A novel method based on combination of semi-in vitro and in vivo conditions in Agrobacterium rhizogenes-mediated hairy root transformation of Glycine species

Lipid droplet proteins regulate the storage and utilisation of intracellular lipids. Evidence is emerging that oocyte lipid utilisation impacts embryo development, but lipid droplet proteins have not been studied in oocytes. The aim of the present study was to characterise the size and localisation of lipid droplets in mouse oocytes during the periovulatory period and to identify lipid droplet proteins as potential biomarkers of oocyte lipid content. Oocyte lipid droplets, visualised using a novel method of staining cumulus–oocyte complexes (COCs) with BODIPY 493/503, were small and diffuse in oocytes of preovulatory COCs, but larger and more centrally located after maturation in response to ovulatory human chorionic gonadotrophin (hCG) in vivo, or FSH + epidermal growth factor in vitro. Lipid droplet proteins Perilipin, Perilipin-2, cell death-inducing DNA fragmentation factor 45-like effector (CIDE)-A and CIDE-B were detected in the mouse ovary by immunohistochemistry, but only Perilipin-2 was associated with lipid droplets in the oocyte. In COCs, Perilipin-2 mRNA and protein increased in response to ovulatory hCG. IVM failed to induce Perilipin-2 mRNA, yet oocyte lipid content was increased in this context, indicating that Perilipin-2 is not necessarily reflective of relative oocyte lipid content. Thus, Perilipin-2 is a lipid droplet protein in oocytes and its induction in the COC concurrent with dynamic reorganisation of lipid droplets suggests marked changes in lipid utilisation during oocyte maturation.

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CircKEAP1 Suppresses the Progression of Lung Adenocarcinoma via the miR-141-3p/KEAP1/NRF2 Axis

Frontiers in Oncology ◽

10.3389/fonc.2021.672586 ◽

2021 ◽

Vol 11 ◽

Author(s):

Yanbo Wang ◽

Fenghai Ren ◽

Dawei Sun ◽

Jing Liu ◽

BenKun Liu ◽

...

Keyword(s):

Tumor Growth ◽

Lung Adenocarcinoma ◽

Expression Profiles ◽

Luciferase Reporter ◽

Tumor Tissues ◽

Tumor Progress ◽

Rna Immunoprecipitation ◽

Novel Method

BackgroundLung cancer is the leading cause of death from cancer, and lung adenocarcinoma (LUAD) is the most common form. Despite the great advances that has been made in the diagnosis and treatment for LUAD, the pathogenesis of LUAD remains unclear. In this study, we aimed to identify the function of circKEAP1 derived from the exon of KEAP1 in LUAD.MethodsThe expression profiles of circRNAs in LUAD tissues and adjacent non-tumor tissues were analyzed by Agilent Arraystar Human CircRNA microarray. The levels and prognostic values of circKEAP1 in tissues and cancer cell lines were determined by quantitative real-time PCR (qRT-PCR). Subsequently, the effects of circKEAP1 on tumor growth were investigated by functional experiments in vitro and in vivo. Mechanistically, the dual luciferase reporter assay, RNA pull-down, and RNA immunoprecipitation experiments were performed to confirm the interaction between circKEAP1 and miR-141-3p in LUAD.ResultsWe found circKEAP1 was significantly downregulated in LUAD tissues and repressed tumor growth both in vitro and in vivo. Mechanistically, circKEAP1 competitively binds to miR-141-3p and relive miR-141-3p repression for its host gene, which activated the KEAP1/NRF2 signal pathway, and finally suppresses the tumor progress. Our findings suggest that circKEAP1 inhibits LUAD progression through circKEAP1/miR-141-3p/KEAP1 axis and it may serve as a novel method for the treatment of LUAD.

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Comparative investigation of sesquiterpene components of essential oils originating from intact plants and hairy root chamomile cultures

GSC Advanced Research and Reviews ◽

10.30574/gscarr.2021.6.2.0016 ◽

2021 ◽

Vol 6 (2) ◽

pp. 028-049

Author(s):

Éva Szőke ◽

Éva Lemberkovics

Keyword(s):

