Nuclear Fraction Research Articles

Protein kinase D1 mitigation against etoposide induced DNA damage in prostate cancer is associated with increased α-Catenin.

The E-cadherin, α- and β-Catenin interaction at the cell adherens junction plays a key role in cell adhesion; alteration in the expression and function of these genes are associated with disease progression in several solid tumors including prostate cancer. The membranous β-Catenin is dynamically linked to the cellular cytoskeleton through interaction with α-Catenin at amino acid positions threonine 120 (T120) to 151 of β-Catenin. Nuclear presence of α-Catenin modulates the sensitivity of cells to DNA damage. The objective of this study is to determine the role of α-Catenin and protein kinase D1 (PrKD1) in DNA damage response. Prostate cancer cells; LNCaP, LNCaP (Sh-PrKD1; silenced PrKD1), C4-2 and C4-2 PrKD1 were used for various sets of experiments to determine the role of DNA damage in PrKD1 overexpression and silencing cells. These cells were treated with compound-10 (100 nM) and Etoposide (30 µM), total cell lysates, cytosolic and nuclear fractions were prepared to observe various protein expressions. We performed single cell gel electrophoresis (COMET assay) to determine the etoposide induce DNA damage in C4-2 and C4-2 PrKD1 cells. The animal experiments were carried out to determine the tolerability of compound-10 by mice and generate preliminary data on efficacy of compound-10 in modulating the α-Catenin and PrKD1 expressions in inhibiting tumor progression. PrKD1, a novel serine threonine kinase, phosphorylates β-Catenin T120. In silico analysis, confirmed that T120 phosphorylation alters β- to α-Catenin binding. Forced expression of PrKD1 in prostate cancer cells increased β- and α-Catenin protein levels associated with reduced etoposide induced DNA damage. Downregulation of α-Catenin abrogates the PrKD1 mitigation of DNA damage. The in vitro results were corroborated in vivo using mouse prostate cancer patient derived xenograft model by inhibition of PrKD1 kinase activity with compound-10, a selective PrKD inhibitor, demonstrating decreased total β- and α-Catenin protein levels, and β-Catenin T120 phosphorylation. Alteration in DNA damage response pathways play major role in prostate cancer progression. The study identifies a novel mechanism of α-Catenin dependent DNA damage mitigation role for PrKD1 in prostate cancer.

EVERY KERATIN-POSITIVE MALIGNANT TUMOR IN THE ADRENAL GLAND IS NOT METASTATIC CARCINOMA- A CASE REPORT

Abstract Introduction/Objective Angiosarcoma is a rare subtype of sarcoma arising from the vascular endothelial cells. Primary adrenal angiosarcoma (PAA) is an exceedingly rare entity, with only 53 cases documented in the literature so far. Methods/Case Report A 78-year-old female presented with left upper quadrant abdominal pain and early satiety. A computed tomography scan of the abdomen revealed a 15 x 13 x 16 cm, heterogeneous enhancing mass with interspersed calcifications, possibly originating from the left adrenal gland. The mass was found to be displacing the spleen and the left kidney. An ultrasound-guided biopsy of the mass was inconclusive, and the patient subsequently underwent resection of the left retroperitoneal mass, left adrenalectomy, splenectomy, and left partial nephrectomy. Gross examination revealed a slightly ruptured, red-brown mass (19.5 x 12.7 x 12.6 cm, 1784g), which was serially sectioned to reveal diffusely hemorrhagic and necrotic cut surfaces. On microscopic examination, the tumor was almost entirely hemorrhagic and necrotic, with a few viable areas that were composed of solid sheets of markedly pleomorphic epithelioid cells with a high nuclear-cytoplasmic ratio, prominent nucleoli, and increased mitotic figures. There were also a few ectatic vascular spaces lined by these markedly pleomorphic epithelioid cells. Immunohistochemical stains of the tumor cells showed positive results for cytokeratins, CD31, ERG, and friend leukemic integration 1 (FLI-1), while Inhibin and SF-1 were negative. A diagnosis of epithelioid angiosarcoma was rendered. Results (if a Case Study enter NA) NA Conclusion PAA can be difficult to diagnose since it is rare, and thus it is not typically considered in the initial differential diagnosis. Most PAA have an epithelioid morphology, and they frequently test positive for cytokeratins. Biopsy diagnosis can also be challenging, as the mass may exhibit extensive necrosis and hemorrhage. Considering its rarity, large adrenal masses with heterogeneous features on imaging PAA should be considered as a differential diagnosis.

