Development and optimization of the VISAGE basic prototype tool for forensic age estimation

The VISAGE (VISible Attributes through GEnomics) consortium aims to develop, optimize and validate prototype tools to broaden the use of DNA intelligence methods in forensic routine laboratories. This includes age estimation based on the quantification of DNA methylation at specific CpG sites. Here, we present the VISAGE basic prototype tool for age estimation targeting 32 CpGs from five genes ELOVL

Iodine prophylaxis in children population on the Wielkopolska Region area from year 1992 to 2005

Wstęp: Celem niniejszych badań była ocena skuteczności profilaktyki jodowej w oparciu o obligatoryjny model jodowania soli. Materiał i metody: Łącznie badaniami objęto 1444 dzieci, zarówno z regionów miejskich, jak i wiejskich, o proporcjonalnym rozkładzie płci i wieku (8-12 lat) - 432 badanych w 1992 roku (przed wprowadzeniem jodowania soli), 558 badanych w 2000 roku i 454 badanych w 2005 roku (po wprowadzeniu jodowania soli w 1996 roku). Wyniki: W badaniach prowadzonych w 1992 roku wykazano wysoką częstość (40%) wola wśród dzieci. Obecnie odsetek ten zmniejszył się do 6% (p < 0,01). Równocześnie mediana stężeń jodu w moczu wzrosła z 44 &#956;g/l do 107 &#956;g/l (p < 0,01). Zaobserwowano niewielkie nasilenie zachorowań na autoimmunologiczne choroby tarczycy, zwłaszcza przewlekłe zapalenia.Wnioski: W badaniach wykazano wysoką skuteczność profilaktyki jodowej na terenie Wielkopolski, jednak nadal w większości pozostaje on rejonem o łagodnym niedoborze jodu.Introduction: The aim of the study was to evaluate of efficiency of iodine prophylaxis based on obligatory model of salt iodization. Material and methods: The study included 1444 children from the rural and urban area, with the proportional sex and age (8-12 years) distribution - 432 children in 1992 (before salt iodization), 558 children in 2000 and 454 children in 2005 (during salt iodization from 1996). Results: The prevalence of goiter detected in children population in 1992 was 40% (recount by current criteria), in 2005 was reduced to 6% (p < 0.01). Parallely, median of urinary iodine concentration increased from 44 mg/l in 1992 to 107 mg/l (p < 0.01) in 2005. The increase of incidence of autoimmunological thyroid diseases is observed, especially chronic thyroiditis.Conclusions: The study proves high efficiency of iodine prophylaxis in Wielkopolska Region, but it is still the area with mild iodine deficiency

Development of an epigenetic age predictor for costal cartilage with a simultaneous somatic tissue differentiation system

Age prediction from DNA has been a topic of interest in recent years due to the promising results obtained when using epigenetic markers. Since DNA methylation gradually changes across the individual's lifetime, prediction models have been developed accordingly for age estimation. The tissue-dependence for this biomarker usually necessitates the development of tissue-specific age prediction models, in this way, multiple models for age inference have been constructed for the most commonly encountered forensic tissues (blood, oral mucosa, semen). The analysis of skeletal remains has also been attempted and prediction models for bone have now been reported. Recently, the VISAGE Enhanced Tool was developed for the simultaneous DNA methylation analysis of 8 age-correlated loci using targeted high-throughput sequencing. It has been shown that this method is compatible with epigenetic age estimation models for blood, buccal cells, and bone. Since when dealing with decomposed cadavers or postmortem samples, cartilage samples are also an important biological source, an age prediction model for cartilage has been generated in the present study based on methylation data collected using the VISAGE Enhanced Tool. In this way, we have developed a forensic cartilage age prediction model using a training set composed of 109 samples (19–74 age range) based on DNA methylation levels from three CpGs in FHL2, TRIM59 and KLF14, using multivariate quantile regression which provides a mean absolute error (MAE) of ± 4.41 years. An independent testing set composed of 72 samples (19–75 age range) was also analyzed and provided an MAE of ± 4.26 years. In addition, we demonstrate that the 8 VISAGE markers, comprising EDARADD, TRIM59, ELOVL2, MIR29B2CHG, PDE4C, ASPA, FHL2 and KLF14, can be used as tissue prediction markers which provide reliable blood, buccal cells, bone, and cartilage differentiation using a developed multinomial logistic regression model. A training set composed of 392 samples (n = 87 blood, n = 86 buccal cells, n = 110 bone and n = 109 cartilage) was used for building the model (correct classifications: 98.72%, sensitivity: 0.988, specificity: 0.996) and validation was performed using a testing set composed of 192 samples (n = 38 blood, n = 36 buccal cells, n = 46 bone and n = 72 cartilage) showing similar predictive success to the training set (correct classifications: 97.4%, sensitivity: 0.968, specificity: 0.991). By developing both a new cartilage age model and a tissue differentiation model, our study significantly expands the use of the VISAGE Enhanced Tool while increasing the amount of DNA methylation-based information obtained from a single sample and a single forensic laboratory analysis. Both models have been placed in the open-access Snipper forensic classification website.</p

