[Lipidomic markers of breast cancer malignant tumor histological types]. / Lipidomnye markery gistologicheskikh tipov zlokachestvennoi opukholi molochnoi zhelezy.

The molecular profile of a tumor is associated with its histological type and can be used both to study the mechanisms of tumor progression and to diagnose it. In this work, changes in the lipid profile of a malignant breast tumor and the adjacent tissue were studied. The potential possibility of determining the histological type of the tumor by its lipid profile was evaluated. Lipid profiling was performed by reverse-phase chromato-mass-spectrometric analysis the tissue of lipid extract with identification of lipids by characteristic fragments. Potential lipid markers of the histological type of tumor were determined using the Kruskal-Wallis test. Impact of lipid markers was calculated by MetaboAnalyst. Classification models were built by support vector machines with linear kernel and 1-vs-1 architecture. Models were validated by leave-one out cross-validation. Accuracy of models based on microenvironment tissue, were 99% and 75%, accuracy of models, based on tumor tissue, were 90% and 40% for the positive ion mode and negative ion mode respectively. The lipid profile of marginal (adjacent) tissue can be used for identification histological types of breast cancer. Glycerophospholipid metabolism pathway changes were statistically significant in the adjacent tissue and tumor tissue.

[Prognostic ability of seminal plasma lipidomic analysis in predicting the success of microTESE in men with azoospermia].

INTRODUCTION: The aim of this work is to assess the possibility of metabolomic (specifically lipidomic) analysis of seminal plasma to identify patients with preserved focal spermatogenesis in the testes who may have a reasonable chance of sperm retrieval during the microTESE procedure. MATERIALS AND METHODS: lipid composition of semen plasma samples from 64 men with azoospermia and 24 fertile control men was analyzed. Lipids were isolated from semen by a modified Folch extraction method. Lipid extracts were analyzed by reverse phase liquid chromatography coupled to mass spectrometry. Lipidomic data were compared with the results of the microTESE procedure. RESULTS: Comparison of two groups revealed a statistically significant difference in concentration for 23 lipids detected in positive ion mode and 37 lipids detected in negative ion mode. Those lipids mainly belong to hexosylceramides, sphingomyelins and phosphatidylcholines, phosphatidylethanolamines and their ethers. In multivariate analysis content of SM d16: 1/18:0 lipid (beta coefficient: -7.23; 95% confidence interval [95% CI]: -11.93 to - 2.53; odds ratio: 7.23e-04; CI for odds ratio: 6.59e-06 to 7.93e-02; Walds test: -3.02; p=0.003), content of TG 14: 1_16 : 0_18: 3 lipid (beta 2.95; 95% CI 0.98 to 4.93; odds ratio: 1.92e + 01; CI for odds ratio: 2.66e + 00 to 1.39e + 02 ; Walds test: 2.93; p=0.003) and testicular volume (beta: 0.14; 95% CI: 0.04 to 2.45; odds ratio: 1.15e + 00; CI odds ratio: from 1.04e + 00 to 1.27e + 00; Walds test: 2.65; p=0.008) were significantly associated with positive MicroTESE outcome. The sensitivity of this regression model was 61%, the specificity was 83%, and the AUC was 0.75. CONCLUSIONS: seminal plasma serves as a rich source of biological markers for identifying patients with preserved focal spermatogenesis in the testes. Seminal plasma lipidomic profile of the of patients in the control group with normal spermatogenesis clearly differs from the profile of patients with azoospermia, also there was a significant difference in content of a difference in lipids between patients with positive and negative microTESE outcomes. These are preliminary results and further research is needed to confirm the validity of the resulting lipid panel.

Lipid Alterations in Early-Stage High-Grade Serous Ovarian Cancer.

