Topical Issues of Theoretical and Clinical Medicine

Abstract book of International scientific and practical conference of students, postgraduates and young scientists, Sumy, October 17-19

ExoPRIME technology for exosomal miRNA analysis and identification of oxidative DNA damage-induced miRNA regulatory network in human astrocytes

The high lipid content of the brain, coupled with its heavy oxygen dependence and relatively weak antioxidant system, makes it highly susceptible to oxidative DNA damage that contributes to neurodegeneration. This study assesses and compares the neurotoxic effects of proton and photon radiation on mitochondrial function and DNA repair capabilities of human astrocytes. Human astrocytes received either proton (0.5 Gy and 3 Gy), photon (0.5 Gy and 3 Gy), or sham-radiation treatment. The mRNA expression level of the human base-excision repair protein, 8-deoxyguanosine DNA glycosylase 1 (hOGG1) was determined via RT-qPCR. Radiation-induced changes in mitochondrial mass and oxidative activity were assessed using fluorescent imaging with MitoTracker™ Green FM and MitoTracker™ Orange CM-H2TMRos dyes, respectively. A significant increase in mitochondrial mass and levels of reactive oxygen species was observed after radiation treatment. This was accompanied by a decreased OGG1 mRNA expression. These results are indicative of a radiation-induced dose-dependent decrease in mitochondrial function, an increase in senescence and astrogliosis, and impairment of the DNA repair capabilities in healthy glial cells. Photon irradiation was associated with a more significant disruption in mitochondrial function and base-excision repair mechanisms in vitro in comparison to the same dose of proton treatment. This study further identifies specific ROS-responsive miRNAs that modulate the expression and activity of the DNA repair proteins in human astrocytes, which could lead to the development of targeted therapeutic strategies for neurological diseases. Oxidative DNA damage was established after treatment of human astrocytes with 10 μM sodium dichromate for 16 hours. Comet assay analysis indicated a significant increase in oxidized guanine lesions. PCR analysis confirmed that sodium dichromate reduced the mRNA expression levels of hOGG1. Small RNAseq was performed on an Ion Torrent™ system and the differentially expressed miRNAs were identified using Partek Flow® software. The biologically significant miRNAs were selected using miRNet 2.0. Oxidative-stressinduced DNA damage was associated with a significant decrease in miRNA expression: 231 downregulated miRNAs and 2 upregulated miRNAs (p \u3c 0.05; \u3e 2-fold). In addition to identifying multiple miRNA-mRNA pairs involved in DNA repair processes, this study uncovered two novel miRNA-mRNA pairs interactions: miR-1248:OGG1 and miR-103a- OGG1. Inhibition of miR-1248 and miR-103a via the transfection of their inhibitors restored the increased expression levels of hOGG1. Therefore, targeting the identified microRNAs could ameliorate the nuclear DNA damage caused by exposure to mutagens. The miRNA candidates identified in this study could serve as potential biomarkers and therapeutics for oxidative stress in the brain to reduce the incidence and improve the treatment of cancer and neurodegenerative disorders. In a parallel but closely related study, we report a direct, one-step exosome sampling technology, for selective capture of CD63+ exosome subpopulations using an immune-affinity protocol. The ExoPRIME microprobe provides a Precise Rapid Inexpensive Mild (non-invasive) and Efficient (i.e. PRIME) alternative to the conventional polymer precipitation-based methods by enriching a comparatively more homogenous exosome population. The tool consists of an inert Serin™ stainless steelz microneedle (300 μm in diameter × 30 mm in height), pre-coated with a thin-film polyelectrolyte layer that serves as a substrate for covalent bonding of biotin. An anti-CD63 steptavidin-conjugated antibody that selectively binds to the corresponding tetraspanin embedded in the lipid bilayer of exosomes was immobilized to the outer surface of the probe. The feasibility of the ExoPRIME technology was validated using two types of biological samples: conditioned astrocyte medium (CAM) and astrocyte-derived exosome suspension (EXO). The study investigated the impact of the temperature (4°C and 22°C) and incubation duration (2h and 16h) on the capture efficiency of the ExoPRIME tool. A fluorescence-based enzymatic assay for exosome quantification was used to assess the probe’s exosomes capture efficiency and the reproducibility of the technology. The low level of non-specific binding initially observed in non-functionalized microneedles was drastically minimized by blocking the ExoPRIME probe with 0.1% BSA. The ExoPRIME microprobe captured exponentially more exosomes than the non-functionalized microneedle that indicates enrichment of CD63-expressing exosomes. A major advantage provided by the ExoPRIME technology over existing platforms is its applicability over a broad dynamic range of temperature and incubation parameters without compromising the purity and viability of exosomal cargoes. The loading capacity of the probe increased after incubation for 16 h at 40C in exosome suspension (24Å~106 exosomes per probe) while the efficiency decreased 10 folds after 2 h at 40C (24Å~105 exosomes per probe). The increase in temperature had an impact on the stability of the reagents that contributed to a 2-fold efficiency reduction after incubation in exosome suspension for 16 h at 220C (12Å~106 exosomes per probe). However, the 2-hour roomtemperature incubation (2 h at 220C) of the ExoPRIME probe yielded an increased capture efficiency (12Å~106 exosomes per probe) when compared to the 2 h at 4°C incubation (24Å~105 exosomes per probe). These results suggest that lower temperatures with extended incubation times constitute the most optimal parameters that ensure high probe loading capacity. Another advantage of the ExoPRIME microprobe is that it captures antigen-specific subpopulation of exosomes directly from conditioned astrocyte medium (CAM), eliminating the requirements for additional filtration and pre-concentration, and thereby cutting down costs and handling time. Besides the relatively reduced number of enriched exosomes, the CAM results are consistent with the trend obtained for EXO incubations, a phenomenon that could be attributed to the presence of various extracellular proteins and cellular debris, which could mask antibodies and compete physically with exosomes for binding. The capabilities to integrate different incubation times, temperatures, and biofluid type thus present exosome researchers with the flexibility to choose the combined parameters that best suit their purpose, the desired factor in clinical and laboratory applications. The developed tool requires very low amounts of antibody, permits the use and reuse of minimal sample volumes (≤ 200 μL), can be multiplexed in arrays to diagnostically profile multiple exosome classes and is amenable to integration into a lab-on-a-chip platform to achieve parallel, high-throughput isolation in a [semi]-automated workstation. Moreover, this platform could provide direct exosomal analysis of biological fluids since it can elegantly interface with existing picomolar-range nucleic acid assays to provide a clinical diagnostic tool at the point of care and facilitate fundamental studies in exosomes functions

