Biotehnološka primjena kvasca Yarrowia lipolytica uzgojenog u uvjetima nedostatka tiamina

During the cultivation of a thiamine-auxotrophic yeast strain Yarrowia lipolytica VKM Y-2412 on ethanol, the growth limitation by thiamine leads to the production of α-ketoglutaric acid. The α-ketoglutaric acid synthesis has been studied in dependence on pH, oxygen supply and ethanol, zinc and iron concentrations. Under optimal conditions, Y. lipolytica produced 88.7 g/L of α-ketoglutaric acid. The culture broth containing α-ketoglutaric acid was subjected to chemical treatment with hydrogen peroxide, which led to the formation of succinic acid in significant quantities (71.7 g/L). Further direct esterification of succinic acid with excess absolute ethanol yielded diethyl succinate. Biomass of Y. lipolytica, a superproducer of α-ketoglutaric acid, was characterized by a high content of protein and essential amino acids, free amino acids, and unusually large amount of γ-aminobutyric acid. The unique amino acid composition of the producer makes it possible to use this biomass as a component of parenteral nutrition mixtures and as a basis for neuroleptics.Auksotrofni kvasac Yarrowia lipolytica VKM Y-2412 uzgojen je na podlozi s etanolom u uvjetima nedostatka tiamina, što je dovelo do nastajanja α-ketoglutarne kiseline. Ispitana je ovisnost njezine sinteze o pH-vrijednosti, opskrbljenosti kisikom i etanolom, te koncentracijama cinka i željeza. U optimalnim je uvjetima kvasac Y. lipolytica proizveo 88,7 g/L α-ketoglutarne kiseline. Podloga za uzgoj koja je sadržavala α-ketoglutarnu kiselinu kemijski je obrađena vodikovim peroksidom, što je dovelo do nastajanja značajne koncentracije sukcinske kiseline (71,7 g/L). Daljnjom izravnom esterifikacijom sukcinske kiseline uz suvišak apsolutnog etanola dobiven je dietil sukcinat. Biomasu kvasca Y. lipolytica, koji proizvodi velike količine α-ketoglutarne kiseline, karakterizira veliki udjel proteina i esencijalnih aminokiselina, slobodnih aminokiselina i neuobičajeno velika količina γ-aminomaslačne kiseline. Jedinstveni aminokiselinski sastav kvasca omogućuje njegovu primjenu u intravenoznoj prehrani i pri liječenju pacijenata antipsihoticima

Differential Methylation of the HPV 16 Upstream Regulatory Region during Epithelial Differentiation and Neoplastic Transformation

High risk human papillomaviruses are squamous epitheliotropic viruses that may cause cervical and other cancers. HPV replication depends on squamous epithelial differentiation. Transformation of HPV-infected cells goes along with substantial alteration of the viral gene expression profile and preferentially occurs at transformation zones usually at the uterine cervix. Methylation of the viral genome may affect regulatory features that control transcription and replication of the viral genome. Therefore, we analyzed the methylation pattern of the HPV16 upstream regulatory region (URR) during squamous epithelial differentiation and neoplastic transformation and analyzed how shifts in the HPV URR methylome may affect viral gene expression and replication. HPV 16 positive biopsy sections encompassing all stages of an HPV infection (latent, permissive and transforming) were micro-dissected and DNA was isolated from cell fractions representing the basal, intermediate, and superficial cell layers, each, as well as from transformed p16INK4a-positive cells. We observed fundamental changes in the methylation profile of transcription factor binding sites in the HPV16 upstream regulatory region linked to the squamous epithelial differentiation stage. Squamous epithelial transformation indicated by p16INK4a overexpression was associated with methylation of the distal E2 binding site 1 leading to hyper-activation of the HPV 16 URR. Adjacent normal but HPV 16-infected epithelial areas retained hyper-methylated HPV DNA suggesting that these viral genomes were inactivated. These data suggest that distinct shifts of the HPV 16 methylome are linked to differentiation dependent transcription and replication control and may trigger neoplastic transformation

Thermal Stability of Iron- and Silicon-Substituted Hydroxyapatite Prepared by Mechanochemical Method

In this study, hydroxyapatite with the substitution of calcium cations by iron and phosphate by silicate groups was synthesized via a mechanochemical method. The as-prepared compounds have the general formula Ca10−xFex(PO4)6−x(SiO4)x(OH)2−xOx/2 with x = 0–1.5. The thermal stability of the as-prepared compounds was studied by ex situ annealing of powders in a furnace. It has been established that, at 800 °C for x ≤ 0.5, a partial decomposition of the substituted apatites occurs with the formation of the β–Ca3(PO4)2 phase. At high “x” values, the formation of this phase starts at the lower temperature of 700 °C, followed by the formation of Fe2O3 at 900 °C. The introduction of iron and silicate ions into the hydroxyapatite lattice was shown to decrease its thermal stability

Biotechnological Potential of Yarrowia lipolytica Grown under Thiamine Limitation