Rare Adult acute leukemia with Coexisting myeloblastic (basophilic differentiation) & B-lymphoblastic subpopulations Case Study

Abstract Introduction/Objective Acute basophilic leukemia is a very rare subgroup of acute myeloid leukemia that is defined in the 2022 WHO classification by presence of at least 20% blast cells in peripheral blood or bone marrow with immature and maturing cells of basophil lineage. The simultaneous occurrence of two hematological malignancies in a middle- aged adult patient is quite a rare event as evidenced by the rare cases reported in the literature. To our knowledge, this is the first case to be reported with concomitant acute basophilic and B-lymphoblastic leukemia in adult patients. these findings necessitates the comprehensive systematic approach to attain accurate diagnosis and plan the appropriate treatment protocol. Methods/Case Report Bone marrow aspiration & biopsy procedure was performed under local anesthesia and marrow samples were subjected to: lieshman staining of marrow films and direct examination. Immunophenotypic analysis of marrow aspirate by FACS Canto II flow cytometer. Cytogenetics studies were done as karyotyping and gene mutation detection by FISH. Results (if a Case Study enter NA) NA Conclusion Morphology of marrow films: 50% Blast cells, group of them are medium to larg-cells with high nucleocytoplasmic ratio and immature chromatin and the other group shows blast cells with sparse basophilic granulations, plus 20% immatue and maturing basophils. IPT shows two populations: the Myeloblast population (basophilic differentiation) is Positive for CD45dim, CD34het, CD13, CD33, CD117prt, CD25het, CD123het, cyt MPO part, HLA-DR partial. the B lineage lymphoblast population is Positive for CD45dim, CD34het, CD19, CD79a, TdT, CD20het, CD38. Cytogenetics studies: hyperploidy 56, (X, Y, 9, 10, 12, 13, 15, 18, 22, mar+ extra-chromosomes) FISH: +ve for FLT3-ITD mutation. the picture is by morphology & IPT consistent with biclonal leukemia, myeloid clone (acute basophilic leukemia) & ProB-ALL. The systematic laboratory approach from careful morphological examination of peripheral blood and bone marrow smears to confirmatory immunophenotypic analysis of marrow specimens by flow cytometry, and the comprehensive cytogenetics analysis, has a vital role in diagnosing such rare and aggressive phenomenon and provide a good chance for planning the appropriate treatment protocol with critical timesaving and cost effectiveness.

Astrocytes initiate autophagic flux and maintain cell viability after internalizing non-active native extracellular α-synuclein

Astrocytes are tasked with regulating the synaptic environment. Early stages of various neurodegenerative diseases are characterized by synapse loss, and astrocytic atrophy and dysfunction has been proposed as a possible cause. α-Synuclein (αS) is a highly expressed neuronal protein located in the synapse that can be released in the extracellular space. Evidence points to astrocytes as being responsible for uptake and degradation of extracellular αS. Therefore, misfolded active fibrillized αS resulting in protein inclusions and aggregates could be due to astrocytic dysfunction. Despite these pathological hallmarks and lines of evidence, the autophagic function of astrocytes in response to monomeric non-active αS to model healthy conditions has not been investigated. Human primary cortical astrocytes were treated with 100 nM of extracellular monomeric non-active αS alone, and in combination with N-terminal binding monomeric γ-synuclein (γS) as a control. Western blot analysis and super resolution imaging of HiLyte-488 labeled αS confirmed successful internalization of αS at 12, 24 and 48 h after treatment, while αS dimers were only observed at 48 h. Western blot analysis also confirmed αS's ability to induce autophagic flux by 48 h. Annexin V/PI flow cytometry results revealed increased early apoptosis at 24 h, but which resolved itself by 48 h, indicating no cell death in cortical astrocytes at all time points, suggesting astrocytes can manage the protein degradation demand of monomeric αS in healthy physiological conditions. Likewise, astrocytes reduced secretion of apolipoprotein (ApoE), a protein involved in pro-inflammatory pathways, synapse regulation, and autophagy by 12 h. Similarly, total c-JUN protein levels, a transcription factor involved in pro-inflammatory pathways increased by 12 h in the nuclear fraction. Therefore, astrocytes are able to respond and degrade αS in healthy physiological conditions, and astrocyte dysfunction could precede detrimental αS accumulation.

Schistosoma japonicum infection-mediated downregulation of lncRNA Malat1 contributes to schistosomiasis hepatic fibrosis by the Malat1/miR-96/Smad7 pathway