Topical Issues of Theoretical and Clinical Medicine

Abstract book of International scientific and practical conference of students, postgraduates and young scientists, Sumy, October 17-19

Preparation, Physico-Chemical Properties and Biomedical Applications of Nanoparticles

Nowadays, the impact of nanotechnology on applications in medicine and biomedical sciences has broader societal and economic effects, enhancing awareness of the business, regulatory, and administrative aspects of medical applications. The selected papers included in the present Special Issue gives readers a critical, balanced and realistic evaluation of existing nanomedicine developments and future prospects, allowing practitioners to plan and make decisions.The topics of this book covers the use of nanoparticles and nanotechnology in medical applications including biomaterials for tissue regeneration, diagnosis and monitoring, surgery, prosthetics, drug delivery systems, nanocarriers, and wound dressing. I would like to express my gratitude to all contributors to this issue, who have given so much of their time and effort to help create this collection of high quality papers

Autoimune antibodies in thyroid diseases

Background: Irrespective of the recommendations to use the measurement of serum TSH as cornerstone of thyroid function testing, the laboratory diagnosis and monitoring of thyroid diseases are based on the thyroid panel including TSH, FT4, TT4, TT3, FT3, anti–TPO and anti-TG. Autoimmune thyroid diseases include Graves' disease, Hashimoto thyroiditis and these types of disorders are caused by immune system malfunction. In other words, instead of protecting the body's healthy tissues, malfunctioning immune cells actually attack them. Methods: Morning serum concentrations of anti-TPO and anti-TG were assessed in a prospective study in 50 subjects with Graves' disease, 50 subjects with Hashimoto thyroiditis and 40 healthy subjects as control 52 BALKAN JOURNAL OF CLINICAL LABORATORY - XXVI, 18, 1 th 26 BCLF 2018 Skopje l October 3 - 5, 2018 group. Serum concentration of anti–TPO and anti-TG were determined by chemiluminescence immunoassay using Immulite 2000 analyzer. Results: The following results were obtained: serum concentration of anti-TPO in the control group was 3.7 IU/mL ± 0.46, in Graves' disease 195 IU/mL ± 0.70 and in Hashimoto thyroiditis 238.5 IU/mL± 0.95. Serum concentration of anti-TG in Hashimoto thyroiditis was highest (333.3 IU/mL ± 0.55). Patients with Graves' disease and Hashimoto thyroiditis showed significantly higher concentrations of anti-TPO and anti-TG compared to healthy individuals (P<0.001). Conclusion: Serum concentrations of anti-TPO and anti-TG organ specific autoantibodies respectively are very precious parameters - markers for reliable diagnosis of autoimune thyroid diseases

Redox-Active Molecules as Therapeutic Agents

Oxidative stress and altered redox signaling have been described in a plethora of pathological conditions. Redox-active molecules can thus potentially be used to modulate the etiology/progression of such diseases. Recent advances in molecular biology and pharmacology have strengthened this area of research by providing novel mechanistic insights. This book compiles a collection of 13 articles, covering a range of topics from in vitro studies to clinical research, focused on the potential therapeutic effects of either natural or synthetic compounds, applicable to different redox-related diseases

Effects of Iodine Intake on Human Health

Iodine, a key component of thyroid hormones, is considered an essential micronutrient for proper health at all life stages. Indeed, an inadequate dietary intake of iodine is responsible for several functional and developmental abnormalities. The most serious consequences of iodine deficiency include hypothyroidism, early abortion, low birth weight, preterm delivery, neurocognitive impairment, and mental retardation. On the other hand, the consequences of mild-to-moderate iodine deficiency, such as goiter, are less well understood but represent an important priority for research and public health practice. Over the last several decades, many countries across the globe have introduced mandatory salt iodization programs, which have dramatically reduced the number of iodine-deficient countries. However, despite substantial progress worldwide, mild-to-moderate deficiency is still prevalent even in many developed countries. Thus, the ongoing monitoring of the population iodine status remains crucially important, and attention may need to be paid to vulnerable life stage groups

Family - Health - Disease

Preface: "The family is the basic social cell and a person’s closest environment; it plays the dominant non-medical role in shaping health as it is the primary source of knowledge about nutrition, life-style and disease prevention. The family may determine both healthy habits and anti-health behaviors; it influences the entire duration of a person’s life. It is important to emphasize the extremely significant role of the family in providing care, supporting treatment processes, and giving company to an ill family member."(...