Epithelial ovarian cancer (OC) ranks first in the number of deaths among diseases of the female reproductive organs. Identification of OC at early stages is highly beneficial for the treatment but is highly challenging due to the asymptomatic or low-symptom disease development. In this study, lipid extracts of venous blood samples from 41 female volunteers, including 28 therapy-naive patients with histologically verified high-grade serous ovarian cancer at different stages (5 patients with I-II stages; 23 patients with III-IV stages) and 13 apparently healthy women of reproductive age, were profiled by high-performance liquid chromatography mass spectrometry (HPLC-MS). Based on MS signals of 128 differential lipid species with statistically significant level variation between the OC patients and control group, an OPLS-DA model was developed for the recognition of OC with 100% sensitivity and specificity R 2 = 0.87 and Q2 = 0.80. The second OPLS-DA model was developed for the differentiation between I-II OC stages and control group with R 2 = 0.97 and Q2 = 0.86 based on the signal levels of 108 differential lipid species. The third OPLS-DA model was developed for the differentiation between I-II OC stages and III-IV stages based on the signal levels of 99 differential lipid species. Various lipid classes (diglycerides, triglycerides, phosphatidylchlorines, ethanolamines, sphingomyelins, ceramides, phosphatidylcholines and phosphoinositols) in blood plasma samples display distinctly characteristic profiles in I-II OC, which indicates the possibility of their use as marker oncolipids in diagnostic molecular panels of early OC stages. Our results suggest that lipid profiling by HPLC-MS can improve identification of early-stage OC and thus increase the efficiency of treatment.

[Lipidomic markers of tumor progress in breast cancer patients]. / Lipidomnye markery opukholevoi progressii u bol'nykh rakom molochnoi zhelezy.

Research of cancer progression mechanisms and their impact on metabolism of tumor cells and tumor microenvironment cells is an important element in drug development for cancer target therapy. In this study, changes in tumor tissue and margin tissue lipid profiles, were associated with the following clinical and morphological characteristics: tumor size, cancer stage, multifocalite, tumor grade, number of lymph node metastasis, Nottingham prognostic index, total malignancy score, level of Ki67 protein. Lipid profiling was performed by reverse-phase chromato-mass spectrometry analysis of lipid tissue extract with lipid identification by characteristic fragments. In the lipid profile of tumor tissue 13 characteristic lipids were selected. Their levels significantly correlated with at least 5 clinical and morphological features. Eight of 13 belonged to phosphatidylcholines. In lipid profile of tumor microenviroment tissue 13 lipid features were selected. Their levels significantly correlated with at least 5 clinical and morphological features. Four of 13 belonged to oxidized lipids, 4 lipid features belonged to sphingomyelins, four of 13 belonged to phosphatidylethanolamines. The tumor microenvironment tissue lipid profile correlated with tumor size, cancer stage, tumor grade, number of axillary metastases, Nottingham prognostic index. The tumor tissue lipid profile correlated with tumor size, tumor grade, total malignant score, and number of axillary metastases.

[Analysis of metabolic pathways in intrauterine growth restriction]. / Analiz metabolicheskikh putei pri zaderzhke rosta ploda.

Objective was to analyze metabolic pathways based on a study of the metabolomic profile of pregnant women with intrauterine growth restriction. The metabolic profile of pregnant women with fetal growth restriction has been analyzed using liquid chromatography-mass spectrometry. At the second stage pathways were identified using SMPDB and MetaboAnalyst databases to clarify the relationship between metabolites. Biological networks allow to determine the effect of proteins on the metabolic pathways involved in pathogenesis of IUGR and determine the epigenetic mechanisms of its formation.

[Features of the urine peptidome under the condition of hypertensive pathologies of pregnancy]. / Osobennosti peptidoma mochi pri gipertenzivnykh patologiiakh beremennykh.

In order to find a peptide panel to differentiate close hypertensive conditions a case-control study was designed for 64 women from 4 groups: preeclampsia (PE), chronic hypertension superimposed with PE, chronic hypertension, and healthy individuals. Chromatography coupled with mass-spectrometry and subsequent bioinformatic analysis showed several patterns in the changes of the urine peptidome. There were 36 peptides common for four groups. Twenty two of them 22 belonged to alpha-1-chain of collagen I, nine peptides were from alpha-1-chain of collagen III, two from alpha-2-chain of collagen I, one from alpha-1/2-chain of collagen I, one from alpha-1-chain of collagen I/XVIII and one from uromodulin. Patients with hypertensive disorders had 34 common peptides: 12 from alpha-1-chain of collagen I, 10 from fibrinogen alpha-chain, eight from alpha-1-chain of collagen III, and 4 per other types of collagen. Comparative analysis revealed 12 peptides, which could be used as a diagnostic panel for confident discrimination of pregnant women with various hypertensive disorders.

Exhaled breath condensate analysis from intubated newborns by nano-HPLC coupled to high resolution MS.

