Staining Of Sections Research Articles

Development of Triphenylmethane Dyes for In Vivo Fluorescence Imaging of Aβ Oligomers.

Detection of amyloid β (Aβ) oligomers, regarded as the most toxic aggregated forms of Aβ, can contribute to the diagnosis and treatment of Alzheimer's disease (AD). Thus, the development of imaging probes for in vivo visualization of Aβ oligomers is crucial. However, the structural uncertainty regarding Aβ oligomers makes it difficult to design imaging probes with high sensitivity to Aβ oligomers against highly aggregated Aβ fibrils. In this study, we developed Aβ oligomer-selective fluorescent probes based on triphenylmethane dyes through screening of commercially available compounds followed by structure-activity relationship (SAR) studies on cyclic or acyclic 4-dialkylamino groups. We synthesized 11 triarylmethane-based Aβ oligomer probe (TAMAOP) derivatives. In vitro evaluation of fluorescence properties, TAMAOP-9, which had bulky 4-diisobutylamino groups introduced into three benzenes of a twisted triphenylmethane backbone, showed marked fluorescence enhancement in the presence of Aβ oligomers and demonstrated high selectivity for Aβ oligomers against Aβ fibrils. In docking studies using the Aβ trimer model, TAMAOP-9 bound to the hydrophobic surface and interacted with the side chain of Phe20. In vitro section staining revealed that TAMAOP-9 could visualize Aβ oligomers in the brains of AD model mice. An in vivo fluorescence imaging study using TAMAOP-9 showed significantly higher fluorescence signals from the brains of AD model mice than those of age-matched wild-type mice, confirmed by ex vivo section observation. These results suggest that TAMAOP-9 is a promising Aβ oligomer-targeting fluorescent probe applicable to in vivo imaging.

Developing a gel-col heterogeneous network hydrogel scaffold for tissue-engineered skin with enhanced basement membrane formation

Tissue-engineered skin is constructed utilizing biocompatible matrix materials as scaffolds, and incorporating human epidermal keratinocytes (HEKs), dermal fibroblasts (HDFs), and bioactive molecules, addressing clinical issues such as skin graft rejection and donor scarcity. Conventional fabrication methods of tissue-engineered skin typically rely on homogeneous single-network scaffolds, which may not adequately address the distinct requirements of epidermal and dermal cells or the critical formation of the basement membrane, potentially impacting the development and functional integrity of the skin tissue. Cell culture studies employing GelMA hydrogels of varied concentrations have elucidated that epidermal cells exhibit increased viability on scaffolds with higher concentrations (15 %), whereas fibroblasts show increased proliferation within scaffolds of lower concentrations (5 %). Consequently, we have engineered a GelMA-collagen (Gel-Col) hydrogel scaffold with a heterogeneous network of dense and sparse matrices, conducive to the growth of both fibroblasts and epidermal cells. Culturing epidermal cells on this innovative scaffold, coupled with histological section staining, RT-PCR, and Western blot analyses, has demonstrated that the scaffold significantly enhances cellular adhesion and promotes the development of the epidermal basement membrane compared to single-network hydrogels. Mouse wound transplantation experiments have further confirmed the scaffold’s effectiveness in accelerating wound repair, supporting the development of the basement membrane, and enhancing epidermal regeneration. This research provides a reference for constructing tissue-engineered skin that more precisely emulates the intricate architecture and cellular dynamics of natural skin, propelling the advancement of tissue-engineered skin grafts in the clinical treatment of skin injuries.

Characterization of ovarian progenitor cells for their potential to generate steroidogenic theca cells in vitro.

Progenitor cells with ovulation-related tissue repair activity were identified with defined markers (LGR5, EPCR, LY6A, and PDGFRA), but their potentials to form steroidogenic cells were not known. This study shows that the cells can generate progenies with different steroidogenic activities. Adult mammalian ovaries contain stem/progenitor cells necessary for folliculogenesis and ovulation-related tissue rupture repair. Theca cells are recruited and developed from progenitors during the folliculogenesis. Theca cell progenitors were not well defined. The aim of current study is to compare the potentials of four ovarian progenitors with defined markers (LY6A, EPCR, LGR5, and PDGFRA) to form steroidogenic theca cells in vitro. The location of the progenitors with defined makers was determined by immunohistochemistry and immunofluorescence staining of ovarian sections of adult mice. Different progenitor populations were purified by magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) techniques from ovarian cell preparation and were tested for their abilities to generate steroidogenic theca cells in vitro. The cells were differentiated with a medium containing LH, ITS, and DHH agonist for 12 days. The results showed that EPCR+ and LGR5+ cells primarily distributed along the ovarian surface epithelium (OSE), while LY6A+ cells distributed in both the OSE and parenchyma. However, PDGFRA+ cells were exclusively located in interstitial compartment. When the progenitors were purified by these markers and differentiated in vitro, LY6A+ and PDGFRA+ cells formed steroidogenic cells expressing both CYP11A1 and CYP17A1 and primarily producing androgens, showing characteristics of theca-like cells, while LGR5+ cells generated steroidogenic cells devoid of CYP17A1 expression and androgen production, showing a characteristic of progesterone-producing cells (granulosa- or lutea-like cells). In conclusion, progenitors from both OSE and parenchyma of adult mice are capable of generating steroidogenic cells with different steroidogenic capacities, showing a possible lineage preference.

