Assessing the performance of quantity and quality metrics using the QIAGEN Investigator® Quantiplex® pro RGQ kit.

The Quantiplex® Pro RGQ kit quantifies DNA in a sample, supports the detection of mixtures and assesses the extent of DNA degradation based on relative ratios of amplified autosomal and male markers. Data show no significant difference in the accuracy and sensitivity of quantification between this and the Promega PowerQuant® System, both detecting the lowest amount of DNA tested, 4 pg. Laboratory controlled mixed male:female DNA samples together with mock sexual assault samples were quantified across a range of mixture ratios. Analysis software detected mixed DNA samples across all ratios for both quantification kits. Subsequent STR analysis using the Investigator® 24Plex QS Kit was able to corroborate mixture detection down to 1:25 male:female DNA ratios, past which point mixtures appeared identical to single-source female samples. Analysis software also detected laboratory degraded DNA samples, with data showing a positive trend between the Degradation Index (DI) and length of time of sonication. When used on ancient remains the assay was able to triage samples for further analysis, and STR profiles were concordant with DNA quantification results in all instances. STR analyses of laboratory-controlled sensitivity, mixture, and degradation studies supports the quality metric obtained from quantification. These data support the use of the Quantiplex® Pro RGQ kit for sample screening and quantification in forensic casework and ancient DNA studies.

Forensic Investigation of a Shawl Linked to the "Jack the Ripper" Murders.

A set of historic murders, known as the "Jack the Ripper murders," started in London in August 1888. The killer's identity has remained a mystery to date. Here, we describe the investigation of, to our knowledge, the only remaining physical evidence linked to these murders, recovered from one of the victims at the scene of the crime. We applied novel, minimally destructive techniques for sample recovery from forensically relevant stains on the evidence and separated single cells linked to the suspect, followed by phenotypic analysis. The mtDNA profiles of both the victim and the suspect matched the corresponding reference samples, fortifying the link of the evidence to the crime scene. Genomic DNA from single cells recovered from the evidence was amplified, and the phenotypic information acquired matched the only witness statement regarded as reliable. To our knowledge, this is the most advanced study to date regarding this case.

SNP Arrays.

The papers published in this Special Issue "SNP arrays" (Single Nucleotide Polymorphism Arrays) focus on several perspectives associated with arrays of this type. The range of papers vary from a case report to reviews, thereby targeting wider audiences working in this field. The research focus of SNP arrays is often human cancers but this Issue expands that focus to include areas such as rare conditions, animal breeding and bioinformatics tools. Given the limited scope, the spectrum of papers is nothing short of remarkable and even from a technical point of view these papers will contribute to the field at a general level. Three of the papers published in this Special Issue focus on the use of various SNP array approaches in the analysis of three different cancer types. Two of the papers concentrate on two very different rare conditions, applying the SNP arrays slightly differently. Finally, two other papers evaluate the use of the SNP arrays in the context of genetic analysis of livestock. The findings reported in these papers help to close gaps in the current literature and also to give guidelines for future applications of SNP arrays.

Acute simulated soccer-specific training increases PGC-1α mRNA expression in human skeletal muscle.

The aim of the current study was to quantify oxygen uptake, heart rate and molecular responses of human skeletal muscle associated with mitochondrial biogenesis following an acute bout of simulated soccer training. Muscle biopsies (vastus lateralis) were obtained from nine active men immediately pre-completion, post-completion and 3 h post-completion of a laboratory-based soccer-specific training simulation on a motorised treadmill. The soccer-specific simulation was a similar intensity (55 ± 6% [Formula: see text]) and duration (60 min) as that observed in professional soccer training (e.g. standing 41%, walking 37%, jogging 11%, high-speed running 9% and sprinting 2%). Post-exercise, muscle glycogen decreased (Pre; 397 ± 86 mmol∙kg(-1) dw, Post; 344 ± 64 mmol∙kg(-1) dw; P = 0.03), plasma lactate increased (P < 0.001) up to ~4-5 mmol∙L(-1), non-esterified fatty acids and glycerol increased (P < 0.001) to values of 0.6 ± 0.2 mmol∙L(-1) and 145 ± 54 µmol∙L(-1), respectively. PGC-1α mRNA increased (P = 0.009) fivefold 3 h post-exercise. We provide novel data by demonstrating that soccer-specific training is associated with increases in PGC-1α mRNA. These data may have implications for practitioners in better understanding the metabolic and muscle responses to soccer-specific training protocols in the field.

Reduced carbohydrate availability enhances exercise-induced p53 signaling in human skeletal muscle: implications for mitochondrial biogenesis.

