Pharmacovariome scanning using whole pharmacogene resequencing coupled with deep computational analysis and machine learning for clinical pharmacogenomics.

BACKGROUND: This pilot study aims to identify and functionally assess pharmacovariants in whole exome sequencing data. While detection of known variants has benefited from pharmacogenomic-dedicated bioinformatics tools before, in this paper we have tested novel deep computational analysis in addition to artificial intelligence as possible approaches for functional analysis of unknown markers within less studied drug-related genes. METHODS: Pharmacovariants from 1800 drug-related genes from 100 WES data files underwent (a) deep computational analysis by eight bioinformatic algorithms (overall containing 23 tools) and (b) random forest (RF) classifier as the machine learning (ML) approach separately. ML model efficiency was calculated by internal and external cross-validation during recursive feature elimination. Protein modelling was also performed for predicted highly damaging variants with lower frequencies. Genotype-phenotype correlations were implemented for top selected variants in terms of highest possibility of being damaging. RESULTS: Five deleterious pharmacovariants in the RYR1, POLG, ANXA11, CCNH, and CDH23 genes identified in step (a) and subsequent analysis displayed high impact on drug-related phenotypes. Also, the utilization of recursive feature elimination achieved a subset of 175 malfunction pharmacovariants in 135 drug-related genes that were used by the RF model with fivefold internal cross-validation, resulting in an area under the curve of 0.9736842 with an average accuracy of 0.9818 (95% CI: 0.89, 0.99) on predicting whether a carrying individuals will develop adverse drug reactions or not. However, the external cross-validation of the same model indicated a possible false positive result when dealing with a low number of observations, as only 60 important variants in 49 genes were displayed, giving an AUC of 0.5384848 with an average accuracy of 0.9512 (95% CI: 0.83, 0.99). CONCLUSION: While there are some technologies for functionally assess not-interpreted pharmacovariants, there is still an essential need for the development of tools, methods, and algorithms which are able to provide a functional prediction for every single pharmacovariant in both large-scale datasets and small cohorts. Our approaches may bring new insights for choosing the right computational assessment algorithms out of high throughput DNA sequencing data from small cohorts to be used for personalized drug therapy implementation.

Six-Gene Signature for Differential Diagnosis and Therapeutic Decisions in Non-Small-Cell Lung Cancer-A Validation Study.

Non-small-cell lung cancer (NSCLC) poses a challenge due to its heterogeneity, necessitating precise histopathological subtyping and prognostication for optimal treatment decision-making. Molecular markers emerge as a potential solution, overcoming the limitations of conventional methods and supporting the diagnostic-therapeutic interventions. In this study, we validated the expression of six genes (MIR205HG, KRT5, KRT6A, KRT6C, SERPINB5, and DSG3), previously identified within a 53-gene signature developed by our team, utilizing gene expression microarray technology. Real-time PCR on 140 thoroughly characterized early-stage NSCLC samples revealed substantial upregulation of all six genes in squamous cell carcinoma (SCC) compared to adenocarcinoma (ADC), regardless of clinical factors. The decision boundaries of the logistic regression model demonstrated effective separation of the relative expression levels between SCC and ADC for most genes, excluding KRT6C. Logistic regression and gradient boosting decision tree classifiers, incorporating all six validated genes, exhibited notable performance (AUC: 0.8930 and 0.8909, respectively) in distinguishing NSCLC subtypes. Nevertheless, our investigation revealed that the gene expression profiles failed to yield predictive value regarding the progression of early-stage NSCLC. Our molecular diagnostic models manifest the potential for an exhaustive molecular characterization of NSCLC, subsequently informing personalized treatment decisions and elevating the standards of clinical management and prognosis for patients.

The role of miRNA molecules in the miscarriage process.

