Ability to determine the nontransferrin-bound iron and total iron in the human placenta using high-performance liquid chromatography method

Iron is one of the most important microelements in the human body. It is a component of haemoglobin, which transports oxygen to all cells in the organism. It is also used in the synthesis of myelin, neurotrans-mitters, and DNA and transfers electrons in biochemical reactions. Iron is also responsible for regular development of the foetus’ central nervous system. Furthermore, as a result of Fenton reactions, iron leads to formation of toxic free radicals. The existence of non-transferrin-bound iron (NTBI) and its part desfer-rioxamine-chelatable iron (DCI) can be used to assess this element in the body. The placenta is an organ transition that is formed during pregnancy in the female organism. It has a dense web of blood vessels in which dynamic exchange of blood between mother and foetus takes place. As a result, a fraction of NTBI may be present in the placenta. The main goal of this work was to develop a method for determining total iron and desferrioxamine-chelatable iron in solid tissues - the human placenta.Iron is one of the most important microelements in the human body. It is a component of hemoglobin which transports oxygen to all cells in the organism. It is also used in a synthesis of myelin, neurotransmitters and DNA, and transfers electrons in the biochemical reactions. Iron is also responsible for regular development of a fetus central nervous system. Furthermore, as a result of Fenton reactions, iron leads to formation of toxic free radicals. Existence of non-transferrin-bound iron (NTBI) and its part - desferrioxamine-chelatable iron (DCI) can be used for assess this element in a body. Placenta is an organ transition which is formed during pregnancy in a female organism. It has a dense web of blood vessels where dynamic exchange of blood between mother and fetus takes place. As a result, fraction of NTBI may be present in the placenta. The main goal of this work was to develop a method for determination of total iron and desferrioxamine-chelatable iron in solid tissues - a human placent

Biobank Łódź® – population based biobank at the University of Łódź, Poland

Biobank Laboratory of the University of Łódź is a unit in the organizational structure of the De- partment of Molecular Biophysics at the Faculty of Biology and Environmental Protection. It was established in 2014 as one of the results of the TESTOPLEK project. One of the main goals of the unit is to collect and share biological material of human origin and related clinical and survey data. Moreover, Biobank Laboratory conducts work in the field of genetics and molecular biology on human biological material. Biobank Laboratory gathers over 40.000 samples such as DNA, FFPE, saliva, together with their data. Data about its material is available for researchers in directories e.g. BBMRI-ERIC Directory 4.0. Since 2014, the unit belongs to the national Consortium BBMRI.pl, and since 2017 it executes a project entitled Research Infrastructure for Biobanks and Biomolecular Resources BBMRI-ERIC, co-creating the Polish Network of Biobanks. Biobank Laboratory is focused on coopera- tion with domestic and foreign scientific institutions and medical units, as well as entities from the local, business and public sector

Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses

Institutional Review Board Statement: The experimental procedures were approved and conducted according to guidelines of the appropriate Polish Local Ethics Commission for Experiments on Animals No. 9 in Lodz (Agreement 9/ŁB87/2018). Acknowledgments: We thank Jeremy Rock and Sarah Fortune for providing us with the pLJR965 vector and detailed instructions for the generation of Cas9-regulated strains in M. tuberculosis. The authors thank the mass spectrometry service at the Institute of Biochemistry and Biophysics PAS in Warsaw for MS analysis. The MS analysis equipment used for the analysis was sponsored in part by the Centre for Preclinical Research and Technology (CePT), a project cosponsored by the European Regional Development Fund and Innovative Economy, the National Cohesion Strategy of Poland.Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level.A.B. was supported by grant “OPUS” from the National Science Centre, Poland, UMO2015/19/B/NZ6/02978. P.P. was supported by grant “OPUS” from the National Science Centre, Poland, UMO-2019/33/B/NZ1/02770

The genetic basis of endometriosis and comorbidity with other pain and inflammatory conditions

