Long-term space missions' effects on the human organism: what we do know and what requires further research.

Space has always fascinated people. Many years have passed since the first spaceflight, and in addition to the enormous technological progress, the level of understanding of human physiology in space is also increasing. The presented paper aims to summarize the recent research findings on the influence of the space environment (microgravity, pressure differences, cosmic radiation, etc.) on the human body systems during short-term and long-term space missions. The review also presents the biggest challenges and problems that must be solved in order to extend safely the time of human stay in space. In the era of increasing engineering capabilities, plans to colonize other planets, and the growing interest in commercial space flights, the most topical issues of modern medicine seems to be understanding the effects of long-term stay in space, and finding solutions to minimize the harmful effects of the space environment on the human body.

The smell of death. State-of-the-art and future research directions.

The decomposition of a body is inseparably associated with the release of several types of odors. This phenomenon has been used in the training of sniffer dogs for decades. The odor profile associated with decomposition consists of a range of volatile organic compounds (VOCs), chemical composition of which varies over time, temperature, environmental conditions, and the type of microorganisms, and insects colonizing the carcass. Mercaptans are responsible for the bad smell associated with corpses; however, there are no unified recommendations for conducting forensic analysis based on the detectable odor of revealed corpses and previous research on VOCs shows differing results. The aim of this review is to systematize the current knowledge on the type of volatile organic compounds related to the decomposition process, depending on a few variables. This knowledge will improve the methods of VOCs detection and analysis to be used in modern forensic diagnostics and improve the methods of training dogs for forensic applications.

Nucleic Acids Persistence-Benefits and Limitations in Forensic Genetics.

The analysis of genetic material may be the only way to identify an unknown person or solve a criminal case. Often, the conditions in which the genetic material was found determine the choice of the analytical method. Hence, it is extremely important to understand the influence of various factors, both external and internal, on genetic material. The review presents information on DNA and RNA persistence, depending on the chemical and physical factors affecting the genetic material integrity. One of the factors taken into account is the time elapsing to genetic material recovery. Temperature can both preserve the genetic material or lead to its rapid degradation. Radiation, aquatic environments, and various types of chemical and physical factors also affect the genetic material quality. The substances used during the forensic process, i.e., for biological trace visualization or maceration, are also discussed. Proper analysis of genetic material degradation can help determine the post-mortem interval (PMI) or time since deposition (TsD), which may play a key role in criminal cases.

Epiglottis Cartilage, Costal Cartilage, and Intervertebral Disc Cartilage as Alternative Materials in the Postmortem Diagnosis of Methanol Poisoning.

Alternative materials for postmortem diagnosis in the case of fatal poisonings are much needed when standard materials, such as blood and urine, are unavailable. The study presents a case of fatal mass methanol intoxication resulting from industrial alcohol consumption. The study aimed to determine methanol and formic acid concentrations in epiglottis cartilage, costal cartilage, and intervertebral disc cartilage and to analyze the correlation between their concentrations in cartilage tissues and the femoral blood. Methanol and formic acid concentrations in samples collected from 17 individuals (n = 17) were estimated using gas chromatography with flame ionization detection (GC-FID). Methanol concentration in the costal cartilage correlated with its concentration in the femoral blood (r = 0.871). Similar correlations were found for epiglottis cartilage (r = 0.822) and intervertebral disc cartilage (r = 0.892). Formic acid concentration in the blood correlated only with its concentration in urine (r = 0.784) and the epiglottis (r = 0.538). Cartilage tissue could serve as an alternative material for methanol analyses in postmortem studies. Formic acid, a methanol metabolite, does not meet the requirements for its presence determination in cartilage tissues.

Fatal Methanol Poisoning Caused by Drinking Industrial Alcohol: Silesia Region, Poland, April-June 2022.