BackgroundSchistosoma japonicum infection causes hepatic fibrosis, a primary cause of morbidity and mortality associated with the disease, and effective treatments are still lacking. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenic process of various tissue fibroses. However, the role of lncRNAs in schistosomiasis hepatic fibrosis (HF) is poorly understood. Understanding the role of lncRNAs in schistosomiasis HF will enhance knowledge of disease processes and aid in the discovery of therapeutic targets and diagnostic biomarkers.MethodsDifferentially expressed lncRNA profiles in primary hepatic stellate cells (HSCs) of mice infected with S. japonicum were identified using high-throughput lncRNA sequencing. Primary HSCs were isolated from infected mice using collagenase digestion and density-gradient centrifugation, cultured in DMEM with 10% fetal bovine serum. Dual-luciferase reporter assays, nuclear cytoplasm fractionation and RIP assays were employed to assess the relationship between Malat1 and miRNA-96. Malat1 lentivirus and ASO-Malat1 were constructed for forced expression and downregulated expression of Malat1. The Malat1-KO mouse was constructed by CRISPR/Cas9 technology. Pathological features of the liver were evaluated by hematoxylin-eosin (HE), Masson’s trichrome staining and immunohistochemistry (IHC). The expression levels of fibrosis-related genes were determined by quantitative real-time PCR (qRT-PCR) and Western blot.ResultsA total of 1561 differentially expressed lncRNAs were identified between infected and uninfected primary HSCs. Among the top altered lncRNAs, the downregulated Malat1 was observed in infected HSCs and verified by qPCR. Treatment of infected mice with praziquantel (PZQ) significantly increased the Malat1 expression. Elevated Malat1 expression in infected primary HSC reduced the expressions of profibrogenic genes, whereas Malat1 knockdown had the opposite effect. Moreover, Malat1 was found to interact with miR-96, a profibrotic miRNA, by targeting Smad7. Forced Malat1 expression reduced miR-96 levels in infected primary HSCs, attenuating fibrogenesis and showing negative correlation between Malat1 expression and the expression levels of miR-96 and profibrogenic genes α-SMA and Col1α1. Notably, in Malat1-KO mice, knockout of Malat1 aggravates schistosomiasis HF, while restored Malat1 expression in the infected HSCs reduced the expression of profibrogenic genes.ConclusionsWe demonstrate that lncRNA is involved in regulation of schistosomiasis HF. Elevated lncRNA Malat1 expression in infected HSCs reduces fibrosis via the Malat1/miR-96/Smad7 pathway, thus providing a novel therapeutic target for schistosomiasis HF. Furthermore, Malat1 expression is sensitive to PZQ treatment, thus offering a potential biomarker for assessing the response to chemotherapy.Graphical abstract

B-328 A Novel Pathway of Akt-Dependent PARP1 Activation Through the Nuclear Localization of EPRS1

Abstract Background Glutamyl-prolyl-tRNA synthetase (EPRS1) is the only bifunctional aminoacyl-tRNA synthetase (aaRS) and is essential for interpreting the genetic code. EPRS1, along with seven other aaRSs, forms the multi-tRNA synthetase complex (MSC). EPRS1 consists of two catalytic domains, GluRS and ProRS, connected by a linker region. Several aaRSs within the MSC, including EPRS1, can dissociate from the MSC in response to specific stimuli. This dissociation enables non-canonical activities distinct from their primary role in protein synthesis. Breast cancer cells harboring a loss-of-function mutation in phosphatase and tensin homolog (PTEN) are known to have more activity in the serine/threonine kinase Akt. Poly-(ADP)-ribose polymerase 1 (PARP1), plays a vital role in DNA damage repair (DDR). PARP1 deposits one or more ADP-Ribose moieties onto damaged DNA or other proteins in a process referred to as PARylation. We hope to connect Akt activation and PARP1 activity through the nuclear localization and noncanonical function of EPRS1. We hypothesize, that when Akt is activated, either through the loss of PTEN function or through stimuli such as heat shock or H2O2 treatment, will stimulate the nuclear localization of EPRS1 and modulate PARP1 activity. Methods To test our hypothesis, we evaluate Akt activation, EPRS1 localization, and PARP1 activity. Using pharmacological activators and inhibitors of Akt, we test if inhibitors and activators alone are sufficient to cause the nuclear localization of EPRS1. Biochemically, we isolate nuclear and cytosolic fractions used in various western blot analysis. We use a variety of assays for PARP1 activity and DDR that include immobilized histones, calculating IC50s of PARP1 inhibitors, comet assay, and confocal microscopy. To ascertain the connection between PARP1 and EPRS1 we use LC-MS/MS analysis and immunoprecipitation. Results We show, in both patient-derived and cultured breast cancer cells, elevated levels of EPRS1 not only in the cytoplasm, but also within the nucleus. We find that nuclear localization EPRS1 is facilitated by a nuclear localization sequence (NLS) located within the linker region of the protein. Breast cancer cells harboring a loss-of-function in PTEN exhibit higher levels of EPRS1 in the nucleus compared to PTEN-positive cells. In EPRS1 knockdown cells, there is diminished expression of multiple tumor-related transcription factors, DNA-damage repair, tumor cell survival, and PARP1 auto-PARylation. We demonstrate that nuclear EPRS1 binds to and shares an interactome with PARP1. Conclusions EPRS1-mediated regulation of PARP1 activity provides a new mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the non-canonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, might provide a new therapeutic approach in breast and other forms of cancer, potentially supplementing existing cancer therapeutics.

Androgen receptor markers do not differ between nonresponders and responders to resistance training-induced muscle hypertrophy.

