A sandwich enzyme immunoassay for human muscle-specific beta-enolase and its application for the determination of skeletal muscle injury

A sensitive sandwich enzyme immunoassay for human beta-enolase was developed and used to examine beta-enolase in blood or bloodstains as a marker for the determination of skeletal muscle injury. Human beta-enolase was purified from human skeletal muscle, and then an antibody against it was prepared. Polystyrene balls coated with rabbit anti-human beta-enolase IgG were incubated with human beta-enolase and then with anti-human beta-enolase Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as a hydrogen donor. The detection limit for human beta-enolase was 2.6 pg (30 amol) per assay. The degree of cross-reaction of the sandwich enzyme immunoassay for other organs except for heart (1/10) was about 1/150 or less. Moreover, the localization of beta-enolase in various human tissues was examined by Northern blot analysis, and this confirmed that beta-enolase was expressed only in skeletal and cardiac muscle. Antigenic activity in bloodstains containing beta-enolase was recovered well after storage for 60 days at room temperature. The ratio of beta-enolase to total protein in bloodstains made from non-traumatic blood, nasal hemorrhage and menstrual blood, was within the normal range. In contrast, the ratio of beta-enolase in bloodstains from traumatic blood was obviously elevated (10-30 fold) in comparison with non-traumatic blood. Furthermore, the ratio of beta-enolase was proved to be higher in stains adhering to weapons that had passed through skeletal muscle, indicating that detection of beta-enolase in bloodstains could be used to distinguish crime weapons. These results suggest that beta-enolase is a useful marker for identification of skeletal muscle injury as well as for detecting the origin of bleeding.     

Identification of remains by sequencing of mitochondrial DNA control region

The maternity of two newborns who were murdered and abandoned >5 and 10 years were analyzed by amplification and direct sequencing of mitochondrial DNA (mtDNA) control regions. Sequences of two hypervariable segments from each femur bone sample and the blood of the putative mother showed four mutations in hypervariable region I and two mutations in addition to two nucleotide insertions in hypervariable region II compared with the reference sequence, and all sequences were identical. The genotype of these individuals is found to be relatively rare in the Japanese population, and it was strongly suggested that both sets of newborn remains really were children of the putative mother. Sexes of the remains were determined to be female and male by amplifying a segment of the X-Y homologous gene, amelogenin. These results demonstrate that sequencing of mtDNA is a useful tool for genetic identification of aged and decomposed materials.     

Marine bacteria comprise a possible indicator of drowning in seawater

To investigate the effectiveness of marine bacteria as a new marker of drowning in seawater, we determined the optimal conditions of media required to selectively detect marine bacteria and applied the technique to drowned cadavers. We incubated model blood samples (n=20 per group) mixed with seawater, river, tap or muddy water on agar plates (Todd Hewitt, TH; Marine 2216, M2216) and determined the NaCl concentration required to selectively detect marine bacteria. We also used TCBS agar plates without manipulation to isolate Vibrio spp. Among the culture media, TH agar was superior. Bioluminescent colonies were detected only in blood mixed with seawater. Blue colonies stained using the cytochrome oxidase test (COT), were detected in blood mixed with both sea and river water. However when the NaCl concentration was above 4%, COT stained colonies were detectable only in blood mixed with seawater. We subsequently used 2, 3 and 4% NaCl in TH and TCBS agar to examine blood from victims who had drowned in seawater (n=8) and in fresh water (n=7), as well as from victims who died near aquatic environments (non drowned; dry-land control, n=7). Bioluminescent colonies were detectable on 2-4% NaCl TH agar only from two victims that drowned in seawater. Bioluminescent colonies did not grow on TCBS agar. Blue colonies from all cadavers that had drowned in seawater (8/8) and in four of those that had drowned in fresh water (4/7) proliferated on TH agar containing 2% and/or 3% NaCl, but at 4% NaCl such colonies were detected only from cadavers that had drowned in seawater (8/8). Colonies from only one cadaver from seawater grew on TCBS agar. Furthermore, neither bioluminescent nor blue colonies were detected on TH agar containing 4% NaCl in samples from two cadavers found in an estuary (brackish water) who were thought to have been carried from areas of fresh water. Homologous analyses of the 16S rRNA gene revealed that the dominant colonies on TH agar containing 4% NaCl were marine bacteria (Photobacterium, Vibrio, Shewanella, Psychrobacter). Thus, proliferating bioluminescent and/or blue colonies detected in the blood of immersed cadavers using 4% NaCl TH agar, could help to establish drowning in seawater.     

