The hero of American and Polish nations: a molecular look at Thaddeus Kosciuszko's cause of death suggests a contribution of endocarditis caused by Cutibacterium acnes infection.

The remains of the heart tissue of Thaddeus Kosciuszko have been investigated as the possible cause of disease and death of the hero of Polish and American nations. Three specimens, DNA isolated from scrappings of wax surface, from the surface of a wooden plate, and from the linen cloth that have had contact with the object were subjected to nanosequencing. From the first two, among all reads identified, only one classified as Propionibacterium acnes (synonymous current name Cutibacterium acnes), had a purported clinical significance. The observed identity between the P. acnes sequences and reference was 89-90% consistent with the hypothesis that the identified reads represent the ancient P. acnes DNA (aDNA), which underwent fragmentation and sequence changes caused by its long-time presence in the environmental conditions conducive to degradation. We present a reasonable and entirely new hypothesis that the analyzed samples could reflect the presence of the bacteria in the original Kosciuszko's heart tissue and that the process of C. acnes infection was progressing inside the organ (endocarditis), not on its surface (pericarditis) leading to rapid deterioration of health and eventually death. We again point out that normal skin and mucosal membranes commensal, a causative agent of common skin acne, may be associated with various severe organ infections posing a threat to health and life.

DNA methylation in adipocytes from visceral and subcutaneous adipose tissue influences insulin-signaling gene expression in obese individuals.

OBJECTIVE: Both obesity and insulin resistance are characterized by severe long-term changes in the expression of many genes of importance in the regulation of metabolism. Because these changes occur throughout life, as a result of external factors, the disorders of gene expression could be epigenetically regulated. MATERIALS/METHODS: We analyzed the relationship between obesity and insulin resistance in enrolled patients by means of evaluation of the expression rate of numerous genes involved in the regulation of adipocyte metabolism and energy homeostasis in subcutaneous and visceral adipose tissue depots. We also investigated global and site-specific DNA methylation as one of the main regulators of gene expression. Visceral and subcutaneous adipose tissue biopsies were collected from 45 patients during abdominal surgery in an age range of 40-60 years. RESULTS: We demonstrated hypermethylation of PPARG, INSR, SLC2A4, and ADIPOQ promoters in obese patients with insulin resistance. Moreover, the methylation rate showed a negative correlation with the expression of the investigated genes. More, we showed a correlation between the expression of PPARG and the expression of numerous genes important for proper insulin action. Given the impact of PPARγ on the regulation of the cell insulin sensitivity through modulation of insulin pathway genes expression, hypermethylation in the PPARG promoter region may constitute one of the epigenetic pathways in the development of insulin resistance in obesity. CONCLUSIONS: Our research shows that epigenetic regulation through excessive methylation may constitute a link between obesity and subsequent insulin resistance.

Identification and characterization of novel rapidly mutating Y-chromosomal short tandem repeat markers.

Short tandem repeat polymorphisms on the male-specific part of the human Y-chromosome (Y-STRs) are valuable tools in many areas of human genetics. Although their paternal inheritance and moderate mutation rate (~10-3 mutations per marker per meiosis) allow detecting paternal relationships, they typically fail to separate male relatives. Previously, we identified 13 Y-STR markers with untypically high mutation rates (>10-2 ), termed rapidly mutating (RM) Y-STRs, and showed that they improved male relative differentiation over standard Y-STRs. By applying a newly developed in silico search approach to the Y-chromosome reference sequence, we identified 27 novel RM Y-STR candidates. Genotyping them in 1,616 DNA-confirmed father-son pairs for mutation rate estimation empirically highlighted 12 novel RM Y-STRs. Their capacity to differentiate males related by 1, 2, and 3 meioses was 27%, 47%, and 61%, respectively, while for all 25 currently known RM Y-STRs, it was 44%, 69%, and 83%. Of the 647 Y-STR mutations observed in total, almost all were single repeat changes, repeat gains, and losses were well balanced; allele length and fathers' age were positively correlated with mutation rate. We expect these new RM Y-STRs, together with the previously known ones, to significantly improving male relative differentiation in future human genetic applications.

Oleic acid influences the adipogenesis of 3T3-L1 cells via DNA Methylation and may predispose to obesity and obesity-related disorders.