Invasiveness of examination and therapy methods is a serious problem for intensive care and nursing of premature infants. Exhaled breath condensate (EBC) is the most attractive biofluid for non-invasive methods development in neonatology for monitoring the status of intubated infants. The aim of the study was to propose an approach for EBC sampling and analysis from mechanically ventilated neonates. EBC collection system with good reproducibility of sampling was demonstrated. Discovery-based proteomic and metabolomic studies were performed using nano-HPLC coupled to high resolution MS. Label-free semi-quantitative data were compared for intubated neonates with congenital pneumonia (12 infants) and left-sided congenital diaphragmatic hernia (12 infants) in order to define disease-specific features. Totally 119 proteins and 164 metabolites were found. A number of proteins and metabolites that can act as potential biomarkers of respiratory diseases were proposed and require further validation.

Effect of Different Levels of Salt Consumption on Urine Protein Composition during 105-Day Isolation Analyzed with the Use of opoSOM Software.

We investigated changes in the urine protein composition of healthy volunteers in controlled conditions during 105-day isolation (Mars-500 program) at different levels of salt consumption. We used newest proteomic techniques based on chromatography-mass spectrometry and various methods of bioinformatics including opoSOM. The period of observation can be divided into three intervals with different dynamics of protein excretion: early (week 1-6), intermediate (week 7-11) and late interval (week 12-15). We identified about 10 different groups of co-detected proteins, which are directly affected by periods with different-levels of salt consumption. We also determined the biological functions of these proteins, tissue specificity and signaling pathways that involve them.

[Determination of proteomic and metabolic composition of exhaled breath condensate of newborns].

Here, the possibility of proteomic and metabolomic analysis of the composition of exhaled breath condensate of neonates with respiratory support. The developed method allows non-invasive collecting sufficient amount of the material for identification of disease-specific biomarkers. Samples were collected by using a condensing device that was incorporated into the ventilation system. The collected condensate was analyzed by liquid chromatography coupled with high resolution mass spectrometry and tandem mass spectrometry. The isolated substances were identified with a use of databases for proteins and metabolites. As a result, a number of compounds that compose the exhaled breath condensate was determined and can be considered as possible biomarkers of newborn diseases or stage of development.

Studying the Proteomic Composition of Expired Air Condensate in Newborns on Breathing Support.

This study was designed to collect and perform a proteomic analysis of expired air condensate in newborns receiving respiratory support at the Department of Resuscitation and Intensive Care. The proteomic composition of expired air condensate was evaluated in newborns at various stages of development and with different abnormalities.

Proteomic Analysis of the Urine for Diagnostics in Newborns.

Proteomic analysis of the urine was used for noninvasive diagnostics of abnormalities in newborns treated in the neonatal intensive care unit. This approach can be used to differentiate between infectious and noninfectious respiratory disorders.

An untargeted approach for the analysis of the urine peptidome of women with preeclampsia.

Preeclampsia (PE) is a pregnancy complication characterized by high blood pressure and proteinuria. The disorder usually occurs after the 20th week of pregnancy and gets worse over time. PE increases the risk of poor outcomes for both the mother and the baby. In the study we applied LC-MS/MS method for the analysis of the urine peptidome of women with PE. Samples were prepared using size-exclusion chromatography method which gives more than twice peptides identities if compared with solid phase extraction. Thirty urine samples from women with mild and severe preeclampsia and the control group were analyzed. In total 1786 peptides were identified using complementary search engines (Mascot, MaxQuant and PEAKS). A high level of agreement in peptide identification was observed with previously published data. Label-free data comparison resulted in 35 peptides which reliably distinguished a particular PE group (severe or mild) from controls. Our results revealed unique identifications (correlate to alpha-1-antitrypsin, collagen alpha-1(I) chain, collagen alpha-1 (III) chain, and uromodulin, for instance) that can potentially serve as early indicators of PE.

PROTEOMIC ANALYSIS OF URINE UNDER CONTROLLED SALT INTAKE IN PROJECT <>.

Specifics of urine proteome is sensitive to a multitude of factors. One is nutrition or entrance in organism of main nutrients including salt (NaCI). Purpose of the investigation was to study the proteomic composition of healthy human urine in the controlled environment of a 105-day isolation experiment (project <>) with various levels of salt intake. Analysis was performed using the present-day proteomics techniques based on chromatography-mass spectrometry and bio-informatics options. An attempt was made to correlate changes in processes and physiological systems with the controlled salt intake. As a result, a list of proteins directly responsible for different salt intake during the experiment and then a list of tissues where these proteins express predominantly were compiled; besides, analysis of the processes these proteins are involved in was performed.

