Quantitative Analysis of Phagocytosis in Whole Blood Using Double Staining and Visualization.

Phagocytosis is an essential innate immunity function in humans and animals. A decrease in the ability to phagocytize is associated with many diseases and aging of the immune system. Assessment of phagocytosis dynamics requires quantification of bacteria inside and outside the phagocyte. Although flow cytometry is the most common method for assessing phagocytosis, it does not include visualization and direct quantification of location of bacteria. Here, we used double-labeled Escherichia coli cells to evaluate phagocytosis by flow cytometry (cell sorting) and confocal microscopy, as well as employed image cytometry to provide high-throughput quantitative and spatial recognition of the double-labeled E. coli associated with the phagocytes. Retention of pathogens on the surface of myeloid and lymphoid cells without their internalization was suggested to be an auxiliary function of innate immunity in the fight against infections. The developed method of bacterial labeling significantly increased the accuracy of spatial and quantitative measurement of phagocytosis in whole blood and can be recommended as a tool for phagocytosis assessment by image cytometry.

LC-MS Analysis Revealed the Significantly Different Metabolic Profiles in Spent Culture Media of Human Embryos with Distinct Morphology, Karyotype and Implantation Outcomes.

In this study we evaluated possible differences in metabolomic profiles of spent embryo culture media (SECM) of human embryos with distinct morphology, karyotype, and implantation outcomes. A total of 153 samples from embryos of patients undergoing in vitro fertilization (IVF) programs were collected and analyzed by HPLC-MS. Metabolomic profiling and statistical analysis revealed clear clustering of day five SECM from embryos with different morphological classes and karyotype. Profiling of day five SECM from embryos with different implantation outcomes showed 241 significantly changed molecular ions in SECM of successfully implanted embryos. Separate analysis of paired SECM samples on days three and five revealed 46 and 29 molecular signatures respectively, significantly differing in culture media of embryos with a successful outcome. Pathway enrichment analysis suggests certain amino acids, vitamins, and lipid metabolic pathways to be crucial for embryo implantation. Differences between embryos with distinct implantation potential are detectable on the third and fifth day of cultivation that may allow the application of culture medium analysis in different transfer protocols for both fresh and cryopreserved embryos. A combination of traditional morphological criteria with metabolic profiling of SECM may increase implantation rates in assisted reproductive technology programs as well as improve our knowledge of the human embryo metabolism in the early stages of development.

N-Acyl Dopamines Induce Apoptosis in Endometrial Stromal Cells from Patients with Endometriosis.

Endometriosis is characterized by the formation and development of endometrial tissues outside the uterus, based on an imbalance between proliferation and cell death, leading to the uncontrolled growth of ectopic foci. The potential target for the regulation of these processes is the endocannabinoid system, which was found to be involved in the migration, proliferation, and survival of tumor cells. In this paper, we investigated the effect of endocannabinoid-like compounds from the N-acyl dopamine (NADA) family on the viability of stromal cells from ectopic and eutopic endometrium of patients with ovarian endometriosis. N-arachidonoyldopamine, N-docosahexaenoyldopamine, and N-oleoyldopamine have been shown to have a five-times-more-selective cytotoxic effect on endometrioid stromal cells. To study the mechanisms of the toxic effect, inhibitory analysis, measurements of caspase-3/9 activity, reactive oxygen species, and the mitochondrial membrane potential were performed. It was found that NADA induced apoptosis via an intrinsic pathway through the CB1 receptor and downstream serine palmitoyltransferase, NO synthase activation, increased ROS production, and mitochondrial dysfunction. The higher selectivity of NADA for endometriotic stromal cells and the current lack of effective drug treatment can be considered positive factors for further research of these compounds as possible therapeutic agents against endometriosis.

Methylation of the PTENP1 pseudogene as potential epigenetic marker of age-related changes in human endometrium.

The processed pseudogene PTENP1 is involved in the regulation of the expression of the PTEN and acts as a tumor suppressor in many types of malignances. In our previous study we showed that PTENP1 methylation is present not only in tumor, but also in normal endometrium tissues of women over 45 years old. Here we used methylation-specific PCR to analyze methylation status of CpG island located near promoter region of PTENP1 in malignant and non-malignant endometrium tissues collected from 236 women of different age groups. To confirm our results, we also analyzed RNA sequencing and microarray data from 431 women with endometrial cancer from TCGA database. We demonstrated that methylation of PTENP1 is significantly increased in older patients. We also found an age-dependent increase in the level of PTENP1 expression in endometrial tissue. According to our data, PTENP1 methylation elevates the level of the pseudogene sense transcript. In turn, a high level of this transcript correlates with a more favorable prognosis in endometrial cancer. The data obtained suggested that PTENP1 methylation is associated with age-related changes in normal and hyperplastic endometrial tissues. We assumed that age-related increase in PTENP1 methylation and subsequent elevation of its expression may serve as a protective mechanism aimed to prevent malignant transformation of endometrial tissue in women during the perimenopause, menopause, and postmenopause periods.

Neuroprotective and neurotoxic effects of endocannabinoid-like compounds, N-arachidonoyl dopamine and N-docosahexaenoyl dopamine in differentiated cultures of induced pluripotent stem cells derived from patients with Parkinson's disease.

