Search

The present study employed nanoparticle tracking analysis, transmission electron microscopy, immunoblotting, RNA sequencing, and quantitative real-time PCR validation to characterize serum-derived small extracellular vesicles (sEVs) from RB patients and age-matched controls. Bioinformatics methods were used to analyze functions, and regulatory interactions between coding and non-coding (nc) sEVs RNAs. The results revealed that the isolated sEVs are round-shaped with a size < 150 nm, 5.3 × 1011 ± 8.1 particles/mL, and zeta potential of 11.1 to −15.8 mV, and expressed exosome markers CD9, CD81, and TSG101. A total of 6514 differentially expressed (DE) mRNAs, 123 DE miRNAs, and 3634 DE lncRNAs were detected. Both miRNA-mRNA and lncRNA-miRNA-mRNA network analysis revealed that the cell cycle-specific genes including CDKNI1A, CCND1, c-MYC, and HIF1A are regulated by hub ncRNAs MALAT1, AFAP1-AS1, miR145, 101, and 16-5p. Protein-protein interaction network analysis showed that eye-related DE mRNAs are involved in rod cell differentiation, cone cell development, and retinol metabolism. In conclusion, our study provides a comprehensive overview of the RB sEV RNAs and regulatory interactions between them.

Background: Exosomes are nano-sized vesicles secreted by various cells into the intra and extracellular space and hence is an integral part of biological fluids including milk. In the last few decades, many research groups have proved the potential of milk exosomes as a sustainable, economical and non-immunogenic drug delivery and therapeutic agent against different pathological conditions. However, its anti-viral properties still remain to be unearthed., Methods: Here, we have been able to isolate, purify and characterize the milk derived exosomes from Cow (CME) and Goat (GME) and further studied its antiviral properties against Dengue virus (DENV), Newcastle Disease Virus strain Komarov (NDV-K) and Human Immunodeficiency Virus (HIV-1) using an in-vitro infection system., Results: TEM, NTA and DLS analysis validated the appropriate size of the isolated cow and goat milk exosomes (30-150 nm). Real-time PCR and immunoblotting results confirmed the presence of several milk exosomal miRNAs and protein markers. Our findings suggest that GME significantly decreased the infectivity of DENV. In addition, we confirmed that GME significantly reduces DENV replication and reduced the secretion of mature virions. Furthermore, heat inactivation of GME did not show any inhibition on DENV infection, replication, and secretion of mature virions. RNase treatment of GME abrogates the anti-viral properties indicating direct role of exosomes in DENV inhibition. In addition GME inhibited the infectivity of NDV-K, but not HIV-1, suggesting that the GME mediated antiviral activity might be virus specific., Conclusion: This study demonstrates the anti-viral properties of milk exosomes and opens new avenues for the development of exosome-based therapies to treat viral diseases., (© 2022. The Author(s).)

Quite recently, milk exosomes have been recognized as efficient drug delivery systems owing to their biocompatibility and easy availability for scale-up technologies. However, there are no reports of comparative studies with regards to drug delivery by milk exosomes derived from different species. In this study, we isolated and characterized milk exosomes of cow, buffalo, and goat by various techniques and tried to understand their drug loading capacity and functional efficiency in HepG2, HCT116, and A549 cells by using doxorubicin. Doxorubicin was loaded to milk exosomes by three methods, that is, incubation, saponin treatment, and sonication. The isolated exosomes were found to be spherical with a size of <200 nm and displayed specific markers, namely, CD81, HSP70, HSC70, and miRNAs. Drug loading studies revealed that goat milk exosomes had the highest loading capacity across all three methods. Doxorubicin-encapsulated goat milk exosomes resulted in the inhibition of cell viability, with low IC 50 values in HepG2, HCT-116, and A549 cells. Doxorubicin-encapsulated goat exosomes displayed better IC 50 values than cow and buffalo milk-derived counterparts. In line with this, the ability of doxorubicin-encapsulated goat milk exosomes to induce apoptosis in HepG2 and HCT-116 cells was higher than that of cow and buffalo milk exosomes and free doxorubicin. Furthermore, unbound goat milk exosomes significantly reduced cell viability as compared to cow and buffalo milk exosomes. The transepithelial transport assay shows that doxorubicin-loaded milk exosomes transport doxorubicin efficiently as compared to free doxorubicin in vitro . Doxorubicin released from milk exosomes shows a biphasic release pattern, burst release followed by sustained release. These observations are important in light of the emerging importance of milk-derived exosomes as drug carriers to treat cancers.