The etiology and pathogenesis of miscarriage, which is the most common pregnancy complication, have not been fully elucidated. There is a constant search for new screening biomarkers that would allow for the early diagnosis of disorders associated with pregnancy pathology. The profiling of microRNA expression is a promising research area, which can help establish the predictive factors for pregnancy diseases. Molecules of microRNAs are involved in several processes crucial for the development and functioning of the body. These processes include cell division and differentiation, programmed cell death, blood vessel formation or tumorigenesis, and the response to oxidative stress. The microRNAs affect the number of individual proteins in the body due to their ability to regulate gene expression at the post-transcriptional level, ensuring the normal course of many cellular processes. Based on the scientific facts available, this paper presents a compendium on the role of microRNA molecules in the miscarriage process. The expression of potential microRNA molecules as early minimally invasive diagnostic biomarkers may be evaluated as early as the first weeks of pregnancy and may constitute a monitoring factor in the individual clinical care of women in early pregnancy, especially after the first miscarriage. To summarize, the described scientific data set a new direction of research in the development of preventive care and prognostic monitoring of the course of pregnancy.

Toward Integration of Pharmacogenomic Tests into Daily Clinical Practice: A General Survey for Polish Healthcare Workers.

BACKGROUND Pharmacogenomics (PGx) has a direct influence on personalized drug therapy for various types of disorders and has been proven to have an important role in the future of medicine. The present study evaluated the awareness of PGx testing of clinicians and healthcare workers in the Republic of Poland. To the best of our knowledge, this is the first direct assessment of Polish healthcare professionals' attitudes toward introducing PGx tests into daily clinical practice. MATERIAL AND METHODS We used a comprehensive anonymous questionnaire with queries on level of education, background knowledge of PGx tests, advantages and barriers for implementation of such tests, and clinicians' desire to order the test that was distributed online to doctors, healthcare workers, related students/Ph.D. students, and administrative staff managing healthcare units. RESULTS We gathered 315 responses. According to the answers, two-thirds of participants had heard about PGx before (64.4%). An overwhelming majority of respondents appreciated the benefits of PGx (93.3%). Indeed, prior knowledge and level of education showed significant associations with positive attitudes toward PGx clinical testing (P≤0.05). However, all participants agreed there are major challenges for including such tests as part of routine clinical practice. CONCLUSIONS While the awareness and interest in PGx clinical testing in Polish healthcare providers are rising, some main barriers for implementation of these tests still need to be addressed in Poland.

miRNA-Seq Tissue Diagnostic Signature: A Novel Model for NSCLC Subtyping.

Non-small cell lung cancer (NSCLC) encompasses distinct histopathological subtypes, namely adenocarcinoma (AC) and squamous cell lung carcinoma (SCC), which require precise differentiation for effective treatment strategies. In this study, we present a novel molecular diagnostic model that integrates tissue-specific expression profiles of microRNAs (miRNAs) obtained through next-generation sequencing (NGS) to discriminate between AC and SCC subtypes of NSCLC. This approach offers a more comprehensive and precise molecular characterization compared to conventional methods such as histopathology or immunohistochemistry. Firstly, we identified 31 miRNAs with significant differential expression between AC and SCC cases. Subsequently, we constructed a 17-miRNA signature through rigorous multistep analyses, including LASSO/elastic net regression. The signature includes both upregulated miRNAs (hsa-miR-326, hsa-miR-450a-5p, hsa-miR-1287-5p, hsa-miR-556-5p, hsa-miR-542-3p, hsa-miR-30b-5p, hsa-miR-4728-3p, hsa-miR-450a-1-3p, hsa-miR-375, hsa-miR-147b, hsa-miR-7705, and hsa-miR-653-3p) and downregulated miRNAs (hsa-miR-944, hsa-miR-205-5p, hsa-miR-205-3p, hsa-miR-149-5p, and hsa-miR-6510-3p). To assess the discriminative capability of the 17-miRNA signature, we performed receiver operating characteristic (ROC) curve analysis, which demonstrated an impressive area under the curve (AUC) value of 0.994. Our findings highlight the exceptional diagnostic performance of the miRNA signature as a stratifying biomarker for distinguishing between AC and SCC subtypes in lung cancer. The developed molecular diagnostic model holds promise for providing a more accurate and comprehensive molecular characterization of NSCLC, thereby guiding personalized treatment decisions and improving clinical management and prognosis for patients.