Abstract PO4-18-03: Combined Treatment with Pyrotinib and Chrysin Synergistically Improve the Autophagy Level in HER2-Positive Breast Cancer by Regulating the miR-16-5p/ZBTB16/G6PD Axis

Abstract Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) has been the most challenging subtype of BC, which consists of 20% of BC with an apparent correlation with poor prognosis. Despite that pyrotinib, a new HER2 inhibitor, has led to dramatic improvements in prognosis outcome, the efficacy of pyrotinib as monotherapy remain largely restricted due to its acquired resistance. Therefore, we aim at identifying a potent antitumor drug incorporated with pyrotinib for amplifying therapeutic efficacy for treating HER2-positive BC. Here, we reported a novel incorporation of pyrotinib in combination with chrysin, and explored its antitumor efficacy and the underlying mechanisms on HER2+ breast cancer. We determined that pyrotinib combined with chrysin yielded a potent synergistic effect to induce apoptosis and inhibit BT-474 and SK-BR-3 tumor cells, and suppressed in vivo tumor growth in tumor-bearing mice models. This may be mechanistically attributed to the induction of enhanced endoplasmic reticulum stress to increase the autophagy level. Furthermore, it was demonstrated that the combined treatment with pyrotinib and chrysin induced ubiquitination and G6PD degradation by regulating zinc finger and BTB/POZ domain-containing family protein 16 (ZBTB16) in tumorigenesis of BC. Besides, we identified that miR-16-5p is a potential upstream regulatory target of ZBTB16. Blocking miR-16-5p overexpression could inhibit HER2-positive tumorigenesis and significantly potentiate the efficacy of pyrotinib in combination with chrysin. Together, these findings demonstrate the utility of combined treatment with pyrotinib and chrysin as a potential option in the target treatment of HER2-positive BC through an unrecognized miR-16-5p/ZBTB16/G6PD axis. The miR-16-5p/ZBTB16/G6PD axis plays a crucial role in the pyrotinib plus chrysin-enabled anti HER2-positive BC Fig.1 a Cell viability of SK-BR-3 cells received various treatments. b Detection of cell cycle arrest of SK-BR-3 cells after various treatments. c Schematic diagram of the established protocol of the animal models. Photograph of resected tumor tissues, tumor volume (d), and tumor weight (e) of mice in different treatment groups during the whole testing period. f H&E, Ki67, and Tunel immunohistochemical staining of tumor sections of mice in various treatment groups. g Autophagy flux of SK-BR-3 cells labeled with mRFP-GFP-LC3 in the different treatment groups. h Expression level of ER stress markers in SK-BR-3 cells after various treatments via RT-qPCR and western blot. *P <0.05, compared with control group (DMSO), #P <0.05, compared with chrysin group, & P<0.05, compared with pyrotinib group. i Western blot analysis of the expression of G6PD in different treatment groups. j Fluorescence images of SK-BR-3 cells subjected to G6PD overexpression for the detection of autophagy level. k Ubibrowser database predicting the E3 ubiquitin ligases that may be involved in the regulation of G6PD ubiquitination. l Western blot analysis of G6PD protein half-life in SK-BR-3 cells with ZBTB16 silence. Cells were co-incubated with cycloheximide (CHX, 50μg/ml) for the indicated time. m Determination of the ubiquitination of G6PD in cells pretreated with 10 μM MG-132 for 3 h. Cells were transfected with ubiquitin after different treatments. The ubiquitinated G6PD was subjected to immunoprecipitation before western blot with ubiquitin antibody. n Dual-luciferase report verifying the targeting of ZBTB16 and miR-16-5p. o Transmission electron microscopy images of autophagosomes of tumors in various treatment groups (× 20000). p Immunohistochemical staining images of tumor slices for the determination of G6PD. q Schematic diagram for the underlying mechanism of pyrotinib combined chrysin against HER2-positive BC. Citation Format: Ting Luo, Xiaorong Zhong, Ping He, Dan Zheng, Yan Cheng, Kunrui Zhu. Combined Treatment with Pyrotinib and Chrysin Synergistically Improve the Autophagy Level in HER2-Positive Breast Cancer by Regulating the miR-16-5p/ZBTB16/G6PD Axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-18-03.

