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).

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.

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.

Original and generic clopidogrel: A comparison of antiplatelet effects and active metabolite concentrations in patients without polymorphisms in the ABCB1 gene and the allele variants CYPC19*2 and *3.

BACKGROUND: Ticagrelor and prasugrel are widely used as antiplatelet therapy after coronary angioplasty. However, there is a group of patients with indications for clopidogrel treatment. This population includes patients with chronic or acute coronary syndrome who are treated invasively and have contraindications to the use of novel antiplatelet drugs due to antithrombotic treatment (particularly with non-vitamin K antagonist oral anticoagulants). A wide range of generic forms of clopidogrel are available on the market. However, it is unclear whether they are as effective as the originator drug. OBJECTIVES: In the current study, we aimed to assess the concentrations of the active metabolite of clopidogrel and its effect on platelet aggregation inhibition in patients receiving the originator drug in comparison with those receiving generic clopidogrel. MATERIAL AND METHODS: We enrolled 22 healthy individuals without polymorphisms in the ABCB1 gene and the allele variants CYPC19*2 and CYPC19*3. All participants received a loading dose of clopidogrel (600 mg), followed by a maintenance dose of 75 mg for the next 3 days. On day 3, blood samples were obtained 1 h after drug administration to assess active metabolite concentrations using liquid chromatography with tandem mass spectrometry. In each participant, platelet aggregation was assessed with light transmission aggregometry after 5-µmol/L and 10-µmol/L adenosine diphosphate (ADP) stimulation. Assays were performed for the originator clopidogrel and 2 different generic groups. RESULTS: The mean ± standard deviation (SD) concentrations of active clopidogrel did not differ between the originator drug and 2 generic products with clopidogrel (12.7±5 pg/µL compared to 13.0 ±4 pg/µL compared to 14.4 ±4 pg/µL). Platelet aggregation inhibition after stimulation with 5 µmol/L and 10 µmol/L ADP was similar for all preparations. CONCLUSIONS: In comparison with original clopidogrel, the use of its generic form does not affect the blood concentrations of the active metabolite or its antiplatelet effect.

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.

Metabolic Differences between Subcutaneous and Visceral Adipocytes Differentiated with an Excess of Saturated and Monounsaturated Fatty Acids.

Obesity is a major health problem in highly industrialized countries. High-fat diet (HFD) is one of the most common causes of obesity and obesity-related disorders. There are considerable differences between fat depots and the corresponding risks of metabolic disorders. We investigated the various effects of an excess of fatty acids (palmitic 16:0, stearic 18:0, and oleic acids 18:1n-9) on adipogenesis of subcutaneous- and visceral-derived mesenchymal stem cells (MSCs) and phenotypes of mature adipocytes. MSCs of white adipose tissue were acquired from adipose tissue biopsies obtained from subcutaneous and visceral fat depots from patients undergoing abdominal surgery. The MSCs were extracted and differentiated in vitro with the addition of fatty acids. Oleic acid stimulated adipogenesis, resulting in higher lipid content and larger adipocytes. Furthermore, oleic acid stimulated adipogenesis by increasing the expression of CCAAT enhancer binding protein ß (CEBPB) and peroxisome proliferator activated receptor γ (PPARG). All of the examined fatty acids attenuated the insulin-signaling pathway and radically reduced glucose uptake following insulin stimulation. Visceral adipose tissue was shown to be more prone to generate inflammatory stages. The subcutaneous adipose tissue secreted a greater quantity of adipokines. To summarize, oleic acid showed the strongest effect on adipogenesis. Furthermore, all of the examined fatty acids attenuated insulin signaling and secretion of cytokines and adipokines.

Smoothbore weapons and ammunition in shooting sports.

The paper discusses the gauge ammunition used in shooting sports, dedicated for Olympic and non-Olympic shooting disciplines. The following disciplines are addressed: trap, skeet, parcour, shooting animal silhouette targets (stationary or moving) and western competition. Ammunition used in these disciplines, especially produced in Poland, is discussed as well.

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.

Polymorphisms of the MTHFR gene in mothers of children with trisomy 21 (Down syndrome) in a Polish population.