The aim of this study was to investigate whether baseline values and acute and chronic changes in androgen receptors (AR) markers, including total AR, cytoplasmic (cAR), and nuclear (nAR) fractions, as well as DNA-binding activity (AR-DNA), are involved in muscle hypertrophy responsiveness by comparing young nonresponder and responder individuals. After 10 wk of resistance training (RT), participants were identified as nonresponders using two typical errors (TE) obtained through two muscle cross-sectional area (mCSA) ultrasound measurements (2 × TE; 4.94%), and the highest responders within our sample were numerically matched. Muscle biopsies were performed at baseline, 24 h after the first RT session (acute responses), and 96 h after the last session (chronic responses). AR, cAR, and nAR were analyzed using Western blotting, and AR-DNA was analyzed using an ELISA-oligonucleotide assay. Twelve participants were identified as nonresponders (ΔmCSA: -1.32%) and 12 as responders (ΔmCSA: 21.35%). There were no baseline differences between groups in mCSA, AR, cAR, nAR, or AR-DNA (P > 0.05). For acute responses, there was a significant difference between nonresponders (+19.5%) and responders (-14.4%) in AR-DNA [effect size (ES) = -1.39; 95% confidence interval (CI): -2.53 to -0.16; P = 0.015]. There were no acute between-group differences in any other AR markers (P > 0.05). No significant differences between groups were observed in chronic responses across any AR markers (P > 0.05). Nonresponders and responders presented similar baseline, acute, and chronic results for the majority of the AR markers. Thus, our findings do not support the influence of AR markers on muscle hypertrophy responsiveness to RT in untrained individuals.NEW & NOTEWORTHY We explored, for the first time, the influence of androgen receptor (AR) through the separation of cytoplasmic and nuclear cell fractions [i.e., cytoplasmic androgen receptor (cAR), nuclear androgen receptor (nAR), and androgen receptor DNA-binding activity (AR-DNA)] on muscle hypertrophy responsiveness to resistance training. The absence of muscle hypertrophy in naïve individuals does not seem to be explained by baseline values, and acute or chronic changes in AR markers.

ЭНДОТЕЛИН-1 В КОРТИКОЦИТАХ НАДПОЧЕЧНИКОВ ПРИ СОЧЕТАНИИ ЧЕРЕПНО-МОЗГОВОЙ ТРАВМЫ И ЭНДОТОКСЕМИИ

Neuroinflammation in traumatic brain injury (TBI) and endotoxemia caused by bacterial lipopolysaccharide have an indirect effect through blood vessels on paracrine and autocrine processes regulating steroidogenesis of adrenal corticocytes. The vasoconstrictor peptide endothelin-1 plays a key role in blood vessel homeostasis. This protein, as a regulator, may be involved in the integration of the listed mechanisms of action on the adrenal glands during endotoxemia and inflammation. Purpose of the study: immunohistochemical characterization of the localization sites and content of endothelin-1 in adrenal corticocytes during a combination of TBI and endotoxemia in the experiment. Materials and methods. Moderate TBI was reproduced in mature rats using a falling weight model weighing 200 g, endotoxemia was induced by intraperitoneal administration of pyrogenal at a total dose of 100 μg/kg body weight. Endothelin-1 was detected in adrenal tissues using the immunohistochemical method and morphometric analysis of the images was performed. Results. In all experimental groups, the stability of the morphometric characteristics of the ratio of the calving cortex and the adrenal medulla was found. With the combination of TBI and endotoxemia, there is a decrease in the area of the nucleus and cytoplasm of adrenal corticocytes and, as a consequence, the nuclear-cytoplasmic ratio. A morphological study revealed an increase in the content of endothelin-1 in the cells of the adrenal cortex, while in the adrenocytes of the medulla, a significant positive reaction to the presence of the peptide was not detected. Conclusion. The results obtained demonstrate that the combination of TBI and endotoxemia causes dysfunction of adrenal cortex cells with an increase in the synthesis of endothelin-1 in them.

Radiofrequency field inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells via modulating the NF-κB signaling pathway

ABSTRACT In this study, we investigated the inhibitory effects of radiofrequency exposure on RANKL-induced osteoclast differentiation in RAW264.7 cells, along with the underlying mechanisms. RAW264.7 cells were subjected to radiofrequency exposure at three distinct power densities: 50 µW/cm2, 150 µW/cm2, and 450 µW/cm2. The results showed that, among the three dosage levels, exposure to 150 µW/cm2 of radiofrequency radiation significantly reduced the proliferation capacity of RAW264.7 cells. RF exposure at three power densities resulted in significant increases in the level of osteoclast apoptosis and notable decreases in osteoclast differentiation. Notably, the most pronounced effects on apoptosis, differentiation in RAW 264.7 cells were observed at the 150 µW/cm2 power density. These effects were accompanied by concurrent decreases in mRNA and protein levels of osteoclast-specific genes, including RANK, NFATc1, and TRACP. Furthermore, radiofrequency exposure at power density of 150 µW/cm2 induced a significant decrease in cytoplasmic NF-κB protein levels while increasing its nuclear fraction, thereby counteracting the effects of RANKL-induced NF-κB activation. These data suggest that radiofrequency exerts inhibitory properties on RANKL-induced NF-κB transcriptional activity, subsequently indirectly suppressing the expression of downstream NF-κB target genes, such as NFATc1 and TRACP. In conclusion, our study demonstrates that radiofrequency radiation effectively inhibits osteoclast differentiation by modulating the NF-κB signaling pathway. These findings have important implications for potential therapeutic interventions in osteoporosis.

