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Dheer Singh

Bio: Dheer Singh is an academic researcher from National Dairy Research Institute. The author has contributed to research in topics: Granulosa cell & Gene expression. The author has an hindex of 18, co-authored 93 publications receiving 1066 citations.


Papers
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Journal ArticleDOI
TL;DR: The synergistic action of IGF1 with FSH is of likely key importance for the development of small antral follicles before selection; subsequently, other factors such as LH may also become necessary for continued cell survival.
Abstract: IGF1, a potent stimulator of cellular proliferation, differentiation and development, regulates granulosa cell steroidogenesis and apoptosis during follicular development. Depending upon species and stage of follicular growth, IGF1 acts on granulosa cell steroidogenesis either alone or together with FSH. We examined the mechanism of action of IGF1 in bovine granulosa cells in serum-free culture without insulin to determine its potential role in the regulation of steroidogenic and apoptotic regulatory gene expression and to investigate the interaction of FSH with IGF1 on this mechanism. Bovine granulosa cells treated with IGF1 demonstrated a significant increase in 17beta-oestradiol (OE(2)) production, cell number and in mRNA expression of CYP11A1, HSD3B1, CYP19A1, BAX, type 1 IGF receptor (IGF1R) and FSHR, while FSH alone had no significant effects. IGF1 or FSH alone or both together had no effect on BCL2 expression. IGF1 with FSH resulted in a synergistic increase in granulosa cell number and in mRNA expression of CYP19A1 and IGF1R without altering OE(2) production. IGF1 stimulated the phosphoinositide 3'-OH kinase (PI3K) but not the MAPK pathway in granulosa cells, as evidenced by increased phosphorylation of AKT but not extracellular-regulated kinase 1/2. Addition of the PI3K pathway inhibitor LY294002 (but not the MAPK pathway inhibitor PD98059) abrogated the increased expression of genes induced by IGF1. IGF1 therefore up-regulates the steroidogenic and apoptotic regulatory genes via activation of PI3K/AKT in bovine granulosa cells. The synergistic action of IGF1 with FSH is of likely key importance for the development of small antral follicles before selection; subsequently, other factors such as LH may also become necessary for continued cell survival.

149 citations

Journal ArticleDOI
TL;DR: The encapsulation of curcumin into the exosomes enhances its stability, solubility, and bioavailability and demonstrated that milk exosome act as stable oral drug delivery vehicles.
Abstract: Exosomes, the extracellular secretary nano-vesicles, act as carriers of biomolecules to the target cells. They exhibit several attributes of an efficient drug delivery system. Curcumin, despite having numerous bioactive and therapeutic properties, has limited pharmaceutical use due to its poor water solubility, stability, and low systemic bioavailability. Hence, this study aims to enhance the therapeutic potential of curcumin, a model hydrophobic drug, by its encapsulation into milk exosomes. In the present study, we investigated the stability of free curcumin and exosomal curcumin in PBS and in vitro digestive processes. Additionally, their uptake and trans-epithelial transport were studied on Caco-2 cells. Curcumin in milk exosomes had higher stability in PBS, sustained harsh digestive processes, and crossed the intestinal barrier than free curcumin. In conclusion, the encapsulation of curcumin into the exosomes enhances its stability, solubility, and bioavailability. Therefore, the present study demonstrated that milk exosomes act as stable oral drug delivery vehicles.

125 citations

Journal ArticleDOI
TL;DR: It is demonstrated that cow milk exosomes protect miRNAs against harsh digestive processes and they are able to cross the intestinal barrier to reach blood circulation for cellular function.

72 citations

Journal ArticleDOI
TL;DR: Overall, nanovesicles are a new class of bioactive compounds from buffalo milk with high proportion of stable immune miRNAs compared to urine and plasma of same animals.
Abstract: Milk is a natural nutraceutical produced by mammals. The nanovesicles of milk play a role in horizontal gene transfer and confer health-benefits to milk consumers. These nanovesicles contain miRNA, mRNA, and proteins which mediate the intercellular communication. In this work, we isolated and characterized the buffalo milk-derived nanovesicles by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), scanning electron microscopy (SEM), Western probing, and Fourier transform infrared (FTIR) spectroscopy. The DLS data suggested a bimodal size distribution with one mode near 50 nm and the other around 200 nm for the nanovesicles. The NTA and SEM data also supported the size of nanovesicles within a range of 50-200 nm. The FTIR measurements of nanovesicles identified some prominent absorption bands attributable to the proteins (1300-1700 cm(-1), amide A and amide B bands), lipids (2800-3100 cm(-1)), polysaccharides, and nucleic acids (900-1200 cm(-1)). The comparative expression profiles of immune miRNA signatures (miR-15b, miR-21, miR-27b, miR-125b, miR-155, and miR-500) in nanovesicles isolated from milk, serum, and urine revealed that these miRNAs are present abundantly (P < 0.05) in milk-derived nanovesicles. Milk miRNAs (miR-21 and 500) that were also found stable under different household storage conditions indicated that these could be biologically available to milk consumers. Overall, nanovesicles are a new class of bioactive compounds from buffalo milk with high proportion of stable immune miRNAs compared to urine and plasma of same animals.

67 citations

Journal ArticleDOI
TL;DR: The encapsulation of siRNA in milk exosomes resists harsh digestive processes, improving intestinal permeability and payload protection and the stable delivery of exosomal AF-488 siRNA along with its transepithelial transport was confirmed by fluorescence microscopy and fluorescence intensity measurements.
Abstract: Milk is not only a composite of nutrients but emerged as a source of exosomes acting as a promising drug delivery vehicle for small interfering RNA (siRNA). siRNA is known for its immense therapeutic potential but has various physiological limitations, including stable delivery. To investigate the suitability of siRNA for physiological stability and oral delivery, we encapsulated scrambled Alexa Fluor (AF)-488 siRNA in milk whey exosomes using lipofection and evaluated stability against the digestive processes along with its uptake and transepithelial transport by intestinal epithelial cells. Milk exosomal siRNA were found resistant to different digestive juices, including saliva, gastric, bile, and pancreatic juices, in vitro and were internalized by Caco-2 cells. The stable delivery of exosomal AF-488 siRNA along with its transepithelial transport was confirmed by fluorescence microscopy and fluorescence intensity measurements. In summary, the encapsulation of siRNA in milk exosomes resists harsh digestive processes, improving intestinal permeability and payload protection.

56 citations


Cited by
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Journal ArticleDOI
TL;DR: Common approaches to 3D culture are reviewed, the significance of 3D cultures in drug resistance and drug repositioning is discussed and some of the challenges of applying 3D cell cultures to high-throughput drug discovery are addressed.
Abstract: Drug development is a lengthy and costly process that proceeds through several stages from target identification to lead discovery and optimization, preclinical validation and clinical trials culminating in approval for clinical use. An important step in this process is high-throughput screening (HTS) of small compound libraries for lead identification. Currently, the majority of cell-based HTS is being carried out on cultured cells propagated in two-dimensions (2D) on plastic surfaces optimized for tissue culture. At the same time, compelling evidence suggests that cells cultured in these non-physiological conditions are not representative of cells residing in the complex microenvironment of a tissue. This discrepancy is thought to be a significant contributor to the high failure rate in drug discovery, where only a low percentage of drugs investigated ever make it through the gamut of testing and approval to the market. Thus, three-dimensional (3D) cell culture technologies that more closely resemble in vivo cell environments are now being pursued with intensity as they are expected to accommodate better precision in drug discovery. Here we will review common approaches to 3D culture, discuss the significance of 3D cultures in drug resistance and drug repositioning and address some of the challenges of applying 3D cell cultures to high-throughput drug discovery.

911 citations

Journal Article
TL;DR: The authors showed that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization, leading to a potent T helper type 1 (TH1)-TH17 response.
Abstract: Polymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (TH1)-TH17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor.

839 citations

01 Jan 2009
TL;DR: In this article, a review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.
Abstract: MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

646 citations

Journal ArticleDOI
TL;DR: The factors that govern follicular growth and atresia, with a special focus on their regulation by granulosa cells, are discussed, in which hormones, growth factors and cytokines, death ligand-receptor system and B cell lymphoma/leukemia 2 (BCL2) family members are further discussed.
Abstract: The mammalian ovary is an extremely dynamic organ in which a large majority of follicles are effectively eliminated throughout their reproductive life. Due to the numerous efforts of researchers, mechanisms regulating follicular growth and atresia in mammalian ovaries have been clarified, not only their systemic regulation by hormones (gonadotropins) but also their intraovarian regulation by gonadal steroids, growth factors, cytokines and intracellular proteins. Granulosa cells in particular have been demonstrated to play a major role in deciding the fate of follicles, serving molecules that are essential for follicular growth and maintenance as well as killing themselves by an apoptotic process that results in follicular atresia. In this review, we discuss the factors that govern follicular growth and atresia, with a special focus on their regulation by granulosa cells. First, ovarian folliculogenesis in adult life is outlined. Then, we explain about the regulation of follicular growth and atresia by granulosa cells, in which hormones, growth factors and cytokines, death ligand-receptor system and B cell lymphoma/leukemia 2 (BCL2) family members (mitochondria-mediated apoptosis) are further discussed.

515 citations

Journal ArticleDOI
TL;DR: DNA methylation of the aromatase promoter may be an essential component of the long-sought-after mechanism connecting environmental temperature and sex ratios in vertebrate species with temperature-dependent sex determination.
Abstract: Sex ratio shifts in response to temperature are common in fish and reptiles. However, the mechanism linking temperature during early development and sex ratios has remained elusive. We show in the European sea bass (sb), a fish in which temperature effects on sex ratios are maximal before the gonads form, that juvenile males have double the DNA methylation levels of females in the promoter of gonadal aromatase (cyp19a), the enzyme that converts androgens into estrogens. Exposure to high temperature increased the cyp19a promoter methylation levels of females, indicating that induced-masculinization involves DNA methylation-mediated control of aromatase gene expression, with an observed inverse relationship between methylation levels and expression. Although different CpGs within the sb cyp19a promoter exhibited different sensitivity to temperature, we show that the increased methylation of the sb cyp19a promoter, which occurs in the gonads but not in the brain, is not a generalized effect of temperature. Importantly, these effects were also observed in sexually undifferentiated fish and were not altered by estrogen treatment. Thus, methylation of the sb cyp19a promoter is the cause of the lower expression of cyp19a in temperature-masculinized fish. In vitro, induced methylation of the sb cyp19a promoter suppressed the ability of SF-1 and Foxl2 to stimulate transcription. Finally, a CpG differentially methylated by temperature and adjacent to a Sox transcription factor binding site is conserved across species. Thus, DNA methylation of the aromatase promoter may be an essential component of the long-sought-after mechanism connecting environmental temperature and sex ratios in vertebrate species with temperature-dependent sex determination.

437 citations