Abstract 2067: Myeloid Cell Lgr4 Contributes To Atherosclerotic Lesion Formation

Background: R-Spondin 2 (RSPO2), a matricellular protein, primarily functions via binding to cell surface receptors including leucine-rich repeat-containing G protein-coupled receptor (LGR) 4, 5, and 6. We previously identified elevated RSPO2 levels in both human and mouse atherosclerotic arteries. Our preliminary experiments demonstrated increased expression of LGR4 in human atherosclerotic aortic segments, identified Lgr4 as the major RSPO2’s receptor in primary murine macrophages, and atherogenic molecule oxLDL stimulated LGR4 levels in macrophages. However, the precise role of macrophage LGR4 in atherosclerosis development is unknown. Methods and Results: We generated novel myeloid cell-specific Lgr4 knockout mice ( Lgr4 fl/fl LysM Cre +/- , Lgr4 ΔM ) by breeding Lgr4 fl/fl mice with LysM Cre +/- mice. AAV8-h PCSK9 -injected Lgr4 ΔM and littermate control Lgr4 fl/fl ( Lgr4 WT ) were fed a Western diet (16 weeks) to investigate for atherosclerosis. Oil red O (ORO) staining of whole aortas (en face) and aortic root sections showed reduced atherosclerosis in male Lgr4 ΔM mice compared with sex-matched Lgr4 WT mice. Further histochemical staining performed on aortic root sections revealed smaller necrotic cores in Lgr4 ΔM mice. However, there were no significant differences in plasma total cholesterol and body weight. Interestingly, male Lgr4 ΔM mice had decreased fat mass percentage, blood glucose, and epididymal adipose tissue weight. Initial studies with female mice ( n = 4/group) demonstrated no differences in atherosclerosis and metabolic parameters. Additional in vitro studies exhibited that RSPO2 treatment induces oxLDL uptake, reduces efferocytic capacity, and promotes activation of VASP protein (inducer of actin aggregates) in macrophages. Further, the deletion of Lgr4 reduces lipid accumulation in macrophages. Conclusions: Taken together, these findings suggest that deletion of myeloid cell Lgr4 suppresses atherosclerotic lesion formation and identify Lgr4 as a novel therapeutic target for atherosclerosis.

The Application of 2d Mxene Nanosheet -Based Thermosensitive Gel Delivery System Loaded with Cisplatin and Imiquimod for Lung Cancer.

Lung cancer's high incidence and dismal prognosis with traditional treatments like surgery and radiotherapy necessitate innovative approaches. Despite advancements in nanotherapy, the limitations of single-treatment modalities and significant side effects persist. To tackle lung cancer effectively, we devised a temperature-sensitive hydrogel-based local injection system with near-infrared triggered drug release. Utilizing 2D MXene nanosheets as carriers loaded with R837 and cisplatin (DDP), encapsulated within a temperature-sensitive hydrogel-forming PEG-MXene@DDP@R837@SHDS (MDR@SHDS), we administered in situ injections of MDR@SHDS into tumor tissues combined with photothermal therapy (PTT). The immune adjuvant R837 enhances dendritic cell (DC) maturation and tumor cell phagocytosis, while PTT induces tumor cell apoptosis and necrosis by converting light energy into heat energy. Material characterization employed transmission electron microscopy, X-ray photoelectron spectroscopy, phase transition temperature, and near-infrared thermography. In vitro experiments assessed Lewis cell proliferation and apoptosis using CCK-8, Edu, and TUNEL assays. In vivo experiments on C57 mouse Lewis transplant tumors evaluated the photothermal effect via near-infrared thermography and assessed DC maturation and CD4+/CD8+ T cell ratios using flow cytometry. The in vivo anti-tumor efficacy of MDR@SHDS was confirmed by tumor growth curve recording and HE and TUNEL staining of tumor sections. The hydrogel exhibited excellent temperature sensitivity, controlled release properties, and high biocompatibility. In vitro experiments revealed that MDR@SHDS combined with PTT had a greater inhibitory effect on tumor cell proliferation compared to MDR@SHD alone. Combining local immunotherapy, chemotherapy, and PTT yielded superior anti-tumor effects than individual treatments. MDR@SHDS, with its simplicity, biocompatibility, and enhanced anti-tumor effects in combination with PTT, presents a promising therapeutic approach for lung cancer treatment, offering potential clinical utility.

Multimodal MRI reveals brainstem connections that sustain wakefulness in human consciousness.