Exosomes are a subgroup of membrane-bound extracellular vesicles secreted by all cell types and present virtually in all biological fluids. The composition of exosomes in the same cell type varies in healthy and disease conditions. Hence, exosomes research is a prime focus area for clinical research in cancer and numerous age-related metabolic syndromes. Functions of exosomes include crucial cell-to-cell communication that mediates complex cellular processes, such as antigen presentation, stem cell differentiation, and angiogenesis. However, very few studies reported the presence and role of exosomes in normal physiological and pathological conditions of specialized ocular tissues of the eye and ocular cancers. The eye being a protected sense organ with unique connectivity with the rest of the body through the blood and natural passages, we believe that the role of exosomes in ocular tissues will significantly improve our understanding of ocular diseases and their interactions with the rest of the body. We present a review that highlights the existence and function of exosomes in various ocular tissues, their role in the progression of some of the neoplastic and non-neoplastic conditions of the eyes.

Aim: Since the evolution of man, microbes are associated with humans, playing a vital role in the maintenance of good health. However, an imbalance in the gut microbial ecosystem is associated with several diseases including colorectal cancer (CRC). The supplementation with probiotics has been proven to be beneficial in improving CRC. In this study, we have evaluated the anticancer effects of 11 probiotic strains on human colorectal carcinoma cell line (HCT-116)., Methods and Results: In this study, HCT-116 cells were treated with various concentrations (0·5, 5, 10, 20 and 200 million CFU per ml) of probiotic strains. The viability was analysed using a MTT assay and IC 50 values were determined. Besides this, we evaluated the expression of multiple genes involved in the apoptosis and stress tolerance by real-time PCR. Lactobacillus reuteri (UBLRu-87), Saccharomyces boulardii (Unique-28), Bacillus clausii (UBBC-07), Bacillus coagulans (Unique-IS2), Streptococcus salivarius (UBSS-01), Lactobacillus fermentum (UBLF-31), Lactobacillus salivarius (UBLS-22), Bifidobacterium bifidum (UBBB-55) and Lactobacillus plantarum (UBLP-40) exhibited potent cytotoxicity on HCT 116 cells. Furthermore, UBLF-31 and Unique-28 induced the expression of CJUN, CFOS and CASP-9, and downregulated the expression of BCL6. UBLRu-87 and UBBB-55 induced the expression of CJUN, CFOS and CASP-9 but not BCL-6. UBLP-40, UBBC-07, UBLS-22, and Unique-IS2 induced the expression of CJUN and CASP-9 and downregulated the expression of BCL-6., Conclusion: These studies indicate the anticancer effects of selected probiotic strains by inducing apoptosis., Significance and Impact of the Study: The probiotic strains with the anticancer effects identified in this study can be proposed as potential candidates in the treatment of CRCs., (© 2021 The Society for Applied Microbiology.)

Background: Metabolic stress, as negative energy balance on one hand or obesity on the other hand can lead to increased levels of free fatty acids in the plasma and follicular fluid of animals and humans. In an earlier study, we showed that increased oleic acid (OA) concentrations affected the function of cultured bovine granulosa cells (GCs). Here, we focus on genome wide effects of increased OA concentrations., Results: Our data showed that 413 genes were affected, of which 197 were down- and 216 up-regulated. Specifically, the expression of FSH-regulated functional key genes, CCND2, LHCGR, INHA and CYP19A1 and 17-β-estradiol (E2) production were reduced by OA treatment, whereas the expression of the fatty acid transporter CD36 was increased and the morphology of the cells was changed due to lipid droplet accumulation. Bioinformatic analysis revealed that associated pathways of the putative upstream regulators "FSH" and "Cg (choriogonadotropin)" were inhibited and activated, respectively. Down-regulated genes are over-represented in GO terms "reproductive structure/system development", "ovulation cycle process", and "(positive) regulation of gonadotropin secretion", whereas up-regulated genes are involved in "circulatory system development", "vasculature development", "angiogenesis" or "extracellular matrix/structure organization"., Conclusions: From these data we conclude that besides inhibiting GC functionality, increased OA levels seemingly promote angiogenesis and tissue remodelling, thus suggestively initiating a premature fulliculo-luteal transition. In vivo this may lead to impeded folliculogenesis and ovulation, and cause sub-fertility.

Exosomes, which facilitate intercellular communication, antigen presentation and shuttling of biological agents, were initially thought as the cell's garbage cargo but today, after about 40 years of their discovery, we are now beginning to understand their potential role in diagnosis and therapy of several diseases including cancers. Various studies over the decades have signified the role of exosomes in different stages of cancer. Exosomes play a key role in colorectal cancer initiation (CRC), promotion of anti- apoptotic signaling pathways, regulating tumor microenvironment, enhancing tumorigenicity, promotion of angiogenesis, stem cell proliferation and endothelial cell migration, establishment of immune suppressive environment, formation of pre- metastatic niche and metastasis. Exosomes also elicits drug resistance. Since, they have the ability to cross the biological barrier, exosomes are now being explored as an efficient target specific drug delivery system that facilitates the shipping of different biomolecules and therapeutic drugs. However, cautious and strong investigative approaches are required before approving exosomes as therapeutics or drug delivery systems. In this review, we summarize the role of exosomes in different stages of CRC and also elaborate on the applications of exosomes in diagnosis and therapy with respect to CRC., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Estrogen is essential for growth and development of ovarian follicles. Infections associated with E. coli or Endotoxin (LPS) suppress estradiol production by the downregulation of CYP19A1 expression. However, the molecular mechanism of its down regulation is not yet known. To elucidate the molecular mechanisms of LPS-mediated downregulation of CYP19A1 gene expression, we studied the effect of LPS and TLR4 signaling pathway inhibitor (OxPAPC, OxPAPC-Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) on CYP19A1 expression, and expression of CEBPB and CEBPB binding on CYP19A1 proximal promoter (CYP19A1 PII) in buffalo granulosa cells in vitro. The results showed that LPS (1μg/ml) significantly declined the expression of CYP19A1 gene. In further experiments, inhibitor studies confirmed the involvement of TLR4 in LPS induced CYP19A1 gene down regulation in buffalo granulosa cells. LPS promoted higher levels of CEBPB at cellular and nuclear level in granulosa cells. Chromatin immunoprecipitation results showed, that LPS induces higher amount of CEBPB binding on the CYP19A1 PII. Further, TLR4 inhibitor attenuated the LPS induced implications. In conclusion, our results demonstrated that CEBPB could be a potential regulator for LPS mediated downregulation of CYP19A1 and decline of 17-beta estradiol levels in buffalo granulosa cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

During negative energy balance, the concentration of different fatty acids, especially of oleic acid (OA) increases in the follicular fluid of cattle. Previously, we showed that OA induced morphological, physiological and molecular changes in cultured bovine granulosa cells. In our present study we analyzed effects of OA on the expression of markers for granulosa and Sertoli cell identity, FOXL2 and SOX9, respectively, in addition to effects on the FOXL2 regulated genes ESR2, FST, PTGS2 and PPARG. The results showed that OA down-regulated FOXL2, ESR2, FST and PPARG but up-regulated PTGS2 and SOX9. From these data we conclude that OA can compromise granulosa cell functionality and may initiate trans-differentiation processes in bovine granulosa cells. This novel mechanism may be causally involved in postpartum fertility problems of lactating dairy cows.

Cell culture models are essential for the detailed study of molecular processes. We analyze the dynamics of changes in a culture model of bovine granulosa cells. The cells were cultured for up to 8 days and analyzed for steroid production and gene expression. According to the expression of the marker genes CDH1, CDH2 and VIM, the cells maintained their mesenchymal character throughout the time of culture. In contrast, the levels of functionally important transcripts and of estradiol and progesterone production were rapidly down-regulated but showed a substantial up-regulation from day 4. FOXL2, a marker for granulosa cell identity, was also rapidly down-regulated after plating but completely recovered towards the end of culture. In contrast, expression of the Sertoli cell marker SOX9 and the lesion/inflammation marker PTGS2 increased during the first 2 days after plating but gradually decreased later on. We conclude that only long-term culture conditions (>4 days) allow the cells to recover from plating stress and to re-acquire characteristic granulosa cell features.