The mechanisms that regulate the enhanced skeletal muscle oxidative capacity observed when training with reduced carbohydrate (CHO) availability are currently unknown. The aim of the present study was to test the hypothesis that reduced CHO availability enhances p53 signaling and expression of genes associated with regulation of mitochondrial biogenesis and substrate utilization in human skeletal muscle. In a repeated-measures design, muscle biopsies (vastus lateralis) were obtained from eight active males before and after performing an acute bout of high-intensity interval running with either high (HIGH) or low CHO availability (LOW). Resting muscle glycogen (HIGH, 467 ± 19; LOW, 103 ± 9 mmol/kg dry wt) was greater in HIGH compared with LOW (P < 0.05). Phosphorylation (P-) of ACC(Ser79) (HIGH, 1.4 ± 0.4; LOW, 2.9 ± 0.9) and p53(Ser15) (HIGH, 0.9 ± 0.4; LOW, 2.6 ± 0.8) was higher in LOW immediately postexercise and 3 h postexercise, respectively (P < 0.05). Before and 3 h postexercise, mRNA content of pyruvate dehydrogenase kinase 4, mitochondrial transcription factor A, cytochrome-c oxidase IV, and PGC-1α were greater in LOW compared with HIGH (P < 0.05), whereas carnitine palmitoyltransferase-1 showed a trend toward significance (P = 0.09). However, only PGC-1α expression was increased by exercise (P < 0.05), where three-fold increases occurred independently of CHO availability. We conclude that the exercise-induced increase in p53 phosphorylation is enhanced in conditions of reduced CHO availability, which may be related to upstream signaling through AMPK. Given the emergence of p53 as a molecular regulator of mitochondrial biogenesis, such nutritional modulation of contraction-induced p53 activation has implications for both athletic and clinical populations.

Protein ingestion does not impair exercise-induced AMPK signalling when in a glycogen-depleted state: implications for train-low compete-high.

The aim of the present study was to test the hypothesis that consuming protein does not attenuate AMPK signalling when exercise is commenced in a glycogen-depleted state. After performing a glycogen-depleting protocol the evening before, the subsequent morning ten active men performed 45 min steady-state cycling at 50 % of peak power output (PPO) followed by an exercise capacity test (1-min intervals at 80 % PPO interspersed with 1-min periods at 40 % PPO). In a repeated measures design, subjects consumed 20 g of a casein hydrolysate solution (PRO) 45 min before exercise, 10 g during and a further 20 g immediately post-exercise, or an equivalent volume of a non-calorie taste matched placebo (PLA). Resting (PRO = 134 ± 29; PLA = 136 ± 28 mmol kg(-1)) and post-exercise muscle glycogen (PRO = 43 ± 16; PLA = 47 ± 18 mmol kg(-1)) was not different (P > 0.05) between trials nor was exercise capacity (PRO = 26 ± 9; PLA = 25 ± 10 min, P > 0.05). Phosphorylation of AMPK(Thr172) increased threefold immediately post-exercise (P < 0.05) and PGC1-mRNA increased sixfold at 3 h post-exercise (P < 0.05), though there were no differences between conditions (P > 0.05). In contrast, there was a trend (P = 0.08) for a divergent response in eEF2(Thr56) phosphorylation such that 1.5 fold increases post- and 3 h post-exercise in PLA were blunted with PRO, thus indicative of greater eEF2 activation. We conclude that athletes who deliberately incorporate training phases with reduced muscle glycogen into their training programmes may consume protein before, during and after exercise without negating signalling through the AMPK cascade.

Matched work high-intensity interval and continuous running induce similar increases in PGC-1α mRNA, AMPK, p38, and p53 phosphorylation in human skeletal muscle.

The aim of the present study was to test the hypothesis that acute high-intensity interval (HIT) running induces greater activation of signaling pathways associated with mitochondrial biogenesis compared with moderate-intensity continuous (CONT) running matched for work done. In a repeated-measures design, 10 active men performed two running protocols consisting of HIT [6 × 3-min at 90% maximal oxygen consumption (Vo(2max)) interspersed with 3-min recovery periods at 50% Vo(2max) with a 7-min warm-up and cool-down period at 70% Vo(2max)] or CONT (50-min continuous running at 70% Vo(2max)). Both protocols were matched, therefore, for average intensity, duration, and distance run. Muscle biopsies (vastus lateralis) were obtained preexercise, postexercise, and 3 h postexercise. Muscle glycogen decreased (P < 0.05) similarly in HIT and CONT (116 ± 11 vs. 111 ± 17 mmol/kg dry wt, respectively). Phosphorylation (P-) of p38MAPK(Thr180/Tyr182) (1.9 ± 0.1- vs. 1.5 ± 0.2-fold) and AMPK(Thr172) (1.5 ± 0.3- vs. 1.5 ± 0.1-fold) increased immediately postexercise (P < 0.05) in HIT and CONT, respectively, and returned to basal levels at 3 h postexercise. P-p53(Ser15) (HIT, 2.7 ± 0.8-fold; CONT, 2.1 ± 0.8-fold), PGC-1α mRNA (HIT, 4.2 ± 1.7-fold; CONT, 4.5 ± 0.9-fold) and HSP72 mRNA (HIT, 4.4 ± 2-fold; CONT, 3.5 ± 1-fold) all increased 3 h postexercise (P < 0.05) although neither parameter increased (P > 0.05) immediately postexercise. There was no difference between trials for any of the above signaling or gene expression responses (P > 0.05). We provide novel data by demonstrating that acute HIT and CONT running (when matched for average intensity, duration, and work done) induces similar activation of molecular signaling pathways associated with regulation of mitochondrial biogenesis. Furthermore, this is the first report of contraction-induced p53 phosphorylation in human skeletal muscle, thus highlighting an additional pathway by which exercise may initiate mitochondrial biogenesis.