Alterations of Length Heteroplasmy in Mitochondrial DNA Under Various Amplification Conditions*

Abstract: There are several areas within mitochondrial DNA that show length heteroplasmy. If the heteroplasmy pattern is unique and consistent for each person, it may be used to support an interpretation of exclusion in identity testing. We investigated whether the length heteroplasmy pattern would be consistent under different amplification conditions. We also determined whether various amplification parameters would affect the homopolymeric cytosine stretches (C‐stretch) in HV1. Monoclonal samples tended to be heteroplasmic after amplification. After several repetitions, C‐stretch patterns of all samples were inconsistent even under the same amplification conditions. Increased PCR cycles and high template concentrations resulted in a more frequent heteroplasmic tendency. These amplification parameters seem to have little effect if samples are not long enough in C‐stretch or total length of the segment from nt 16180 to nt 16193. It is suggested that the pattern of length heteroplasmy cannot be used as an additional polymorphic marker.     

The Heterogeneous Effects of a Food Price Crisis on Child School Enrolment and Labour: Evidence from Pakistan

Using a panel survey, this paper investigates how food price increases in Pakistan in 2008–2010 affect children’s school enrolment and labour. The causal identification relies on the geographical variations in food (wheat) price. The results show that the negative impacts of food price increase on school enrolment differ by gender, economic status and the presence of siblings. The negative effects on school do not directly correspond to the increase in child labour because the transition from being idle to labour activity or from school to being idle is significant, particularly among poor girls. The results also show that children in households with access to agricultural lands are not affected by higher food prices. The analyses reveal a more dynamic picture of the impact of food price increase on child status and contribute to broader policy discussion to mitigate the impact of crises on child education.     

Reaching out to the people? Assessing the relationship between parliament and citizens in Finland

Despite the Open Government (Parliament) initiatives and notions of a ‘democratic parliament’, the relationship between legislatures and citizens remains seriously under-researched. This article introduces a comprehensive analytical framework, combining the normative principles of visibility, accessibility, and permeability with practical indicators (parliament as public space, sharing of information, contact with MPs, media and digital engagement, transparency of legislative process, and actual participation in legislative decision-making) for assessing the public engagement of parliaments. Applying this framework to the Finnish Eduskunta, the authors show that despite recent reforms that have partially ‘opened up’ parliamentary proceedings and attempted to connect citizens to democratic process, there remains scope for reforms and innovations. The Eduskunta should embrace a more positive approach towards new forms of civic participation, particularly regarding how its influential committees operate. The findings reflect the tensions between, or the difficulties in reconciling, traditional forms of representative democracy with alternative and more direct channels of political participation.     

Mitochondrial DNA and STR typing of matter adhering to an earphone

STR typing and mitochondrial DNA (mtDNA) sequencing were performed on the matter adhering to an earphone found at a crime scene. Experimental studies were carried out using the earphones provided by volunteers. By means of immunohistochemistry, keratinocytes and a portion of nucleated epithelial cells were proven to exist in the contents from the earphones. DNA was extracted by means of the phenol/chloroform method, and the low quantity of extracted DNA was found to be highly degraded. Six STR loci, CSFIPO, TPOX, TH01, F13A01, FESFPS and vWA, were PCR amplified and typed by using two triplex systems (CTT and FFv Multiplexes, Promega, WI), and an amelogenin locus was determined as well. Although partial profiles were observed in some experimental samples, all STR loci could be typed when a considerable amount of high molecular weight DNA was obtained (>0.5 ng/microL). Amplification and sequencing of mtDNA hypervariable region I(15997-16401) and hypervariable region 11(29-408) were all successful. The mitochondrial DNA sequence of the actual case sample, comprising two hypervariable regions and a total of 785 base pairs, showed eight mutations and two insertions with respect to the standard published reference sequence. The genotype was unique in the three published Japanese databases. These results suggest that it is possible to analyze mtDNA from minute amounts of materials and from degraded materials more effectively and routinely in forensic practice.