BACKGROUND: Adipogenesis is the process of adipocytes formation from unspecialized progenitor cells called mesenchymal stromal cells. Numerous mechanisms including epigenetic regulation modulate the correct progress of this process. Dietary exposures occurring over a specific period of time might cause long-lasting and even permanent changes in gene expression regulated by epigenetic mechanisms. For that reason, we investigated the adipogenesis of 3 T3-L1 cells with the excess of saturated and monounsaturated fatty acids and their influence on global and site-specific DNA methylation in these cells. MATERIALS AND METHODS: 3T3-L1 cells were cultured in vitro to obtain 100% of confluence, then the adipogenesis was induced by a differentiation cocktail with the addition of the excess of 0.25 mM and 0.5 mM of palmitic (16:0), stearic (18:0) and oleic (18:1n-9) acids. DNA and RNA were extracted at five-time points to assess the adipogenesis process. The phenotype of mature adipocytes (insulin sensitivity, adipokines secretion, fat content) was estimated in fully mature adipocytes. DNA methylation was investigated both during adipogenesis and in mature adipocytes. RESULTS: Oleic acids stimulated expression of C/ebpα and Pparγ, which was correlated with lower methylation levels at promoters sites. Furthermore, cells cultured with an excess of oleic acid were characterized by higher lipid accumulation rate, higher leptin, and lower adiponectin secretion. Moreover, in all experimental cells, insulin signaling and glucose utilization were impaired. CONCLUSION: Oleic acid affected the methylation of Pparγ and C/ebpα promoters, what correlated with higher expression. Furthermore, examined free fatty acids influenced the phenotype of mature adipocytes, especially insulin signaling pathway and adipokine secretion.

Specialty gauge ammunition.

This paper, as the third of the series, is devoted to gauge ammunition, which may be a problem in ballistic opinions. The paper reviews existing cartridges of the type used in smoothbore weapons (bullet cartridges, non-penetrating, compressed powder, flash-bang, signal, adapter-type, and training cartridges). The paper also presents homemade special cases of gauge ammunition, as well as ammunition used for "peculiar" purposes. The second part of the paper discusses other uses of specialty gauge ammunition, specifically its use as mortar ammunition, propelling charges, pyrotechnic tools, and to start combustion engines.

Preliminary analysis of polymorphism of STR markers in the population of domestic cats (Felis catus) in Lower Silesia.

AIM OF THE STUDY: Genetic tests play a crucial role in the crime investigation process and often provide the strongest evidence for case resolution. Although the majority of genetic analyses in the field of criminalistics focus on the human DNA, genetic identification of animals is becoming an increasingly common procedure. Domestic animals, which live around people, may be silent witnesses and even victims of criminal activity. Their typically limited value as evidence in such cases could radically change thanks to the possibility of using animal biological material present at the crime scene. In addition to forensic medicine, genetic identification methods of this type may also become a valuable tool in many other areas of life. Recently, there has been an increase in public interest in verifying the pedigree of animals, investigating poaching and illegal shooting of animals, e.g. protected wildcats and lynx, as well as illegal trade in animals. The main aims of the studies reported in this paper were to assess the degree of polymorphism of the analyzed STR markers in feline genetic material, and to perform a preliminary evaluation of their suitability for developing an original feline genetic identification test. MATERIAL AND METHODS: The studies involved an analysis of genetic material samples obtained from a population consisting of 123 unrelated cats representing various domestic cat breeds, living in the Lower Silesia region. The material collected from individual cats in the form of blood drops or buccal swabs was subjected to an analysis of five STR markers forming a single multiplex assay (FCA742, FCA744, F124, FCA732, FCA749). RESULTS: The results obtained for each marker separately were analyzed statistically and, using the 2 test, the concordance of the study population with the Hardy-Weinberg principle was evaluated. CONCLUSIONS: The findings demonstrate a significant potential of the analyzed markers for the development of genetic identification tests.

Smoothbore hunting ammunition - a historical overview.

This article deals with traditional hunting amunitions in paper cases, intended for smooth-bore shotgun. The paper describes the development of gunshot cartridges in historical terms, with particular emphasis on the origins and development of systems, initiating the combustion of subsequent generations of propelling cargo. The work gives an author's look into the uprising process of an integrating gunshot cartridge. The article contains a number of information about historical cartridges, currently not to be found, their dimensions, markings and ignition systems. The described cartridges are often examples of rare and long-lasting constructional solutions, that have been made to create a modern cartridge in weight scale calibers. The presented work includes a number of numerical data as well as ballistic, obsolete and archaic today's cartridges, with dimensions and ballistic properties. Some of the theses and data in this paper contained, according to the author's knowledge, are for the first time presented in the literature, and others can be found, but it is very tedious because of their dispersion in the literature or the availability of hard to find and unstable internet sources.