Methanol poisonings caused by drinking industrial alcohol remain a severe problem worldwide. Education on types of alcohol and their harmfulness and legal regulations limiting the industrial alcohol trade seem to be the keys to reducing the number of poisonings. Methanol distribution in different tissues after absorption is not well understood. This research aimed to quantify the methanol and formic acid distribution in body fluids and tissue material in post-mortem samples collected from 19 fatal victims of massive intoxication with industrial alcohol in the Silesia Region (Poland) who died between April and June 2022. The samples were analyzed using a gas chromatography-flame ionization detector (GC-FID), and correlation coefficients for methanol and formic acid were determined. The results show a wide distribution of methanol and formic acid in human post-mortem biological fluids (blood, urine, vitreous humor, bile, and cerebrospinal fluid) and tissues (muscle, kidney, liver, spleen, lung, and brain). The strongest correlation for methanol concentration in blood and body fluids/tissues was obtained in the cerebrospinal fluid (r = 0.997) and for formic acid in muscle tissue (r = 0.931). The obtained results may be a valuable tool in toxicological analysis and improve medical standards of early diagnosis and targeted treatment.

The intervertebral discs' fibrocartilage as a DNA source for genetic identification in severely charred cadavers.

Identifying charred human remains poses a challenge to forensic laboratories. High temperature completely incinerates the superficial tissues and partially destroys bones, forcing the forensics to seek an alternative, for bones and teeth, forensic material that should quickly and cheaply deliver DNA of sufficient quantity and quality. We sought, other than rib cartilage, types of cartilages that could serve as a DNA source. DNA was isolated from the fibrous cartilage of a fibrous ring of intervertebral L1-L2 discs sampled from charred cadavers or charred body fragments: 5 victims of car fires, 1 victim of combustion during a residential house gas explosion, and 3 victims of nitroglycerin explosion. DNA was isolated by the column method. DNA quality and concentration were assessed by RT-PCR and multiplex PCR for 23 autosomal and 17 Y chromosome STR loci. STR polymorphism results obtained by capillary electrophoresis served for likelihood ratio (LR) calculations. DNA concentration in relation to the cadaver's age and post-mortem interval (PMI) were analyzed. All samples (n = 9) yielded good-quality DNA in quantities (0.57-17.51 ng/µL for T. Large autosomal sequence) suitable for STR-based amplification. The isolated DNA characterized a low degradation index (0.80-1.99), and we were able to obtain complete genetic profiles. In each of the nine cases, the genotyping results allowed identifying the victims based on comparative material from the immediate family. The results demonstrate the usefulness of human intervertebral disc fibrocartilage as an alternative DNA source for the genetic identification of charred bodies or charred torso fragments.

Post-tracheostomy complications: respiratory failure caused by authologic foreign body-case report.

Tracheostomy is performed frequently as a palliative treatment in patients with end-stage respiratory failure (RF). However, in patients requiring prolonged mechanical ventilation it may be difficult to recognize and can often lead to life-threatening RF. We present two cases of acute-on-chronic respiratory failure (ACRF) occurring in patients who had undergone tracheostomy [one with percutaneous dilatational tracheostomy (PDT) and the second with surgical tracheostomy (ST)]. The first case was admitted due to ACRF several months after previous successful decannulation and the second case after failure of several attempts of weaning from tracheal cannula. In both cases, noninvasive mechanical ventilation assisted flexible bronchoscopy (NIV-FB) was able to identify and solve the tracheal stenosis secondary to stiff banana-shaped whitish foreign bodies. Histology sampling and genetic testing confirmed autologous foreign body formation-tracheal cartilage calcification. NIV-FB was found to be safe and effective in both diagnosis and treatment of the tracheal stenosis. Life-threatening RF connected with tracheal stenosis may be caused by rupture of tracheal cartilage ossification in patients with a history of ST and PDT. Bronchofiberoscopy performed with NIV will be a useful procedure to evaluate and treat the respiratory tract in patients with RF with suspected tracheal stenosis.