Endometriosis is a common condition associated with debilitating pelvic pain and infertility. A genome-wide association study meta-analysis, including 60,674 cases and 701,926 controls of European and East Asian descent, identified 42 genome-wide significant loci comprising 49 distinct association signals. Effect sizes were largest for stage 3/4 disease, driven by ovarian endometriosis. Identified signals explained up to 5.01% of disease variance and regulated expression or methylation of genes in endometrium and blood, many of which were associated with pain perception/maintenance (SRP14/BMF, GDAP1, MLLT10, BSN and NGF). We observed significant genetic correlations between endometriosis and 11 pain conditions, including migraine, back and multisite chronic pain (MCP), as well as inflammatory conditions, including asthma and osteoarthritis. Multitrait genetic analyses identified substantial sharing of variants associated with endometriosis and MCP/migraine. Targeted investigations of genetically regulated mechanisms shared between endometriosis and other pain conditions are needed to aid the development of new treatments and facilitate early symptomatic intervention

The genetic basis of endometriosis and comorbidity with other pain and inflammatory conditions

Endometriosis is a common condition associated with debilitating pelvic pain and infertility. A genome-wide association study meta-analysis, including 60,674 cases and 701,926 controls of European and East Asian descent, identified 42 genome-wide significant loci comprising 49 distinct association signals. Effect sizes were largest for stage 3/4 disease, driven by ovarian endometriosis. Identified signals explained up to 5.01% of disease variance and regulated expression or methylation of genes in endometrium and blood, many of which were associated with pain perception/maintenance (SRP14/BMF, GDAP1, MLLT10, BSN and NGF). We observed significant genetic correlations between endometriosis and 11 pain conditions, including migraine, back and multisite chronic pain (MCP), as well as inflammatory conditions, including asthma and osteoarthritis. Multitrait genetic analyses identified substantial sharing of variants associated with endometriosis and MCP/migraine. Targeted investigations of genetically regulated mechanisms shared between endometriosis and other pain conditions are needed to aid the development of new treatments and facilitate early symptomatic intervention

Variation of genes encoding proteins from the ABC superfamily (ATP-binding cassette) in the Polish population in comparison to other populations

Białka ABC, zawierające kasetę wiążącą ATP (ATP-Binding Cassette), stanowią największą nadrodzinę transporterów błonowych. U ludzi kodowane są przez 48 genów, które zostały podzielone na siedem rodzin (ABCA – ABCG). Białka te biorą udział w wielu podstawowych procesach komórkowych i regulują homeostazę endogennych i egzogennych związków, w tym ksenobiotyków. Niektóre mutacje w genach ABC prowadzą zatem do wielu poważnych chorób wrodzonych. Wiele transporterów ABC ma także szczególne znaczenie w procesie usuwania leków, a ich aktywność jest jedną z głównych przyczyn powstania niekorzystnego zjawiska oporności wielolekowej, najczęściej obserwowanej w leczeniu nowotworów. Niektóre warianty w genach ABC istotnie więc wpływają na różnice w odpowiedzi na farmakoterapię. Co więcej, częstość występowania wielu z nich istotnie różni się pomiędzy populacjami o różnym pochodzeniu etnicznym. Głównym celem pracy była zatem analiza zmienności tych genów w populacji polskiej i porównanie jej z populacjami, zarówno o odrębnym pochodzeniu etnicznym, jak i również tylko z tymi o pochodzeniu europejskim. Wyniki zmienności genów ABCC1 i ABCG2 w populacji polskiej uzyskano z wykorzystaniem metody topnienia produktów PCR w wysokiej rozdzielczości (HRM), a dla całej nadrodziny wykorzystano dane uzyskane z genotypowania mikromacierzami Illumina. Zdecydowaną większość wykrytych wariantów w genach ABC stanowiły warianty rzadkie, z częstością występowania rzadszego allelu <0,05. Stwierdzono, że zmienność genów ABC w populacji polskiej istotnie różni się od populacji o innym pochodzeniu etnicznym, z kolei nie różni się istotnie od innych populacji referencyjnych o pochodzeniu europejskim. Prace badawcze w niniejszej rozprawie są pierwszymi, które w tak kompleksowy sposób podjęły temat zmienności genów kodujących całą nadrodzinę białek ABC w polskiej populacji.1. „TESTOPLEK – Rola transporterów oporności wielolekowej w farmakokinetyce i toksykologii – testy in vitro w praktyce farmaceutycznej i klinicznej”. Projekt POIG.01.01.02-10-005/08 współfinansowany przez Unię Europejską z Europejskiego Funduszu Rozwoju Regionalnego. 2. „Cyfrowe udostępnianie zasobów biomolekularnych i opisowych Biobanku i Katedry Antropologii Uniwersytetu Łódzkiego – charakterystyka populacji zamieszkujących tereny dzisiejszej Polski na przestrzeni dziejów. Platforma informacyjna e-Czlowiek.pl”. Projekt POPC.02.03.01-00-0012/17 współfinansowany przez Unię Europejską w ramach Programu Operacyjnego Polska Cyfrowa. 3. Projekt „Utworzenie sieci biobanków w Polsce w obrębie Infrastruktury Badawczej Biobanków i Zasobów Biomolekularnych BBMRI-ERIC”, na podstawie Decyzji Ministra Nauki i Szkolnictwa Wyższego nr DIR/WK/2017/01