Consciousness is composed of arousal (i.e., wakefulness) and awareness. Substantial progress has been made in mapping the cortical networks that underlie awareness in the human brain, but knowledge about the subcortical networks that sustain arousal in humans is incomplete. Here, we aimed to map the connectivity of a proposed subcortical arousal network that sustains wakefulness in the human brain, analogous to the cortical default mode network (DMN) that has been shown to contribute to awareness. We integrated data from ex vivo diffusion magnetic resonance imaging (MRI) of three human brains, obtained at autopsy from neurologically normal individuals, with immunohistochemical staining of subcortical brain sections. We identified nodes of the proposed default ascending arousal network (dAAN) in the brainstem, hypothalamus, thalamus, and basal forebrain. Deterministic and probabilistic tractography analyses of the ex vivo diffusion MRI data revealed projection, association, and commissural pathways linking dAAN nodes with one another and with DMN nodes. Complementary analyses of in vivo 7-tesla resting-state functional MRI data from the Human Connectome Project identified the dopaminergic ventral tegmental area in the midbrain as a widely connected hub node at the nexus of the subcortical arousal and cortical awareness networks. Our network-based autopsy methods and connectivity data provide a putative neuroanatomic architecture for the integration of arousal and awareness in human consciousness.

Fungal colonization and penetration of mortar as a suitable simulant for concrete: Implications for fungal biodeterioration in the built environment

A range of fungal species showed variable abilities to colonize and penetrate a mortar substrate. Calcium biomineralization was a common feature with calcium-containing crystals deposited in the microenvironment or encrusting hyphae, regardless of the specific mortar composition. Several species caused significant damage to the mortar surface, exhibiting burrowing and penetration, surface etching, and biomineralization. In some cases, extensive biomineralization of hyphae, probably by carbonatization, resulted in the formation of crystalline tubes after hyphal degradation on mortar blocks, including those amended with Co or Sr carbonate. Ca was the only metal detected in the biomineralized formations with Co or Sr undetectable. Aspergillus niger, Stemphylium sp. and Paecilomyces sp. could penetrate mortar with differential responses depending on the porosity. Fluorescent staining of thin sections recorded penetration depths of ∼530 um for A. niger and ∼620 um for Stemphylium sp. Penetration depth varied inversely with porosity and greater penetration depths were achieved in mortar with a lower porosity (lower water/cement ratio). These results have provided further understanding of biodeteriorative fungal interactions with cementitious substrates that can clearly affect structural integrity. The potential significance of fungal colonization and such biodeteriorative phenomena should not be overlooked in built environment contexts, including radionuclide storage and surface decontamination.

TRV027 REDUCES BLOOD PRESSURE IN SHR AND RESTORES VENTRICULAR HYPERTROPHY BY AFFECTING CONNEXIN 43 PATHWAY

Objective: Hypertension is one of the common causes of cardiovascular diseases. Angiotensin II (AngII) plays a crucial role in hypertension and hypertension-induced cardiac hypertrophy. Whether TRV027, an AngII type 1 receptor biased agonist, could lower blood pressure and treat hypertensive cardiac hypertrophy is unknown. Design and method: Spontaneously hypertensive rats and Wistar-Kyoto rats were selected as the study subjects. Some SHRs were giving TRV027. H9c2 cells were selected as the in vitro study subjects to explore the mechanism of action of TRV027. Results: When compared to the WKY group, the SHR group displayed a pronounced increase in blood pressure. However, after giving subcutaneous pump of TRV027, blood pressure exhibited a marked reduction. Based on the Doppler ultrasound data, prior to the intervention, the SHR group had higher values for LVAWs, LVAWd, LVPWs, and LVPWd when contrasted with the WKY group. Moreover, the left ventricular mass in the SHR group also increased significantly (P<0.05). After the intervention with TRV027 in the SHR group, all the above-mentioned cardiac Doppler parameters were lower than those in the SHR group. HE staining of cardiac sections revealed a slight disarray in myocardial arrangement and significant myocardial hypertrophy in the SHR group. Compared with the WKY group, the level of Cx43 in the heart tissue of the SHR group was decreased, and the phosphorylation of MAPK and ERK2 was increased. After administration of TRV027, the expression levels of these proteins were restored. In the cell intervention, the expression level of Cx43 was significantly decreased, the phosphorylation levels of MAPK and ERK2 were increased, and the levels of MHC and ANP were increased in the AngII intervention group. After the intervention of TRV027 the expressions of Cx43 and MAPK/ERK pathways and MHC and ANP were restored. When Cx43 inhibitor was given to H9C2 cells,along with the decrease of Cx43 expression, the phosphorylation levels of MAPK and ERK2 were increased, while the levels of MHC and ANP were significantly increased. Conclusions: TRV027 lower blood pressure in SHR and ameliorate cardiac hypertrophy induced by hypertension. This effect is likely achieved through the Cx43 pathway.