Recombinant Human Prolidase (rhPEPD) Induces Wound Healing in Experimental Model of Inflammation through Activation of EGFR Signalling in Fibroblasts.

The potential of recombinant human prolidase (rhPEPD) to induce wound healing in an experimental model of IL-1ß-induced inflammation in human fibroblasts was studied. It was found that rhPEPD significantly increased cell proliferation and viability, as well as the expression of the epidermal growth factor receptor (EGFR) and downstream signaling proteins, such as phosphorylated PI3K, AKT, and mTOR, in the studied model. Moreover, rhPEPD upregulated the expression of the ß1 integrin receptor and its downstream signaling proteins, such as p-FAK, Grb2 and p-ERK 1/2. The inhibition of EGFR signaling by gefitinib abolished rhPEPD-dependent functions in an experimental model of inflammation. Subsequent studies showed that rhPEPD augmented collagen biosynthesis in IL-1ß-treated fibroblasts as well as in a wound healing model (wound closure/scratch test). Although IL-1ß treatment of fibroblasts increased cell migration, rhPEPD significantly enhanced this process. This effect was accompanied by an increase in the activity of MMP-2 and MMP-9, suggesting extracellular matrix (ECM) remodeling during the inflammatory process. The data suggest that rhPEPD may play an important role in EGFR-dependent cell growth in an experimental model of inflammation in human fibroblasts, and this knowledge may be useful for further approaches to the treatment of abnormalities of wound healing and other skin diseases.

Development of an extensive workflow for comprehensive clinical pharmacogenomic profiling: lessons from a pilot study on 100 whole exome sequencing data.

This pilot study is aimed at implementing an approach for comprehensive clinical pharmacogenomics (PGx) profiling. Fifty patients with cardiovascular diseases and 50 healthy individuals underwent whole-exome sequencing. Data on 1800 PGx genes were extracted and analyzed through deep filtration separately. Theoretical drug induced phenoconversion was assessed for the patients, using sequence2script. In total, 4539 rare variants (including 115 damaging non-synonymous) were identified. Four publicly available PGx bioinformatics algorithms to assign PGx haplotypes were applied to nine selected very important pharmacogenes (VIP) and revealed a 45-70% concordance rate. To ensure availability of the results at point-of-care, actionable variants were stored in a web-hosted database and PGx-cards were developed for quick access and handed to the study subjects. While a comprehensive clinical PGx profile could be successfully extracted from WES data, available tools to interpret these data demonstrated inconsistencies that complicate clinical application.

LC-PDA-MS and GC-MS Analysis of Scorzonera hispanica Seeds and Their Effects on Human Breast Cancer Cell Lines.

Scorzonera hispanica is an herbaceous perennial cultivated in Central and Southern Europe. This study aimed to qualitatively and quantitatively evaluate the composition of oil, extracts, and fractions (SH1-SH12) obtained from S. hispanica seeds. Furthermore, an evaluation of biological activities in breast cancer cell lines was also performed. GC-MS analysis revealed that the primary components of the seed oil (SH12) were fatty acids and ß-sitosterol. In the evaluation of extracts (SH1-SH3, SH8-SH10) and fractions (SH4-SH7, SH11) composition, the presence of apigenin, derivatives of p-coumaric and caffeic acids, was reported. In the biological assays, methanolic extract (SH1), diethyl ether (SH4), and chloroform (SH11) fractions exhibited cytotoxicity toward cells. The highest activity was observed for fatty acids- and 3,4-dimethoxycinnamate-rich SH11 (IC50: 399.18 µg/mL for MCF-7, 781.26 µg/mL for MDA-MB-231). SH11 was also observed to induce apoptosis in MCF-7 cells (52.4%). SH1, SH4, and SH11 attenuate signaling pathways and affect the expression of apoptosis-, autophagy-, and inflammation-related proteins. SH12 was non-toxic toward either cancer or normal cell lines in concentrations up to 1 mg/mL. The results suggest that S. hispanica seeds exhibit a wide range of potential uses as a source of oil and bioactive compounds for complementary therapy of breast cancer.

