Suggested mechanism of CCR5Δ32, CCR2-64I and SDF 1-3'A allele frequency change in Polish and Lithuanian gene pools from the perspective of passing time.

The study aimed to determine the frequency of the alleles associated with hereditary immune response in 16 historical populations and assess which evolutionary forces may have contributed to the observed frequency fluctuation. The analysed polymorphic sites are located in three genes - CCR5, CCR2 and SDF 1 (CXCL12). Protein products are involved in the innate immune response and are also involved in various types of infections, autoimmune diseases and tumours. The frequency of the alleles found in the DNA of the studied individuals was determined by the Sanger methodology and was compared with the data obtained for modern populations. To confirm the authenticity of the obtained results, mtDNA HVRI haplotypes of all the studied samples were obtained and compared with the genetic database of the laboratory personnel who came into contact with the studied material. Based on the variability of allele frequency, advanced biostatistical analysis was used to distinguish the effect of natural selection from genetic drift, i.e. the forces operating on the polymorphic sites studied. All procedures were performed according to the guidelines for working with ancient DNA to avoid contamination with modern DNA molecules. 681 samples from 39 archaeological sites in Poland and Lithuania dated to the 40th century BC and the 19th century were studied. The biostatistical analysis showed that the fluctuations in the frequency of CCR5Δ32 in the analysed time interval could be mainly the effect of genetic drift. Nevertheless, for CCR2-64I and SDF 1-3'A, the results confirm the suggestion of negative selection as the mechanism involved. Since all the polymorphic sites encode the elements of innate immune response that are indirectly associated with the process of an HPV infection and the development of cervical cancer, the human papillomavirus may be a good candidate for a selection coefficient affecting the frequency of CCR2-64I and SDF 1-3'A. However, for CCR5Δ32, selection was not detected despite its proven role in the molecular mechanism involved in the response to an HPV infection. The presented work seems to be the first in which the problem of the pattern of CCR5Δ32, CCR2-64I and SDF 1-3'A frequency fluctuations in a temporal perspective was discussed, proposing HPV as a factor influencing the occurrence of the CCR2 and SDF1 alleles.

Plasma Concentration of Cortisol Negatively Associates with Platelet Reactivity in Older Subjects.

The interaction of platelets with steroid hormones is poorly investigated. Age is one of the factors that increase the risk of pathological platelet reactivity and thrombosis. The aim of this study was to assess whether there were associations between platelet reactivity and plasma cortisol levels in volunteers aged 60-65 years. For this purpose, impedance aggregometry in whole blood measured after arachidonic acid, collagen, or ADP stimulation was used to estimate platelet reactivity and mass spectrometry was used to measure peripheral plasma cortisol concentration. Statistically significant negative correlations were observed between cortisol concentration and platelet reactivity in response to arachidonic acid and ADP, but not to collagen. The presented results suggest for the very first time that cortisol is a new endogenous modulator of platelet reactivity in the elderly population.

Canonical, Non-Canonical and Atypical Pathways of Nuclear Factor кb Activation in Preeclampsia.

Although higher nuclear factor κB (NFκB) expression and activity is observed in preeclamptic placentas, its mechanism of activation is unknown. This is the first study to investigate whether the canonical, non-canonical, or atypical NFκB activation pathways may be responsible for the higher activation of NFκB observed in preeclamptic placentas. The study included 268 cases (130 preeclamptic women and 138 controls). We studied the expression of the genes coding for NFκB activators (NIK, IKKα, IKKß, and CK2α) and inhibitors (IκBα and IκBß) using RT-PCR in real time. The RT-PCR results were verified on the protein level using ELISA and Western blot. To determine the efficiency of the pathways, the ratios of activator(s) to one of the inhibitors (IκBα or IκBß) were calculated for each studied pathway. The preeclamptic placentas demonstrated significantly lower IKKα and CK2α but higher IκBα and IκBß protein levels. In addition, the calculated activator(s) to inhibitor (IκBα or IκBß) ratios suggested that all studied pathways might be downregulated in preeclamptic placentas. Our results indicate that preeclamptic placentas may demonstrate mechanisms of NFκB activation other than the canonical, non-canonical, and atypical forms. In these mechanisms, inhibitors of NFκB may play a key role. These observations broaden the existing knowledge regarding the molecular background of preeclampsia development.

