scholarly journals Pre-ovulatory intercellular regulation of miR-125a-3p within mouse ovarian follicles

Reproduction ◽  
2020 ◽  
Vol 159 (2) ◽  
pp. 215-225 ◽  
Author(s):  
Hadas Grossman ◽  
Efrat Har-Paz ◽  
Natalie Gindi ◽  
Irit Miller ◽  
Ruth Shalgi
Keyword(s):  
Culture Media ◽  
Ex Vivo ◽  
Cumulus Cells ◽  
Negative Control ◽  
Follicular Cells ◽  
Transcription Regulator ◽  
Ovulatory Follicles ◽  
Intercellular Communications ◽  
Gap Junction Inhibitor

miR-125a-3p, a post-transcription regulator of Fyn kinase, is expressed in mouse pre-ovulatory follicles; its expression within the follicle decreases toward ovulation. Our aim was to follow the synthesis of miR-125a-3p and regulation of its expression in all follicular compartments, focusing on intercellular communication. Mural granulosa cells (GCs) or cumulus cells (CCs) were transfected with either scrambled-miR (negative control) or miR-125a-3p mimic. Freshly isolated GCs or CCs were incubated overnight in culture media conditioned by transfected cells. To examine a possible role of gap junctions in the regulation of miR-125a-3p, we incubated large antral follicles in the presence of carbenoxolone, a gap-junction inhibitor, and triggered them to mature with hGC. Levels of miR-125a family members in GCs, CCs, oocytes, and culture media were measured by qPCR. We showed that miR-125a-3p is synthesized by all follicular components, but is regulated within the follicle as a whole. It is secreted by mural-GCs and taken up by CCs, where it remains functional, and vice versa, mural-GCs can take up miR-125a-3p secreted by CCs. miR-125a-3p is transcribed and accumulated in oocytes throughout oogenesis. Transcriptionally quiescent GV oocytes utilize their accompanying follicular cells to monitor the level of miR-125a-3p within them, as indicated in an ex vivo follicle culture. Our study reveals that miR-125a-3p expression is modulated by a network of intercellular communications within pre-ovulatory follicles, thus enabling a coordinated decrease of miR-125a-3p toward ovulation.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

scholarly journals Evaluating Targeted Therapies in Ovarian Cancer Metabolism: Novel Role for PCSK9 and Second Generation mTOR Inhibitors

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3727
Author(s):  
Dafne Jacome Sanz ◽  
Juuli Raivola ◽  
Hanna Karvonen ◽  
Mariliina Arjama ◽  
Harlan Barker ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Lipid Metabolism ◽  
Cell Survival ◽  
Drug Testing ◽  
Cancer Metabolism ◽  
Ex Vivo ◽  
Specific Inhibitor ◽  
Low Grade ◽  
Serous Ovarian Cancer

Background: Dysregulated lipid metabolism is emerging as a hallmark in several malignancies, including ovarian cancer (OC). Specifically, metastatic OC is highly dependent on lipid-rich omentum. We aimed to investigate the therapeutic value of targeting lipid metabolism in OC. For this purpose, we studied the role of PCSK9, a cholesterol-regulating enzyme, in OC cell survival and its downstream signaling. We also investigated the cytotoxic efficacy of a small library of metabolic (n = 11) and mTOR (n = 10) inhibitors using OC cell lines (n = 8) and ex vivo patient-derived cell cultures (PDCs, n = 5) to identify clinically suitable drug vulnerabilities. Targeting PCSK9 expression with siRNA or PCSK9 specific inhibitor (PF-06446846) impaired OC cell survival. In addition, overexpression of PCSK9 induced robust AKT phosphorylation along with increased expression of ERK1/2 and MEK1/2, suggesting a pro-survival role of PCSK9 in OC cells. Moreover, our drug testing revealed marked differences in cytotoxic responses to drugs targeting metabolic pathways of high-grade serous ovarian cancer (HGSOC) and low-grade serous ovarian cancer (LGSOC) PDCs. Our results show that targeting PCSK9 expression could impair OC cell survival, which warrants further investigation to address the dependency of this cancer on lipogenesis and omental metastasis. Moreover, the differences in metabolic gene expression and drug responses of OC PDCs indicate the existence of a metabolic heterogeneity within OC subtypes, which should be further explored for therapeutic improvements.