An RNA expression method for aging forensic hair samples.

A common limitation to most forensic trace evidence analysis is the ability to determine the time at which the evidence was deposited at the crime scene. This issue of timing is vitally important as it may not only reveal when the crime occurred, but could exclude potential suspects from the investigation. Using a reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, we monitored the relative expression ratio (RER) of two different RNA species (18S and ß-actin) in hair samples that were aged naturally over a period of 3 months. No gender or age-of-donor biases were observed, and results were linear up to 60 days. After 60 days, the results were more variable and gave unreliable estimates of time since deposition. Overall, the results presented in this paper suggest that the age of hair samples containing follicular tags can be approximated using a second-order polynomial, although with limitations: Age = 3.31RER(2) - 2.85RER - 0.54 (R(2) = 0.98).

RNA expression profiling of normal and tumor cells following photodynamic therapy with 5-aminolevulinic acid-induced protoporphyrin IX in vitro.

Photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX synthesis as a photosensitizing reagent is an encouraging modality for cancer treatment. Understanding the mechanism of tumor phototoxicity is important to provide a basis for combinatory therapy regimens. A normal cell line (UROtsa, urothelial) and two tumor cell lines (RT4, urothelial; HT29, colonic) were treated with cell line-specific LD50 doses of light after exposure to 5-aminolevulinic acid (100 microg/mL), and harvested for RNA extraction 0, 10, and 30 minutes after irradiation. The RNA was hybridized to the metg001A Affymetrix GeneChip containing 2,800 genes, focusing on cancer-related and growth regulatory targets. Comparing the gene expression profiles between the different samples, 40 genes (e.g., SOD2, LUC7A, CASP8, and DUSP1) were identified as significantly altered in comparison with the control samples, and grouped according to their gene ontology. We selected caspase-8 (CASP8) and dual specificity phosphatase 1 (DUSP1) for further validation of the array findings, and compared their expression with the expression of the immediate early gene FOS by quantitative reverse transcription-PCR. RNA expression of CASP8 stayed unchanged whereas DUSP1 RNA was up-regulated in normal and tumor cells starting 30 minutes after irradiation. In contrast, FOS RNA was found continuously up-regulated over time in all three cell lines. Induction of DUSP1 protein expression was clearly shown after 1 hour using Western blot analysis. Interestingly, no changes of caspase-8 protein expression but activation of catalytic activity was detected only in UROtsa cells starting 1 hour after photodynamic therapy, whereas no changes were seen in both tumor cell lines. According to caspase-8, the active caspase 3 fragment was found only in the normal urothelial cell line (UROtsa) 1 hour after photodynamic therapy. Combined data analysis suggests that photodynamic therapy in vitro (LD50) leads to apoptosis in UROtsa and to necrosis in the tumor cell lines, respectively. RNA expression profiling of normal and tumor cell lines following photodynamic therapy with 5-aminolevulinic acid gave insight into the major molecular mechanisms induced by photodynamic therapy.

High-resolution deletion mapping of 15q13.2-q21.1 in transitional cell carcinoma of the bladder.

Deletions found in several types of human tumor, including carcinomas of the colorectum, breast, and lung, suggest the presence of a potential tumor suppressor gene(s) on chromosome 15. Common regions of deletion in these tumors are at 15q15 and 15q21. Here, we have analyzed loss of heterozygosity (LOH) on chromosome 15 to ascertain its potential involvement in the development and progression of transitional cell carcinoma (TCC) of the bladder. A panel of 26 polymorphic markers, spanning 15q12-15q22, were used to map regions of LOH in 51 TCCs. LOH was found for at least one marker in the region 15q14-15q15.3 in 20 of 51 (39%) tumors. Deletion mapping defined two minimum regions of deletion: a distal region between the markers D15S514 and D15S537 at 15q15.1-15q15.3 (estimated as 3 Mb) and a more proximal region between the markers D15S971 and D15S1042 at 15q14 (estimated as 1.1 Mb). Analysis of a panel of 33 bladder tumor cell lines revealed regions of contiguous homozygosity for markers in 15q15, indicating likely LOH. Fluorescence in situ hybridization analysis demonstrated that mitotic recombination is the predicted mechanism of LOH in two of these. These regions of LOH on 15q may contain tumor suppressor genes the loss or inactivation of which is associated with TCC development. The DNA repair gene RAD51 at 15q15.1 represents a candidate 15q tumor suppressor gene. Expression analysis of rad51 protein in tumor cell lines revealed variable levels of expression but no significant loss of expression in cell lines with likely 15q LOH.