Improving the differentiation of closely related males by RMplex analysis of 30 Y-STRs with high mutation rates.

The discovery of rapidly mutating (RM) Y-STRs started to move the field of forensic Y-STR analysis from male lineage identification towards male individual identification. Previously, the forensic value of RM Y-STRs for differentiating male relatives was limited due to the modest number of 13 identified RM Y-STRs. Recently, new RM Y-STRs were discovered, with strong expectations for significantly improving male relative differentiation; however, empirical evidence is missing yet. More recently, the genotyping method RMplex for efficiently analyzing 30 Y-STRs with high mutation rates, including all 26 currently known RM Y-STRs, was introduced. Here, we applied RMplex as well as the current state-of-the-art commercial Y-STR kit: Yfiler™ Plus PCR Amplification kit, to several hundreds of DNA-confirmed father-son pairs. Newly established estimates confirmed the high mutation rates of novel and previous RM Y-STRs. By combining current with previous data, we provide updated consensus estimates of mutation rates for all 49 Y-STRs targeted with both methods. Based on RMplex, 42% of 499 father-son pairs were differentiated, while 14% of 530 pairs based on Yfiler™ Plus, and 48% of 499 pairs based on both methods combined. Regarding brothers, RMplex also clearly outperformed Yfiler™ Plus, with differentiation rates of 62% and 33%, respectively. By combining both methods 72.9% of the brothers showed at least one mutation. For unrelated males, both methods achieved a discrimination capacity of 99.8% and a haplotype diversity of 0.999991, since all males had different haplotypes, except for two, perhaps indicating a hidden paternal relationship. Overall, this study underlines the value of RM Y-STRs in general and RMplex in particular for differentiating male relatives highly relevant in forensic genetics. It provides the first empirical evidence on the high value of RMplex for differentiating close male relatives, which for father-son pairs was almost 60% higher than with the initial set of 13 RM Y-STRs and three times higher than with Yfiler™ Plus. Based on our results from closely related males, we expect RMplex to also improve the differentiation of more distantly related males significantly, which needs empirical demonstration in future studies. We encourage the forensic community to apply RMplex in all forensic cases where a match with a commercial Y-STR kit was obtained between the male suspect and the evidence material, or to solely use RMplex in such cases, aiming to find out if the male suspect or any of his male paternal relatives left the evidence material at the crime scene.

Suitability of specific soft tissue swabs for the forensic identification of highly decomposed bodies.

When decomposition of a recovered body is fairly advanced, identification based on common morphologic features is often impossible. In these cases, short tandem repeat (STR) marker genotyping has established itself as a convenient and reliable alternative. However, at very progressed stages of decomposition, postmortem tissue putrefaction processes can decrease DNA yields considerably. Hence, not all types of tissue are equally suitable for successful STR marker-based postmortem identification. Bone or dental material is often analysed in corpses with advanced decompositional changes. However, processing of these materials is very elaborate and time and resource consuming. We have therefore focused on the suitableness of various types of soft tissue swabs, where DNA extraction is easier and faster. By sampling 28 bodies at various stages of decomposition, we evaluated the suitability of different tissues for genotyping at varying degrees of physical decay. This was achieved by a systematic classification of the sampled bodies by morphological scoring and subsequent analysis of multiple tissue swabs of the aortic wall, urinary bladder wall, brain, liver, oral mucosa and skeletal muscle. In summary, we found variable degrees of suitability of different types of soft tissue swabs for DNA-based identification. Swabs of the aortic wall, the urinary bladder wall and brain tissue yielded the best results - in descending order - even at advanced levels of decay.

Acid- and Volume-Sensitive Chloride Currents in Human Chondrocytes.