Systematic Study of Solid-State Fluorescence and Molecular Packing of Methoxy-<i>trans</i>-Stilbene Derivatives, Exploration of Weak Intermolecular Interactions Based on Hirshfeld Surface Analysis

In recent years, fluorescent compounds that emit efficiently in the solid state have become particularly interesting, especially those that are easily prepared and inexpensive. Hence, exploring the photophysical properties of stilbene derivatives, supported by a detailed analysis of molecular packing obtained from single-crystal X-ray diffraction data, is a relevant area of research. A complete understanding of the interactions to determine the molecular packing in the crystal lattice and their effect on the material’s physicochemical properties is essential to tune various properties effectively. In the present study, we examined a series of methoxy-trans-stilbene analogs with substitution pattern-dependent fluorescence lifetimes between 0.82 and 3.46 ns and a moderate-to-high fluorescence quantum yield of 0.07–0.69. The relationships between the solid-state fluorescence properties and the structure of studied compounds based on X-ray analysis were investigated. As a result, the QSPR model was developed using PLSR (Partial Least Squares Regression). Decomposition of the Hirshfeld surfaces (calculated based on the arrangement of molecules in the crystal lattice) revealed the various types of weak intermolecular interactions that occurred in the crystal lattice. The obtained data, in combination with global reactivity descriptors calculated using HOMO and LUMO energy values, were used as explanatory variables. The developed model was characterized by good validation metrics (RMSECAL = 0.017, RMSECV = 0.029, R2CAL = 0.989, and R2CV = 0.968) and indicated that the solid-state fluorescence quantum yield of methoxy-trans-stilbene derivatives was mainly dependent on weak intermolecular C…C contacts corresponding to π-π stacking and C…O/O…C interactions. To a lesser extent and inversely proportional, the fluorescence quantum yield was affected by the interactions of the type O…H/H…O and H…H and the electrophilicity of the molecule

High Resolution Melting (HRM) for High-Throughput Genotyping—Limitations and Caveats in Practical Case Studies

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup

Functional Analysis of the rs774872314, rs116171003, rs200231898 and rs201107751 Polymorphisms in the Human RORγT Gene Promoter Region

RAR-related orphan receptor gamma RORγT, a tissue-specific isoform of the RORC gene, plays a critical role in the development of naive CD4+ cells into fully differentiated Th17 lymphocytes. Th17 lymphocytes are part of the host defense against numerous pathogens and are also involved in the pathogenesis of inflammatory diseases, including autoimmune disorders. In this study, we functionally examined four naturally occurring polymorphisms located within one of the previously identified GC-boxes in the promoter region of the gene. The single nucleotide polymorphisms (SNPs) rs774872314, rs116171003 and rs201107751 negatively influenced the activity of the RORγT promoter in a gene reporter system and eliminated or reduced Sp1 and Sp2 transcription factor binding, as evidenced by the electrophoretic mobility shift assay (EMSA) technique. Furthermore, we investigated the frequency of these SNPs in the Polish population and observed the presence of rs116171003 at a frequency of 3.42%. Thus, our results suggest that polymorphisms within the RORγT promoter occurring at significant rates in populations affect promoter activity. This might have phenotypic effects in immune systems, which is potentially significant for implicating pathogenetic mechanisms under certain pathological conditions, such as autoimmune diseases and/or primary immunodeficiencies (e.g., immunoglobulin E (IgE) syndrome)