Surgical Management of Canine Transmissible Venereal Tumor with Adjunctive Chemotherapy Combination

One male and a female 2-year-old Neapolitan Mastiff dogs weighing 41 and 40 kg, respectively, were presented to the University Veterinary Teaching Hospital, Jos, Nigeria, with complaints of continuous bleeding from the prepuce and vulva which were first observed about 2 months before presentation. Clinical examination revealed preputial bleeding of the male dog and serosanguinous vulva discharges with the presence of palpable masses with multiple nodules that were cauliflower-like. Cytological evaluations of smears from preputial and vaginal swabs revealed characteristic round cells with large eccentric nuclei, large nuclear-cytoplasmic ratio, and intracytoplasmic vacuolations as well as anisocytosis and anisokaryosis. Based on these cytological features, a diagnosis of canine transmissible venereal tumor (CTVT) was made in each case. Blood samples were collected for hematology and clinical chemistry. Debulking of the masses was carried out under general anesthesia using atropine sulfate and chlorpromazine as pre-anesthetics and Ketamine hydrochloride as induction and maintenance anesthetics. Following debulking, the resulting defect was reconstructed and the surgical wound was managed by daily wound cleaning and the administration of vincristine sulfate (0.025 mg/kg) slowly intravenously, ivermectin (0.3 mg/kg) subcutaneously, and piroxicam (0.3 mg/kg) and 20% amoxicillin (20 mg/kg) intramuscularly. There were no observed known vincristine sulfate-associated adverse effects as the dogs recovered and were discharged 3-week post-tumor debulking with no recurrence within 6 months of follow-up examinations. The debulked mass histologically showed several sheets of round neoplastic cells with prominent nuclei, mitotic figures, and infiltrating lymphocytes. Based on these findings, a diagnosis of CTVT was further confirmed. The hemogram showed moderate leukocytosis at day 1 in the bitch with a mild left shift at days 1 and 14 while clinical chemistry showed a marked increase in alkaline phosphatase, moderate increase in serum creatinine, and mild hypoproteinemia. In conclusion, management of CTVT with adjunctive vincristine sulfate and ivermectin combination resulted in complete healing with amelioration of known associated adverse effects.

The BRD4 Inhibitor I-BET-762 Reduces HO-1 Expression in Macrophages and the Pancreas of Mice.

In pancreatic cancer, the tumor microenvironment (TME) accounts for up to 90% of the tumor mass. Pancreatitis, characterized by the increased infiltration of macrophages into the pancreas, is a known risk factor for pancreatic cancer. The NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor regulates responses to oxidative stress and can promote cancer and chemoresistance. NRF2 also attenuates inflammation through the regulation of macrophage-specific genes. Heme oxygenase 1 (HO-1) is expressed by anti-inflammatory macrophages to degrade heme, and its expression is dependent on NRF2 translocation to the nucleus. In macrophages stimulated with conditioned media from pancreatic cancer cells, HO-1 protein levels increased, which correlated with higher NRF2 expression in the nuclear fraction. Significant differences in macrophage infiltration and HO-1 expression were detected in LSL-KrasG12D/+; Pdx-1-Cre (KC) mice, Nrf2 whole-body knockout (KO) mice and wildtype mice with pancreatitis. Since epigenetic modulation is a mechanism used by tumors to regulate the TME, using small molecules as epigenetic modulators to activate immune recognition is therapeutically desirable. When the bromodomain inhibitor I-BET-762 was used to treat macrophages or mice with pancreatitis, high levels of HO-1 were reduced. This study shows that bromodomain inhibitors can be used to prevent physiological responses to inflammation that promote tumorigenesis.