15th Anniversary of the Molecular Techniques Unit at the Department of Forensic Medicine at Wroclaw Medical University.

Molecular Techniques Unit at the Department of Forensic Medicine, Wroclaw Medical University has been operating since December 2003. Soon it will be 15 years since its establishment. This anniversary become an inspiration to write down the story of this institution whose origins illustrate the immense changes that have taken place in forensic genetics. The aim of our work was also to consolidate the professional achievements of Professor Tadeusz Dobosz, chief of the Unit, one of the pioneers of introducing DNA testing technology into Polish forensic medicine. The most important achievements of the Unit include participation in two EU research projects, the development of a non-destructive method of extraction of genetic material, research in field of gene therapy and certification of the Laboratory of the Molecular Techniques Unit by the Polish Accreditation Center (PCA) confirming compliance with the requirements of the PN-EN ISO/IEC 17025:2005 standard.

Hunting shot - evolution of manufacturing technology

Hunting shot are 1.2-10 mm diameter balls, usually made of lead alloys, forming a cluster projectile used in smoothbore hunting shotguns. Shot may also be used in pistol and revolver ammunition, in which it can constitute structural element of the projectile. Shot pellets may also be made of other materials and have other shapes. The aim of this paper is to aggregate information on the topic available from a number of different sources. It is hoped that such information will be useful for forensic ballistics experts. Historical development of pellets and their manufacturing technology from the 15th century is presented.

First successful DNA isolation and profiling from bone using an approach that is non-destructive toward bone surface

Commonly used destructive DNA isolation techniques consist of pulverization which leads to the complete destruction of smaller bones or irreversible damage to larger bones through the cutting of extensive fragments. The procedure is totally unacceptable when handling bones which are valuable for anthropological or religious reasons, as museum exhibits or due to emotional factors. Since the Department's team has already resolved this problem in the case of human teeth (A. Pilecka), efforts have been undertaken to develop a similar non-destructive technique for the isolation of DNA from human bones. To the best knowledge of the authors of the report, the study has yielded the world's first STR profile derived from DNA isolated from human bones by a technique that is non-destructive toward the bone surface.

The role of FGF2 in migration and tubulogenesis of endothelial progenitor cells in relation to pro-angiogenic growth factor production.

In recent years, special attention has been paid to finding new pro-angiogenic factors which could be used in gene therapy of vascular diseases such as critical limb ischaemia (CLI). Angiogenesis, the formation of new blood vessels, is a complex process dependent on different cytokines, matrix proteins, growth factors and other pro- or anti-angiogenic stimuli. Numerous lines of evidence suggest that key mediators of angiogenesis, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) together with fibroblast growth factor2 (FGF2) are involved in regulation of the normal and pathological process of angiogenesis. However, less information is available on the complex interactions between these and other angiogenic factors. The aim of this study was to characterise the effect of fibroblast growth factor2 on biological properties of human endothelial progenitor cells with respect to the expression level of other regulatory cytokines. Ectopic expression of FGF2 in EP cells stimulates their pro-angiogenic behaviour, leading to increased proliferation, migration and tube formation abilities. Moreover, we show that the expression profile of VEGF and other pro-angiogenic cytokines, such as HGF, MCP2, and interleukins, is affected differently by FGF2 in EPC. In conclusion, we provide evidence that FGF2 directly affects not only the biological properties of EP cells but also the expression pattern and secretion of numerous chemocytokines. Our results suggest that FGF2 could be applied in therapeutic approaches for CLI and other ischaemic diseases of the vascular system in vivo.

Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

Mutability of Y-chromosomal microsatellites: rates, characteristics, molecular bases, and forensic implications.

Nonrecombining Y-chromosomal microsatellites (Y-STRs) are widely used to infer population histories, discover genealogical relationships, and identify males for criminal justice purposes. Although a key requirement for their application is reliable mutability knowledge, empirical data are only available for a small number of Y-STRs thus far. To rectify this, we analyzed a large number of 186 Y-STR markers in nearly 2000 DNA-confirmed father-son pairs, covering an overall number of 352,999 meiotic transfers. Following confirmation by DNA sequence analysis, the retrieved mutation data were modeled via a Bayesian approach, resulting in mutation rates from 3.78 × 10(-4) (95% credible interval [CI], 1.38 × 10(-5) - 2.02 × 10(-3)) to 7.44 × 10(-2) (95% CI, 6.51 × 10(-2) - 9.09 × 10(-2)) per marker per generation. With the 924 mutations at 120 Y-STR markers, a nonsignificant excess of repeat losses versus gains (1.16:1), as well as a strong and significant excess of single-repeat versus multirepeat changes (25.23:1), was observed. Although the total repeat number influenced Y-STR locus mutability most strongly, repeat complexity, the length in base pairs of the repeated motif, and the father's age also contributed to Y-STR mutability. To exemplify how to practically utilize this knowledge, we analyzed the 13 most mutable Y-STRs in an independent sample set and empirically proved their suitability for distinguishing close and distantly related males. This finding is expected to revolutionize Y-chromosomal applications in forensic biology, from previous male lineage differentiation toward future male individual identification.