Essential Oil ◽

Essential Oils ◽

Hairy Root ◽

Folk Medicine ◽

Root Culture ◽

Hairy Root Culture ◽

Comparative Investigation ◽

Biologically Active

The importance of chamomile (Chamomilla recutita) inflorescence is widely known in classical and folk medicine, with the largest group of its effective constituents forming the essential oil (chamazulene, a-bisabolol, α-farnesene, trans-β-farnesene, spathulenol, cis/trans-en-in-dicycloethers). Among cultivated species, the Hungarian BK-2 contains more chamazulene in its essential oil than the German Degumil type, which is mainly cultivated for its a-bisabolol. Both components have important antiinflammatory activities. Wild populations can be easily distinguished from cultivated ones by their high amount of bisaboloides, particularly the flower of Hungarian Szabadkígyós wild type, which contained on average 48 % of the biologically active (-)-a-bisabolol. The population of Szabadkígyós has good salt tolerance which is important owing to global warming, because the proportion of saline areas is increasing worldwide. To keep the genome of Szabadkígyós having high (-)-a-bisabolol content, Szőke and research team used biotechnological methods. Sterile plantlets, were infected by Agrobacterium rhizogenes strains #A-4, #15834, #R-1601. The hairy root clones possessing the best growing and biosynthetical potential were multiplied for phytochemical investigations. Pharmacologically important compounds of their essential oils were followed in great detail. The amount of in vitro cultured terpenoids and polyin compounds was compared with that of in vivo plants. GC-MS studies showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristics of the mother plant. Berkheyaradulene, geranyl-isovalerat and cedrol as new components were identified in these sterile cultures. The main component of hairy root cultures (D/400, D/1, D/100 and Sz/400) was tr-b-farnesene and in addition one new compound: a-selinene was identified. Hairy root culture originated from chamomile collected in Szabadkígyós was intensive increased the essential oil content and pharmacological active compounds: (-) -α-bisabolol and β-eudesmol was also synthetized in large quantity. Furthermore, in vitro organized cultures were made from this population to obtain propagation material containing numerous active substances.

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An in-vitro assay using human spermatozoa to detect toxicity of biologically active substances

Scientific Reports ◽

10.1038/s41598-019-50929-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Tino Vollmer ◽

Börje Ljungberg ◽

Vera Jankowski ◽

Joachim Jankowski ◽

Griet Glorieux ◽

...

Keyword(s):

Ex Vivo ◽

Toxicity Testing ◽

Biologically Active ◽

Human Spermatozoa ◽

Vitro Assay ◽

Cellular Behavior ◽

Novel Method ◽

Set Up

Abstract Identifying the key toxic players within an in-vivo toxic syndrome is crucial to develop targeted therapies. Here, we established a novel method that characterizes the effect of single substances by means of an ex-vivo incubation set-up. We found that primary human spermatozoa elicit a distinct motile response on a (uremic) toxic milieu. Specifically, this approach describes the influence of a bulk toxic environment (uremia) as well as single substances (uremic toxins) by real-time analyzing motile cellular behavior. We established the human spermatozoa-based toxicity testing (HSTT) for detecting single substance-induced toxicity to be used as a screening tool to identify in-vivo toxins. Further, we propose an application of the HSTT as a method of clinical use to evaluate toxin-removing interventions (hemodialysis).

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A novel method for measuring absolute coronary blood flow and microvascular resistance in patients with ischaemic heart disease

Cardiovascular Research ◽

10.1093/cvr/cvaa220 ◽

2020 ◽

Cited By ~ 2

Author(s):

Paul D Morris ◽

Rebecca Gosling ◽

Iwona Zwierzak ◽

Holli Evans ◽

Louise Aubiniere-Robb ◽

...

Keyword(s):

Blood Flow ◽

Heart Disease ◽

Coronary Flow ◽

Microvascular Disease ◽

Flow Reserve ◽

Catheter Laboratory ◽

Novel Method ◽

Microvascular Resistance

Abstract Aims Ischaemic heart disease is the reduction of myocardial blood flow, caused by epicardial and/or microvascular disease. Both are common and prognostically important conditions, with distinct guideline-indicated management. Fractional flow reserve (FFR) is the current gold-standard assessment of epicardial coronary disease but is only a surrogate of flow and only predicts percentage flow changes. It cannot assess absolute (volumetric) flow or microvascular disease. The aim of this study was to develop and validate a novel method that predicts absolute coronary blood flow and microvascular resistance (MVR) in the catheter laboratory. Methods and results A computational fluid dynamics (CFD) model was used to predict absolute coronary flow (QCFD) and coronary MVR using data from routine invasive angiography and pressure-wire assessment. QCFD was validated in an in vitro flow circuit which incorporated patient-specific, three-dimensional printed coronary arteries; and then in vivo, in patients with coronary disease. In vitro, QCFD agreed closely with the experimental flow over all flow rates [bias +2.08 mL/min; 95% confidence interval (error range) −4.7 to +8.8 mL/min; R2 = 0.999, P < 0.001; variability coefficient <1%]. In vivo, QCFD and MVR were successfully computed in all 40 patients under baseline and hyperaemic conditions, from which coronary flow reserve (CFR) was also calculated. QCFD-derived CFR correlated closely with pressure-derived CFR (R2 = 0.92, P < 0.001). This novel method was significantly more accurate than Doppler-wire-derived flow both in vitro (±6.7 vs. ±34 mL/min) and in vivo (±0.9 vs. ±24.4 mmHg). Conclusions Absolute coronary flow and MVR can be determined alongside FFR, in absolute units, during routine catheter laboratory assessment, without the need for additional catheters, wires or drug infusions. Using this novel method, epicardial and microvascular disease can be discriminated and quantified. This comprehensive coronary physiological assessment may enable a new level of patient stratification and management.