Differential expression of lung adenocarcinoma transcriptome with signature of tobacco exposure.

Smoking accounts for almost 80-90% of lung cancer cases, which is also the most frequent cause of cancer-related deaths in humans. With over 60 carcinogens in tobacco smoke, cells dividing at the time of carcinogen exposure are at particular risk of neoplasia. The present study aimed to investigate global gene expression differences in lung adenocarcinoma (LUAD) tumour samples of current smokers and non-smokers, in an attempt to elucidate biological mechanisms underlying divergent smoking effects. Current and non-smoker tumour samples were analysed using bioinformatics tools, examining differences in molecular drivers of cancer initiation and progression, as well as evaluating the effect of smoking and sex on epithelial mesenchymal transition (EMT). As a result, we identified 1150 differentially expressed genes showing visible differences in the expression profiles between the smoking subgroups. The genes were primarily involved in cell cycle, DNA replication, DNA repair, VEGF, GnRH, ErbB and T cell receptor signalling pathways. Our results show that smoking clearly affected E2F transcriptional activity and DNA repair pathways including mismatch repair, base excision repair and homologous recombination. We observed that sex could modify the effects of PLA2G2A and PRG4 in LUAD tumour samples, whereas sex and smoking status might possibly have a biological effect on the EMT-related genes: HEY2, OLFM1, SFRP1 and STRAP. We also identified potential epigenetic changes smoking solely might have on EMT-related genes, which may serve as potential diagnostic and prognostic biomarkers for LUAD patients.

Evaluate Cutpoints: Adaptable continuous data distribution system for determining survival in Kaplan-Meier estimator.

BACKGROUND AND OBJECTIVE: Growing evidence of transcriptional and metabolomic differentiation induced many studies which analyze such differentiation in context of outcome of disease progression, treatment or influence of many different factors affecting cellular and tissue metabolism. Particularly, cancer researchers are looking for new biomarkers that can serve as a diagnostic/prognostic factor and its further corresponding relationship regarding clinical effects. As a result of the increasing interest in use of dichotomization of continuous variables involving clinical or epidemiological data (gene expression, biomarkers, biochemical parameters, etc.) there is a large demand for cutoff point determination tools with simultaneous lack of software offering stratification of patients based on continuous and binary variables. Therefore, we developed "Evaluate Cutpoints" application offering wide set of statistical and graphical methods for cutpoint optimization enabling stratification of population into two or three groups. METHODS: Application is based on R language including algorithms of packages such as survival, survMisc, OptimalCutpoints, maxstat, Rolr, ggplot2, GGally and plotly offering Kaplan-Meier plots and ROC curves with cutoff point determination. RESULTS: All capabilities of Evaluate Cutpoints were illustrated with example analysis of estrogen, progesterone and human epidermal growth factor 2 receptors in breast cancer cohort. Through ROC curve the cutoff points were established for expression of ESR1, PGR and ERBB2 in correlation with their immunohistochemical status (cutoff: 1301.253, 243.35, 11,434.438, respectively; sensitivity: 94%, 85%, 64%, respectively; specificity: 93%, 86%, 91%, respectively). Through disease-free survival analysis we divided patients into two and three groups regarding expression of ESR1, PGR and ERBB2. Example algorithm cutp showed that lowered expression of ESR1 and ERBB2 was more favorable (HR = 2.07, p = 0.0412; HR = 2.79, p = 0.0777, respectively), whereas heightened PGR expression was correlated with better prognosis (HR = 0.192, p = 0.0115). CONCLUSIONS: This work presents application Evaluate Cutpoints that is freely available to download at http://wnbikp.umed.lodz.pl/Evaluate-Cutpoints/. Currently, many softwares are used to split continuous variables such as Cutoff Finder and X-Tile, which offer distinct algorithms. Unlike them, Evaluate Cutpoints allows not only dichotomization of populations into groups according to continuous variables and binary variables, but also stratification into three groups as well as manual selection of cutoff point thus preventing potential loss of information.