scholarly journals Extracellular Vesicles: Novel Opportunities to Understand and Detect Neoplastic Diseases

2021 ◽  
pp. 030098582199932
Author(s):  
Laura Bongiovanni ◽  
Anneloes Andriessen ◽  
Marca H. M. Wauben ◽  
Esther N. M. Nolte-’t Hoen ◽  
Alain de Bruin
Keyword(s):  
Extracellular Vesicles ◽  
Biologically Active ◽  
Liquid Biopsies ◽  
Donor Cells ◽  
Recent Developments ◽  
Veterinary Pathology ◽  
Cell Components ◽  
Potential Applications ◽  
Intercellular Communications

With a size range from 30 to 1000 nm, extracellular vesicles (EVs) are one of the smallest cell components able to transport biologically active molecules. They mediate intercellular communications and play a fundamental role in the maintenance of tissue homeostasis and pathogenesis in several types of diseases. In particular, EVs actively contribute to cancer initiation and progression, and there is emerging understanding of their role in creation of the metastatic niche. This fact underlies the recent exponential growth in EV research, which has improved our understanding of their specific roles in disease and their potential applications in diagnosis and therapy. EVs and their biomolecular cargo reflect the state of the diseased donor cells, and can be detected in body fluids and exploited as biomarkers in cancer and other diseases. Relatively few studies have been published on EVs in the veterinary field. This review provides an overview of the features and biology of EVs as well as recent developments in EV research including techniques for isolation and analysis, and will address the way in which the EVs released by diseased tissues can be studied and exploited in the field of veterinary pathology. Uniquely, this review emphasizes the important contribution that pathologists can make to the field of EV research: pathologists can help EV scientists in studying and confirming the role of EVs and their molecular cargo in diseased tissues and as biomarkers in liquid biopsies.

scholarly journals Pharmacological ablation of astrocytes reduces Aβ degradation and synaptic connectivity in an ex vivo model of Alzheimer’s disease

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Nicola Davis ◽  
Bibiana C. Mota ◽  
Larissa Stead ◽  
Emily O. C. Palmer ◽  
Laura Lombardero ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Culture Media ◽  
Ex Vivo ◽  
Wild Type ◽  
Insulin Degrading Enzyme ◽  
Ex Vivo Model ◽  
Model Of Alzheimer’S Disease ◽  
Aβ Clearance ◽  
5Xfad Mice

Abstract Background Astrocytes provide a vital support to neurons in normal and pathological conditions. In Alzheimer’s disease (AD) brains, reactive astrocytes have been found surrounding amyloid plaques, forming an astrocytic scar. However, their role and potential mechanisms whereby they affect neuroinflammation, amyloid pathology, and synaptic density in AD remain unclear. Methods To explore the role of astrocytes on Aβ pathology and neuroinflammatory markers, we pharmacologically ablated them in organotypic brain culture slices (OBCSs) from 5XFAD mouse model of AD and wild-type (WT) littermates with the selective astrocytic toxin L-alpha-aminoadipate (L-AAA). To examine the effects on synaptic circuitry, we measured dendritic spine number and size in OBCSs from Thy-1-GFP transgenic mice incubated with synthetic Aβ42 or double transgenics Thy-1-GFP/5XFAD mice treated with LAAA or vehicle for 24 h. Results Treatment of OBCSs with L-AAA resulted in an increased expression of pro-inflammatory cytokine IL-6 in conditioned media of WTs and 5XFAD slices, associated with changes in microglia morphology but not in density. The profile of inflammatory markers following astrocytic loss was different in WT and transgenic cultures, showing reductions in inflammatory mediators produced in astrocytes only in WT sections. In addition, pharmacological ablation of astrocytes led to an increase in Aβ levels in homogenates of OBCS from 5XFAD mice compared with vehicle controls, with reduced enzymatic degradation of Aβ due to lower neprilysin and insulin-degrading enzyme (IDE) expression. Furthermore, OBSCs from wild-type mice treated with L-AAA and synthetic amyloid presented 56% higher levels of Aβ in culture media compared to sections treated with Aβ alone, concomitant with reduced expression of IDE in culture medium, suggesting that astrocytes contribute to Aβ clearance and degradation. Quantification of hippocampal dendritic spines revealed a reduction in their density following L-AAA treatment in all groups analyzed. In addition, pharmacological ablation of astrocytes resulted in a decrease in spine size in 5XFAD OBCSs but not in OBCSs from WT treated with synthetic Aβ compared to vehicle control. Conclusions Astrocytes play a protective role in AD by aiding Aβ clearance and supporting synaptic plasticity.

scholarly journals Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

2020 ◽  
Author(s):  
Lina Y Alkaissi ◽  
Martin E Winberg ◽  
Stéphanie DS Heil ◽  
Staffan Haapaniemi ◽  
Pär Myrelid ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Ussing Chambers ◽  
E Coli ◽  
Follicle Associated Epithelium ◽  
Vitro Model ◽  
Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

scholarly journals Optimizing cisplatin delivery to triple-negative breast cancer through novel EGFR aptamer-conjugated polymeric nanovectors

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Lisa Agnello ◽  
Silvia Tortorella ◽  
Annachiara d’Argenio ◽  
Clarissa Carbone ◽  
Simona Camorani ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Tumor Targeting ◽  
Triple Negative ◽  
Ex Vivo ◽  
Treatment Options ◽  
Negative Control ◽  
Therapeutic Effects ◽  
Metastatic Setting

Abstract Background Management of triple-negative breast cancer (TNBC) is still challenging because of its aggressive clinical behavior and limited targeted treatment options. Cisplatin represents a promising chemotherapeutic compound in neoadjuvant approaches and in the metastatic setting, but its use is limited by scarce bioavailability, severe systemic side effects and drug resistance. Novel site-directed aptamer-based nanotherapeutics have the potential to overcome obstacles of chemotherapy. In this study we investigated the tumor targeting and the anti-tumorigenic effectiveness of novel cisplatin-loaded and aptamer-decorated nanosystems in TNBC. Methods Nanotechnological procedures were applied to entrap cisplatin at high efficacy into polymeric nanoparticles (PNPs) that were conjugated on their surface with the epidermal growth factor receptor (EGFR) selective and cell-internalizing CL4 aptamer to improve targeted therapy. Internalization into TNBC MDA-MB-231 and BT-549 cells of aptamer-decorated PNPs, loaded with BODIPY505-515, was monitored by confocal microscopy using EGFR-depleted cells as negative control. Tumor targeting and biodistribution was evaluated by fluorescence reflectance imaging upon intravenously injection of Cyanine7-labeled nanovectors in nude mice bearing subcutaneous MDA-MB-231 tumors. Cytotoxicity of cisplatin-loaded PNPs toward TNBC cells was evaluated by MTT assay and the antitumor effect was assessed by tumor growth experiments in vivo and ex vivo analyses. Results We demonstrate specific, high and rapid uptake into EGFR-positive TNBC cells of CL4-conjugated fluorescent PNPs which, when loaded with cisplatin, resulted considerably more cytotoxic than the free drug and nanovectors either unconjugated or conjugated with a scrambled aptamer. Importantly, animal studies showed that the CL4-equipped PNPs achieve significantly higher tumor targeting efficiency and enhanced therapeutic effects, without any signs of systemic toxicity, compared with free cisplatin and untargeted PNPs. Conclusions Our study proposes novel and safe drug-loaded targeted nanosystems for EGFR-positive TNBC with excellent potential for the application in cancer diagnosis and therapy.

scholarly journals A Tumbling Magnetic Microrobot System for Biomedical Applications