[Proteomic analysis of exhaled breath condensate for diagnosis of pathologies of the respiratory system].

Study of the proteomic composition of exhaled breath condensate (EBC), is a promising non-invasive method for the diagnosis of the respiratory tract diseases in patients. In this study the EBC proteomic composition of the 79 donors, including patients with different pathologies of the respiratory system has been investigated. Cytoskeletal keratins type II (1, 2, 3, 4, 5, 6) and cytoskeletal keratins the type I (9, 10, 14, 15, 16) were invariant for all samples. Analyzing the frequency of occurrence of proteins in different groups of examined patients, several categories of protein have been recognized: found in all pathologies (Dermcidin, Alpha-1-microglobulin, SHROOM3), found in several pathologies (CSTA, LCN1, JUP, PIP, TXN), and specific for a single pathology (PRDX1, Annexin A1/A2). The EBC analysis by HPLC-MS/MS can be used to identify potential protein markers characteristic for pathologies such as chronic obstructive pulmonary disease (PRDX1) and pneumonia (Annexin A1/A2).

[Identification of the minimal zinc-binding center in natural isoforms of amyloid-beta domain 1-16 using ESI-MS].

Alzheimer's disease, is a lethal neurodegenerative pathology, characterized by the formation of soluble neurotoxic oligomers of the human amyloid-beta peptide Abeta which get accumulated forming polymeric extracellular aggregates (so-called amyloid plaques). The isomerized at aspartate 7 isoform of the human Abeta (isoAbeta) is the main component of these plaques and is considered as the potential pathogenic agent of AD. Besides this, there is a possible generation mechanism for this isoform from a genetically deficient D7N Abeta variant (Tottori mutation). On the contrary the rodent Abeta (rat Abeta), which has three amino acid substitutions in its metal-binding domain, is not susceptible to pathogenic aggregation in vivo, unlike the other known natural isoforms of Abeta. Interactions with zinc ions play a crucial role in the aggregation of monomeric human Abeta in vitro and in vivo. In the presented article using high resolution ESI-MS methods it was shown that domains 1-16 of isoAbeta and D7NAbeta bind zinc ions in the exactly the same manner as the normal human Abeta1-16, whereas ratAbeta has significant differences in structure of its minimal zinc binding center. These results confirm the overall interaction mechanism between zinc ions and the humanAbeta isoforms and allows to suppose that perhaps modulation of the structure of region 6-14 of Abeta can be used as a promising therapeutic approach to AD treatment.

[Identification of transthyretin posttranslational modifications 1n human blood using mass-spectrometric methods].

Transthyretin, one of the major plasma proteins, has a number of posttranslational modifications and mutations, some of which are associated with the development of severe diseases, for instance, familial amyloid neuropathy and Alzheimer's disease. In order to investigate the role of modified forms in the development of these diseases a complex analytical platform, based on two mass-spectrometric approaches (bottom-up and op-down) has been developed. The high efficiency of this method was shown using 10 plasma samples obtained from patients with Alzheimer's disease and healthy individuals.

[Expression of genes for metalloproteinases (MMP-1, MMP-2, MMP-9, and MMP-12) associated with psoriasis].

Using real-time polymerase chain reaction (RT-PCR), we measured mRNA amounts of matrix metalloproteinases (MMPs): MMP-1, MMP-2, MMP-9, and MMP-12 genes in psoriatic lesions and unaffected skin of the same patients. We observed significant (about 15-fold) increase in the expression level of matrix metalloproteinase MMP-1 and MMP-12 genes associated with psoriasis. The results of our studies of MMP gene expression in cultured primary human keratinocytes treated with interleukin (IL-17) have shown upregulation of MMP gene expression both in cultured keratinocytes and in psoriatic skin lesions. Therefore, upregulation of MMP genes in the skin affected by psoriasis could result from IL-17 effects on skin cells.

Comparative study of the expression of ATF-3 and ATF-4 genes in vessels involved into atherosclerosis process and in psoriatic skin.

Expression of ATF-3 and ATF-4 genes was examined quantitatively by real-time PCR and changes in the expression of these genes in atherosclerotic lesions and in psoriatic skin were demonstrated. It was found that concomitant pathologies do not affect the expression of these genes. Opposite changes in the expression of ATF-3 and ATF-4 genes in atherosclerotic and psoriatic samples were revealed and a hypothesis was put forward that this parameter could be a criterion of pathological process in both diseases.