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Polyprenols in hairy roots of Coluria geoides

Biochemical Society Transactions ◽

10.1042/bst0280790 ◽

2000 ◽

Vol 28 (6) ◽

pp. 790-791 ◽

Cited By ~ 5

Author(s):

K. Skorupińska-Tudek ◽

V. S. Hung ◽

O. Olszowska ◽

M. Furmanowa ◽

T. Chojnacki ◽

...

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Lipid Content ◽

Mammalian Cells ◽

Root Culture ◽

Dry Weight ◽

Hairy Root Culture ◽

Living Organisms ◽

Long Chain

Long-chain polyisoprenoid alcohols built from several up to more than 100 isoprenoid units are common constituents of all living organisms. They were found mostly in plants, bacteria, yeasts and mammalian cells. In vitro hairy root culture of Coluria geoides was obtained from plants transformed with Agrobacterium rhizogenes. Growth was optimal at 0.75% (w/v) glucose and at 22 °C. Dry samples of roots were extracted and lipid content was analysed by HPLC. According to our estimation, polyprenols are accumulated in roots of C. geoides cultivated in vitro as a mixture of several prenologues with the dominating prenol composed of 16 isoprenoid units. The content of polyprenols in tissue was approx. 300 μg/g of dry weight.

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NOVEL POLYETHYLENIMINE NANOGELS AS POTENTIAL GENE CARRIERS PRODUCED VIA PHOTOCHEMISTRY IN SURFACTANT-FREE AQUEOUS SOLUTION

International Journal of Nanoscience ◽

10.1142/s0219581x06005108 ◽

2006 ◽

Vol 05 (06) ◽

pp. 753-756 ◽

Cited By ~ 7

Author(s):

DONGMEI XU ◽

JIAHUI YU ◽

YONGBIAO LIU ◽

HANWEN SUN ◽

JINGYING XU ◽

...

Keyword(s):

Aqueous Solution ◽

Transfection Efficiency ◽

In Vitro Tests ◽

Gene Carriers ◽

Polymeric Particles ◽

Potential Applications ◽

Novel Method ◽

Low Toxicity

Cationic polymer nanogels, positively-charged submicrometer polymeric particles that swell in water, have attracted an increasing research attentions in recent years because of their potential applications as gene carriers. In this paper, we report a novel method to synthesize polyethylenimine (PEI) nanogels with sizes ranging from 80 nm to 200 nm via UV irradiation at room temperature in aqueous solution without adding any kind of surfactants. The morphology of the nanoparticles is determined to be spherical. The nanogels are of high stability, high transfection efficiency, low toxicity and low immunogenicity, as having been confirmed by in vivo tests with mice as an animal model, and by in vitro tests with human lung and liver cancer cells as well.

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In-vivo targeted tagging of RNA isolates cell specific transcriptional responses to environmental stimuli and identifies liver-to-adipose RNA transfer

10.1101/670398 ◽

2019 ◽

Author(s):

J. Darr ◽

M. Lassi ◽

Archana Tomar ◽

R. Gerlini ◽

F. Scheid ◽

...

Keyword(s):

Transcriptional Response ◽

Cellular Communication ◽

Lipid Homeostasis ◽

Tissue Architecture ◽

Transcriptional Responses ◽

Rna Transcripts ◽

Novel Method ◽

Endocrine Signaling

AbstractBio-fluids contain various circulating cell-free RNA transcripts (ccfRNAs). The composition of these ccfRNAs varies between bio-fluids and constitute tantalizing biomarker candidates for several pathologies. ccfRNAs have also been demonstrated as mediators of cellular communication, yet little is known about their function in physiological and developmental settings and most works are limited to in-vitro studies. Here, we have developed iTAG-RNA, a novel method for the unbiased tagging of RNA transcripts in mice in-vivo. We used this method to isolate hepatocytes and kidney proximal epithelial cells-specific transcriptional response to a dietary challenge without interfering with the tissue architecture, and to identify multiple hepatocyte-secreted ccfRNAs in plasma. We also identified transfer of these hepatic derived ccfRNAs to adipose tissue, where they likely serve as a buffering mechanism to maintain cholesterol and lipid homeostasis. Our findings directly demonstrate in-vivo transfer of RNAs between tissues and highlight its implications for endocrine signaling and homeostasis.

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