Abstract 1044: Single Cell RNA-sequencing Of Endothelial Cells In Atherosclerosis Reveals A Single Phenotypic Transition To A Mesenchymal Cell State

Introduction: Coronary artery disease (CAD) is the leading cause of mortality in the United States. Multiple cell types contribute to the atherosclerotic plaques causing CAD, influencing disease burden. Endothelial-to-mesenchymal transition (EndMT), the process by which endothelial cells (ECs) acquire characteristics of mesenchymal cells and loose canonical endothelial features, is correlated with an unstable plaque phenotype, although this transition remains poorly characterized. Hypothesis: A specific population of ECs may undergo EndMT, potentially contributing to the progression of atherosclerosis. Methods: Endothelial-specific lineage-tracing was induced in Tg Cdh5-CreERT2 , ApoE -/- mice, followed by 16 weeks of high-fat diet (HFD) to produce atherosclerosis. Aortic roots were collected from male and female disease-condition animals (n=13) and a baseline cohort that was not fed HFD (n=6). Single cell RNA sequencing was performed using 10X Genomics and analyzed with the Seurat package. RNAscope staining of sections (n=10 per genotype) was performed to characterize the lesion. Results: Multiple endothelial subtypes in the aortic root were identified. One population of arterial ECs, characterized by Klk10 expression, appears to give rise to a disease-specific population of ECs expressing Edn1 . These ECs appear to acquire a mesenchymal phenotype, consistent with the occurrence of EndMT. RNAscope staining revealed Edn1 + cells at the luminal surface and deep within the plaque. Upstream regulator analysis comparing Edn1+ and Klk10+ populations predict EndMT-associated transcription regulators including SNAI2, TWIST2, STAT3, SNAI1 and YAP1 as drivers of this transition. This analysis predicts upregulation of both fibrotic and cytokine signaling. In contrast to a previous study of carotid ECs under disturbed flow that identified multiple disparate EC phenotypes, we observed a single disease-associated EC transition. Conclusion: We identified ECs undergoing EndMT in a HFD-induced mouse model of atherosclerosis and determined that this transition is the primary EC cell state transition during disease. Thus, the process of EndMT may account for both mesenchymal and inflammatory changes observed in ECs during atherosclerosis.

Nicotinamide Mononucleotide and Nicotinamide Riboside Reverse Ovarian Aging in Rats Via Rebalancing Mitochondrial Fission and Fusion Mechanisms

PurposeThis study examined the effects of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) on folliculogenesis and mitochondrial dynamics (fission and fusion mechanisms) in ovaries of middle-aged female rats.MethodsExperimental groups were young, middle-aged (control), middle-aged + NMN and middle-aged + NR. NMN was administered at a concentration of 500 mg/kg intraperitoneally but NR at a concentration of 200 mg/kg by gavage. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels were analyzed by ELISA. Hematoxylin-eosin staining sections were used for histopathological examination and follicles-counting. Expression levels of mitochondrial fission (Drp1, Mff and Fis1) and fusion (Mfn1, Mfn2, Opa1, Fam73a and Fam73b) genes as well as Sirt1 gene were analyzed by RT-PCR. Expression levels of fission-related proteins (DRP1, MFF, FIS1 and SIRT1) were analyzed by Western Blot.ResultsHigher ovarian index, more corpus luteum and antral follicles were detected in NMN and NR groups compared to the control. NMN or NR could rebalance LH/FSH ratio. The control group was determined to possess higher expression levels of fission genes and lower expression levels of fusion genes when compared the young group. In comparison with the control group, both NMN and NR group were found to exhibit less mitochondrial fission but more mitochondrial fussion. Higher gene and protein levels for Sirt1 were measured in NMN and NR groups compared to the control group.ConclusionThis study reveals that NMN alone or NR alone can rebalance mitochondrial dynamics by decreasing excessive fission in middle-aged rat ovaries, thus alleviating mitochondrial stress and correcting aging-induced folliculogenesis abnormalities.

Titanium dioxide nanoparticles oral exposure induce osteoblast apoptosis, inhibit osteogenic ability and increase lipogenesis in mouse