Changes in the liver of Djungarian hamsters under conditions of a three-month supply of water-soluble silicon of various concentrations

BACKGROUND: Silicon enters the human body through drinking water, air and food. Silicon nanoparticles used in the cosmetic, pharmaceutical and food industries are known to have biological activity. Taking into account the widespread prevalence of silicon compounds, the issue of the safety of its use is becoming more urgent. AIM: To study the effect of water-soluble silicon on the morphological structure of the liver of Djungarian hamsters for three months. MATERIALS AND METHODS: The experiment was carried out on Djungarian hamsters kept in normal vivarium conditions under natural light. The animals were divided into three groups: control, which received bottled drinking water; the first experimental group, which received the same water, but with the addition of sodium metasilicate nine-hydrate at a concentration of 10 mg/l in terms of silicon; the second experimental group, which also received the same water, but with the concentration of sodium metasilicate nine-hydrate doubled (up to 20 mg/l). After three months, the animals were removed from the experiment. Sections were processed by general histological (hematoxylin and eosin, Van Gieson method, toluidine blue), histochemical (monoamine oxidase-positive cells) methods. RESULTS: In the liver of hamsters from the experimental groups, changes in the micromorphological structure were revealed, such as an increase in the nuclear area, nuclear-cytoplasmic ratio of hepatocytes, and the diameter of sinusoidal capillaries. Moreover, more pronounced changes were observed in the liver of hamsters of the second experimental group, such as polymorphic cell infiltration of the portal tracts, an increase in the number of eosinophils, deformation of hepatocyte nuclei and the appearance of apoptotic bodies. A decrease in the area of mast cells and an increase in their number, as well as the number of monoamine oxidase-positive cells in the liver of hamsters of both experimental groups were recorded. CONCLUSIONS: An increase in the concentration of silicon supplied with drinking water in both cases is reflected in the micromorphological structure of the liver of hamsters. Moreover these changes are more pronounced in the liver of hamsters of the second experimental group.

Activation of the Keap1/Nrf2/HO-1 Pathway by "Tianyu" Pairing: Implications for Inflammation and Oxidative Stress in Rheumatoid Arthritis.

The objective of this study was to examine the impact of "Tianyu" Pairing on oxidative stress in the development of Rheumatoid arthritis (RA) and approach its potential mechanism using cell experiments. A cell model of RA was developed by stimulating rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) with tumor necrosis factor-α (TNF-α). This model aimed to assess the impact of serum containing Rhodiola rosea-Euonymus alatus drug pair (TYP) on inflammation and oxidative stress in the development of RA, specifically through the Keap1/Nrf2/HO-1 pathway. The findings from the in vitro experiment demonstrated that the presence of TYP in the serum effectively suppressed the proliferation of RA-FLS induced by TNF-α. Additionally, TYP facilitated the apoptosis of afflicted cells, attenuated the migratory and invasive capabilities of diseased cells, and decreased the levels of Kelch ECH associating protein 1 (Keap1), reactive oxygen species (ROS), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) (p < 0.01). The influence of inflammation and oxidative stress in RA-FLS cells was reduced by increasing the nuclear-cytoplasmic ratio of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) and levels of phosphorylated Nrf2, Heme Oxygenase 1 (HO-1), and Superoxide Dismutase (SOD) (p < 0.01). TYP can regulate inflammation and oxidative stress in RA-FLS cells by activating the Keap1/Nrf2/HO-1 pathway.

CLDN6 inhibits breast cancer growth and metastasis through SREBP1-mediated RAS palmitoylation

BackgroundBreast cancer (BC) ranks as the third most fatal malignant tumor worldwide, with a strong reliance on fatty acid metabolism. CLDN6, a candidate BC suppressor gene, was previously identified as a regulator of fatty acid biosynthesis; however, the underlying mechanism remains elusive. In this research, we aim to clarify the specific mechanism through which CLDN6 modulates fatty acid anabolism and its impact on BC growth and metastasis.MethodsCell function assays, tumor xenograft mouse models, and lung metastasis mouse models were conducted to evaluate BC growth and metastasis. Human palmitic acid assay, triglyceride assay, Nile red staining, and oil red O staining were employed to investigate fatty acid anabolism. Reverse transcription polymerase chain reaction (RT–PCR), western blot, immunohistochemistry (IHC) assay, nuclear fractionation, immunofluorescence (IF), immunoprecipitation and acyl–biotin exchange (IP-ABE), chromatin immunoprecipitation (ChIP), dual luciferase reporter assay, and co-immunoprecipitation (Co-IP) were applied to elucidate the underlying molecular mechanism. Moreover, tissue microarrays of BC were analyzed to explore the clinical implications.ResultsWe identified that CLDN6 inhibited BC growth and metastasis by impeding RAS palmitoylation both in vitro and in vivo. We proposed a unique theory suggesting that CLDN6 suppressed RAS palmitoylation through SREBP1-modulated de novo palmitic acid synthesis. Mechanistically, CLDN6 interacted with MAGI2 to prevent KLF5 from entering the nucleus, thereby restraining SREBF1 transcription. The downregulation of SREBP1 reduced de novo palmitic acid synthesis, hindering RAS palmitoylation and subsequent endosomal sorting complex required for transport (ESCRT)-mediated plasma membrane localization required for RAS oncogenic activation. Besides, targeting inhibition of RAS palmitoylation synergized with CLDN6 to repress BC progression.ConclusionsOur findings provide compelling evidence that CLDN6 suppresses the palmitic acid-induced RAS palmitoylation through the MAGI2/KLF5/SREBP1 axis, thereby impeding BC malignant progression. These results propose a new insight that monitoring CLDN6 expression alongside targeting inhibition of palmitic acid-mediated palmitoylation could be a viable strategy for treating oncogenic RAS-driven BC.Graphical

A nuclear jamming transition in vertebrate organogenesis.

Jamming of cell collectives and associated rigidity transitions have been shown to play a key role in tissue dynamics, structure and morphogenesis. Cellular jamming is controlled by cellular density and the mechanics of cell-cell contacts. However, the contribution of subcellular organelles to the physical state of the emergent tissue is unclear. Here we report a nuclear jamming transition in zebrafish retina and brain tissues, where physical interactions between highly packed nuclei restrict cellular movements and control tissue mechanics and architecture. Computational modelling suggests that the nuclear volume fraction and anisotropy of cells control the emerging tissue physical state. Analysis of tissue architecture, mechanics and nuclear movements during eye development show that retina tissues undergo a nuclear jamming transition as they form, with increasing nuclear packing leading to more ordered cellular arrangements, reminiscent of the crystalline cellular packings in the functional adult eye. Our results reveal an important role of the cell nucleus in tissue mechanics and architecture.

Fuchs endothelial corneal dystrophy: aspects of pathogenesis

Fuchs endothelial corneal dystrophy (FECD) – multigenic bilateral disorder, characterized by dysfunction of corneal endothelium cells (CECs) that eventually results in loss of transparency. Purpose: To evaluate the pathogenesis of FECD with histological and immunohistochemical methods, immunofluorescence, scanning electron microscopy (SEM). Methods: Endothelium–Descemet membrane (EDM) complexes, corneal buttons were obtained from 76 patients (85 eyes) with FECD during keratoplasty and divided into 1A, 2A, 3A groups, 15 EDMs (donor tisuue) – 1B, 2B, 3B groups (control). Morphological (hematoxylin/eosin staining) and immunohistochemical (primary antibodies to cytokeratin AE1/AE3 and vimentin) studies (1A, 1B groups), phase-contrast microscopy and immunofluorescence analysis of tight junction protein ZO-1 (2A, 2B groups), SEM (3A, 3B groups) were performed. Results: FECD is characterized by decline of CECs, cell and nuclear enlargement. Nuclear-cytoplasmic ratio of CECs is relative to control. CECs expression ZO-1 is decreased in the area of guttae ingrowth. Coexpression of cytokeratin AE1/AE3 and vimentin is found. Morphological and immunohistochemical studies show DM’ thickening, stromal and epithelial edema and local fibrosis, vimentin (stromal cells) and cytokeratin AE1/AE3 (epithelium) expression in FECD. Conclusion: Pathogenesis of FECD include CECs’ epithelial-mesenchymal transition, followed by guttae formation – the excrescences, which destroy cytosketon and lead to progressive loss of CECs. Changes in permeability of endothelium cause edema and fibrosis of stroma and epithelial layer.

Features of reactions to stress in rats with passive-defense behavior after light desynchronosis and physical activity

An assessment was made of hematological parameters of peripheral blood and structural and functional changes in the adrenal glands in stress-unresistant rats (passive-defensive type of behavior in the open field test) after exposure to light deprivation and physical activity during the spring equinox. The experiment showed that light deprivation for 10 days reduces the total number of leukocytes, the absolute content of monocytes, granulocytes, lymphocytes and the level of corticosterone in the peripheral blood compared to the intact group, that was on a natural lighting regime. Histological analysis of the adrenal glands of this group showed, that in the zona fasciculata under such conditions there was nuclear hypertrophy, an increase in the nuclear-cytoplasmic ratio of adrenocorticocytes and a decrease in the size of the zona glomerulosa of the adrenal cortex of rats in relation to the intact group. Physical activity in the form of forced swimming until complete fatigue for 5 days in a row in natural light in stress-unresistant rats did not change the parameters of the peripheral blood of animals, however it led to an increase in the area of the cytoplasm, the nuclear-cytoplasmic ratio, as well as the formation of hypertrophy of the nuclei of adrenocorticocytes in the fascicle adrenal zones, which indicated the preparation of cells for increased synthetic activity. Keeping rats for 10 days in complete darkness before forced swimming every day for 5 days, on the one hand, formed a hypoxic state and exhaustion of the adrenal glands, on the other hand, stabilized the leukocyte pool of peripheral blood compared to similar indicators in intact rats.

Light-Dependent Circadian Rhythm Governs O-GlcNAc Cycling to Influence Cognitive Function in Adult Zebrafish.

