Genomic, transcriptomic, and metabolic characterization of 2-Phenylethanol-resistant Saccharomyces cerevisiae obtained by evolutionary engineering.

2-Phenylethanol is an aromatic compound commonly used in the food, cosmetic, and pharmaceutical industries. Due to increasing demand for natural products by consumers, the production of this flavor by microbial fermentation is gaining interest, as a sustainable alternative to chemical synthesis or expensive plant extraction, both processes relying on the use of fossil resources. However, the drawback of the fermentation process is the high toxicity of 2-phenylethanol to the producing microorganism. The aim of this study was to obtain a 2-phenylethanol-resistant Saccharomyces cerevisiae strain by in vivo evolutionary engineering and characterize the adapted yeast at the genomic, transcriptomic and metabolic levels. For this purpose, the tolerance to 2-phenylethanol was developed by gradually increasing the concentration of this flavor compound through successive batch cultivations, leading to an adapted strain that could tolerate 3.4 g/L of 2-phenylethanol, which was about 3-times better than the reference strain. Genome sequencing of the adapted strain identified point mutations in several genes, notably in HOG1 that encodes the Mitogen-Activated Kinase of the high-osmolarity signaling pathway. As this mutation is localized in the phosphorylation lip of this protein, it likely resulted in a hyperactive protein kinase. Transcriptomic analysis of the adapted strain supported this suggestion by revealing a large set of upregulated stress-responsive genes that could be explained in great part by HOG1-dependent activation of the Msn2/Msn4 transcription factor. Another relevant mutation was found in PDE2 encoding the low affinity cAMP phosphodiesterase, the missense mutation of which may lead to hyperactivation of this enzyme and thereby enhance the stressful state of the 2-phenylethanol adapted strain. In addition, the mutation in CRH1 that encodes a chitin transglycosylase implicated in cell wall remodeling could account for the increased resistance of the adapted strain to the cell wall-degrading enzyme lyticase. Finally, the potent upregulation of ALD3 and ALD4 encoding NAD+ -dependent aldehyde dehydrogenase together with the observed phenylacetate resistance of the evolved strain suggest a resistance mechanism involving conversion of 2-phenylethanol into phenylacetaldehyde and phenylacetate implicating these dehydrogenases.

Genomic, transcriptomic and physiological analyses of silver-resistant Saccharomyces cerevisiae obtained by evolutionary engineering.

Silver is a non-essential metal used in medical applications as an antimicrobial agent, but it is also toxic for biological systems. To investigate the molecular basis of silver resistance in yeast, we employed evolutionary engineering using successive batch cultures at gradually increased silver stress levels up to 0.25-mM AgNO3 in 29 populations and obtained highly silver-resistant and genetically stable Saccharomyces cerevisiae strains. Cross-resistance analysis results indicated that the silver-resistant mutants also gained resistance against copper and oxidative stress. Growth physiological analysis results revealed that the highly silver-resistant evolved strain 2E was not significantly inhibited by silver stress, unlike the reference strain. Genomic and transcriptomic analysis results revealed that there were mutations and/or significant changes in the expression levels of the genes involved in cell wall integrity, cellular respiration, oxidative metabolism, copper homeostasis, endocytosis and vesicular transport activities. Particularly the missense mutation in the RLM1 gene encoding a transcription factor involved in the maintenance of cell wall integrity and with 707 potential gene targets might have a key role in the high silver resistance of 2E, along with its improved cell wall integrity, as confirmed by the lyticase sensitivity assay results. In conclusion, the comparative physiological, transcriptomic and genomic analysis results of the silver-resistant S. cerevisiae strain revealed potential key factors that will help understand the complex molecular mechanisms of silver resistance in yeast.

Evolutionary Engineering of an Iron-Resistant Saccharomyces cerevisiae Mutant and Its Physiological and Molecular Characterization.

Iron plays an essential role in all organisms and is involved in the structure of many biomolecules. It also regulates the Fenton reaction where highly reactive hydroxyl radicals occur. Iron is also important for microbial biodiversity, health and nutrition. Excessive iron levels can cause oxidative damage in cells. Saccharomyces cerevisiae evolved mechanisms to regulate its iron levels. To study the iron stress resistance in S. cerevisiae, evolutionary engineering was employed. The evolved iron stress-resistant mutant "M8FE" was analysed physiologically, transcriptomically and by whole genome re-sequencing. M8FE showed cross-resistance to other transition metals: cobalt, chromium and nickel and seemed to cope with the iron stress by both avoidance and sequestration strategies. PHO84, encoding the high-affinity phosphate transporter, was the most down-regulated gene in the mutant, and may be crucial in iron-resistance. M8FE had upregulated many oxidative stress response, reserve carbohydrate metabolism and mitophagy genes, while ribosome biogenesis genes were downregulated. As a possible result of the induced oxidative stress response genes, lower intracellular oxidation levels were observed. M8FE also had high trehalose and glycerol production levels. Genome re-sequencing analyses revealed several mutations associated with diverse cellular and metabolic processes, like cell division, phosphate-mediated signalling, cell wall integrity and multidrug transporters.

Evolutionary engineering and molecular characterization of a caffeine-resistant Saccharomyces cerevisiae strain.

Caffeine is a naturally occurring alkaloid, where its major consumption occurs with beverages such as coffee, soft drinks and tea. Despite a variety of reports on the effects of caffeine on diverse organisms including yeast, the complex molecular basis of caffeine resistance and response has yet to be understood. In this study, a caffeine-hyperresistant and genetically stable Saccharomyces cerevisiae mutant was obtained for the first time by evolutionary engineering, using batch selection in the presence of gradually increased caffeine stress levels and without any mutagenesis of the initial population prior to selection. The selected mutant could resist up to 50 mM caffeine, a level, to our knowledge, that has not been reported for S. cerevisiae so far. The mutant was also resistant to the cell wall-damaging agent lyticase, and it showed cross-resistance against various compounds such as rapamycin, antimycin, coniferyl aldehyde and cycloheximide. Comparative transcriptomic analysis results revealed that the genes involved in the energy conservation and production pathways, and pleiotropic drug resistance were overexpressed. Whole genome re-sequencing identified single nucleotide polymorphisms in only three genes of the caffeine-hyperresistant mutant; PDR1, PDR5 and RIM8, which may play a potential role in caffeine-hyperresistance.

Genomic and transcriptomic analysis of a coniferyl aldehyde-resistant Saccharomyces cerevisiae strain obtained by evolutionary engineering.

Phenolic inhibitors in lignocellulosic hydrolysates interfere with the performance of fermenting microorganisms. Among these, coniferyl aldehyde is one of the most toxic inhibitors. In this study, genetically stable Saccharomyces cerevisiae mutants with high coniferyl aldehyde resistance were successfully obtained for the first time by using an evolutionary engineering strategy, based on the systematic application of increasing coniferyl aldehyde stress in batch cultures. Among the selected coniferyl aldehyde-resistant mutants, the highly resistant strain called BH13 was also cross-resistant to other phenolic inhibitors, vanillin, ferulic acid and 4-hydroxybenzaldehyde. In the presence of 1.2 mM coniferyl aldehyde stress, BH13 had a significantly reduced lag phase, which was less than 3 h and only about 25% of that of the reference strain and converted coniferyl aldehyde faster. Additionally, there was no reduction in its growth rate, either. Comparative transcriptomic analysis of a highly coniferyl aldehyde-resistant mutant revealed upregulation of the genes involved in energy pathways, response to oxidative stress and oxidoreductase activity in the mutant strain BH13, already under non-stress conditions. Transcripts associated with pleiotropic drug resistance were also identified as upregulated. Genome re-sequencing data generally supported transcriptomic results and identified gene targets that may have a potential role in coniferyl aldehyde resistance.

Different propolis samples, phenolic content, and breast cancer cell lines: Variable cytotoxicity ranging from ineffective to potent.

Researchers have started focusing on investigating the anticarcinogenic effects of natural products with the slightest side effects possible, because current breast cancer treatment approaches are unable to achieve absolute success especially on aggressive subtypes. Propolis is among these products with its antimicrobial, antifungal, anti-inflammatory, and anticancer effects. Therefore, seven different samples were collected from different regions (Argentina, China, and Istanbul-Turkey) and applied on nonaggressive breast cancer cell line (BCCL) MCF-7 and aggressive cell lines SK-BR-3, and MDA-MB-231. Initially, the phenolic/flavonoid constituents of the propolis ethanol extracts were investigated by liquid chromatography-mass spectrometry-mass spectrometry (LS-MS/MS) and high-performance liquid chromatography (HPLC) analyses. Then, the anticarcinogenic effects of the propolis samples on MCF-7, SK-BR-3, MDA-MB-231 were evaluated by WST1 analysis and only selected ones on MCF-10A and hPdLF. According to the LS-MS/MS and HPLC analysis, Turkey originated propolis (Turkey3) were found to be richer than the other propolis samples in terms of phenolic/flavonoid compounds. Turkey propolis significantly inhibited cell proliferation in both nonaggressive and aggressive BCCL (P < 0.01). Therefore, Turkey3 propolis was selected for further evaluation using Annexin V-PI apoptosis detection assays. In addition, selected compounds among the propolis contents such as galangin, caffeic acid, apigenin, quercetin, and ferulic acid were applied to the MCF-7 cell line to detect cytotoxic and apoptotic effects. Galangin, caffeic acid, apigenin, and quercetin remarkably induced cell proliferation inhibition at all time intervals, whereas ferulic acid was found non efficient on the MCF-7 cell line. Annexin V-PI assay clarified that all cell proliferation inhibitions were markedly apoptotic. Our findings indicated that the inhibition effect of propolis on breast cancer cell proliferation was in a propolis type-, dose- and time-dependent fashion. Turkey3 propolis showed statistically significant cytotoxic effects on both the nonaggressive and aggressive BCCL. These findings were consistent with the effects of its rich phenolic and flavonoid contents, in terms of variety. © 2018 IUBMB Life, 71(5):619-631, 2019.

Physiological and Transcriptomic Analysis of a Chronologically Long-Lived Saccharomyces cerevisiae Strain Obtained by Evolutionary Engineering.

High-throughput aging studies with yeast as a model organism involve transposon-mutagenesis and yeast knockout collection, which have been pivotal strategies for understanding the complex cellular aging process. In this study, a chronologically long-lived Saccharomyces cerevisiae mutant was successfully obtained by using another high-throughput approach, evolutionary engineering, based on systematic selection in successive batch cultures under gradually increasing levels of caloric restriction. Detailed comparative physiological and transcriptomic analyses of the chronologically long-lived mutant and the reference strain revealed enhanced levels of respiratory metabolism, upregulation of genes related to carbohydrate metabolic processes, glycogen-trehalose pathways, stress response, and repression of protein synthesis-related genes in the long-lived mutant SRM11, already in the absence of caloric restriction. Interestingly, SRM11 had also significantly higher resistance to copper stress, and higher resistance to silver, ethanol, and 2-phenylethanol stresses than the reference strain. It also had lower ethanol production levels and an enhanced ethanol catabolism. To conclude, evolutionary engineering is another powerful high-throughput method for aging research, in addition to its widespread use in industrial strain development. Additionally, the interesting results revealed by this study about the potential relationship between longevity and various cellular properties are yet to be investigated further at molecular level.

Anatolian honey is not only sweet but can also protect from breast cancer: Elixir for women from artemis to present.

Natural products with bioactive components are widely studied on various cancer cell lines for their possible cytotoxic effects, recently. Among these products, honey stands out as a valuable bee product containing many active phenolic compounds and flavonoids. Numerous types of multifloral honey and honeydew honey are produced in Turkey owing to its abundant vegetation. Therefore, in this study, we investigated the cytotoxic effects of particular tree-originated honeys from chestnut, cedar, pine, and multifloral honey on cell lines representing different types of the most common cancer of women, breast cancer, MCF7, SKBR3, and MDAMB-231, and fibrocystic breast epithelial cell line, MCF10A as a control. All honey samples were analyzed biochemically. The dose- (1, 2.5, 5, 7.5, and 10 µg/mL) and time (24th, 48th, and 72nd hours)-dependent effects of ethanol/water solutions of the honey samples were scrutinized. Cell viability/cytotoxicity was evaluated by the water soluble tetrazolium Salt-1 (WST-1) method. Apoptotic status was detected by Annexin V-PI assay using FACSCalibur. The statistical analysis was performed using GraphPad Prism 6 and the clustering data analysis with the R programming language. The biochemical analyses of the honey samples showed that the tree-originated honey samples contained more total phenolic compounds than the multifloral honey. Phenolic content of the honey types increases in order of multifloral, pine, cedar, and chestnut, respectively, which is compatible with their cytotoxic affectivity and dark color. In addition, the antioxidant capacity of the studied honey types was observed to increase in order of multifloral < pine < cedar ≅ chestnut. According to the WST-1 data, chestnut honey induced cytotoxicity over 50% on all the cell lines, including the control MCF10A cells, even with low doses (honey concentrations starting from 1 µg/mL) (P < 0.0001). Similarly, Cedar honey was observed to be the second most effective honey in this study. Cedar honey, with the dose of 1 µg/mL, was detected statistically highly significant on MCF10A, MCF7, and SKBR3. In contrast, pine honey showed dramatically significant cytotoxicity only on the MDAMB 231 cells with a 1 µg/mL dose at the same time point (P = 0.018). While pine honey caused an anticancer effect on the MCF-7 and SKBR3 cancer cell lines with a 2.5-5 µg/mL dose (P < 0.0001), like cedar and chestnut honeys, it increased the viability of the MCF10A control cells with the doses of 2.5-5 µg/mL. It only showed cytotoxicity with higher doses (10 µg/mL) on the MCF10A cell line (P < 0.0001). Moreover, we have observed that the multifloral and artificial honey samples were mostly ineffective or increased cell viability with the doses of 1-5 µg/mL. Apoptotic effects of the other honey samples on the MCF-7 cell line were found as chestnut> pine> cedar> multifloral in the Annexin V-propidium iodide (PI) analysis. Chestnut, cedar, and pine honey displayed a remarkably cytotoxic effect on breast cancer cell lines, MCF7, SKBR3, and even on the most aggressive MDAMB 231, representing the triple negative breast cancer, which lacks of targeted anticancer therapy. The chestnut and cedar honeys stand out to be the most cytotoxic on all cell lines, while pine honey was found to be the least toxic on control cells with appropriate toxicity on the cancer cells. © 2017 IUBMB Life, 69(9):677-688, 2017.

Expression profiling of maternal plasma and placenta microRNAs in preeclamptic pregnancies by microarray technology.

Preeclampsia (PE) is one of the leading causes of maternal and fetal morbidity and mortality, occurring usually in the second half of pregnancy and affecting approximately 5-8% of pregnancies in the world. miRNAs play critical role in the regulation of placental development processes. We aimed to determine specific novel miRNAs for early diagnosis of preeclampsia which is one of the most dangerous pregnancy diseases. In this study 72 samples, maternal age 22 ≤ and ≤36, have been analyzed; maternal plasma and placental miRNAs were isolated from 18 severe preeclampsia (sPE) patients and 18 controls, respectively. Profiling of human miRNAs (1368 probe) was performed in samples with Agilent v16 microarrays for detection of the differences in miRNA expression between two groups. The results were validated by using TaqMan RT-qPCR method. The analysis indicated that 406 of these miRNAs in all placentas and 42 of these miRNAs in all maternal plasma were expressed. The relative expression analysis has shown that 12 miRNAs (p < 0.05 and >2-fold) in maternal plasma were differentially expressed in PE and control group. However, five miRNAs were validated by qRT-PCR. Once validated miRNAs have been searched in databases for their target genes and function, it has been shown that there are some preeclampsia related pathways as a target such as angiogenesis, cardiovascular, hypertension, placental abruption and preeclampsia disorders. Differentially expressed and validated plasma miRNAs might be used as notable biomarkers for non-invasive early diagnosis of preeclampsia and treatment of disease.

Evaluation of Local CYP17A1 and CYP19A1 Expression Levels as Prognostic Factors in Postmenopausal Invasive Ductal Breast Cancer Cases.