Micromachines ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 861
Author(s):  
Elizabeth E. Niedert ◽  
Chenghao Bi ◽  
Georges Adam ◽  
Elly Lambert ◽  
Luis Solorio ◽  
...  
Keyword(s):  
Ultrasound Imaging ◽  
Biomedical Applications ◽  
Imaging System ◽  
Ex Vivo ◽  
Negative Control ◽  
Circular Path ◽  
Rotational Axis ◽  
Significant Difference

A microrobot system comprising an untethered tumbling magnetic microrobot, a two-degree-of-freedom rotating permanent magnet, and an ultrasound imaging system has been developed for in vitro and in vivo biomedical applications. The microrobot tumbles end-over-end in a net forward motion due to applied magnetic torque from the rotating magnet. By turning the rotational axis of the magnet, two-dimensional directional control is possible and the microrobot was steered along various trajectories, including a circular path and P-shaped path. The microrobot is capable of moving over the unstructured terrain within a murine colon in in vitro, in situ, and in vivo conditions, as well as a porcine colon in ex vivo conditions. High-frequency ultrasound imaging allows for real-time determination of the microrobot’s position while it is optically occluded by animal tissue. When coated with a fluorescein payload, the microrobot was shown to release the majority of the payload over a 1-h time period in phosphate-buffered saline. Cytotoxicity tests demonstrated that the microrobot’s constituent materials, SU-8 and polydimethylsiloxane (PDMS), did not show a statistically significant difference in toxicity to murine fibroblasts from the negative control, even when the materials were doped with magnetic neodymium microparticles. The microrobot system’s capabilities make it promising for targeted drug delivery and other in vivo biomedical applications.

scholarly journals Novel role of endothelial BKCa channels in altered vasoreactivity following hypoxia

2010 ◽  
Vol 299 (5) ◽  
pp. H1439-H1450 ◽  
Author(s):  
Jennifer M. Hughes ◽  
Melissa A. Riddle ◽  
Michael L. Paffett ◽  
Laura V. Gonzalez Bosc ◽  
Benjimen R. Walker
Keyword(s):  
Ex Vivo ◽  
Barometric Pressure ◽  
Channel Blockers ◽  
Channel Activity ◽  
Resistance Arteries ◽  
Bkca Channels ◽  
Oxide Synthase ◽  
Increased Activity ◽  
Small Conductance

The systemic vasculature exhibits attenuated vasoconstriction following hypobaric chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased activity of endothelial cell (EC) large-conductance, calcium-activated potassium (BKCa) channels contributes to this response. Gracilis resistance arteries from hypobaric CH (barometric pressure = 380 mmHg for 48 h) rats demonstrated reduced myogenic reactivity and hyperpolarized VSM membrane potential ( Em) compared with controls under normoxic ex vivo conditions. These differences were eliminated by endothelial disruption. In the presence of cyclooxygenase and nitric oxide synthase inhibition, combined intraluminal administration of the intermediate and small-conductance, calcium-activated K+ channel blockers TRAM-34 and apamin was without effect on myogenic responsiveness and VSM Em in both groups; however, these variables were normalized in CH arteries by intraluminal administration of the BKCa inhibitor iberiotoxin (IBTX). Basal EC Em was hyperpolarized in arteries from CH rats compared with controls and was restored by IBTX, but not by TRAM-34/apamin. K+ channel blockers were without effect on EC basal Em in controls. Similarly, IBTX blocked acetylcholine-induced dilation in arteries from CH rats, but was without effect in controls, whereas TRAM-34/apamin eliminated dilation in controls. Acetylcholine-induced EC hyperpolarization and calcium responses were inhibited by IBTX in CH arteries and by TRAM-34/apamin in controls. Patch-clamp experiments on freshly isolated ECs demonstrated greater K+ current in cells from CH rats that was normalized by IBTX. IBTX was without effect on K+ current in controls. We conclude that hypobaric CH induces increased endothelial BKCa channel activity that contributes to reduced myogenic responsiveness and EC and VSM cell hyperpolarization.

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