Chondrocytes face extreme alterations of extracellular osmolarity and pH, which force them to appropriately regulate their cell volume (CV) and cellular pH. Perturbations of these mechanisms lead to chondrocyte death and ultimately to osteoarthritis (OA), the most common chronic joint diseases worldwide. OA hallmarks are altered cartilage hydration and severe fluid acidification. Impaired CV regulation and acidotoxicity contribute to disease progression and volume-sensitive anion channels are upregulated in OA. This study assessed the effect of hypotonicity and extracellular acidification on chondrocyte Cl- conductances and CV regulation. Cl- currents and membrane potentials were measured in human C28/I2 cells and primary human chondrocytes using the patch clamp technique. Intracellular pH was assessed by BCECF fluorescence, CV measurements were performed using the Coulter method, and cell viability/cell death by a resazurin assay. Hypotonic cell swelling caused activation of a volume-sensitive outwardly rectifying (VSOR) Cl- current followed by a regulatory volume decrease (RVD), which was attenuated by the Cl- channel blocker DCPIB. Extracellular, but not intracellular acidification to pH ≤ 5.0 elicited an acid-sensitive outwardly rectifying (ASOR) Cl- conductance. Activation of either current depolarized the cell membrane potential. Under simultaneous hypotonic and acidic stimulation, VSOR and ASOR currents transiently coactivated, giving rise to a mixed current phenotype. Over time the VSOR current gradually vanished and the residual conductance showed a pure ASOR current phenotype. Extracellular acidification caused an isotonic CV gain and a complete suppression of RVD under hypotonic conditions. The results suggest that deactivation of the VSOR current under acidic conditions impairs CV regulation in chondrocytes, which is likely to compromise chondrocyte viability.

The applicability of forensic time since death estimation methods for buried bodies in advanced decomposition stages.

Estimation of the postmortem interval in advanced postmortem stages is a challenging task. Although there are several approaches available for addressing postmortem changes of a (human) body or its environment (ecologically and/or biochemically), most are restricted to specific timeframes and/or individual and environmental conditions. It is well known, for instance, that buried bodies decompose in a remarkably different manner than on the ground surface. However, data on how established methods for PMI estimation perform under these conditions are scarce. It is important to understand whether and how postmortem changes are affected under burial conditions, if corrective factors could be conceived, or if methods have to be excluded for respective cases. We present the first multi-methodological assessment of human postmortem decomposition carried out on buried body donors in Europe, at the Amsterdam Research Initiative for Sub-surface Taphonomy and Anthropology (ARISTA) in the Netherlands. We used a multidisciplinary approach to investigate postmortem changes of morphology, skeletal muscle protein decomposition, presence of insects and other necrophilous animals as well as microbial communities (i.e., microbiomes) from August to November 2018 associated with two complete body exhumations and eight partial exhumations. Our results clearly display the current possibilities and limitations of methods for PMI estimation in buried remains and provide a baseline for future research and application.

Acid- and Volume-Sensitive Chloride Currents in Microglial Cells.

Many cell types express an acid-sensitive outwardly rectifying (ASOR) anion current of an unknown function. We characterized such a current in BV-2 microglial cells and then studied its interrelation with the volume-sensitive outwardly rectifying (VSOR) Cl- current and the effect of acidosis on cell volume regulation. We used patch clamp, the Coulter method, and the pH-sensitive dye BCECF to measure Cl- currents and cell membrane potentials, mean cell volume, and intracellular pH, respectively. The ASOR current activated at pH ≤ 5.0 and displayed an I- > Cl- > gluconate- permeability sequence. When compared to the VSOR current, it was similarly sensitive to DIDS, but less sensitive to DCPIB, and insensitive to tamoxifen. Under acidic conditions, the ASOR current was the dominating Cl- conductance, while the VSOR current was apparently inactivated. Acidification caused cell swelling under isotonic conditions and prevented the regulatory volume decrease under hypotonicity. We conclude that acidification, associated with activation of the ASOR- and inactivation of the VSOR current, massively impairs cell volume homeostasis. ASOR current activation could affect microglial function under acidotoxic conditions, since acidosis is a hallmark of pathophysiological events like inflammation, stroke or ischemia and migration and phagocytosis in microglial cells are closely related to cell volume regulation.

The Cancer Stem Cell Inhibitor Napabucasin (BBI608) Shows General Cytotoxicity in Biliary Tract Cancer Cells and Reduces Cancer Stem Cell Characteristics.

Biliary tract cancer is a devastating disease with limited therapeutic options. The involvement of cancer stem cells in biliary tract cancer is likely. Napabucasin is a previously described cancer stem cell inhibitor that is currently being used in clinical trials. However, data regarding napabucasin and biliary tract cancer are not available yet. We tested the general cytotoxic effect of napabucasin on a comprehensive biliary tract cancer in vitro model, using resazurin assay and Annexin V/7-AAD staining. The effect of napabucasin on functional cancer stem cell characteristics was analyzed via soft agar assay, aldehyde-dehydrogenase-1 assay, measurement of surface CD326 expression, and measurement of clonogenic growth. The evaluation of the effect of napabucasin on cancer stem cell protein and gene expression was performed using Western blot and reverse transcription-PCR-based human cancer stem cell array. Napabucasin showed a concentration- and cell line-dependent cytotoxic effect, and increased the apoptotic and necrotic cell fractions. Treatment with napabucasin significantly reduced the formation of tumor spheres and clonogenic growth, as well as CD326 surface expression. Expression of cancer stem cell markers were reduced following napabucasin treatment on the protein and mRNA levels. Our study provides first data regarding napabucasin as a promising substance for the treatment of biliary tract cancer.

Continuous, label-free, 96-well-based determination of cell migration using confluence measurement.

Cellular migration is essential in diverse physiological and pathophysiological processes. Here, we present a protocol for quantitative analysis of migration using confluence detection allowing continuous, non-endpoint measurement with minimal hands-on time under cell incubator conditions. Applicability was tested using substances which enhance (EGF) or inhibit (cytochalasin D, ouabain) migration. Using a gap-closure assay we demonstrate that automated confluence detection monitors cellular migration in the 96-well microplate format. Quantification by % confluence, % cell free-area or % confluence in cell-free area against time, allows detailed analysis of cellular migration. The study describes a practicable approach for continuous, non-endpoint measurement of migration in 96-well microplates and for detailed data analysis, which allows for medium/high-throughput analysis of cellular migration in vitro.

Neuronal connectivity in major depressive disorder: a systematic review.

BACKGROUND: The causes of major depressive disorder (MDD), as one of the most common psychiatric disorders, still remain unclear. Neuroimaging has substantially contributed to understanding the putative neuronal mechanisms underlying depressed mood and motivational as well as cognitive impairments in depressed individuals. In particular, analyses addressing changes in interregional connectivity seem to be a promising approach to capture the effects of MDD at a systems level. However, a plethora of different, sometimes contradicting results have been published so far, making general conclusions difficult. Here we provide a systematic overview about connectivity studies published in the field over the last decade considering different methodological as well as clinical issues. METHODS: A systematic review was conducted extracting neuronal connectivity results from studies published between 2002 and 2015. The findings were summarized in tables and were graphically visualized. RESULTS: The review supports and summarizes the notion of an altered frontolimbic mood regulation circuitry in MDD patients, but also stresses the heterogeneity of the findings. The brain regions that are most consistently affected across studies are the orbitomedial prefrontal cortex, anterior cingulate cortex, amygdala, hippocampus, cerebellum and the basal ganglia. CONCLUSION: The results on connectivity in MDD are very heterogeneous, partly due to different methods and study designs, but also due to the temporal dynamics of connectivity. While connectivity research is an important step toward a complex systems approach to brain functioning, future research should focus on the dynamics of functional and effective connectivity.

Pharmacological Inhibition of Class IIA HDACs by LMK-235 in Pancreatic Neuroendocrine Tumor Cells.

Histone deacetylases (HDACs) play a key role in epigenetic mechanisms in health and disease and their dysfunction is implied in several cancer entities. Analysis of expression patterns in pancreatic neuroendocrine tumors (pNETs) indicated HDAC5 to be a potential target for future therapies. As a first step towards a possible treatment, the aim of this study was to evaluate the in vitro cellular and molecular effects of HDAC5 inhibition in pNET cells. Two pNET cell lines, BON-1 and QGP-1, were incubated with different concentrations of the selective class IIA HDAC inhibitor, LMK-235. Effects on cell viability were determined using the resazurin-assay, the caspase-assay, and Annexin-V staining. Western Blot and immunofluorescence microscopy were performed to assess the effects on HDAC5 functionality. LMK-235 lowered overall cell viability by inducing apoptosis in a dose- and time-dependent manner. Furthermore, acetylation of histone-H3 increased with higher LMK-235 concentrations, indicating functional inhibition of HDAC4/5. Immunocytochemical analysis showed that proliferative activity (phosphohistone H3 and Ki-67) decreased at highest concentrations of LMK-235 while chromogranin and somatostatin receptor 2 (SSTR2) expression increased in a dose-dependent manner. HDAC5 expression was found to be largely unaffected by LMK-235. These findings indicate LMK-235 to be a potential therapeutic approach for the development of an effective and selective pNET treatment.

HDAC-Linked "Proliferative" miRNA Expression Pattern in Pancreatic Neuroendocrine Tumors.

Epigenetic factors are essentially involved in carcinogenesis, tumor promotion, and chemoresistance. Two epigenetic key players are miRNAs and histone deacetylases (HDACs). As previously shown by own theoretical databank analysis, the crosstalk between miRNAs and HDACs is relevant in different human chronic diseases and cancerogenic pathways. We aimed to investigate a potential connection between the expression of a well-defined subset of "proliferation-associated" miRNAs and the expression of HDACs as well as clinical parameters in pancreatic neuroendocrine tumors (pNETs). MATERIALS AND METHODS: Expression levels of miRNA132-3p, miRNA145-5p, miRNA183-5p, miRNA34a-5p, and miRNA449a in 57 pNETs resected between 1997 and 2015 were measured and linked to the immunohistochemical expression pattern of members of the four HDAC classes on human tissue microarrays. All pNET cases were clinically and pathologically characterized according to published guidelines. Correlation analysis revealed a significant association between expression of specific miRNAs and two members of the HDAC family (HDAC3 and HDAC4). Additionally, a linkage between miRNA expression and clinico-pathological parameters like grading, TNM-staging, and hormone activity was found. Moreover, overall and disease-free survival is statistically correlated with the expression of the investigated miRNAs. Overall, we demonstrated that specific miRNAs could be linked to HDAC expression in pNETs. Especially miRNA449a (associated with HDAC3/4) seems to play an important role in pNET proliferation and could be a potential prognostic factor for poor survival. These first data could help, to improve our knowledge of the complex interactions of the epigenetic drivers in pNETs for further therapeutic approaches.

The histone methyltransferase G9a: a new therapeutic target in biliary tract cancer.

The histone methyltransferase G9a (EHMT2) is a key enzyme for dimethylation of lysine 9 at histone 3 (H3K9me2), a suppressive epigenetic mark. G9a is over-expressed in tumor cells and contributes to cancer aggressiveness. Biliary tract cancer (BTC) is a rare cancer with dismal prognosis due to a lack of effective therapies. Currently, there are no data on the role of G9a in BTC carcinogenesis. We analyzed G9a expression in n=68 BTC patient specimens and correlated the data with clinicopathological and survival data. Moreover, we measured G9a expression in a panel of BTC cell lines and evaluated the cytotoxic effect of G9a inhibition in BTC cells using established small-molecule G9a inhibitors. G9a was considerably expressed in about half of BTC cases and was significantly associated with grading and tumor size. Additionally, we observed significant differences of G9a expression between growth type and tumor localization groups. G9a expression diametrically correlated with Vimentin (positive) and E-Cadherin (negative) expression. Importantly, survival analysis revealed G9a as a significant prognostic factor of poor survival in patients with BTC. In BTC cells, G9a and H3K9me2 were detectable in a cell line-dependent manner on mRNA and/or protein level, respectively. Treatment of BTC cells with established small-molecule G9a inhibitors resulted in reduction of cell viability as well as reduced G9a and H3K9me2 protein levels. The present study strongly suggests that G9a contributes to BTC carcinogenesis and may represent a potential prognostic factor as well as a therapeutic target.

In Vitro Cell Death Discrimination and Screening Method by Simple and Cost-Effective Viability Analysis.

BACKGROUND/AIMS: For in vitro cytotoxicity testing, discrimination of apoptosis and necrosis represents valuable information. Viability analysis performed at two different time points post treatment could serve such a purpose because the dynamics of metabolic activity of apoptotic and necrotic cells is different, i.e. a more rapid decline of cellular metabolism during necrosis whereas cellular metabolism is maintained during the entire execution phase of apoptosis. This study describes a straightforward approach to distinguish apoptosis and necrosis. METHODS: A431 human epidermoid carcinoma cells were treated with different concentrations/doses of actinomycin D (Act-D), 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), Ro 31-8220, H2O2 and photodynamic treatment (PDT). The resazurin viability signal was recorded at 2 and 24 hrs post treatment. Apoptosis and necrosis were verified by measuring caspase 3/7 and membrane integrity. RESULTS: Calculation of the difference curve between the 2 and 24 hrs resazurin signals yields the following information: a positive difference signal indicates apoptosis (i.e. high metabolic activity at early time points and low signal at 24 hrs post treatment) while an early reduction of the viability signal indicates necrosis. For all treatments, this dose-dependent sequence of cellular responses could be confirmed by independent assays. CONCLUSION: Simple and cost-effective viability analysis provides reliable information about the dose ranges of a cytotoxic agent where apoptosis or necrosis occurs. This may serve as a starting point for further in-depth characterisation of cytotoxic treatments.

Quercetin Stimulates Insulin Secretion and Reduces the Viability of Rat INS-1 Beta-Cells.

BACKGROUND/AIMS: Previously we described insulinotropic effects of Leonurus sibiricus L. plant extracts used for diabetes mellitus treatment in Traditional Mongolian Medicine. The flavonoid quercetin and its glycoside rutin, which exert anti-diabetic properties in vivo by interfering with insulin signaling in peripheral target tissues, are constituents of these extracts. This study was performed to better understand short- and long-term effects of quercetin and rutin on beta-cells. METHODS: Cell viability, apoptosis, phospho-protein abundance and insulin release were determined using resazurin, annexin-V binding assays, Western blot and ELISA, respectively. Membrane potentials (Vmem), whole-cell Ca2+ (ICa)- and ATP-sensitive K+ (IKATP) currents were measured by patch clamp. Intracellular Ca2+ (Cai) levels were measured by time-lapse imaging using the ratiometric Ca2+ indicator Fura-2. RESULTS: Rutin, quercetin and the phosphoinositide-3-kinase (PI3K) inhibitor LY294002 caused a dose-dependent reduction in cell viability with IC50 values of ∼75 µM, ∼25 µM and ∼3.5 µM, respectively. Quercetin (50 µM) significantly increased the percentage of Annexin-V+ cells within 48 hrs. The mean cell volume (MCV) of quercetin-treated cells was significantly lower. Within 2 hrs, quercetin significantly decreased basal- and insulin-stimulated Akt(T308) phosphorylation and increased Erk1/2 phosphorylation, without affecting P-Akt(S473) abundance. Basal- and glucose-stimulated insulin release were significantly stimulated by quercetin. Quercetin significantly depolarized Vmem by ∼25 mV which was prevented by the KATP-channel opener diazoxide, but not by the L-type ICa inhibitor nifedipine. Quercetin significantly stimulated ICa and caused a 50% inhibition of IKATP. The effects on Vmem, ICa and IKATP rapidly reached peak values and then gradually diminished to control values within ∼1 minute. With a similar time-response quercetin induced an elevation in Cai which was completely abolished in the absence of Ca2+ in the bath solution. Rutin (50 µM) did not significantly alter the percentage of Annexin-V+ cells, MCV, Akt or Erk1/2 phosphorylation, insulin secretion, or the electrophysiological behavior of INS-1 cells. CONCLUSION: We conclude that quercetin acutely stimulates insulin release, presumably by transient KATP channel inhibition and ICa stimulation. Long term application of quercetin inhibits cell proliferation and induces apoptosis, most likely by inhibition of PI3K/Akt signaling.

Investigations on the effect of caffeine on cerebral venous vessel contrast by using susceptibility-weighted imaging (SWI) at 1.5, 3 and 7 T.

Caffeine lowers the blood oxygenation level-dependent (BOLD) signal by acting as an adenosine antagonist, thus decreasing the cerebral blood flow (CBF). The aims of this study were to demonstrate the sensitivity of susceptibility-weighted imaging (SWI) to caffeine-induced changes in CBF and to investigate the time course and magnitude of signal change in caffeine-habituated and -abstinent volunteers. High-resolution susceptibility-weighted images were acquired with both groups at 1.5 T using a fully velocity compensated 3D gradient echo sequence. Following a native scan, subjects were given a tablet containing 200 mg of caffeine. Scans were repeated for about 1 h and the acquired 3D data sets were co-registered to each other. BOLD signal changes of several venous vessels were analyzed in dedicated ROIs. Maps of relative signal change clearly visualized the caffeine-induced signal response of veins. Only very weak signal changes of about -2+/-1% were found in both, grey and white matter and -1+/-2% in the ventricles. Maximum signal decrease of veins occurred 40-50 min after caffeine ingestion. The signal decrease was -16.5+/-6.5% and -22.7+/-8.3% for the caffeine users group and abstainers, respectively. The signal difference of both groups was statistically significant (Student's t-test, t=2.16, p=0.021). Data acquired at 1.5, 3 and 7 T with echo times scaled to the respective field strength display very similar temporal signal behavior.