Titanium dioxide nanoparticles (TiO2-NPs) are widely used in food, paint, coating, cosmetic, and composite orthodontic material. As a common food additive, TiO2-NPs can accumulate in various organs of human body, but the effect and underlying mechanism of bone remain unclear. Here mice were exposed to TiO2-NPs by oral gavage, and histological staining of femoral sections showed that TiO2-NPs reduced bone formation and enhanced osteoclast activity and lipogenesis, contributing to decreased trabecula bone. Transmission electron microscope (TEM) as well as biochemical and flow cytometry analysis of osteoblast exhibited that TiO2-NPs accumulated in osteoblast cytoplasm and impaired mitochondria ultrastructure with increased reactive oxygen species (ROS) and lipid hyperoxide, resulting in osteoblast apoptosis. In terms of mechanism, TiO2-NPs treatment inhibited expression of AKT and then increased pro-apoptotic protein Bax expression which was failure to form heterodimers with decreased anti-apoptotic Bcl-2, activating downstream Caspase-9 and Caspase-3 and inducing apoptosis. Additionally, TiO2-NPs suppressed Wnt3a level and then activated anti-Glycogen synthesis kinase (GSK-3β) phosphorylation, and ultimately resulted in degradation of β-catenin which down-regulated Runt-related transcription factor 2 (Runx2) and Osterix, inhibiting expression of osteogenic related proteins. Together, these results revealed that exposure of TiO2-NPs induced apoptosis and inhibited osteoblast differentiation through suppressing PI3K/AKT and Wnt/β-catenin signaling pathways, resulting in reduction of trabecula bone.

MALDI Imaging Mass Spectrometry Visualizes the Distribution of Antidepressant Duloxetine and Its Major Metabolites in Mouse Brain, Liver, Kidney, and Spleen Tissues.

Imaging mass spectrometry (IMS) is a powerful tool for mapping the spatial distribution of unlabeled drugs and metabolites that may find application in assessing drug delivery, explaining drug efficacy, and identifying potential toxicity. This study focuses on determining the spatial distribution of the antidepressant duloxetine, which is widely prescribed despite common adverse effects (liver injury, constant headaches) whose mechanisms are not fully understood. We used high-resolution IMS with matrix-assisted laser desorption/ionization to examine the distribution of duloxetine and its major metabolites in four mouse organs where it may contribute to efficacy or toxicity: brain, liver, kidney, and spleen. In none of these tissues is duloxetine or its metabolites homogeneously distributed, which has implications for both efficacy and toxicity. We found duloxetine to be similarly distributed in spleen red pulp and white pulp but differentially distributed in different anatomic regions of the liver, kidney, and brain, with dose-dependent patterns. Comparison with hematoxylin and eosin staining of tissue sections reveals that the ion images of endogenous lipids help delineate anatomic regions in the brain and kidney, while heme ion images assist in differentiating regions within the spleen. These endogenous metabolites may serve as a valuable resource for examining the spatial distribution of other drugs in tissues when staining images are not available. These findings may facilitate future mechanistic studies of the therapeutic and adverse effects of duloxetine. In the current work, we did not perform absolute quantification of duloxetine, which will be reported in due course. SIGNIFICANCE STATEMENT: The study utilized imaging mass spectrometry to examine the spatial distribution of duloxetine and its primary metabolites in mouse brain, liver, kidney, and spleen. These results may pave the way for future investigations into the mechanisms behind duloxetine's therapeutic and adverse effects. Furthermore, the mass spectrometry images of specific endogenous metabolites such as heme could be valuable in analyzing the spatial distribution of other drugs within tissues in scenarios where histological staining images are unavailable.

Human Infrapatellar Fat Pad Mesenchymal Stem Cell-derived Extracellular Vesicles Purified by Anion Exchange Chromatography Suppress Osteoarthritis Progression in a Mouse Model.

Human Infrapatellar Fat Pad Mesenchymal Stem Cell-derived Extracellular Vesicles Purified by Anion Exchange Chromatography Suppress Osteoarthritis Progression in a Mouse Model.

Morphological changes in the testicle in "Sertoli cell only" syndrome in men with non-obstructive azoospermia

BACKGROUND: The relevance of morphological studying of testicular biopsies in infertile men is explained by the small amount of information and research in this area, which is associated with limited indications for obtaining a lifetime biopsy in this disease.
 AIM: To evaluate morphological changes in the testicle in Sertoli cell-only syndrome (SCOS).
 MATERIALS AND METHODS: Using staining of sections with hematoxylin and eosin, toluidine blue, a morphological assessment of convoluted seminal tubules with SCOS was performed using light microscopy in 9 men with non-obstructive azoospermia.
 RESULTS: Mature Sertoli cells were found in 48.2% of convoluted seminal tubules, Sertoli cells with signs of dystrophy were in 33.6% of seminal tubules, Sertoli cells with signs of apoptosis were found in 18.2% of seminal tubules. The average number of Sertoli cells in one convoluted seminal tubule was 15.4±6.1. The diameter of the convoluted seminal tubule was on average 33.8±9.6 microns. The thickness of the shell of the convoluted seminal tubule in SCOS was on average 0.63 ± 0.18 microns. The number of mast cells in the testicular interstitium was on average 8.5±0.3 per 1 mm2. The relationship between the thickness of the shell of the convoluted seminal tubule and the number of Sertoli cells was strong and inverse (r=-0.87). The relationship between the thickness of the membrane of the convoluted seminal tubule and the number of mast cells in the testicular interstitium was strong and direct (r=0.83).
 CONCLUSIONS: SCOS occurs in 47.3% of patients with non-obstructive azoospermia. An increase in the thickness of the shell of the convoluted seminal tubule contributes to a violation of the architectonics of the hematotesticular barrier. This study showed that mast cells have a direct effect on the wall thickness of the convoluted seminal tubule, which demonstrates the possible pathological effect of these cells on the permeability of the hematotesticular barrier and impaired spermatogenesis.