Proline Dehydrogenase/Proline Oxidase (PRODH/POX) Is Involved in the Mechanism of Metformin-Induced Apoptosis in C32 Melanoma Cell Line.

The role of proline dehydrogenase/proline oxidase (PRODH/POX) in the mechanism of antineoplastic activity of metformin (MET) was studied in C32 melanoma cells. PRODH/POX is a mitochondrial enzyme-degrading proline that is implicated in the regulation of cancer cell survival/apoptosis. The enzyme is activated by AMP kinase (AMPK). It has been found that MET induced a significant decrease in cell viability and DNA biosynthesis accompanied by an increase in the expressions of AMPK and PRODH/POX in C32 cells. The mechanism for MET-dependent cytotoxicity on C32 cells was found at the level of PRODH/POX-induced ROS generation and activation of Caspase-3 and Caspase-9 expressions in these cells. The effects were not observed in MET-treated PRODH/POX knock-out C32 cells. Of interest is an MET-dependent increase in the concentration of proline, which is a substrate for PRODH/POX. This phenomenon is due to the MET-dependent inhibition of collagen biosynthesis, which is the main proline-utilizing process. It has been found that the underlying mechanism of anticancer activity of MET involves the activation of AMPK, PRODH/POX, increase in the cytoplasmic concentration of proline, inhibition of collagen biosynthesis, and stimulation of PRODH/POX-dependent ROS generation, which initiate the apoptosis of melanoma cells.

LDGs versus NDGs in patients with oral squamous cell carcinoma (OSCC).

BACKGROUND: The significant role played by neutrophils in cancer biology is indisputable; yet, their subpopulations may exhibit a contrasting role. The phenomenon of polarization of neutrophils and signaling modulators in the course of a neoplastic process has gained increased attention in recent times. The present study's objective was to quantitatively assess low-density neutrophils (LDNs) and normal-density neutrophils (NDNs) populations including IL-17 expression in confrontation with Th17 lymphocytes in patients with oral squamous cell carcinoma (OSCC). The neutrophil to lymphocyte ratio (NLR) biomarker value was determined. Besides, the influence of rhIL-17 on the proliferation level of the squamous cell carcinoma (SCC) malignant line cells was tested. METHODS: Leukocytes were isolated in the density gradient and the CD16+ population was magnetically sorted. The percentages of neutrophil subpopulations, lymphocyte Th17, and IL-17 expression in the studied cells were determined on a flow cytometer. Squamous cell carcinoma proliferation was assessed with the MTT test. RESULT: The existence of two populations of human neutrophils was determined: LDNs and NDNs. A higher percentage of LDNs and Th17 was observed with the concomitant lower percentage of NDNs in patients with OSCC as compared with the control group. NLR was elevated in patients with cancer. The highest IL-17 expression was obtained in the LDNs population in these patients. However, no influence of IL-17 on SCC proliferation could be determined. CONCLUSION: The present study demonstrated a strong relationship between IL-17 concentration and the count of LDNs or Th17 in the course of OSCC, which may serve as a reference point for new therapies. Moreover, the obtained LDNs/NDNs and NLR values in patients with cancer prove their usefulness in diagnostic and prognostic in patients with OSCC.

Platelet-Rich Plasma Promotes the Proliferation of Human Keratinocytes via a Progression of the Cell Cycle. A Role of Prolidase.