The prominent protective effects in diverse neuron injury paradigms exerted by cannabinoids and in particular their endogenously produced species render the endocannabinoid system a promising molecular target in the treatment of neurodegenerative diseases. However, the effects of individual endocannabinoids in human cells remain poorly investigated. Neural derivatives of human induced pluripotent stem cells (iPSC) offer unique opportunities for studying the neuroprotective compounds and development of patient-specific treatment. For the first time the cytotoxic and neuroprotective effects endocannabinoids N-arachidonoyl dopamine (N-ADA) and N-docosahexaenoyl dopamine (N-DDA) were assessed in human neural progenitors and dopamine neurons derived from iPSCs of healthy donors and patients with Parkinson's disease. While the short-term treatment with the investigated compounds in 0.1-10 µM concentration range exerted no toxicity in these cell types, the long-term exposure to 0.1-5 µM N-ADA or N-DDA reduced the survival of human neural progenitors. At the same time, both N-ADA and N-DDA protected neural progenitors and terminally differentiated neurons both from healthy donors and patients with Parkinson's disease against oxidative stress induced by hydrogen peroxide. The observed dramatic difference in the mode of action of N-acyl dopamines points on the possible existence of novel pathogenic mechanism of neurodegeneration induced by prolonged uncompensated production of these substances within neuronal tissue and should also be considered as a precaution in the future development of N-acyl dopamine-based therapeutic drugs.

SeqURE - a new copy-capture based method for sequencing of unknown Retroposition events.

BACKGROUND: Retroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (< 1%) subclonal cancer specific RE insertions is complicated. RESULTS: Here we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions. CONCLUSIONS: This study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.

Archaeal cyclopentane fragment in a surfactant's hydrophobic tail decreases the Krafft point.

Archaea are prokaryotic microorganisms famous for their ability to adapt to extreme environments, including low and high temperatures. Archaeal lipids often are macrocycles with two polar heads and a hydrophobic core that contains methyl groups and in-line cycles. Here we present the design of novel general-purpose surfactants that have inherited features of archaeal lipids. These are C12 and C14 carboxylic acids containing in-line cyclopentanes. The cyclopentanes disturb the chain packing, which results in remarkable expansion of the operational range of the surfactant into the low-temperature region. We report synthesis and properties of these novel archaea-like surfactants and details of their chain packing derived from thermodynamics model predictions, molecular dynamics simulations, and experimental data on CMC and Krafft points.

Quantum dot semiconductor disk laser at 1.3 µm.

We present a semiconductor disk laser (SDL) emitting at the wavelength of 1.3 µm. The active region of the SDL comprises InAs quantum dots (QDs) that are embedded into InGaAs quantum wells (QWs). An output power over 200 mW is obtained at 15°C, which represents the highest output power reported from QD-based SDLs in this wavelength range. The results demonstrate the feasibility of QD-based gain media for fabricating SDLs emitting at 1.3 µm.

Antioxidant and neuroprotective properties of N-arachidonoyldopamine.

N-Acyldopamines were recently described as putative endogenous substances in the rat brain. Among them, N-arachidonoyldopamine (AADA) was characterized as cannabinoid CB1 and vanilloid TRPV1 receptor ligand. The physiological significance of such compounds is yet poorly understood. In this study, we describe the novel properties of AADA as antioxidant and neuroprotectant. Antioxidant potential of AADA and its analogs were first tested in the galvinoxyl assay. It was found that N-acyldopamines are potent antioxidants and that the number of free hydroxyl groups in the phenolic moiety of dopamine is essential for the activity. AADA dose dependently (0.1-10 microM) protected cultured cerebellar granule neurons (CGN) in the model of oxidative stress induced by hydrogen peroxide. N-Oleoyldopamine, another endogenous substance, was much less potent in these conditions while the natural antioxidant alpha-tocopherol was inactive. In this test, AADA decreased the peroxide level in CGN preparations and its neuroprotection was independent of cannabinoid/vanilloid receptors blockade. AADA (10 microM) also protected CGN from death induced by K(+)/serum deprivation and glutamate exitotoxicity. These data indicate that AADA may act as endogenous antioxidant in different pathological conditions.

Different pharmacological profile of two closely related endocannabinoid ester analogs.

The pharmacological and neuroprotective properties of two ester analogs of the endocannabinoids, arachidonoylethyleneglycol (AA-EG) and alpha,alpha,-dimethyl arachidonoylethyleneglycol (DMA-EG), were investigated. We examined the interaction of both compounds with cannabinoid receptors (CB1 and CB2) and their efficacy in functional assays. In competition binding assays, AA-EG and DMA-EG had low potency to displace the CB1/CB2 agonist [3H]CP-55,940 in membrane preparations expressing rodent or human receptors. Binding data correlate with low efficacy of both compounds as regards to inhibition of adenylyl cyclase activity. It was also shown that DMA-EG resists hydrolysis by rat brain membranes while AA-EG undergo complete splitting under these conditions. In the cannabinoid tetrad, AA-EG induced hypomotility, analgesia, catalepsy and decreased rectal temperature indicating cannabimimetic activity. By contrast, DMA-EG was completely inactive in the same models. DMA-EG and AA-EG potently protected rat cortical neurons in culture against oxygen deprivation at nanomolar concentrations. In glutamate-induced damage, the compounds were less active protecting neurons at micromolar concentrations. The data obtained indicate that the ester endocannabinoid template can be used for the development of new compounds with potent biological activity lacking some of the undesirable behavioral side effects.

Hemisynthesis and preliminary evaluation of novel endocannabinoid analogues.

Three new endocannabinoid analogues in which amide moiety was replaced either by oxomethylene group or ester moiety with simultaneous substitution of both alpha-hydrogens with methyl groups were synthesized and their abilities to interact with CB1-receptor and FAAH were investigated.