Platelet and Red Blood Cell Counts, as well as the Concentrations of Uric Acid, but Not Homocysteinaemia or Oxidative Stress, Contribute Mostly to Platelet Reactivity in Older Adults.

PURPOSE: The goal of this study was to estimate the hierarchical contribution of the most commonly recognized cardiovascular risk factors associated with atherogenesis to activation and reactivity of blood platelets in a group of men and women at ages 60-65. METHODS: Socioeconomic and anthropometric data were taken from questionnaires. Blood morphology and biochemistry were measured with standard diagnostic methods. Plasma serum homocysteine was measured by immunochemical method. Plasma concentrations of VCAM, ICAM, total antioxidant status, and total oxidant status were estimated with commercial ELISA kits. Markers of oxidative stress of plasma and platelet proteins (concentrations of protein free thiol and amino groups) and lipids (concentrations of lipid peroxides) and generation of superoxide anion by platelets were measured with colorimetric methods. Platelet reactivity was estimated by impedance aggregometry with arachidonate, collagen, and ADP as agonists. Expression of selectin-P and GPIIb/IIIa on blood platelets was tested by flow cytometry. RESULTS: Platelet aggregation associated significantly negatively with HGB and age and significantly positively with PLT, MPV, PCT, PDW, and P-LCR. When platelet reactivity ("cumulative platelet reactivity_aggregation") was analyzed in a cumulated manner, the negative association with serum concentration of uric acid (R s = -0.169, p = 0.003) was confirmed. Multivariate analysis revealed that amongst blood morphological parameters, platelet count, plateletcrit, and number of large platelets and uric acid are the most predictive variables for platelet reactivity. CONCLUSIONS: The most significant contributors to platelet reactivity in older subjects are platelet morphology, plasma uricaemia, and erythrocyte morphology.

Testosterone and dihydrotestosterone reduce platelet activation and reactivity in older men and women.

The cardiovascular effects of testosterone and dihydrotestosterone are generally attributed to their modulatory action on lipid and glucose metabolism. However, no ex vivo studies suggest that circulating androgen levels influence the activation and reactivity of blood platelets - one of the main components of the haemostasis system directly involved in atherosclerosis. The levels of testosterone, dihydrotestosterone and oestradiol in plasma from men and women aged from 60 to 65 years were measured by LC-MS; the aim was to identify any potential relationships between sex steroid levels and the markers of platelet activation (surface membrane expression of GPII/IIIa complex and P-selectin) and platelet reactivity in response to arachidonate, collagen or ADP, monitored with whole blood aggregometry and flow cytometry. The results of the ex vivo part of the study indicate that the concentrations of testosterone and its reduced form, dihydrotestosterone are significantly negatively associated with platelet activation and reactivity. These observations were confirmed in an in vitro model: testosterone and dihydrotestosterone significantly inhibited platelet aggregation triggered by arachidonate or collagen. Our findings indicate that testosterone and dihydrotestosterone are significant haemostatic steroids with inhibitory action on blood platelets in older people.

Reduced fecundity and cellular changes in Acheta domesticus after multigenerational exposure to graphene oxide nanoparticles in food.