BACKGROUND: Down syndrome (DS) is the most frequent cause of intellectual disability. In 95% of cases, it is caused by simple trisomy of chromosome 21 resulting from nondisjunction of chromosomes in meiotic division. Currently, the molecular and cellular mechanisms responsible for the phenomenon of nondisjunction are unknown. OBJECTIVES: To investigate the incidence of 5 single-nucleotide polymorphisms (SNPs) of the MTHFR gene in a population of Polish mothers who had given birth to children with trisomy 21 in comparison with a control group of women with healthy offspring. MATERIAL AND METHODS: The test material comprised venous blood collected from mothers who had given birth to a child with DS (study group, n = 130) as well as from women who had given birth to children without trisomy 21 (control group, n = 88). DNA was isolated using a kit manufactured by Qiagen. Amplification was carried out using a Qiagen Multiplex PCR Kit (Qiagen); genotyping was performed using SNaPshot Genotyping MasterMix (Applied Biosystems). RESULTS: No statistically significant differences were observed in the frequency of genotypes between the examined groups in terms of the polymorphisms of the MTHFR gene. CONCLUSIONS: In the Polish population studied, no relationship was found between the occurrence of particular genotypes of the MTHFR gene, i.e., 677CT, 1298AC, rs3737964, rs4846048, and rs1994798, in women and the birth of children with trisomy 21. The results contradict the validity of research on polymorphisms of the MTHFR gene as potential predisposing factors for the occurrence of trisomy 21 in children.

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.

Obesity-induced insulin resistance via changes in the DNA methylation profile of insulin pathway genes.

BACKGROUND: Obesity has been shown to play a key role in the development of insulin resistance (IR). Abundant data implicate obesity in DNA hypermethylation at global and site-specific levels, including genes regulating insulin sensitivity. Deregulation of epigenetic marks implicates gene expression and changes in cell metabolism. OBJECTIVES: Our previous reports demonstrated that the strongest risk factor in the development of IR is BMI; accordingly, the objective of this study was to investigate the effect of obesity on DNA methylation and insulin sensitivity. MATERIAL AND METHODS: A study was carried out on lymphocytes (N-34) and visceral adipose tissue (VAT; N-35) of insulin-resistant subjects and healthy controls. Genetic material (DNA and RNA) was extracted from cells. Global and site-specific DNA methylation was analyzed with the use of restriction enzymes followed by real-time polymerase chain reaction (PCR). Gene expression was analyzed as relative mRNA level normalized to a housekeeping gene. RESULTS: Global DNA methylation increased in both types of tissue in obese and insulin-resistant individuals and correlated positively with IR. Two of the 3 investigated promoters of insulin pathway genes were hypermethylated, which correlated negatively with gene expression and positively with IR. The DNMT3a gene was upregulated in obese insulin-resistant individuals in both types of tissues and correlated positively with global DNA methylation. CONCLUSIONS: DNA methylation profile changed depending on body mass index (BMI) and influenced glucose metabolism and insulin sensitivity in VAT.

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.

Disease not genetic but infectious: multiple tuberculomas and fibrinous pericarditis as symptoms pathognomonic for tuberculosis of Frederic Chopin.

Chopin's heart, generally enlarged, presented morphologic features pathognomonic for fibrinous pericarditis presumably of tubercular origin: multiple nodular hyalinization foci-tuberculomas and fibrillary coating covering the whole surface of pericardium ("frosted heart"). We show that these features differ significantly from post mortem-formed inorganic crystalline deposits, mold colonies, or fat deposits known from various preserved anatomical objects stored for a long period of time. In our opinion, these pathologies fully justify the claim that chronic cavernous pulmonary, laryngeal, and intestinal tuberculosis presents itself as the most plausible diagnosis of Frederic Chopin and that rapidly progressing tubercular pericarditis became the immediate cause of his death.

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.

Serotonin-Related Gene Variants in Patients with Irritable Bowel Syndrome and Depressive or Anxiety Disorders.

AIM: To assess the association of six polymorphisms in serotonin-related genes with depressive or anxiety disorders in patients with irritable bowel syndrome (IBS). METHODS: The lifetime prevalence of depressive and anxiety disorders was assessed in 95 IBS patients (85% women) using the Munich version of the Composite International Diagnostic Interview (CIDI). IBS was diagnosed according to the Rome III criteria. SCL6A4 HTTLPR polymorphism (rs4795541) was determined using PCR-based method. Single-nucleotide polymorphisms in HTR1A (rs6295), HTR2A (rs6313 and rs6311), HTR2C (rs6318), and TPH1 (rs1800532) were detected by minisequencing method. RESULTS: IBS patients with depressive disorders were characterized by higher frequency of 5-HTTLPR L allele in comparison to IBS patients with anxiety disorders. The lower frequency of 1438 A allele in HTR2A was found in IBS patients with depressive disorders in comparison to IBS patients without mental disorders. The lower G allele frequency in HTR2C rs6318 polymorphism among IBS patients with anxiety disorders was also observed. CONCLUSIONS: Our results provide further evidence for the involvement of SLC6A4 rs4795541 and HTR2A rs6311 polymorphisms in the pathophysiology of depressive disorders in IBS patients. The new findings indicate that HTR2C rs6318 polymorphism may be associated with the susceptibility to anxiety disorders in IBS patients.