During the cultivation of a thiamine-auxotrophic yeast strain Yarrowia lipolytica VKM Y-2412 on ethanol, the growth limitation by thiamine leads to the production of α-ketoglutaric acid. The α-ketoglutaric acid synthesis has been studied in dependence on pH, oxygen supply and ethanol, zinc and iron concentrations. Under optimal conditions, Y. lipolytica produced 88.7 g/L of α-ketoglutaric acid. The culture broth containing α-ketoglutaric acid was subjected to chemical treatment with hydrogen peroxide, which led to the formation of succinic acid in significant quantities (71.7 g/L). Further direct esterification of succinic acid with excess absolute ethanol yielded diethyl succinate. Biomass of Y. lipolytica, a superproducer of α-ketoglutaric acid, was characterized by a high content of protein and essential amino acids, free amino acids, and unusually large amount of γ-aminobutyric acid. The unique amino acid composition of the producer makes it possible to use this biomass as a component of parenteral nutrition mixtures and as a basis for neuroleptics

The Phenomenon of the Cross-Resistance of Breast Cancer to Target and Hormonal Drugs: The Role of Epigenetic Reconstruction

The rearrangement of molecular pathways and the activation of bypass signaling determine the progression of tumor cell resistance to various drugs that specifically block target signaling proteins. The present work was performed on the MCF-7 breast cancer cells and established sublines, resistant to mTOR inhibitor rapamycin or antiestrogen tamoxifen, developed under prolonged cell treatment with rapamycin or tamoxifen, respectively. We have shown that both resistant sublines demonstrate the cross-resistance to rapamycin and tamoxifen and are characterized with the common signaling changes, namely—blocking of the estrogen receptor α (ERα) transcriptional activity and constitutive activation of Akt signaling. Analysis of the epigenetic machinery revealed the drastic suppression of the level of DNA methyltransferase 3A (DNMT3A) in both the resistant sublines that were correlated with the demethylation of the LINE-1 repeats. Knockdown of the DNMT3A via siRNA results in the progression of partial resistance of MCF-7 cells to both tamoxifen and rapamycin, supporting the important role of DNA methylation in the formation of the resistant phenotype. Totally, the results obtained highlight the possible mechanism of the tumor cell resistance to targeting/hormonal drugs based on the rearrangement of DNA methylation profile and activation of the bypass signaling pathways

Possibilities of Mechanochemical Synthesis of Apatites with Different Ca/P Ratios

Apatite is widely used in medicine as a biomaterial for bone tissue restoration. Properties of apatite depend on its composition, including the Ca/P ratio. This paper shows what range of Ca/P ratio can be attained in apatite by the mechanochemical method of synthesis, providing fast formation of a single-phase product. The synthesis was carried out from a reaction mixture of CaHPO4 and CaO at different Ca/P ratios in the range of 1.17–2.10. The products were studied by PXRD, FTIR and NMR spectroscopy, HRTEM, and STA. In mixtures with a low initial Ca/P ratio (1.17–1.48), directly in the mill, the formation of calcium orthophosphate with whitlockite structure containing an HPO42− group and structural water is shown for the first time. This phosphate has structure similar to that of whitlockites of hydrothermal origin and differs from high-temperature β-tricalcium phosphate that has composition Ca3(PO4)3. A series of samples of apatite was obtained with varied composition, which depends on the initial Ca/P ratio. At Ca/P 1.67, the existence of two types of calcium-rich apatite is confirmed

Possibilities of Mechanochemical Synthesis of Apatites with Different Ca/P Ratios

Apatite is widely used in medicine as a biomaterial for bone tissue restoration. Properties of apatite depend on its composition, including the Ca/P ratio. This paper shows what range of Ca/P ratio can be attained in apatite by the mechanochemical method of synthesis, providing fast formation of a single-phase product. The synthesis was carried out from a reaction mixture of CaHPO4 and CaO at different Ca/P ratios in the range of 1.17–2.10. The products were studied by PXRD, FTIR and NMR spectroscopy, HRTEM, and STA. In mixtures with a low initial Ca/P ratio (1.17–1.48), directly in the mill, the formation of calcium orthophosphate with whitlockite structure containing an HPO42− group and structural water is shown for the first time. This phosphate has structure similar to that of whitlockites of hydrothermal origin and differs from high-temperature β-tricalcium phosphate that has composition Ca3(PO4)3. A series of samples of apatite was obtained with varied composition, which depends on the initial Ca/P ratio. At Ca/P < 1.67, the formation of two types of calcium-deficient apatite was documented. At Ca/P > 1.67, the existence of two types of calcium-rich apatite is confirmed

Extracellular Vesicles from Uterine Aspirates Represent a Promising Source for Screening Markers of Gynecologic Cancers

Extracellular vesicles (EVs), including exosomes, are key factors of intercellular communication, performing both local and distant transfers of bioactive molecules. The increasingly obvious role of EVs in carcinogenesis, similarity of molecular signatures with parental cells, precise selection and high stability of cargo molecules make exosomes a promising source of liquid biopsy markers for cancer diagnosis. The uterine cavity fluid, unlike blood, urine and other body fluids commonly used to study EVs, is of local origin and therefore enriched in EVs secreted by cells of the female reproductive tract. Here, we show that EVs, including those corresponding to exosomes, could be isolated from individual samples of uterine aspirates (UA) obtained from epithelial ovarian cancer (EOC) patients and healthy donors using the ultracentrifugation technique. First, the conducted profiling of small RNAs (small RNA-seq) from UA-derived EVs demonstrated the presence of non-coding RNA molecules belonging to various classes. The analysis of the miRNA content in EVs from UA performed on a pilot sample revealed significant differences in the expression levels of a number of miRNAs in EVs obtained from EOC patients compared to healthy individuals. The results open up prospects for using UA-derived EVs as a source of markers for the diagnostics of gynecological cancers, including EOC