This study explores the 24-h rhythmic cycle of protein O-GlcNAcylation within the brain and highlights its crucial role in regulating the circadian cycle and neuronal function based on zebrafish as an animal model. In our experiments, disruption of the circadian rhythm, achieved through inversion of the light-dark cycle or daytime melatonin treatment, not only impaired the rhythmic changes of O-GlcNAcylation along with altering expression patterns of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in zebrafish brain but also significantly impeded learning and memory function. In particular, circadian disruption affected rhythmic expression of protein O-GlcNAcylation and OGT in the nuclear fraction. Notably, the circadian cycle induces rhythmic alterations in O-GlcNAcylation of H2B histone protein that correspond to changes in H3 trimethylation. Disruption of the cycle interfered with these periodic histone code alterations. Pharmacological inhibition of OGT with OSMI-1 disrupted the wake-sleep patterns of zebrafish without affecting expression of circadian rhythm-regulating genes. OSMI-1 inhibited the expression of c-fos, bdnf, and calm1, key genes associated with brain function and synaptic plasticity, and decreased the binding of O-GlcNAcylated H2B and OGT to promoter regions of these genes. The collective findings support the potential involvement of circadian cycling of the O-GlcNAc histone code in regulating synaptic plasticity and brain function. Overall, data from this study provide evidence that protein O-GlcNAcylation serves as a pivotal posttranslational mechanism integrating circadian signals and neuronal function to regulate rhythmic physiology.

Simultaneous induction of systemic hyperglycaemia and stress impairs brain redox homeostasis in the adult zebrafish

For diabetic patients it is crucial to constantly monitor blood glucose levels to mitigate complications due to hyperglycaemia, including neurological issues and cognitive impairments. This activity leads to psychological stress, called “diabetes distress,” a problem for most patients living with diabetes. Diabetes distress can exacerbate the hyperglycaemia effects on brain and negatively impact the quality of life, but the underlying mechanisms remain poorly explored. We simulated diabetes distress in adult zebrafish by modelling hyperglycaemia, through exposure to dextrose solution, along with chronic unpredictable mild stress (CUMS), and evaluated brain redox homeostasis by assessing reactive oxygen species (ROS) content, the antioxidant system, and effects on mitochondrial biogenesis and fission/fusion processes. We also evaluated the total, cytosolic and nuclear content of nuclear factor erythroid 2-related factor 2 (NRF2), a critical regulator of redox balance, in the whole brain and total NRF2 in specific brain emotional areas. The combined CUMS + Dextrose challenge, but not the individual treatments, reduced total NRF2 levels in the entire brain, but strongly increased its levels in the nuclear fraction. Compensatory upregulation of antioxidant genes appeared inadequate to combat elevated levels of ROS, leading to lowering of the reduced glutathione content and total antioxidant capacity. CUMS + Dextrose treatment also upregulated transcription factors implicated in mitochondrial biogenesis and dynamics with a predominance of fission, which is consistent with increased oxidative stress. In conclusion, this study highlights the close interplay between hyperglycaemia and psychological distress causing overriding oxidative stress in the brain, rendering the organism vulnerable to the development of disease complications.

Cytomorphologic comparison of upper urinary tract urothelial carcinomas and renal cell carcinomas on urine cytology.

Compared to urothelial carcinomas (UCs), the cytomorphology of renal cell carcinomas (RCCs) is underdescribed. This study aims to investigate whether UCs and RCCS of the upper urinary tract can be differentiated cytologically, and to identify distinguishing cytomorphological features. Consecutive urine cytology specimens with atypical/C3, suspicious/C4 or malignant/C5 diagnoses matched with a nephrectomy or ureterectomy specimen with UC or RCC over a 15-year period were reviewed for cellularity, architecture, background composition and cytomorphologic features. Totally 132 specimens were retrieved, comprising 24 RCCs and 108 UCs. Clear cell RCC (CCRCC) (n = 18) was the most common RCC. Urine cytology specimens from UC showed a trend of higher cellularity (p = 0.071) against RCC and was significant in subgroup analysis with CCRCC (p < .001). Epithelial structures in sheets, tubules, and papillae were exclusive in specimens of UC (p < .05). For background features, squamous cells were more common for RCC (p = .006) including CCRCC (p = .003), whereas polymorphs (p = .011) and necrotic material (p = .010) were associated with UC. Average nuclear size was larger and nuclear size variation (p < .001) and nuclear-cytoplasmic ratio (p = .001) were greater in UC (p = .001) than RCC. Comparing RCC to high-grade UCs only, nuclear-cytoplasmic ratio maintained statistical significance (p = .006) while average nuclear size showed a trend (p = .063). A clean background free of tumor necrosis and polymorphs, and the lack of complex tumor fragments favors RCC. UCs also display larger nuclear size, higher nuclear size variation and nuclear-cytoplasmic ratio. These cytomorphological features with corroboration of clinical/radiological findings, can aid in raising a diagnosis of RCC.