Although the role of platelet-rich plasma (PRP) in tissue regeneration has been confirmed in many studies, the mechanism of this process is still not fully understood. Human keratinocytes (HaCaT) cells were used as an experimental model for studies on the effects of PRP on cell proliferation, migration, collagen biosynthesis, prolidase activity, and its expression and anabolic signaling. The activation of epidermal growth factor receptor (EGFR), ß1-integrin, and insulin-like growth factor-1 receptor (IGF-1R) by PRP were investigated by western blot and immunocytochemistry. It has been found that PRP induced keratinocytes migration and proliferation through activation of cell cycle progression and EGFR downstream signaling. Similar biological effects were achieved by an addition to the culture medium of prolidase (PEPD), a ligand of EGFR (PRP is a rich source of PEPD-2 ng/mL). PRP-dependent stimulation of collagen biosynthesis was accompanied by an increase in the expression of NF-κß, IGF-1R-downstream signaling proteins, and PEPD activity. The data suggest that PRP activates a complex of growth factors and adhesion receptors that stimulate cell proliferation, migration, and collagen biosynthesis. PRP induces PEPD-dependent human keratinocyte proliferation through activation of the EGFR receptor. Our study provides a novel mechanism of PRP-dependent wound healing.

Extracellular Prolidase (PEPD) Induces Anabolic Processes through EGFR, ß1-integrin, and IGF-1R Signaling Pathways in an Experimental Model of Wounded Fibroblasts.

The role of prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR) was studied in an experimental model of wound healing in cultured fibroblasts. The cells were treated with PEPD (1-100 nM) and analysis of cell viability, proliferation, migration, collagen biosynthesis, PEPD activity, and the expressions of EGFR, insulin-like growth factor 1 (IGF-1), and ß1-integrin receptor including downstream signaling proteins were performed. It has been found that PEPD stimulated proliferation and migration of fibroblasts via activation of the EGFR-downstream PI3K/Akt/mTOR signaling pathway. Simultaneously, PEPD stimulated the expression of ß1-integrin and IGF-1 receptors and proteins downstream to these receptors such as FAK, Grb2, and ERK1/2. Collagen biosynthesis was increased in control and "wounded" fibroblasts under PEPD treatment. The data suggest that PEPD-induced EGFR signaling may serve as a new attempt to therapy wound healing.

Cancers Cells in Traps? The Pathways of NETs Formation in Response to OSCC in Humans-A Pilot Study.

The aim of the experiment was to evaluate the process of neutrophil extracellular traps (NETs) formation in patients with oral squamous cell carcinoma (OSCC) in response to direct or indirect contact with SCC cells in comparison to results obtained in the cells of healthy subjects. To fulfill study objectives CAL 27 cell line and blood were obtained from cancer patients and control subjects. Parameters related to NETs formation were analyzed utilizing flow cytometry, fluorescence microscopy, and ELISA-type tests. The expression of selected phosphorylated proteins of the PI3K/Akt/PBK pathway in neutrophils was evaluated using the Western blot method. An increase in NETs formation was observed in a coculture of neutrophils with SCC cells, with the largest amount of NETs formed after stimulation with a supernatant obtained from the SCC culture. The enhanced process of NETs formation was accompanied by changes in the expression of proteins from the PI3K/Akt/PBK pathway. The obtained results prove the existence of interactions between neutrophils and cancer cells resulting in NETosis with the participation of the PI3K/Akt/PBK pathway in patients with OSCC.

Current Understanding of the Emerging Role of Prolidase in Cellular Metabolism.