Filter mask as a new candidate of personal belonging used in cadaver identification - a case report.

The case report presents an identification process based on DNA isolated from personal belongings, including a filter mask. In May 2021, an unidentified 65-year-old male corpse was revealed by the city's outskirts road. Since it was impossible to use material from living relatives for comparative analysis, the samples of personal belongings of the alleged victim were used instead: clippings of the filtering face piece type 2 (FFP2) face mask (parts adhering to the nose and the earlobes, the central part of the mask), swabs from the razor (blade and shaft), toothbrush shaft, and toothbrush filaments clippings. The presented case indicates the need for collecting a wide range of samples for genetic analyses, including filter masks as an alternative item of personal belonging.

Gene Expression in Amnion-Derived Cells Cultured on Recombinant Laminin 332-A Preliminary Study.

Human amniotic cells (hAC) exhibit characteristics of undifferentiated cells and immunomodulatory properties. Recognition of the relationship between amniotic cells and components of the extracellular matrix is an important condition for their ex vivo preparation and further successful clinical application in regenerative medicine and transplantology. Laminin 332 (LN-332), as a natural component of the basement membrane of amniotic epithelial cells and a ligand for integrin receptors, may strongly influence the phenotype and fate of amniotic cells. We investigated the impact of recombinant LN-332 on hAC viability and expression of markers for pluripotency, early differentiation, adhesion, and immunomodulatory properties. During 14 days of culture, hAC were quantified and qualified by light microscopy, immunohistochemistry, immunocytochemistry, and flow cytometry. Gene expression was assessed with real-time polymerase chain reaction (RT-PCR) arrays and compared with differentiated cells originated from the three germ layers. LN-332 caused an over 2-fold increase in the total number of hAC, accompanied by a 75% reduction of SSEA-4-positive cells and an increase in HLA-ABC-positive cells. In particular, we observed that the presence of laminin 332 in the medium of a short-time culture modifies the effect of culture duration on hAC, enhancing time-dependent inhibition of expression of certain genes, including pluripotency and differentiation markers, laminin 332 subunits (which may be part of self-regulation of LN-332 synthesis by amniotic cells), and integrins. The changes observed in hAC were more distinct with respect to differentiated mesenchymal cells, resulting in more comparable phenotypes than those represented by differentiated endo- and ectodermal cells. We concluded that laminin 332 present in the culture medium influences to a certain extent proliferation, adhesion, and differentiation of amniotic cells in culture.

Sodium nitrite detection in costal cartilage and vitreous humor - Case report of fatal poisoning with sodium nitrite.

Medico-legal case reports very rarely describe sodium nitrite poisonings, but when they do most often they describe fatal suicide attempts. The case report presents a suicidal attempt with sodium nitrite of unknown provenance and the first attempt to detect nitrite ions in costal cartilage and vitreous humor samples. In February 2020, the corpse of a 23-year-old man was revealed in a student apartment. According to the prosecutor's office, the deceased had an incomplete IT (Information Technology) degree. The onsite inspection revealed the body on the bathroom floor, an opened container with sodium nitrite III in the bathroom cabinet, and a farewell letter in the apartment. The autopsy showed the hypoxia symptoms. The blood and urine of the deceased showed no trace of ethyl alcohol or psychoactive substances. Analyses showed the presence of nitrite ions in the blood (0.2 µg/ml) and urine (24.6 µg/ml) of the deceased. Additional analyses revealed nitrites presence in the gastric contents (2200 µg/ml), liver tissue (0.3 µg/g), kidney tissue (3.6 µg/g) and, for the first time, in costal cartilage (3.4 µg/g) and vitreous humor (57.7 µg/ml). The autopsy concluded that the cause of death was an acute cardio-respiratory failure in the course of suicidal sodium nitrite poisoning. The presented case indicates the need for collecting a wide range of samples for toxicological analyses. It also proves that both costal cartilage and vitreous humor may serve as an alternative forensic material in sodium nitrite poisonings.