Role of PCR in Helicobacter pylori diagnostics and research--new approaches for study of coccoid and spiral forms of the bacteria.

Helicobacter pylori since Marshall and Warren's discovery has been an object of interest of gastroenterologists and many researchers of other specialties. What needs to be highlighted is also the growing interest of dentists in the role of oral residue of H. pylori in oral pathologies such as burning mouth syndrome, periodontitis and gingivitis. With the development of medical techniques more studies using highly specific diagnostic methods are performed in order to determine the transmission pattern of bacterial infection. Suggested faecal-oral and oral-oral routes of bacterial transmission raised interest in molecular biology methods as tools for the study of these environments. Additionally, co-existence of helical and coccoidal forms of H. pylori in the mentioned niches raised the question whether the latter is potentially pathogenic. This is why molecular biology is now giving a great opportunity to explore parts of the human body that could not have been diagnosed before using only gold standard diagnostic methods. Molecular techniques have shown their usefulness in examining the potential virulence of coccoid forms of bacterium. This review was created also to summarize the knowledge about molecular methods, especially different PCR techniques, as diagnostic tools that can help medical teams during regular diagnosis of gastritis. 

Transcriptional expression of selected genes associated with excretion of carboxylic acids from aci mutants of Saccharomyces cerevisiae.

INTRODUCTION: Saccharomyces cerevisiae is an excellent model organism for studies of transcriptional regulation of metabolic processes in other eukaryotic cells including human cells. Cellular acid-base balance can be disturbed in pathologic situations such as renal acidosis or cancer. The extracellular pH of malignant solid tumors is acidic in the range of 6.5-6.9. EG07 and EG37 aci mutants of Saccharomyces cerevisiae excessively excrete carboxylic acids to glucose-containing media or distilled water. The excreted acids are Krebs and/or glyoxylate cycle intermediates. The genes restoring the wild-type phenotype have function that does not easily explain theAci+ phenotype. MATERIAL/METHODS: In this study, using real-time PCR we measured relative mRNA expression, in the mutants compared to the wild-type strain, of selected genes associated with both carboxylic acid cycles and two cell transporters, Pma1 and Pdr12, of organic acids. RESULTS: Unexpectedly, we found that the relative expression of the selected Krebs cycle and glyoxylate cycle genes did not change significantly. However, the expression of the two transporter genes was strongly elevated in EG37 and moderately increased in EG07. CONCLUSION: These results indicate that the induction of the two cell transporterg enes plays an important role in acid excretion by the aci mutants.

DNA Content of Various Fluids and Tissues of the Human Body.

Due to the scarcity of literature data on the DNA content of different human tissues, this study aimed to isolate DNA from different tissues and fluids of the human body together with the determination of its content in the samples studied. Material was collected and tests were performed between 1990 and 2010, during autopsies performed for prosecutor's offices in the Department of Forensic Medicine. Goiter and thyroid cancer tissues were obtained from the Department of General Surgery, Gastroenterology and Endocrinology of Wroclaw Medical University. Isolated samples were measured spectrophotometrically, yielding an R 260/280 nm between 1.5 and 1.6. In some cases (when a sufficiently pure preparation could not be obtained), isolation was continued using the silica-based commercial QIAquick PCR Purification Kit (Qiagen). If the sampling tissues showed signs of decomposition such as bad odour or colour, the results were calibrated by Real-Time PCR, using the Quantifiler DNA assay (Thermo Fisher Scientific, Applied Biosystems). The results have shown that the maximum amount of genetic material was obtained from hair roots, adrenal glands, gonads and lymph nodes. The lowest DNA content per gram or milliliter of tissue or body fluid was found in adipose tissue, blood, saliva, bile, sweat, tears and the vitreous body of the eye. The presented findings indicate the best sources of high-quality DNA from the human body: gonads, kidneys, muscle (including heart), blood and bones (after decalcification).

