The Polish Genetic Database of Victims of Totalitarianisms.

This paper describes the creation of the Polish Genetic Database of Victims of Totalitarianism and the first research conducted under this project. On September 28th 2012, the Pomeranian Medical University in Szczecin and the Institute of National Remembrance-Commission for Prosecution of Crimes against the Polish Nation agreed to support the creation of the Polish Genetic Database of Victims of Totalitarianism (PBGOT, www.pbgot.pl). The purpose was to employ state-of-the-art methods of forensic genetics to identify the remains of unidentified victims of Communist and Nazi totalitarian regimes. The database was designed to serve as a central repository of genetic information of the victim's DNA and that of the victim's nearest living relatives, with the goal of making a positive identification of the victim. Along the way, PGBOT encountered several challenges. First, extracting useable DNA samples from the remains of individuals who had been buried for over half a century required forensic geneticists to create special procedures and protocols. Second, obtaining genetic reference material and historical information from the victim's closest relatives was both problematic and urgent. The victim's nearest living relatives were part of a dying generation, and the opportunity to obtain the best genetic and historical information about the victims would soon die with them. For this undertaking, PGBOT assembled a team of historians, archaeologists, forensic anthropologists, and forensic geneticists from several European research institutions. The field work was divided into five broad categories: (1) exhumation of victim remains and storing their biological material for later genetic testing; (2) researching archives and historical data for a more complete profile of those killed or missing and the families that lost them; (3) locating the victim's nearest relatives to obtain genetic reference samples (swabs), (4) entering the genetic data from both victims and family members into a common database; (5) making a conclusive, final identification of the victim. PGBOT's first project was to identify victims of the Communist regime buried in hidden mass graves in the Powazki Military Cemetery in Warsaw. Throughout 2012 and 2013, PGBOT carried out archaeological exhumations in the Powazki Military Cemetery that resulted in the recovery of the skeletal remains of 194 victims in several mass graves. Of the 194 sets of remains, more than 50 victims have been successfully matched and identified through genetic evidence.

A new dimension of the forensic DNA expertise - the need for training experts and expertise recipients.

Forensic genetics is a rapidly developing discipline. Nowadays, human genetic identification relies on the application of complex solutions ensuring high sensitivity and resistance to the inhibition and degradation of biological traces, and revealing maximum information which has relevance for the justice system. However, recent improvements in forensic DNA identification testing are associated with problems including secondary transfer, DNA mixtures and incompleteness of DNA profiles, which were formerly less significant. It also seems that the potential of the national DNA database in Poland has not been fully developed, and it is necessary to implement an appropriate information policy in order to improve it. Novel methods that can be applied at the level of investigation include analysis of biogeographic ancestry, prediction of visible traits, and estimation of human chronological age. Moreover, next-generation sequencing has a potential to entirely replace capillary electrophoresis in forensic genetics. Further works are necessary to ensure a proper implementation of uniform standards of data interpretation and evaluation of DNA evidence in forensic genetics. In order to maintain proper standards of forensic DNA assessment, continuous training of DNA experts and appropriate information policy for recipients of DNA assessments are required.

Association of polymorphic sites in the OCA2 gene with eye colour using the tree scanning method.

A number of genes are considered to affect normal variation in human pigmentation. Recent studies have indicated that OCA2 is the crucial gene involved in the high variation of iris colour present among populations of European descent. In this study, eleven polymorphisms of the OCA2 gene were examined in search of their association with different pigment traits. The evolutionary tree scanning method indicated that the strongest phenotypic eye colour variation is associated with the branch defined by nonsynonymous change rs1800407, which refers to amino acid causing change Arg419Gln located in exon 13. Single SNP analysis indicated that allele 419Gln is associated with green/hazel iris colour (p < 0.001). According to tree scanning analysis, the proportion of eye colour variation explained by this nucleotide position is merely 4%. Thus, additional variation present in the OCA2 gene and perhaps some other pigment related genes must be taken into account in order to explain the high phenotypic variation in iris colour.

Effects of estrone on N-methyl-D-aspartic acid- and staurosporine-induced changes in caspase-3-like protease activity and lactate dehydrogenase-release: time- and tissue-dependent effects in neuronal primary cultures.