The anti-colorectal cancer effect and metabolites of Agrimonia pilosa Ledeb

Ethnopharmacological relevanceAgrimonia pilosa Ledeb. (Rosaceae, A. pilosa) has been used in traditional medicine in China, Japan, Korea, and other Asian countries for treatment of acute and chronic enteritis and diarrhea. Secondary metabolites have been isolated and tested for biological activities. It remains unclear in terms of its potential components of anti-colorectal cancer properties. Aim of the studyThe study aimed to how extracts from A. pilosa and their components influenced tumor microenvironment and the colorectal tumor growth in vivo on AOM/DSS induced colorectal cancer mice, the metabolites of A. pilosa was also been studied. Materials and methodsDifferent methods have been used to extract different parts of A. pilosa. And the anti-proliferation effect of these extracts on colon cancer cells have been tested. The components of A. pilosa and its metabolites in vivo were analyzed by UPLC-QTOF-MS/MS. The anti-colorectal cancer (CRC) effects of A. pilosa and its components in vivo were studied on AOM/DSS induced CRC mice. The effects of constituents of A. pilosa on the composition of immune cells in tumor microenvironment (TME) were analyzed by flow cytometry. 16 S rDNA technology was used to analyze the effect of administration on the composition of intestinal microflora. Pathological section staining was used to compare the morphological changes and molecular expression of intestinal tissue in different groups. ResultsThe constituent exists in root of A. pilosa showed the strongest anti-proliferation ability on colon cancer cells in vitro. The extract from the root of A. pilosa could attenuate the occurrence of colorectal tumors induced by AOM/DSS in a concentration-dependent manner. Administration of the extract from the root of A. pilosa could affect the proportion of γδT cells, tumor associated macrophages and myeloid derived suppressor cells in TME, increasing the proportion of anti-tumor immune cells and decrease the immunosuppressive cells in the TME to promote the anti-tumor immune response. The administration of the extract adjusted the composition of gut microbiota and its components Agrimoniin and Agrimonolide-6-o-glucoside showed the strongest anti-CRC effect in vivo with adjusting the gut microbiota differently. ConclusionsThe extract from root of A. pilosa showed anti-colorectal cancer effects in vivo and in vitro, affecting the composition of gut microbiota and the anti-tumor immune response. Within all components of A. pilosa, Agrimoniin and Agrimonolide-6-o-glucoside showed remarkable anti-CRC efficiency in vivo and in vitro. Besides, the metabolites of extract from root of A. pilosa in gastrointestinal tract mainly composed of two parts: Agrimonolide-related metabolites and Urolithins. The extract from root of A. pilosa could contribute to potential drugs for assisting clinical anti-colon cancer therapy.

Pilose antler extract promotes hair growth in androgenic alopecia mice by promoting the initial anagen phase

Androgenetic alopecia (AGA) is a prevalent disease in worldwide, local application or oral are often used to treat AGA, however, effective treatments for AGA are currently limited. In this work, we observed the promoting the initial anagen phase effect of pilose antler extract (PAE) on hair regeneration in AGA mice. We found that PAE accelerated hair growth and increased the degree of skin blackness by non-invasive in vivo methods including camera, optical coherence tomography and dermoscopy. Meanwhile, HE staining of sagittal and coronal skin sections revealed that PAE augmented the quantity and length of hair follicles, while also enhancing skin thickness and hair papilla diameter. Furthermore, PAE facilitated the shift of the growth cycle from the telogen to the anagen phase and expedited the proliferation of hair follicle stem cells and matrix cells in mice with AGA. This acceleration enabled the hair follicles to enter the growth phase at an earlier stage. PAE upregulated the expression of the sonic hedgehog (SHH), smoothened receptor, glioma-associated hemolog1 (GLI1), and downregulated the expression of bone morphogenetic protein 4 (BMP4), recombinant mothers against decapentaplegic homolog (Smad) 1 and 5 phosphorylation. This evidence suggests that PAE fosters hair growth and facilitates the transition of the growth cycle from the telogen to the anagen phase in AGA mice. This effect is achieved by enhancing the proliferation of follicle stem cells and matrix cells through the activation of the SHH/GLI pathway and suppression of the BMP/Smad pathway.