When DNA profiling is not enough? A case of same-sex siblings identification by odontological assessment after gas explosion-related building collapse.

We aimed to show the usefulness of odontological assessment in forensic investigation. Charred remains of two female siblings were found in a collapsed building after a gas explosion. Due to thermal damage of the bodies, the facial characteristics, fingerprints, height and weight could not be used to distinguish between siblings. Since the victims, 4 and 10-year-old, died simultaneously and all personal belongings were lost, DNA profiling performed with their parents only confirmed the relationship. As dental charts of siblings were not found, we could not easily discriminate which remains would be of the elder and which of the younger sister. The odontological examination enabled us to discriminate between the siblings based on differences in deciduous and permanent dentition. We conclude that although DNA profiling is becoming a standard method of personal identification in some cases it should be supported by additional methods to deliver comprehensive forensic reports.

Cadaveric Stem Cells: Their Research Potential and Limitations.

In the era of growing interest in stem cells, the availability of donors for transplantation has become a problem. The isolation of embryonic and fetal cells raises ethical controversies, and the number of adult donors is deficient. Stem cells isolated from deceased donors, known as cadaveric stem cells (CaSCs), may alleviate this problem. So far, it was possible to isolate from deceased donors mesenchymal stem cells (MSCs), adipose delivered stem cells (ADSCs), neural stem cells (NSCs), retinal progenitor cells (RPCs), induced pluripotent stem cells (iPSCs), and hematopoietic stem cells (HSCs). Recent studies have shown that it is possible to collect and use CaSCs from cadavers, even these with an extended postmortem interval (PMI) provided proper storage conditions (like cadaver heparinization or liquid nitrogen storage) are maintained. The presented review summarizes the latest research on CaSCs and their current therapeutic applications. It describes the developments in thanatotranscriptome and scaffolding for cadaver cells, summarizes their potential applications in regenerative medicine, and lists their limitations, such as donor's unknown medical condition in criminal cases, limited differentiation potential, higher risk of carcinogenesis, or changing DNA quality. Finally, the review underlines the need to develop procedures determining the safe CaSCs harvesting and use.

Concentrations of volatile substances in costal cartilage in relation to blood and urine - preliminary studies.

Aim: The study aimed to examine whether volatile substances (ethanol, isopropanol, and acetone) can be detected in costal cartilage and also if concentrations of detected substances reliably reflect their concentrations in the peripheral blood - the standard forensic material for toxicological analyses. Such knowledge can be useful in cases when a cadaver's blood is unavailable or contaminated. Material and methods: Ethanol, isopropanol, and acetone concentrations were determined in samples of unground costal cartilage (UCC), ground costal cartilage (GCC), femoral venous blood, and urine. The samples were analysed by gas chromatography (GC) with a flame ionization detector using headspace analysis. Results: Volatile substances were detected in 12 out of 100 analysed samples. There was a strong positive correlation between ethanol concentration in the blood and urine (r = 0.899, p < 0.001), UCC (r = 0.809, p < 0.01), and GCC (r = 0.749, p < 0.01). A similar strong correlation was found for isopropanol concentration in the blood and urine (r = 0.979, p < 0.001), UCC (r = 0.866, p < 0.001), and GCC (r = 0.942, p < 0.001). Acetone concentration in the blood strongly correlated only with its concentration in urine (r = 0.960, p < 0.001). Conclusions: We demonstrated for the first time the possibility of detecting volatile substances: ethanol, isopropanol and acetone in a human costal cartilage. Also, the study showed that higher volatiles concentrations were better determined in ground samples.

Mutation analysis of short tandem repeats in a population sample from Upper Silesia (southern Poland).