Prolidase [EC 3.4.13.9], known as PEPD, cleaves di- and tripeptides containing carboxyl-terminal proline or hydroxyproline. For decades, prolidase has been thoroughly investigated, and several mechanisms regulating its activity are known, including the activation of the ß1-integrin receptor, insulin-like growth factor 1 receptor (IGF-1) receptor, and transforming growth factor (TGF)-ß1 receptor. This process may result in increased availability of proline in the mitochondrial proline cycle, thus making proline serve as a substrate for the resynthesis of collagen, an intracellular signaling molecule. However, as a ligand, PEPD can bind directly to the epidermal growth factor receptor (EGFR, epidermal growth factor receptor 2 (HER2)) and regulate cellular metabolism. Recent reports have indicated that PEPD protects p53 from uncontrolled p53 subcellular activation and its translocation between cellular compartments. PEPD also participates in the maturation of the interferon α/ß receptor by regulating its expression. In addition to the biological effects, prolidase demonstrates clinical significance reflected in the disease known as prolidase deficiency. It is also known that prolidase activity is affected in collagen metabolism disorders, metabolic, and oncological conditions. In this article, we review the latest knowledge about prolidase and highlight its biological function, and thus provide an in-depth understanding of prolidase as a dipeptidase and protein regulating the function of key biomolecules in cellular metabolism.

Prolidase Stimulates Proliferation and Migration through Activation of the PI3K/Akt/mTOR Signaling Pathway in Human Keratinocytes.

Recent reports have indicated prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR). Since this receptor is involved in the promotion of cell proliferation, growth, and migration, we aimed to investigate whether prolidase may participate in wound healing in vitro. All experiments were performed in prolidase-treated human keratinocytes assessing cell vitality, proliferation, and migration. The expression of downstream signaling proteins induced by EGFR, insulin-like growth factor 1 (IGF-1), transforming growth factor ß1 (TGF-ß1), and ß1-integrin receptors were evaluated by Western immunoblotting and immunocytochemical staining. To determine collagen biosynthesis and prolidase activity radiometric and colorimetric methods were used, respectively. Proline content was determined by applying the liquid chromatography coupled with mass spectrometry. We found that prolidase promoted the proliferation and migration of keratinocytes through stimulation of EGFR-downstream signaling pathways in which the PI3K/Akt/mTOR axis was involved. Moreover, PEPD upregulated the expression of ß1-integrin and IGF-1 receptors and their downstream proteins. Proline concentration and collagen biosynthesis were increased in HaCaT cells under prolidase treatment. Since extracellular prolidase as a ligand of EGFR induced cell growth, migration, and collagen biosynthesis in keratinocytes, it may represent a potential therapeutic approach for the treatment of skin wounds.

Development of an LC-MS Targeted Metabolomics Methodology to Study Proline Metabolism in Mammalian Cell Cultures.

A growing interest in metabolomics studies of cultured cells requires development not only untargeted methods capable of fingerprinting the complete metabolite profile but also targeted methods enabling the precise and accurate determination of a selected group of metabolites. Proline metabolism affects many crucial processes at the cellular level, including collagen biosynthesis, redox balance, energetic processes as well as intracellular signaling. The study aimed to develop a robust and easy-to-use targeted metabolomics method for the determination of the intracellular level of proline and the other two amino acids closely related to proline metabolism: glutamic acid and arginine. The method employs hydrophilic interaction liquid chromatography followed by high-resolution, accurate-mass mass spectrometry for reliable detection and quantification of the target metabolites in cell lysates. The sample preparation consisted of quenching by the addition of ice-cold methanol and subsequent cell scraping into a quenching solution. The method validation showed acceptable linearity (r > 0.995), precision (%RSD < 15%), and accuracy (88.5-108.5%). Pilot research using HaCaT spontaneously immortalized human keratinocytes in a model for wound healing was performed, indicating the usefulness of the method in studies of disturbances in proline metabolism. The developed method addresses the need to determine the intracellular concentration of three key amino acids and can be used routinely in targeted mammalian cell culture metabolomics research.

Simultaneous analysis of bisphenol A fractions in maternal and fetal compartments in early second trimester of pregnancy.