Development of a procedure involving artifact examination to determine the species affiliation of its biological material, as illustrated by an attempt to demystify an alleged Nazi lampshade made from human skin.

Objectives: The main purpose of the study was to identify the species origin of the material from which the incriminating lampshade bought at a flea market had been made. Methods: The histological and molecular biology methods commonly used in forensic genetics were selected to achieve this goal. The DNA for the research was isolated using a QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's protocol for tissues. The quantitative and qualitative evaluation of genetic material was carried out by the real-time PCR method with a Quantifiler Duo DNA Quantification Kit (Applied Biosystems). Specific genetic markers of mtDNA of cattle, equines, deer, wild boar, and sheep were selected to identify species. Results: Histological tests showed that the lampshade had been made from intestinal flaps. The DNA from sample tested positive for cattle. The test results dispelled the suspicion that the researched lampshade had been made from human skin.-hour journey. The second case is a 55-year-old male assaulted with double punches in his chest and declared dead on arrival at the hospital after 30 minutes. A medicolegal autopsy and thorough investigation, in both cases, revealed cardiac tamponade due to ventricular rupture with no underlying pathology. Conclusion: The proposed testing method can be used to verify the origin of the artifacts misleadingly described as made from human skin. To our knowledge, such artifacts can be found in museums and private collections. Further-more, it has been widely believed until now that human-skin products, mainly lampshades, were mass-produced in Nazi concentration camps, mainly in Buchenwald.

Inheritance vs. infectivity as a mechanism of malady and death of Frederic Chopin.

Based on a macroscopic analysis of the heart of Frederic Chopin performed in 2014, it can be stated with high probability that the composer suffered from a long lasting tuberculosis as a primary disease, which was the cause of progressive deterioration of his physical condition and numerous symptoms mainly from the respiratory tract. Tuberculous pericarditis rapidly progressing within a rather short period of time, a relatively rare complication of diffuse tuberculosis, might have been an immediate cause of death. This would aptly coincide with a startling opinion that in an autopsy picture the composer's heart had been more affected by the disease than the lungs.

PPARG Hypermethylation as the First Epigenetic Modification in Newly Onset Insulin Resistance in Human Adipocytes.

Insulin acts by binding with a specific receptor called an insulin receptor (INSR), ending up with glucose transporter activation and glucose uptake. Insulin resistance (IR) is a state when the physiological amount of insulin is not sufficient to evoke proper action, i.e., glucose uptake. Epigenetic modifications associated with obesity and IR are some of the main mechanisms leading to IR pathogenesis. The mesenchymal stem cells of adipose tissue (subcutaneous (SAT) and visceral (VAT)) were collected during abdominal surgery. IR was induced ex vivo by palmitic acid. DNA methylation was determined at a global and site-specific level. We found higher global DNA methylation in IR adipocytes after 72 h following IR induction. Furthermore, numerous genes regulating insulin action (PPARG, SLC2A4, ADIPOQ) were hypermethylated in IR adipocytes; the earliest changes in site-specific DNA methylation have been detected for PPARG. Epigenetic changes appear to be mediated through DNMT1. DNA methylation is an important component of IR pathogenesis; the PPARG and its epigenetic modification appear to be the very first epigenetic modification in newly onset IR and are probably of the greatest importance.

Histone modifications influence the insulin-signaling genes and are related to insulin resistance in human adipocytes.

Insulin resistance (IR) is a state when the physiological amount of insulin is not sufficient to evoke proper action, that is, glucose uptake. Numerous conditions lead to IR, including epigenetic components. Epigenetic modifications, associated with obesity and IR are one of the main mechanisms leading to IR pathogenesis. The adipose tissue samples (subcutaneous (SAT) and visceral (VAT)) were collected during abdominal surgery from 40 patients of a wide range of BMI, age, and insulin resistance ratios (F = 9, M = 31). IR was induced in 3T3-L1 adipocytes and human adipocytes collected from SAT and VAT of healthy subjects. Global and site-specific histone modifications (H3K4me3 and H3K9/14ac) were determined. We found lower histone modifications in adipose tissue of IR patients. Furthermore, numerous genes regulating insulin action (PPARG, SLC2A4, ADIPOQ) were differently marked by histone methylation and acetylation. Moreover, we noticed that epigenetic changes appear as soon as 72 h following IR induction. The epigenetic changes appeared to be mediated through the SIRT family. Based on obtained results, the histone marks related to insulin resistance mostly concerned PPARG and SLC2A4 genes. Furthermore, our results proved a vital role of the SIRT family in insulin action and IR pathogenesis.