Despite the fact that the demand for graphene and its derivatives in commercial applications is still growing, many aspects of its toxicity and biocompatibility are still poorly understood. Graphene oxide, which is released into the environment (air, soil and water) as so-called nanowaste or nanopollution, is able to penetrate living organisms. It is highly probable that, due to its specific nature, it can migrate along food chains thereby causing negative consequences. Our previous studies reported that short-term exposure to graphene oxide may increase the antioxidative defense parameters, level of DNA damage, which results in numerous degenerative changes in the gut and gonads. The presented research focuses on reproductive dysfunction and cellular changes in Acheta domesticus after exposure to GO nanoparticles in food (concentrations of 20 and 200 µg·g-1 of food) throughout their entire life cycle. The results showed that long-term exposure to GO caused a significant decrease in the reproductive capabilities of the animals. Moreover, the next generation of A. domesticus had a lower cell vitality compared to their parental generation. It is possible that graphene oxide can cause multigenerational harmful effects.

Searching for signals of recent natural selection in genes of the innate immune response - ancient DNA study.

The last decade has seen sharp progress in the field of human evolutionary genetics and a great amount of genetic evidence of natural selection has been provided so far. Since host-pathogen co-evolution is difficult to trace due to the polygenic nature of human susceptibility to microbial diseases, of particular interest is any signal of natural selection in response to the strong selective pressure exerted by pathogens. Analysis of ancient DNA allows for the direct insight into changes of a gene pool content over time and enables monitoring allele frequency fluctuations. Among pathogenic agents, mycobacteria are proved to have remained in an intimate, long-lasting relation with humans, reflected by the current high level of host resistance. Therefore, we aimed to investigate the prevalence of several polymorphisms within innate immune response genes related to susceptibility to mycobacterial diseases (in SLC11A1, MBL2, TLR2, P2RX7, IL10, TNFA) in time series data from North and East Poland (1st-18th century AD, n = 207). The comparison of allele frequencies over time revealed a predominant role of genetic drift in shaping past gene pool of small, probably isolated groups, which was explained by the high level of population differentiation and limited gene flow. However, the trajectory of frequency fluctuations of two SNPs suggested the possibility of their non-neutral evolution and the results of applied forward simulations further strengthened the hypothesis of natural selection acting on those loci. However, we observed an unusual excess of homozygosity in the profile of several SNPs, which pinpoints to the necessity of further research on temporally and spatially diverse samples to support our inference on non-stochastic evolution, ideally employing pathway-based approaches. Nevertheless, our study confirms that time series data could help to decipher very recent human adaptation to life-threatening pathogens and assisting demographic events.

Hunting for the LCT-13910*T allele between the Middle Neolithic and the Middle Ages suggests its absence in dairying LBK people entering the Kuyavia region in the 8th millennium BP.

Populations from two medieval sites in Central Poland, Stary Brzesc Kujawski-4 (SBK-4) and Gruczno, represented high level of lactase persistence (LP) as followed by the LCT-13910*T allele's presence (0.86 and 0.82, respectively). It was twice as high as in contemporaneous Cedynia (0.4) and Sródka (0.43), both located outside the region, higher than in modern inhabitants of Poland (0.51) and almost as high as in modern Swedish population (0.9). In an attempt to explain the observed differences its frequency changes in time were followed between the Middle Neolithic and the Late Middle Ages in successive dairying populations on a relatively small area (radius ∼60km) containing the two sites. The introduction of the T allele to Kuyavia 7.4 Ka BP by dairying LBK people is not likely, as suggested by the obtained data. It has not been found in any of Neolithic samples dated between 6.3 and 4.5 Ka BP. The identified frequency profile indicates that both the introduction and the beginning of selection could have taken place approx. 4 millennia after first LBK people arrived in the region, shifting the value of LP frequency from 0 to more than 0.8 during less than 130 generations. We hypothesize that the selection process of the T allele was rather rapid, starting just after its introduction into already milking populations and operated via high rates of fertility and mortality on children after weaning through life-threatening conditions, favoring lactose-tolerant individuals. Facing the lack of the T allele in people living on two great European Neolithization routes, the Danubian and Mediterranean ones, and based on its high frequency in northern Iberia, its presence in Scandinavia and estimated occurrence in Central Poland, we propose an alternative Northern Route of its spreading as very likely. None of the successfully identified nuclear alleles turned out to be deltaF508 CFTR.