Background Bisphenol A (BPA) is an estrogenic, endocrine-disrupting compound widely used in the industry. It is also a ubiquitous environmental pollutant. Its presence was confirmed in human fetuses, which results from maternal exposure during pregnancy. The mechanisms behind maternal-fetal transfer, and relationships between pregnant women and fetal exposures remain unclear. The aim of this study was to assess the impact of maternal exposure to BPA on the exposure of the fetus. Methods Maternal plasma and amniotic fluid samples were collected from 52 pregnant women undergoing amniocentesis for prenatal diagnosis of chromosomal abnormalities. BPA was measured by gas chromatography-mass spectrometry (GC-MS). The permeability factor - a ratio of fetal-to-maternal BPA concentration - was used as a measure delineating the transplacental transfer of BPA. Results The median concentration of maternal plasma BPA was 8 times higher than the total BPA concentration in the amniotic fluid (8.69 ng/mL, range: 4.3 ng/mL-55.3 ng/mL vs. median 1.03 ng/mL, range: 0.3 ng/mL-10.1 ng/mL). There was no direct relationship between the levels of BPA in maternal plasma and amniotic fluid levels. The permeability factor, in turn, negatively correlated with fetal development (birth weight) (R = -0.54, P < 0.001). Conclusion Our results suggest that the risk of fetal BPA exposure depends on placental BPA permeability rather than the levels of maternal BPA plasma concentration and support general recommendations to become aware and avoid BPA-containing products.

Maternal Plasma Metabolomic Profiles in Spontaneous Preterm Birth: Preliminary Results.

OBJECTIVE: To profile maternal plasma metabolome in spontaneous preterm birth. METHOD: In this retrospective case-control study, we have examined plasma of patient with preterm birth (between 22 and 36 weeks of pregnancy (n = 57)), with threatened preterm labor (between 23 and 36 weeks of pregnancy (n = 49)), and with term delivery (n = 25). Plasma samples were analysed using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) in positive and negative polarity modes. RESULTS: We found 168 differentially expressed metabolites that were significantly distinct between study groups. We determined 51 metabolites using publicly available databases that could be subdivided into one of the five groups: amino acids, fatty acids, lipids, hormones, and bile acids. PLS-DA models, verified by SVM classification accuracy, differentiated preterm birth and term delivery groups. CONCLUSIONS: Maternal plasma metabolites are different between term and preterm parturitions. Part of them may be related with preterm labor, while others may be affected by gestational age or the beginning of labor. Metabolite profile can classify preterm or term delivery groups raising the potential of metabolome as a biomarker to identify high-risk pregnancies. Metabolomic studies are also a tool to detect individual compounds that may be further tested in targeted researches.

Novel Gel Formulations as Topical Carriers for the Essential Oil of Bidens tripartita for the Treatment of Candidiasis.

The genus Bidens L. (Asteraceae) refers to several species of plants used in traditional phytotherapeutic preparations. B. tripartita, also known as bur marigold, is the most familiar plant and has been known as a remedy for chronic dysentery. The hydrodistilled essential oil of the aerial parts of the Polish B. tripartita was analyzed using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) techniques. To exclude any potential toxic effects of the oil on human dermal fibroblasts, the MTT test (methyl thiazolyl tetrazolium) and COMET assay (single-cell gel electrophoresis) were performed. Novel gel formulations as topical carriers for essential oil obtained from B. tripartita were developed and characterized. The bioadhesive properties of the designed preparations in the ex vivo model using the skin of hairless mice were also evaluated. The therapeutic efficacy of the topical formulations is influenced by active phytoconstituents and vehicle characteristics. The antifungal properties of the essential oil of B. tripartita were also tested against Candida species, and this oil appears to be a promising topical anticandidal agent.

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model.

BACKGROUND/AIMS: The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. METHODS: We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. RESULTS: PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. CONCLUSION: PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways.