Brief communication: Minimally invasive bone sampling method for DNA analysis

Obtaining a bone sample for DNA analysis has traditionally been a destructive practice, which has resulted in reluctance on behalf of curators for skeletal collections to allow invasive testing. A novel minimally invasive bone sampling method for DNA analysis is presented here. This method uses a conventional hand drill wherein the bone sample is extracted from the intercondylar fossa of the femur; it does not interfere with any known anthropometric landmarks and only leaves a small hole on the surface of the bone. The temperature of the drill is documented and it was established due to the minor increase in temperature, that this should not affect the molecular integrity of the sample. This method is easily replicated and is suitable for both human and other animal skeletal material and can be applied to rare specimens with little risk. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Extensive carbon isotopic heterogeneity among methane seep microbiota

To assess and study the heterogeneity of δ¹³C values for seep microorganisms of the Eel River Basin, we studied two principally different sample sets: sediments from push cores and artificial surfaces colonized over a 14 month in situ incubation. In a single sediment core, the δ¹³C compositions of methane seep-associated microorganisms were measured and the relative activity of several metabolisms was determined using radiotracers. We observed a large range of archaeal δ¹³C values (> 50‰) in this microbial community. The δ¹³C of ANME-1 rods ranged from −24‰ to −87‰. The δ¹³C of ANME-2 sarcina ranged from −18‰ to −75‰. Initial measurements of shell aggregates were as heavy as −19.5‰ with none observed to be lighter than −57‰. Subsequent measurements on shell aggregates trended lighter reaching values as ¹³C-depleted as −73‰. The observed isotopic trends found for mixed aggregates were similar to those found for shell aggregates in that the initial measurements were often enriched and the subsequent analyses were more ¹³C-depleted (with values as light as −56‰). The isotopic heterogeneity and trends observed within taxonomic groups suggest that ANME-1 and ANME-2 sarcina are capable of both methanogenesis and methanotrophy. In situ microbial growth was investigated by incubating a series of slides and silicon (Si) wafers for 14 months in seep sediment. The experiment showed ubiquitous growth of bacterial filaments (mean δ¹³C = −38 ± 3‰), suggesting that this bacterial morphotype was capable of rapid colonization and growth.     

Comparison of two single-platform ISHAGE-based CD34 enumeration protocols on BD FACSCalibur and FACSCanto flow cytometers

Background aimsEnumeration of viable CD34⁺ cells provides critical information for the bone marrow (BM) transplant physician. The single-platform ISHAGE protocol is the most reliable method currently available to quantitate accurately this important subset of cells. Previous studies have shown that 5 CD34⁺ cells/µL blood predicts the collection of at least 0.5 × 10⁶ CD34⁺ cells/kg patient weight. From the apheresis product, infusion of 2.5 × 10⁶ viable CD34⁺ cells (measured pre-cryopreservation)/kg patient weight will reliably permit engraftment of the hematopoietic system (as measured by the time to 20000 platelets/µL) by day 12–14 post-infusion.MethodsWe compared the CD34⁺ cell numbers derived from Flow Count-based Stem-Kit?; (Beckman Coulter) and Trucount? tube-based stem cell enumeration (SCE) kit (BD Biosciences) ISHAGE templates on BD FACSCalibur? and BD FACSCanto? cytometers on 12 granulocyte–colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) and 10 peripheral blood stem cell (PBSC) samples.ResultsComparison of results showed that there was no statistical difference between samples run with Stem-Kit on the FACSCalibur versus SCE kit-based assays on either the FACSCalibur or FACSCanto. Mean results for the Stem-Kit/Calibur combination were 137, for SCE kit/Calibur 140 and for SCE kit/Canto 137 cells/µL. Pair-wise comparison of data based on rank order showed no statistically significant difference and all correlation coefficients had an R²>0.98.ConclusionsThe two kits generated very similar data on a range of fresh samples regardless of instrument platform. These results confirm and extend the utility of the single-platform ISHAGE protocols with a variety of reagent kits and instrument platforms.     

Ancestry Analysis in the 11-M Madrid Bomb Attack Investigation

The 11-M Madrid commuter train bombings of 2004 constituted the second biggest terrorist attack to occur in Europe after Lockerbie, while the subsequent investigation became the most complex and wide-ranging forensic case in Spain. Standard short tandem repeat (STR) profiling of 600 exhibits left certain key incriminatory samples unmatched to any of the apprehended suspects. A judicial order to perform analyses of unmatched samples to differentiate European and North African ancestry became a critical part of the investigation and was instigated to help refine the search for further suspects. Although mitochondrial DNA (mtDNA) and Y-chromosome markers routinely demonstrate informative geographic differentiation, the populations compared in this analysis were known to show a proportion of shared mtDNA and Y haplotypes as a result of recent gene-flow across the western Mediterranean, while any two loci can be unrepresentative of the ancestry of an individual as a whole. We based our principal analysis on a validated 34plex autosomal ancestry-informative-marker single nucleotide polymorphism (AIM-SNP) assay to make an assignment of ancestry for DNA from seven unmatched case samples including a handprint from a bag containing undetonated explosives together with personal items recovered from various locations in Madrid associated with the suspects. To assess marker informativeness before genotyping, we predicted the probable classification success for the 34plex assay with standard error estimators for a naïve Bayesian classifier using Moroccan and Spanish training sets (each n = 48). Once misclassification error was found to be sufficiently low, genotyping yielded seven near-complete profiles (33 of 34 AIM-SNPs) that in four cases gave probabilities providing a clear assignment of ancestry. One of the suspects predicted to be North African by AIM-SNP analysis of DNA from a toothbrush was identified late in the investigation as Algerian in origin. The results achieved illustrate the benefit of adding specialized marker sets to provide enhanced scope and power to an already highly effective system of DNA analysis for forensic identification.     

Effects of antagonists and inhibitors of ethylene biosynthesis on maize root elongation

During the first days of development, maize roots showed considerable variation in the production of ethylene and the rate of elongation. As endogenous ethylene increases, root elongation decreases. When these roots are treated with the precursor of ethylene aminocyclopropane- 1-carboxylic acid (ACC), or inhibitors of ethylene biosynthesis 2-aminoethoxyvinyl glycine (AVG) or cobalt ions, the root elongation is also inhibited. Because of root growth diminishes at high or reduced endogenous ethylene concentrations, it appears that this phytohormone must be maintained in a range of concentrations to support normal root growth. In spite of its known role as inhibitor of ethylene action, silver thiosulphate (STS) does not change significantly the root elongation rate. This suggests that the action of ethylene on root elongation should occur, at least partially, by interaction with other growth regulators.Key words: 2-aminoethoxyvinyl glycine, cobalt, ethylene, root elongation, silver thiosulphate, Zea mays