There is growing attention focused on local estrogen production in the breast tissue and its possible role in breast cancer initiation and progression. Understanding the underlying mechanisms for estrogen synthesis and the microenvironment consisting of tumor and its surrounding adipose tissue might open new avenues in breast cancer prevention, prognosis and treatment. In order to obtain insight, we compared peritumoral and tumor tissue expressions of CYP17A1 and CYP19A1 genes, which play an important role in estrogen biosynthesis. The paired tissue samples of 20 postmenopausal ER+/PR+ patients diagnosed with invasive ductal breast cancer were studied. In addition, 12 breast tissue samples obtained from premenopausal women without a history of breast cancer were also investigated as representative of normal conditions. Peritumoral adipose tissues expressed CYP19A1 approximately threefold higher than tumor itself (p = 0.001). A nonsignificant trend toward low expression of CYP17A1 was observed in peritumoral compared to tumor tissue (p = 0.687). Clinicopathological parameters and patient characteristics which are accepted as risk factors for breast cancer were also associated with individual and combined expressions of CYP17A1 and CYP19A1. This study offers that evaluation of CYP17A1 and CYP19A1 local expression levels might be useful for deciding on personalized treatment approaches and more accurate diagnosis, when evaluated together with several clinicopathological and disease risk factors. Considering the key role of these CYPs in estrogen synthesis, determining their expression levels may be useful as a postdiagnostic marker and for choosing the right treatment method in addition to the conventional approach.

Preliminary Study: Prominent miRNAs of Breast Malignant Tissues Compared to Normal Tissues in Turkish Patients with Breast Cancer.

miRNA involvement has been observed in almost every type of cancer, including breast cancer. The etiology of abnormal expression of miRNAs in cancer is still not clearly understood. In order to obtain insight into miRNA de-regulation in breast cancer, we analyzed expression levels of five breast cancer-related miRNAs, miRNA21, miRNA155, miRNA19a, miRNA17-5p and let7a miRNA, in both malignant and neighboring non-tumoral paraffin-embedded tissues of 47 patients with invasive ductal breast cancer. The targeted miRNAs, and a reference snRNA, U6, were analyzed by real-time polymerase chain reaction. let7a Levels were significantly lower in patients with lymphatic invasion than in those without (p=0.047). miR21 was down-regulated in 93.3% of patients with necrosis [p=0.017 (Fisher's exact test (FE))], while at least one oncogenic miRNA was up-regulated in 87.3% of the patients with invasive ductal carcinoma [p=0.009 (FE)]. In addition, tumor-suppressor miRNA was down-regulated or unaltered in 65.8% of the patients with tumor grade 2 or 3 and in all with grade 1 [p=0.047 (FE)]. Based on this preliminary study, we suggest that these miRNAs, especially let7a and miRNA21, might be useful markers in follow-up of breast cancer and in prognosis.

The effect of CYP1A1 and GSTM1 gene polymorphisms in bladder cancer development in a Turkish population.

BACKGROUND: The aim of this study was to investigate a possible association of the CYP1A1 Ile462Val and GSTM1 null polymorphisms with the risk of developing bladder cancer in a Turkish population. PATIENTS AND METHODS: The study constituted 176 patients with bladder cancer and 97 healthy individuals. Evaluation of CYP1A1 Ile462Val gene polymorphism was performed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). GSTM1 null gene polymorphism was exclusively determined by PCR. Our results were examined by statistical analyses. RESULTS: There were no significant differences in CYP1A1 genotype frequencies between patients and controls. Furthermore, the frequency of GSTM1 null genotype was higher in patients compared to controls, but it did not reach significance (p=0.622 χ(2)=0.243 OR=0.94 95% CI=0.75-1.18). Significance was discovered in combined analysis of CYP1A1 and GSTM1 genotypes. In the present study, GSTM1 null genotype with CYP1A1 Ile/Ile genotype combination was significantly more frequent in the patient group than in controls (p=0,04, χ(2)=4.217). At the same time, possessing both GSTM1 null genotype and CYP1A1 Val variants (Ile/Val+Val/Val) were significantly higher in control group than in patients (p=0.017, χ(2)=5.468). When the pathological tumor grades were assessed, the frequency of CYP1A1 Val mutant variant with GSTM1 null genotype combination was higher in patients with medium and high-grade tumors than in those with low-grade tumors (p=0.06, χ(2)=3.527, OR=1.36 95% CI=1.03-1.78). CONCLUSION: We suggest that the CYP1A1 Ile/Ile genotype with GSTM1 null genotype combination may contribute to the development of bladder cancer in this Turkish population.