A growing body of evidence indicates that estrogens affect apoptotic processes in neuronal cells. However, their effects seem to depend on type of neuronal tissue, stage of development and apoptosis inducing factors. In the present study we compared effects of estrone (100 and 500 nM) on N-methyl-D-aspartic acid (NMDA) (1 mM)- and staurosporine (1 microM)-induced caspase-3-like activity and lactate dehydrogenase (LDH)-release in primary cultures of rat hippocampal and neocortical neurons. Fluorometric and colorimetric determination of enzyme activity was performed 6 h, 14 h, and 24 h after exposure to apoptotic agents. In the hippocampal cell cultures on 7 days in vitro (DIV), a time-dependent NMDA-induced activation of caspase-3-like proteases was accompanied by increased LDH-release. In neocortical cell cultures on 7 DIV NMDA did not affect caspase activity and decreased LDH-release. In neocortical cell cultures on 12 DIV NMDA inhibited spontaneous caspase activity, but was toxic to neurons after 24 h exposure suggesting that these cells underwent necrotic rather than apoptotic death. Estrone has attenuated both pro- and anti-apoptotic NMDA-induced changes in rat primary neuronal cultures acting independently of estrogen receptors, as detected with ICI 182, 780. In hippocampal neurons estrone antagonized not only the NMDA-induced caspase-3-like activity, but also NMDA-mediated LDH-release. However, in neocortical neurons estrone either attenuated NMDA-induced inhibition of caspase-3-like activity (12 DIV) or partly blocked NMDA-mediated decrease in LDH-release (7 DIV). In contrast to NMDA, staurosporine elevated caspase-3-like activity and LDH-release in a time-dependent manner in all used culture systems. Estrone inhibited pro-apoptotic effects of staurosporine in neocortical neurons, but only at later stage of development in vitro, which points to the protective role of estrogens during the brain tissue maturation. Since estrone triggered its effects via non-genomic mechanisms, it suggests that the other estradiol metabolites exhibiting low affinity to hormone receptors may be potent neuroprotective agents, which could retain the favorable and minimize the adverse side effects of estrogens.

STR data for AmpF/STR Profiler Plus loci in south Poland.

Frequency data of short tandem repeats (STR) loci included in the AmpF/STR Profiler Plus kit were collected from a sample of 253 random, unrelated individuals born in the south Poland region. All loci met Hardy-Weinberg expectation.

Stability of Adderall in extemporaneously compounded oral liquids.

The short-term stability of Adderall in three extemporaneously compounded oral liquids was studied. Three suspensions of Adderall 1 mg/mL were prepared from commercially available 10-mg Adderall tablets with Ora-Sweet, Ora-Plus, and a 1:1 mixture of Ora-Sweet and Ora-Plus. Each suspension was stored in the dark in a stability chamber at 25 degrees C and 60% relative humidity for 30 days. The stability of the active drug (a mixture of levoamphetamine and dextroamphetamine salts) in each of the three vehicles was determined immediately after preparation and at 10, 20, and 30 days by using gas chromatography-mass spectrometry (GCMS). No significant changes in concentrations of either amphetamine isomer occurred during the 30-day study period. Visual inspection of samples revealed no changes in color or odor. Extemporaneously compounded liquid oral formulations of Adderall 1 mg/mL in Ora-Sweet, Ora-Plus, or a 1:1 mixture of Ora-Sweet and Ora-Plus were stable for at least 30 days at 25 degrees C and 60% relative humidity.

Impact of hypericum (St.-John's-wort) given prenatally on cognition of mice offspring.

This study investigated the cognitive impact of prenatal exposure to the herbal antidepressant hypericum in CD-1 mice. Hypericum (182 mg/kg/day) or a placebo was consumed in food bars for 2 weeks before mating and throughout gestation. The hypericin content in our hypericum formulation was in the middle range of standardized hypericum products. One offspring per gender from each litter (hypericum 13, placebo 12) was tested on each of the following tasks: juvenile runway with adult memory, adult Morris maze, adult passive avoidance, or adult straight water runway followed by a dry Cincinnati maze. Learning occurred in both genders in all tasks (P<.003) with no significant differences between treatments at the final trial. Female offspring exposed to hypericum, rather than to a placebo, required more time to learn the Morris maze task (P<.05). Postlearning sessions did not show any significant differences. In conclusion, prenatal exposure to a therapeutic dose of hypericum did not have a major impact on certain cognitive tasks in mice offspring.

Impact of antenatal exposure of mice to fenfluramine on cardiac development and long-term growth of the offspring.