After parturition, one of the major problems related to nutritional management that is faced by the majority of dairy cows is negative energy balance (NEB). During NEB, excessive lipid mobilization takes place and hence the levels of free fatty acids, among them oleic acid, increase in the blood, but also in the follicular fluid. This accumulation can be associated with serious metabolic and reproductive disorders. In the present study, we analyzed the effects of physiological concentrations of oleic acid on cell morphology, apoptosis, necrosis, proliferation and steroid production, and on the abundance of selected transcripts in cultured bovine granulosa cells. Increasing oleic acid concentrations induced intracellular lipid droplet accumulation, thus resulting in a foam cell-like morphology, but had no effects on apoptosis, necrosis or proliferation. Oleic acid also significantly reduced the transcript abundance of the gonadotropin hormone receptors, FSHR and LHCGR, steroidogenic genes STAR, CYP11A1, HSD3B1 and CYP19A1, the cell cycle regulator CCND2, but not of the proliferation marker PCNA. In addition, treatment increased the transcript levels of the fatty acid transporters CD36 and SLC27A1, and decreased the production of 17-beta-estradiol and progesterone. From these data it can be concluded that oleic acid specifically affects morphological and physiological features and gene expression levels thus altering the functionality of granulosa cells. Suggestively, these effects might be partly due to the reduced expression of FSHR and thus the reduced responsiveness to FSH stimulation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Precise regulation of cell type-specific gene expression profiles precedes the profound morphological reorganization of somatic cell layers during folliculogenesis, ovulation and luteinization. Cell culture models are essential to the study of corresponding molecular mechanisms of gene regulation. In a recent study, it was shown that an increased cell plating density can largely change gene expression profiles of cultured bovine granulosa cells. In our present study, we comparatively analyzed cell plating density effects on cultured bovine and buffalo granulosa cells. Cells were isolated from small- to medium-sized follicles (2-6 mm) and cultured under serum-free conditions at different plating densities. The abundance of selected marker transcripts and associated miRNA candidates was determined by quantitative real-time RT-PCR. We found in both species that the abundance of CYP19A1, CCNE1 and PCNA transcripts was remarkably lower at a high plating density, whereas VNN2 and RGS2 transcripts significantly increased. In contrast, putative regulators of CYP19A1, miR-378, miR-106a and let-7f were significantly higher in both species or only in buffalo, respectively. Also miR-15a, a regulator of CCNE1, was upregulated in both species. Thus, increased plating density induced similar changes of mRNA and miRNA expression in granulosa cells from buffalo and cattle. From these data, we conclude that specific miRNA species might be involved in the observed density-induced gene regulation.

Background: Bovine granulosa cell culture models are important to understand molecular mechanisms of ovarian function. Folliculogenesis and luteinization are associated with increasing density of cells and local hypoxic conditions. The current study identified two reliable housekeeping genes useful for gene normalization in granulosa cells under different in vitro conditions., Methods: During the current experiments cells were subjected to different biological and physical stimuli, follicle stimulating hormone, different initial cell plating density and hypoxia. Transcript abundance of seven housekeeping genes was quantified by real-time RT-PCR with co-amplification of the respective external standard., Results: Three of the genes, GAPDH, HMBS, and HPRT1 were found to be regulated by initial cell plating density, five of them, GAPDH, HMBS, HPRT1, RPLP0 and RPS18 under hypoxic conditions, but none of them after FSH stimulation. In detail, GAPDH was up regulated, but HPRT1 and HMBS were down regulated at high density and under hypoxia. Expression of RPLP0 and RPS18 was inconsistent, but was significantly down-regulated in particular at high cell density combined with hypoxia. In contrast, TBP and B2M genes were neither regulated under different plating density conditions nor by hypoxia as they showed similar expression levels under all conditions analyzed., Conclusions: The present data indicate that TBP and B2M are appropriate housekeeping genes for normalization of transcript abundance measured by real-time RT-PCR in granulosa cells subjected to different plating densities, oxygen concentrations and FSH stimulation.

Problem: In granulosa cells, TLR4-mediated LPS-induced immune response interferes with ovarian granulosa cell function., Method of Study: LPS-induced pro-inflammatory gene expression was monitored by real-time PCR and NF-κB was determined by the immuno-blotting and immuno-staining in granulosa cells in vitro., Results: The LPS (1 μg/mL) increased pro-inflammatory gene expression which was reverted back by treatment with TLR4 signaling inhibitor. Cotreatment of CLA (10 μm) with LPS did not show any effect on LPS-induced pro-inflammatory gene expression but granulosa cells pre-treated with CLA for 24 hr, attenuated LPS-induced pro-inflammatory gene expression and nuclear NF-κB. GW9662, a PPARγ-inhibitor, further increased the expression of pro-inflammatory genes., Conclusion: The present findings reiterated that pre-treatment with CLA can prevent LPS-induced granulosa cells dysfunction., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)