In paternity testing, DNA polymorphism analysis not only settles explicitly disputed paternity issue but also provides information on mutation frequencies in STR loci. In this study, insertion or deletion of one repetitive unit was observed in 38 of 32,391 meiotic transfers analysed in 953 paternity testing cases. Parentage samples from Upper Silesia (southern Poland) were examined in 2008-2014 with the use of three commercially available amplification kits: AmpFlSTR Identifiler (Applied Biosystems), PowerPlex 16 HS (Promega) and PowerPlex ESX 17 (Promega). The rate of paternal mutations was 4.6 times higher than that of maternal ones. The highest mutation rate was noted at VWA locus. Interpopulation comparisons showed statistically significant differences in mutation rates of several STRs between Upper Silesia and populations from Brazil and China. There were no differences in occurrence of mutations between a population from Upper Silesia and another southern Polish population from a region of Lesser Poland. Our results suggest that knowledge of STR mutation rates in different populations may be important for calculations of probability of relationship in disputed paternity testing and that such calculations should be based on population-specific mutation rates, at least for some STR markers used commonly in forensic genetics.

An experimental approach to the generation of human embryonic stem cells equivalents.

Recently, particular attention has been paid to the human embryonic stem cells (hESC) in the context of their potential application in regenerative medicine; however, ethical concerns prevent their clinical application. Induction of pluripotency in somatic cells seems to be a good alternative for hESC recruitment regarding its potential use in tissue regeneration, disease modeling, and drug screening. Since Yamanaka's team in 2006 restored pluripotent state of somatic cells for the first time, a significant progress has been made in the area of induced pluripotent stem cells (iPSC) generation. Here, we review the current state of knowledge in the issue of techniques applied to establish iPSC. Somatic cell nuclear transfer, cell fusion, cell extracts reprogramming, and techniques of direct reprogramming are described. Retroviral and lentiviral transduction are depicted as ways of cell reprogramming with the use of integrating vectors. Contrary to them, adenoviruses, plasmids, single multiprotein expression vectors, and PiggyBac transposition systems are examples of non-integrative vectors used in iPSC generation protocols. Furthermore, reprogramming with the delivery of specific proteins, miRNA or small chemical compounds are presented. Finally, the changes occurring during the reprogramming process are described. It is concluded that subject to some limitations iPSC could become equivalents for hESC in regenerative medicine.

Expression and co-expression of surface markers of pluripotency on human amniotic cells cultured in different growth media.

OBJECTIVES: Despite constant advances in the field of biology and medical application of human embryonic stem cells, the molecular mechanism of pluripotency remains largely unknown. So far, definitions of pluripotent stem cells (SC) have been based on a limited number of antigenic markers and have not allowed for unambiguous determination of the homogeneity of each subpopulation. Moreover, the use of some crucial pluripotency markers such as SSEA-3 and SSEA-4 has recently been questioned due to the possibility that the pattern of surface glycans may be changed depending on the content of the cell culture medium. AIM: Quantitative analysis of amniotic SC subpopulations cultured in different media, based on the following pluripotency surface markers: SSEA-3, SSEA-4, TRA- 1-60 and TRA- 1-81 expression and co-expression. MATERIAL AND METHODS: Immunofluorescence and fluorescence microscopy were used to identify and localize SC within a normal human placenta at term. The number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells with co-expression of the above mentioned markers, cultured in media containing different protein supplements of animal origin, was counted by flow cytometry RESULTS AND CONCLUSIONS: Cells with characteristics of embryonic SC were identified in the amniotic epithelium and the chorion, but not in the decidua basalis. Amniotic epithelium contained various types of SC, with SSEA-4+ as the most numerous. Disproportion in the number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells characterized by co-expression of these antigens, as well as lack of quantitative differences between SC subpopulations cultured in different media, was observed. In conclusion, the amniotic epithelium is composed of SC at different stages of the development but human amnion might become an alternative source of SSEA-4+ embryonic-like SC. The composition of the evaluated media, characterized by different content of animal-derived proteins, does not influence the number of cells identified within the SC subpopulations.