Divergent COVID-19 vaccine policies: policy mapping of ten European countries

Background: The COVID-19 pandemic highlighted the fragmented nature of governmental policy decisions in Europe. However, the extent to which COVID-19 vaccination policies differed between European countries remains unclear. Here, we mapped the COVID-19 vaccination policies that were in effect in January 2022 as well as booster regulations in April 2022 in Austria, Denmark, England, France, Germany, Ireland, Italy, the Netherlands, Poland, and Spain. Methods: National public health and health policy experts from these ten European nations developed and completed an electronic questionnaire. The questionnaire included a series of questions that addressed six critical components of vaccine implementation, including (1) authorization, (2) prioritization, (3) procurement and distribution, (4) data collection, (5) administration, and (6) mandate requirements. Results: Our findings revealed significant variations in COVID-19 vaccination policies across Europe. We observed critical differences in COVID-19 vaccine formulations authorized for use, as well as the specific groups that were provided with priority access. We also identified discrepancies in how vaccination-related data were recorded in each country and what vaccination requirements were implemented. Conclusion: Each of the ten European nations surveyed in this study reported different COVID-19 vaccination policies. These differences complicated efforts to provide a coordinated pandemic response. These findings might alert policymakers in Europe of the need to coordinate their efforts to avoid fostering divergent and socially disruptive policies

Development of the VISAGE enhanced tool and statistical models for epigenetic age estimation in blood, buccal cells and bones

DNA methylation is known as a biomarker for age with applications in forensics. Here we describe the VISAGE (VISible Attributes through GEnomics) Consortium’s enhanced tool for epigenetic age estimation in somatic tissues. The tool is based on eight DNA methylation markers (44 CpGs), bisulfite multiplex PCR followed by sequencing on the MiSeq FGx platform, and three statistical prediction models for blood, buccal cells and bones. The model for blood is based on six CpGs from ELOVL2, MIR29B2CHG, KLF14, FHL2, TRIM59 and PDE4C, and predicts age with a mean absolute error (MAE) of 3.2 years, while the model for buccal cells includes five CpGs from PDE4C, MIR29B2CHG, ELOVL2, KLF14 and EDARADD and predicts age with MAE of 3.7 years, and the model for bones has six CpGs from ELOVL2, KLF14, PDE4C and ASPA and predicts age with MAE of 3.4 years. The VISAGE enhanced tool for age estimation in somatic tissues enables reliable collection of DNA methylation data from small amounts of DNA using a sensitive multiplex MPS assay that provides accurate estimation of age in blood, buccal swabs, and bones using the statistical model tailored to each tissue

Production of the rat polyclonal anti-hTNF antibody

Czynnik martwicy nowotworów (TNF) jest cytokiną prozapalną indukowaną infekcją bakteryjną. Wpływa na ekspresję wielu genów o charakterze pro- jak i anty-apoptotycznym. W niniejszej pracy przedstawiono poszczególne etapy procedury mającej na celu otrzymanie szczurzych poliklonalnych przeciwciał anty-hTNF. W tym celu prowadzono hodowle komórek produkujących hTNF, oczyszczano białko wykorzystując chromatografię powinowactwa oraz przygotowanym antygenem immunizowano szczura. Wynikiem dwóch procesów oczyszczania było uzyskanie łącznie 82 µg hTNF. Uzyskany antygen użyto do immunizacji szczura. Proces immunizacji przebiegł pomyślnie i szczur został przygotowany do izolacji poliklonalnych przeciwciał anty-hTNF z surowicy.Tumor necrosis factor (TNF) is a pro-inflammatory cytokine-induced bacterial infection. It affects the expression of multiple genes of a pro- or anti-apoptotic character. This experiments present the various stages of procedure in order to obtain the rat polyclonal anti-hTNF antibody. For this purpose, cell cultures was carried out, purified hTNF using affinity chromatography and immunized a rat. Finally, purified 82 µg hTNF, as a result of the experiment. The obtained antigen was used for the rat immunization. The immunization process was successful and the rat was prepared to isolation polyclonal anti-hTNF antibody from serum