الصفات الظاهرية والدقيقة لـ Citrus reticulata Blanco المزروع في شرق ليبيا

Citrus reticulate Blanco is economically and medically important and cultivated in Al-Marj city (Libya). In this study, the morphological, epidermal and anatomical characteristics of Citrus reticulate were considered in a laboratory experiment through staining of cut sections and microscope visualization with the aim of providing detailed description of characteristics of this plant species. Morphological characteristics of leaves, fruits, and seed are very important in comparing between Citrus species. Epidermal attributes such as a variety of cell shapes, hypostomatic, anamocytic stomata, and the presence of secretory cavities on the upper surface of the epidermis are valuable characteristics in distinguishing Citrus species. Also, the mesophyll structure, the shape of vascular bundle, the presence of druses and the secretory cavities features have taxonomic value in the Citrus genus. Additionally, the petiole features, the presence of trichomes and druses and characteristics of the vascular bundles are important for determining phylogenetic relationships between Citrus species. Key words: Citrus reticulata, morphology, anatomy, secertory cavities and druses, Al- Marj (Libya).

Determination of the Relationship Between the Number of Clusters and Primary Bud Size in Vitis vinifera and Vitis labrusca Grapevines by Histological Sectioning Method

This study was conducted to reveal the relationship between the number of clusters and of primary buds size in dormant grapevine winter buds. In addition, it has been tried to determine how the size of the primary bud and the cluster primordials in it change according to the position on one-year-old shoots. In this study, Vitis vinifera L. cv. ‘Karaerik’ and Vitis labrusca L. cv. 'Ülkemiz' varieties was used. Primary buds are separated from other shoot beds (secondary and tertiary buds) with the a scalpel, and fixation, vacuuming, paraffin impregnation, paraffin embedding, freezing, sectioning and tissue staining were performed, respectively. The stained samples were examined microscopically, images were taken and these images were transferred to the computer and histological analyzes was made. In the study, it was determined that bud sizes including three clusters were statistically different (p < 0.05) from other bud sizes (without clusters, one cluster and two clusters of buds). It was determined that there were statistical differences between cultivars in terms of both bud size and the number of cluster. In addition, it was determined that the middle (0.77) buds of the ‘Karaerik’ variety contained more clusters than the basal (0.66) and apical (0.59) buds, whereas the apical (1.19) buds of ‘Ülkemiz’ variety contained more clusters than the basal (0.88) and middle (0.98) buds. It is very important to know the structural features of the buds in order to make the right pruning application suitable for the variety.

Low-intensity pulsed ultrasound reduces alveolar bone resorption during orthodontic treatment via Lamin A/C-Yes-associated protein axis in stem cells.

The bone remodeling during orthodontic treatment for malocclusion often requires a long duration of around two to three years, which also may lead to some complications such as alveolar bone resorption or tooth root resorption. Low-intensity pulsed ultrasound (LIPUS), a noninvasive physical therapy, has been shown to promote bone fracture healing. It is also reported that LIPUS could reduce the duration of orthodontic treatment; however, how LIPUS regulates the bone metabolism during the orthodontic treatment process is still unclear. To investigate the effects of LIPUS on bone remodeling in an orthodontic tooth movement (OTM) model and explore the underlying mechanisms. A rat model of OTM was established, and alveolar bone remodeling and tooth movement rate were evaluated via micro-computed tomography and staining of tissue sections. In vitro, human bone marrow mesenchymal stem cells (hBMSCs) were isolated to detect their osteogenic differentiation potential under compression and LIPUS stimulation by quantitative reverse transcription-polymerase chain reaction, Western blot, alkaline phosphatase (ALP) staining, and Alizarin red staining. The expression of Yes-associated protein (YAP1), the actin cytoskeleton, and the Lamin A/C nucleoskeleton were detected with or without YAP1 small interfering RNA (siRNA) application via immunofluorescence. The force treatment inhibited the osteogenic differentiation potential of hBMSCs; moreover, the expression of osteogenesis markers, such as type 1 collagen (COL1), runt-related transcription factor 2, ALP, and osteocalcin (OCN), decreased. LIPUS could rescue the osteogenic differentiation of hBMSCs with increased expression of osteogenic marker inhibited by force. Mechanically, the expression of LaminA/C, F-actin, and YAP1 was downregulated after force treatment, which could be rescued by LIPUS. Moreover, the osteogenic differentiation of hBMSCs increased by LIPUS could be attenuated by YAP siRNA treatment. Consistently, LIPUS increased alveolar bone density and decreased vertical bone absorption in vivo. The decreased expression of COL1, OCN, and YAP1 on the compression side of the alveolar bone was partially rescued by LIPUS. LIPUS can accelerate tooth movement and reduce alveolar bone resorption by modulating the cytoskeleton-Lamin A/C-YAP axis, which may be a promising strategy to reduce the orthodontic treatment process.