Evaluation of the effect of ozone disinfection on forensic identification of blood, saliva, and semen stains.

Good laboratory practice minimizes the biological hazard posed by potentially infectious casework samples. In certain scenarios, when the casework sample is contaminated with highly contagious pathogens, additional safety procedures such as disinfection might be advised. It was previously proven that ozone gas treatment does not hamper STR analysis, but there is no data on how the disinfection affects other steps of the forensic analysis. In this study, we aimed to assess the interference of ozone disinfection with forensic tests used to identify biological stains. A dilution series of blood, saliva, and semen samples were pipetted onto cotton fabric and let completely dry. Half of the samples were subjected to ozone treatment, while the rest served as controls. All the samples were tested with specific lateral flow immunochromatographic assays and for specific RNA markers with quantitative real-time PCR. Additionally, luminol test was carried out on blood spots, Phadebas® Amylase Test on saliva stains, and semen stains were examined with STK Lab kit and light microscope following Christmas Tree or Hematoxylin-Eosin staining. Ozone treatment had no detrimental effect on the microscopic identification of sperm cells. Undiluted blood samples were detected with luminol and immunoassay, but at higher dilution, the sensitivity of the test decreased after disinfection. The same decrease in sensitivity was observed in the detection of semen stains using STK Lab kit from STK® Sperm Tracker, and in the case of the immunoassay specific for prostate-specific antigen (PSA). Ozone treatment almost completely inhibited the enzymatic activity of amylase. The sensitivity of antibody-based detection of amylase was also greatly reduced. RNA markers showed degradation but remained detectable in blood and semen samples after incubation in the presence of ozone. In saliva, the higher Ct values of the mRNA markers were close to the detection limit, even before ozone treatment.

Michaelis-Menten kinetic modeling of hemoglobin A1c status facilitates personalized glycemic control.

INTRODUCTION: Discrepancy between measured HbA1c and HbA1c calculated from plasma glucose is associated with higher risk for diabetic complications. However, quantification of this difference is inaccurate due to the imperfect linear conversion models. We propose to introduce a mathematical formula that correlates with the observational data and supports individualized glycemic control. METHODS: We analysed 175,437 simultaneous plasma glucose and HbA1c records stored in our laboratory database. Employing the Michaelis-Menten (MM) equation, we compared the calculated HbA1c levels to the measured HbA1c levels. Data from patients with multiple records were used to establish the patients' glycemic status and to assess the predictive power of our MM model. RESULTS: HbA1c levels calculated with the MM equation closely matched the population's average HbA1c levels. The Michaelis constant (Km) had a negative correlation with HbA1c (r2 = 0.403). Using personalized Km values in the MM equation, 85.1% of HbA1c predictions were within 20% error (ADAG calculation: 78.4%). MM prediction also performed better in predicting pathologic HbA1c levels (0.904 AUC vs. 0.849 AUC for ADAG). CONCLUSION: MM equation is an improvement over linear models and could be readily employed in routine diabetes management. Km is a reliable and quantifiable marker to characterize variations in glucose tolerance.

Effect of ozone disinfection on forensic STR profiling.

While good laboratory practice in a forensic lab minimises the chance of infection of laboratory personnel, in certain cases, possible contamination of pieces of evidence with highly contagious pathogens might call for additional precautions. The number of potential disinfection methods that might be suitable for forensic genetics are surprisingly limited. First and foremost, the ideal technique should not inhibit DNA amplification, it should be effective against a host of pathogens, and it should be applicable on porous surfaces. We examined ozone treatment on extracted DNA samples and mock casework samples. Ozone-treated and control specimens were amplified with Qiagen Investigator ESSplex SE QS kit. Detected allele counts were compared between the treated and untreated sample groups. Following disinfection, concentration of ozone-treated DNA was about half of the control samples, but full STR profiles were recovered. In the case of mock casework samples (disposable surgical masks), there was no significant difference (p = 0.513) between the detected allele counts of control and ozone-treated samples. Sampling location of surgical masks (earloop, nosepiece) showed a statistically significant difference (p = 0.011), though. Comparing the effect of contributors on STR profiling, a significant difference (p = 0.001) was observed, which could be explained with the differences between individuals including shedding capacity, head size or shape. According to our pilot study, ozone treatment does not encumber the routine forensic DNA analysis, the sampling position or the contributor affected the allele counts more than the ozone treatment.

PCR-based identification of drowning: four case reports.

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.

