Post-mortem interval (PMI) is the amount of time elapsed since the time of death. Over the years, many methods were developed to assess PMI, but their precision and time frame of applicability are often limited. Our present pilot study aimed to prove if RNA degradation of human dental pulp can be used for PMI estimation. RNA was isolated from the pulps of healthy wisdom teeth and premolars. RNA degradation was determined as RNA integrity number (RIN) with Agilent Bioanalyzer and subsequently by amplification of different length products by PCR after reverse transcription. The RNA integrity analysis allowed us to determine the time of post-mortem interval with high confidence level in the first 21 days. With the PCR-based method, we were able to perform a crude estimation of incubation time of teeth between 20 and 42 days post extraction. These results show that this method might be a promising new tool for PMI estimation despite the limitations.

Hepcidin is the only hormone directly involved in iron regulation. It is synthesized as an 84-amino-acid (AA) preprohormone, and is present in the plasma as a mature 25-AA peptide and as a 60-AA prohormone form. Maturation is facilitated by the serine peptidase furin. The aim of this study was to determine whether prepro-and prohormones show significant interactions with proteins, which may affect the maturation of the hormone in the cell and its cleavage to active hormone in blood.Iron is one of the essential trace elements in living organisms. In vertebrates, the plasma iron level is in the micromolar range and circulating iron is associated predominantly with the transport protein transferrin. The blood iron level and the saturation of transferrin are frequently used indicators of the body Recent discoveries have indicated that the hormone hepcidin plays a major role in the control of iron homeostasis. Hepcidin regulates the iron level in the blood through the interaction with ferroportin, an iron exporter molecule, causing its internalization and degradation. As a result, hepcidin increases cellular iron sequestration, and decreases the iron concentration in the plasma. Only mature hepcidin (result of the cleavage of prohepcidin by furin proteases) has biological activity; however, prohepcidin, the prohormone form, is also present in the plasma. In this study, we aimed to identify new protein-protein interactions of preprohepcidin, prohepcidin and hepcidin using the BacterioMatch two-hybrid system. Screening assays were carried out on a human liver cDNA library. Preprohepcidin screening gave the following results: a-1 antitrypsin, transthyretin and a-1-acid glycoprotein showed strong interactions with preprohepcidin. We further confirmed and examined the a-1 antitrypsin binding in vitro (glutathione S-transferase, pull down, coimmunoprecipitation, MALDI-TOF) and in vivo (ELISA, cross-linking assay). Our results demonstrated that the serine protease inhibitor a-1 antitrypsin binds preprohepcidin within the cell during maturation. Furthermore, a-1 antitrypsin binds prohepcidin significantly in the plasma. This observation may explain the presence of prohormone in the circulation, as well as the post-translational regulation of the mature hormone level in the blood. In addition, the lack of cleavage protection in patients with a-1 antitrypsin deficiency may be the reason for the disturbance in their iron homeostasis.Abbreviations A1AT, a-1

Proper diagnosis in drowning victims is often difficult due to the lack of signs specific to drowning. The diatom test is a widely used procedure for the diagnosis. Some types of water contain only minimal amounts of diatom cells which may provide false-negative results, while a negative diatom test result does not exclude drowning. In proving drowning, we used a polymerase chain reaction (PCR)-based biological method in addition to the conventional methods. DNA was extracted from postmortem spleen tissues and water of the drowning site. Samples were tested with algae (diatoms and small green algae)- and cyanobacteria (blue-green algae)-specific primers. We present here multiple drowning cases in which diatom tests of the postmortem tissue samples and the water were negative. In each case, the presence of phytoplanktonic DNA strengthened the autopsy diagnosis of drowning even in the absence of visible diatoms. In the future, the PCR method may be of consideration as a possible supplement of the diatom test in the examination of presumed drowning cases.

O‐linked β‐N‐acetlyglucosamine or O‐GlcNAc modification is a dynamic post‐translational modification occurring on the Ser/Thr residues of many intracellular proteins. The chronic imbalance between phosphorylation and O‐GlcNAc on tau protein is considered as one of the main hallmarks of Alzheimer's disease. In recent years, many studies also showed that O‐GlcNAc levels can elevate upon acute stress and suggested that this might facilitate cell survival. However, many consider chronic stress, including oxidative damage as a major risk factor in the development of the disease. In this study, using the neuronal cell line SH‐SY5Y we investigated the dynamic nature of O‐GlcNAc after treatment with 0.5 mM H2O2 for 30 min. to induce oxidative stress. We found that overall O‐GlcNAc quickly increased and reached peak level at around 2 hrs post‐stress, then returned to baseline levels after about 24 hrs. Interestingly, we also found that tau protein phosphorylation at site S262 showed parallel, whereas at S199 and PHF1 sites showed inverse dynamic to O‐Glycosylation. In conclusion, our results show that temporary elevation in O‐GlcNAc modification after H2O2‐induced oxidative stress is detectable in cells of neuronal origin. Furthermore, oxidative stress changes the dynamic balance between O‐GlcNAc and phosphorylation on tau proteins.

Hepcidin is the major regulatory peptide hormone of iron metabolism, encoded by the HAMP (hepcidin antimicrobial peptide) gene. Hepcidin is expressed mainly in hepatocytes, but is also found in the blood in both a mature and prohormone form. Although, the function of mature hepcidin and the regulation of the HAMP gene have been extensively studied, the intracellular localization and the fate of prohepcidin remains controversial. In the present study, we propose a novel role for prohepcidin in the regulation of its own transcription. Using indirect immunofluorescence and mCherry tagging, a portion of prohepcidin was detected in the nucleus of hepatocytes. Prohepcidin was found to specifically bind to the STAT3 (signal transducer and activator of transcription 3) site in the promoter of HAMP. Overexpression of prohepcidin in WRL68 cells decreased HAMP promoter activity, whereas decreasing the amount of prohepcidin caused increased promoter activity measured by a luciferase reporter-gene assay. Moreover, overexpression of the known prohepcidin-binding partner, α-1 antitrypsin caused increased HAMP promoter activity, suggesting that only the non-α-1 antitrypsin-bound prohepcidin affects the expression of its own gene. The results of the present study indicate that prohepcidin can bind to and transcriptionally regulate the expression of HAMP, suggesting a novel autoregulatory pathway of hepcidin gene expression in hepatocytes.