The objective of this investigation was to determine, in a placebo-controlled manner, whether antenatal exposure to formulations of fenfluramine and dexfenfluramine impacted cardiac development and long-term growth of exposed mice offspring. One hundred forty-four CD-1 mice were randomized to six treatment groups (n=23 or 25) to obtain, per group, 5 gravids for killing on gestational day (GD) 15 and < or =10 deliveries for assessing growth of the offspring. Either fenfluramine preparation was administered in feed bars in two doses: 1 and 3.2 times the equivalent human daily dosage according to body surface area. The drugs were given from 2 weeks before mating until GD 15. The mice ingested each drug at target values, averaging 10.5+/-0.3 and 31.8+/-1.9 mg/kg/d for fenfluramine and 5.0+/-0.2 and 16.2+/-0.4 mg/kg/d for dexfenfluramine. The drug concentration was about 36% in the fetal brain compared with the adult brain. The maternal and the offspring hearts, including mitral and aortic valves, of fenfluramine-exposed mice were indistinguishable from the placebo-exposed mice. The duration of gestation and the litter size were the same between the treatment groups. The mean body weights, body lengths, and head circumferences and early functional testing did not differ significantly between the fenfluramine or dexfenfluramine-exposed offspring and the placebo-exposed offspring. There were no significant treatment differences in growth measured as body weights to PND 120. Neither fenfluramine formulation, given before conception and during gestation, impacted cardiac development and long-term growth of the mice offspring.

Effect of antenatal exposure to paroxetine (paxil) on growth and physical maturation of mice offspring.

OBJECTIVE: Our purpose was to determine, in a placebo-controlled manner, whether antenatal exposure to paroxetine affected long-term growth and physical maturation of mice offspring. METHODS: Forty-one CD-1 mice consumed paroxetine (n = 21) or a placebo (n = 20) for 2 weeks before conception and throughout gestation. The daily dose of paroxetine (Paxil; 30 mg/kg/d) was known to achieve concentrations in the serum equivalent to the upper therapeutic level in humans and in the fetal brain equivalent to that of the adult mouse. Growth and physical maturation of the offspring were compared by paired t-test, Welch's corrected test, and Fisher's exact test. RESULTS: The maternal weight gain, litter sizes, number of fetal resorptions, and gestational age at delivery were not different between the paroxetine and the placebo-exposed offspring. Newborn pups exposed to paroxetine were more likely to have low birthweights (1.65 gm vs. 1.70 gm; P < 0.05) and narrower heads (7.7 mm vs. 8.1 mm; P < 0.05). Body weight, body length, and head circumference measurements increased in a manner that was indistinguishable between the two groups of offspring, regardless of gender. No differences in achievement of physical milestones (lower incisor eruption, eye opening, and development of external genitalia) were noted between the two groups. The reproductive capability and the perinatal outcomes of the second-generation offspring were unaffected by paroxetine exposure. CONCLUSION: A clinically relevant dose of paroxetine, when given throughout gestation, did not affect long-term growth and physical maturation of mice offspring.

Dry-heat sterilization of parenteral oil vehicles.

The purpose of this study was to evalutate the effect of temperature and time on the dry-heat sterilization conditions of cottonseed, peanut and sesame-seed oils used as vehicles for parenteral drugs. The three oils were inidividually spiked wiht Bacillus subtilis spores and exposed to dry heat at four temperatures (150, 160, 170, and 180 deg C) for three different time intervals (one, 1.5 and two hours). Following inoculation and dry-heat sterilization, samples were placed in a laminar airflow hood and processed according to 71, "Sterility Tests" of the USP XXIV/NF 19 using thioglycolate broth and fluid D. The specimens were then placed into an incubator at 30 deg C and observed for three, five and seven days for bacterial growth. All tests were performed in triplicate. Positive and negative controls were conducted with each group. All three oils were found to be free of viable Bacillus subtilis following dry-heat sterilization at 150, 160, 170 and 180 deg C for one, 1.5 and two hours after incubation for seven days. The positive controls had no observed bacterial growth. Dry-heat sterilization of the three oils at 150 deg C for one hour appeared to be sufficient for time and temperature conditions. However, the authors recommend dry-heat sterilization procedures be validated for each product.

Y-chromosomal polymorphic loci DYS19, DYS390, DYS393 in a population sample from northern Poland.

Short tandem repeats (STR) are presently of particular use in the field of forensic science, evolution and anthropology. Y-chromosomal STR systems are widely used for population genetics, population history, and for male identification in forensic cases. Here we present an examination of DYS19, DYS390 and DYS393 allele frequencies in a northern Polish population sample. The calculated indices reflect the potential of these markers for application in forensic casework. Statistically significant differences were observed between most western European populations and the Polish cohort when comparing homogeneity of distribution of DYS19 and DYS390 alleles. For three analyzed loci in a sample of 176 males, 43 haplotypes were observed. The studies revealed some rare alleles and a new allele, allele 16, in the DYS393 system. Sequencing by capillary electrophoresis (PE ABI 310) showed the presence of 16 GATA repetitive elements, confirming results obtained after capillary electrophoresis of the DNA fragment. Additionally, sequencing revealed the presence of a novel transversion (A->C) in the analyzed sample.