The rate of RNA degradation in human dental pulp reveals post-mortem interval.

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.

Drowning-related fatalities during a 5-year period (2008-2012) in South-West Hungary--a retrospective study.

The purpose of the present study was to investigate all the drowning-related cases in South-West Hungary between 2008 and 2012. It is a retrospective and descriptive study of 114 drowning-related deaths during that 5-year period. The investigation includes both unintentional and intentional drowning cases. We found that the most considerable risk factor of drowning in this area of Hungary is alcohol consumption, because in more than half of the cases the victims were under the influence of alcohol. We also concluded that more than two third of the cases included males. The cause of drowning in younger victims was mostly accident. In both genders subjects aged 50-70 had the highest risk of drowning in the period investigated. Seniors frequently committed suicide by drowning. Drowning deaths occurred in all types of water, mostly in lakes, rivers, canals and other types of catchment in the vicinity of victims' homes (most commonly in wells). This study might help to understand the circumstances and the causes leading to drowning and it may draw the attention to the possible preventive interventions.

Lithium induces ER stress and N-glycan modification in galactose-grown Jurkat cells.

We previously reported that lithium had a significant impact on Ca(2+) regulation and induced unfolded protein response (UPR) in yeast cells grown on galactose due to inhibition of phosphoglucomutase (PGM), however the exact mechanism has not been established yet. In this study, we analysed lithium's effect in galactose-fed cells to clarify whether these ER-related changes are the result of a relative hypoglycemic state. Furthermore, we investigated whether the alterations in galactose metabolism impact protein post-translational modifications. Thus, Jurkat cells were incubated in glucose or galactose containing media with or without lithium treatment. We found that galactose-fed and lithium treated cells showed better survivability than fasting cells. We also found higher UDP-Hexose and glycogen levels in these cells compared to fasting cells. On the other hand, the UPR (X-box binding protein 1 mRNA levels) of galactose-fed and lithium treated cells was even greater than in fasting cells. We also found increased amount of proteins that contained N-linked N-acetyl-glucosamine, similar to what was reported in fasting cells by a recent study. Our results demonstrate that lithium treatment of galactose-fed cells can induce stress responses similar to hypoglycemia, however cell survival is still secured by alternative pathways. We propose that clarifying this process might be an important addition toward the better understanding of the molecular mechanisms that regulate ER-associated stress response.

Prohepcidin binds to the HAMP promoter and autoregulates its own expression.

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.

Serum prohepcidin levels in chronic inflammatory bowel diseases.

BACKGROUND AND AIMS: One of the major symptoms of chronic inflammatory bowel diseases is anemia. The two most common diseases are Crohn's disease and ulcerative colitis. Anemia may develop due to intestinal bleeding, iron absorption disturbances, or high levels of inflammatory cytokines. It is not clear whether or not hepcidin, the only known hormone regulating cellular iron uptake in mammals is involved. The transcription of hepcidin is controlled by the iron status of the body, hypoxia, and/or inflammation. This study was meant to find relationship between serum prohepcidin levels and clinical parameters of iron homeostasis or inflammatory state in patients suffering from Crohn's disease or ulcerative colitis. METHODS: Serum prohepcidin levels were measured with ELISA in 72 patients diagnosed with ulcerative colitis and 30 patients suffering from Crohn's disease. RESULTS: In both groups serum iron levels were lower, while levels of C-reactive protein were higher than in the healthy controls. Serum prohepcidin levels showed no significant differences compared to those in the control group. In the affected patients only weak correlations were observed between prohepcidin levels and diagnostic parameters: in Crohn's disease prohepcidin levels correlated positively with transferrin levels, total iron-binding capacity, transferrin saturation, activity index, and serum albumin levels, while in ulcerative coltitis prohepcidin levels were related to transferrin levels and transferrin saturation. CONCLUSION: It seems obvious that serum prohepcidin level determination in itself is not a satisfactory diagnostic or prognostic measure in anemia of chronic inflammatory bowel diseases.

Alpha-1 antitrypsin binds preprohepcidin intracellularly and prohepcidin in the serum.

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: alpha-1 antitrypsin, transthyretin and alpha-1-acid glycoprotein showed strong interactions with preprohepcidin. We further confirmed and examined the alpha-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 alpha-1 antitrypsin binds preprohepcidin within the cell during maturation. Furthermore, alpha-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 alpha-1 antitrypsin deficiency may be the reason for the disturbance in their iron homeostasis.