In vitro model of mucopolysaccharidosis IIIB

W niniejszej pracy magisterskiej przedstawiono szereg wyników, które mają na celu scharakteryzowanie mysiego modelu komórkowego choroby MPS IIIB. Badania prowadzono na komórkach unieśmiertelnionych pochodzących od myszy o genotypie Naglu-/- (KO, ang. knockout) oraz z myszy typu dzikiego (WT), jak i na komórkach pierwotnych izolowanych z myszy obu genotypów.Przedstawione badania dotyczą jednej z najważniejszych cech dotyczących lizosomalnych chorób spichrzeniowych, czyli gromadzeniu glikozaminoglikanów. Do sprawdzenia ilości GAG w komórkach KO i WT wykorzystano trzy barwniki, a badania z ich zastosowaniem pokazały wyższy poziom GAG w komórkach KO. Dzięki zastosowaniu kilku technik potwierdzono fibroblastyczny fenotyp komórek. Zbadano tempo proliferacji i dokonano porównania lizosomów między tymi dwoma typami komórek. Kolejną liczną grupą doświadczeń było sprawdzenie odpowiedzi prozapalnej oraz wrażliwości komórek na cytokiny. Ważnym celem tej grupy badań było stwierdzenie, że komórki po procesie unieśmiertelnienia zachowują cechy komórek pierwotnych. Przy wykorzystaniu techniki Western blot oraz zastosowaniu pomiaru aktywności enzymatycznej Naglu potwierdzono, że komórki KO nie produkują tego enzymu. Zaprezentowane wyniki potwierdzają, że komórki KO mogą być modelem MPS IIIB w badaniach in vitro.The aim of this master thesis was to characterize the murine in vitro model of MPS IIIB. The study was carried out using immortalized cells derived from WT and Naglu-/- mice, as well as, primary cells isolated from mice of both genotypes.One of the most important features of lysosomal storage diseases is GAG accumulation. In this study, three complexing compounds were used to test GAG levels. The experiments showed differences in GAGs between KO (Naglu-/-) and WT cell types. Several techniques have been used to confirm the fibroblastic phenotype of the cells. The results of the study present a comparison of proliferation rate and lysosomal staining between the two cell types. A number of experiments have been performed, testing pro-inflammatory response and cell sensitivity to cytokines. An important goal of this research was to find out if cells after the immortalization retain the characteristics of primary cells. Using Western blot techniques and measurement of Naglu enzyme activity, it was confirmed that the KO cells did not produce this enzyme. All these results confirm that the KO cells can be an in vitro model of MPS IIIB

The role of CYP19A1 and ESR2 gene polymorphisms in female androgenetic alopecia in the Polish population

INTRODUCTION: Androgenetic alopecia is the most common type of non-cicatricial alopecia both in male and female patients. The mechanism that leads to hair loss is similar in both sexes, but the underlying cause, and especially the role of genes and sex hormones in the pathogenesis of the disease in women has not fully been explained as of yet. So far, a few attempts have been made to assess selected SNPs for CYP19A1 and ESR2 genes, but their results are not unequivocal and fully reproducible. AIM: To investigate the association of 13 CYP19A1 and 11 ESR2 gene SNPs with female androgenetic alopecia (FAGA) in a population of Polish patients, including some already genotyped SNPs of possible importance for FAGA pathophysiology in other populations. MATERIAL AND METHODS: Twenty-four genetic polymorphisms were analysed for the ESR2 and CYP19A1 genes in 117 patients with FAGA and 128 healthy subjects treated at the Department of Dermatology in Krakow. RESULTS: In the studied Polish population, none of the selected SNPs, frequently detected in the Caucasian population and linked with the transformation pathway of sex hormones, showed a significant association with FAGA. CONCLUSIONS: Further studies into the genetic background of androgenetic alopecia are needed. Ethnic differences as well as the size of the studied population may be of great significance for the obtained results