Stereochemical determination of selegiline metabolites in postmortem biological specimens.

In this study, findings related to an aircraft accident are reported. Biological specimens collected at autopsy from the pilot of the fatal accident and two types of tablets found at the accident scene were submitted for toxicological evaluation. It was determined that the pilot was dead at the crash site and the cause of death was multiple traumatic injuries. The tablets were identified as selegiline and levodopa, commonly prescribed for the treatment of Parkinson's disease. Selegiline, a stereospecific compound, is biotransformed into (-)-N-desmethylselegiline, (-)-methamphetamine, and (-)-amphetamine. The latter two levorotatory metabolites cannot be easily distinguished by routine analysis from their dextrorotatory isomers, which are controlled substances. It was, therefore, prudent to differentiate these isomers to determine if they resulted from the ingestion of a controlled substance, (+)-methamphetamine. Initial immunoassay drug screenings revealed the presence of amphetamine class drugs (867 ng/mL) in urine, amphetamine/methamphetamine (261 ng/mL) in urine, and methamphetamine (46 ng/mL) in blood. The gas chromatography-mass spectrometry (GC/MS) results revealed the presence of methamphetamine in the concentrations of 76 ng/mL of blood and 685 ng/mL of urine. The concentration of amphetamine was 52 ng/mL in blood and 320 ng/mL in urine. To determine the stereospecificity of these amines, the isolated amines from the biosamples were derivatized by a stereospecific agent, (S)-(-)-N-(trifluoroacetyl)-prolyl chloride, and characterized by a GC/MS method to be levorotatory. The 2.14 ratio of (-)-methamphetamine to (-)-amphetamine concentrations in the urine was consistent with a selegiline study in the recent literature. The stereospecific analysis, in conjunction with the history of the pilot being on Parkinson's medications, suggests that the source of these amines was selegiline. This conclusion substantiates the importance of the identification of enantiomers in evaluating and interpreting related analytical results for accident investigations.

Vaccinia virus-regulated acute phase cytokine production in human fibroblasts, U937 cells and endothelium.

The production of acute phase cytokines, interleukin 6 (IL-6), tumour necrosis factor (TNFalpha) and interleukin 1 (IL-1beta), was studied in primary cultures of human skin fibroblasts, human monocytic cell line U937 and primary cultures of human umbilical vein endothelial cells (HUVEC) after in vitro infection with vaccinia virus. Significant increase in IL-6 mRNA followed by enhanced protein secretion into the culture media was found in fibroblasts, U937 cells, and HUVEC. TNFalpha increased production in vaccinia virus infected U937 cells resembled closely the pattern of IL-6 production observed in the infected cells. Transient increase in NF-kappaB binding activity was found in the infected U937 (at 90 min) and endothelial (at 30 min) cells. Vaccinia virus induced cytokine production appeared to be transcriptional.

Postmortem alcohol production in fatal aircraft accidents.

During 1989 and 1990, the Civil Aeromedical Institute received specimens from 975 victims of fatal aircraft accidents. The maximum concentration of ethanol allowed under FAA regulations (0.04%, 40 mg/dL) was exceeded in 79 of these cases (8%). It was determined based on the distribution of ethanol in urine, vitreous humor, blood, and tissue that 21 of the positive cases (27%) were from postmortem alcohol production. Twenty-two of the positive cases (28%) were found to be from the ingestion of ethanol. In 36 cases (45%), no determination could be made regarding the origin of the ethanol. In two cases, postmortem alcohol production exceeded 0.15% (150 mg/dL). The opinion held by some toxicologists that postmortem alcohol production can be inferred from the presence of acetaldehyde, acetone, butanol, and other volatiles was found to be incorrect. Several cases with postmortem ethanol had no other volatiles. Volatile compounds were found in several cases where no ethanol was present. In addition a case was found in which the relative ethanol concentrations in blood, bile, and vitreous humor were solely consistent with the ingestion of ethanol, but acetaldehyde, acetone, and 2-butanol were also found in blood. This clearly indicates that the presence or absence of other volatiles does not establish postmortem ethanol production.