dysfunction, hypertrophy, lung edema, fibrosis, and gene expression changes relative to wild-type mice. JP2CR preserves the integrity of calpain-sensitive E-C coupling-related proteins, including ryanodine receptor 2, CaV1.2, and sarcoplasmic reticulum calcium ATPase 2a, by attenuating transverse aortic constriction-induced increases in calpain activity. Furthermore, JP2CR gene therapy after the onset of cardiac dysfunction was found to be effective at slowing the progression of HF and superior to wild-type JP2. The data presented here demonstrate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain cleavage of JP2 offers multifaceted beneficial effects, conferring cardiac protection against stress-induced proteolysis, hypertrophy, and HF. Our data also indicate that specifically targeting the primary calpain cleavage site of JP2 by gene therapy approaches holds great therapeutic potential as a novel precision medicine for treating HF.

Application of small animal ultrasound imaging technology for identification of polycystic ovary syndrome in a mouse model

Background and AimsPolycystic ovary syndrome (PCOS) is a hormonal disorder common among women of reproductive age, characterized by irregular menstrual periods, elevated levels of androgens, and polycystic ovaries, leading to various symptoms and complications such as infertility, metabolic issues, and increased risk of diabetes and heart disease. This study aimed to compare traditional histological methods and ultrasound imaging for consistency in identifying PCOS in a mouse model. The shortest time to construct the PCOS model using letrozole was determined. MethodsFemale C57/BL mice were randomly divided into three groups: Group A received normal saline and a regular diet; Group B received 1 mg/kg/day of letrozole with a regular diet; and Group C received 1 mg/kg/day of letrozole with a high-fat diet. All mice were administered letrozole by intragastric gavage daily for five weeks. The traditional identification method included measuring body weight, examining vaginal smears, monitoring the estrous cycle, measuring serum androgen levels, and performing H&E staining of ovarian tissues. The PCOS model was evaluated using ultrasound imaging to identify and monitor follicles. The significance of the difference between the traditional identification method and the ultrasonic method was calculated using the nonparametric McNemar test, and consistency between the two methods was assessed with the kappa-coefficient test. On this basis, the ultrasound imaging technology was used to monitor the model-making process for 2, 3 and 4 weeks, and to monitor the parameters of the ovary and follicles to judge the shortest time that gavage letrozole caused the appearance of vesicular follicles in the mouse ovary. ResultsThe traditional identification method showed no PCOS phenotype in group A mice, while groups B and C showed multiple ovarian cystic follicles, indicating successful model induction. The ultrasound imaging results were consistent with the traditional method, showing no PCOS in group A and multiple cystic follicles in groups B and C. The McNemar test revealed no significant difference between the traditional and ultrasonic identification methods. The kappa-coefficient test assessed consistency, yielding a value of 0.903, indicating strong agreement between the methods. The ovarian area, diameter, and the number and diameter of cystic follicles were not significantly changed at two weeks in the letrozole group compared with the control group. At three weeks, there were significant increases in the number and in the diameter of vesicular follicles compared with control cells. At four weeks, the number and diameter, the maximum cross-sectional area and diameter of the ovary were significantly increased compared with the control group. ConclusionsThe ultrasound and traditional methods provide consistent results for identifying PCOS in a mouse model. Construction of the PCOS model by letrozole gavage takes at least three weeks.

Progress on three-dimensional visualizing skin architecture with multiple immunofluorescence staining and tissue-clearing approaches.

The skin forms the external covering of the body and is its largest organ, comprising many different cell types. Although the diversity of these cells has been widely studied with various histological methods, our understanding of skin architecture is mainly established on thin tissue sections, which restricted the information available to two dimensions. The development of innovative techniques to induce optical transparency ("clearing") in biological tissues has enabled researchers to visualize the three-dimensional reconstruction of intact organs and thick tissue sections at a cellular resolution. With the aid of tissue-clearing treatment, the labeled cutaneous nerve fibers and blood vessels can be followed for a longer distance on the thicker skin section or the whole mount skin under a fluorescence microscopy or a confocal microscopy. It is beneficial for demonstrating the morphological characteristics of nerve fibers and blood vessels themselves, as well as their spatial interconnection. In this review, we provide a brief summary of the literature on the use of tissue optical clearing methods and describe our experience of multiple fluorescent staining and tissue clearing approaches on thicker skin sections and whole-mount skin in our laboratory. Given the existing conventional methods, we expected to provide a more effective approach to comprehensively study skin architecture.

Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

Abnormal SUMOylation is implicated in non-alcoholic fatty liver disease (NAFLD) progression. Forkhead box protein A1 (FoxA1) has been shown to protect liver from steatosis, which was down-regulated in NAFLD. This study elucidated the role of FoxA1 deSUMOylation in NAFLD. NAFLD models were established in high-fat diet (HFD)-induced mice and palmitate acid (PAL)-treated hepatocytes. Hepatic steatosis was evaluated by biochemical and histological methods. Lipid droplet formation was determined by BODIPY and Oil red O staining. Target molecule levels were analyzed by RT-qPCR, Western blotting, and immunohistochemistry staining. SUMOylation of FoxA1 was determined by Ni-NTA pull-down assay and SUMOylation assay Ultra Kit. Protein interaction and ubiquitination were detected by Co-IP. Gene transcription was assessed by ChIP and dual luciferase reporter assays. Liver FoxA1 knockout mice developed severe liver steatosis, which could be ameliorated by sirtuin 6 (Sirt6) overexpression. Nutritional stresses reduced Sumo2/3-mediated FoxA1 SUMOylation at lysine residue K6, which promoted lipid droplet formation by repressing fatty acid β-oxidation. Moreover, Sirt6 was a target gene of FoxA1, and Sirt6 transcription activity was restrained by deSUMOylation of FoxA1 at site K6. Furthermore, nutritional stresses-induced deSUMOylation of FoxA1 promoted the ubiquitination and degradation of FoxA1 with assistance of murine double minute 2 (Mdm2). Finally, activating FoxA1 SUMOylation delayed the progression of NAFLD in mice. DeSUMOylation of FoxA1 at K6 promotes FoxA1 degradation and then inhibits Sirt6 transcription, thereby suppressing fatty acid β-oxidation and facilitating NAFLD development. Our findings suggest that FoxA1 SUMOylation activation might be a promising therapeutic strategy for NAFLD.

In Vitro Cytotoxic Potential and In Vivo Antitumor Effects of NOS/PDK-Inhibitor T1084.

Previously, we showed the antitumor activity of the new NOS/PDK inhibitor T1084 (1-isobutanoyl-2-isopropylisothiourea dichloroacetate). The present study included an assessment of in vitro cytotoxicity against human malignant and normal cells according to the MTT-test and in vivo antitumor effects in solid tumor models in comparison with precursor compounds T1023 (NOS inhibitor; 1-isobutanoyl-2-isopropylisothiourea hydrobromide) and Na-DCA (PDK inhibitor; sodium dichloroacetate), using morphological, histological, and immunohistochemical methods. The effects of T1084 and T1023 on the in vitro survival of normal (MRC-5) and most malignant cells (A375, MFC-7, K562, OAW42, and PC-3) were similar and quantitatively equal. At the same time, melanoma A375 cells showed 2-2.5 times higher sensitivity (IC50: 0.39-0.41 mM) to the cytotoxicity of T1023 and T1084 than other cells. And only HeLa cells showed significantly higher sensitivity to the cytotoxicity of T1084 compared to T1023 (IC50: 0.54 ± 0.03 and 0.81 ± 0.02 mM). Comparative studies of the in vivo antitumor effects of Na-DCA, T1023, and T1084 on CC-5 cervical cancer and B-16 melanoma in mice were conducted with subchronic daily i.p. administration of these agents at an equimolar dose of 0.22 mmol/kg (33.6, 60.0, and 70.7 mg/kg, respectively). Cervical cancer CC-5 fairly quickly evaded the effects of both Na-DCA and T1023. So, from the end of the first week of Na-DCA or T1023 treatment, the tumor growth inhibition (TGI) began to decrease from 40% to an insignificant level by the end of the observation. In contrast, in two independent experiments, CC-5 showed consistently high sensitivity to the action of T1084: a significant antitumor effect with high TGI (43-58%) was registered throughout the observation, without any signs of neoplasia adaptation. The effect of precursor compounds on melanoma B-16 was either minimal (for Na-DCA) or moderate (for T1023) with TGI only 33%, which subsequently decreased by the end of the experiment. In contrast, the effect of T1084 on B-16 was qualitatively more pronounced and steadily increasing; it was accompanied by a 3-fold expansion of necrosis and dystrophy areas, a decrease in proliferation, and increased apoptosis of tumor cells. Morphologically, the T1084 effect was 2-fold superior to the effects of T1023-the TGI index reached 59-62%. This study suggests that the antitumor effects of T1084 develop through the interaction of NOS-dependent and PDK-dependent pathophysiological effects of this NOS/PDK inhibitor. The NOS inhibitory activity of T1084 exerts an anti-angiogenic effect on neoplasia. At the same time, the PDK inhibitory activity of T1084 enhances the cytotoxicity of induced intratumoral hypoxia and suppresses the development of neoplasia adaptation to anti-angiogenic stress. Such properties allow T1084 to overcome tumor resistance and realize a stable synergistic antitumor effect.

Evaluation of Ultrasound and Intraoperative Histological Methods in the Diagnosis of Sentinel Lymph Node Metastases in Various Biological Subtypes of Breast Cancer

Evaluation of Ultrasound and Intraoperative Histological Methods in the Diagnosis of Sentinel Lymph Node Metastases in Various Biological Subtypes of Breast Cancer