Analysis of two meteorite fragments (lunar and martian) using X-Ray microfluorescence and X-Ray computed microtomography techniques.

Meteorites have been arousing the curiosity of mankind since antiquity. However, the interest in these objects goes far beyond mere curiosity in the study of such materials, which has great importance due essentially to the information they can provide. The importance of studying meteorites is associated about the earliest conditions and processes during the formation and earliest history of the solar system. So, in this study, the characterization of two meteorite fragments was performed using X-ray computed microtomography (micro-CT) and X-ray microfluorescence (micro-XRF). These techniques were used for their non-destructive characteristics and the ability to provide information about the structure and composition the meteorites. The micro-CT images showed encrusted structures within both samples. However, while in Lunar meteorites spheroidal structures very similar to small grains internally grouped in clusters were found, in the Martian meteorite a very peculiar structure was identified. Besides that, the micro-CT it was also possible to evaluate the different density materials that compose the samples. The micro-XRF results accounted for the presence of the elements Si, Ca, Ti, Cr, Mn, Fe, Ni and Sr in the Lunar sample, as well as of Si, S, K, Ca, Ti, Cr, Mn, Fe, Cu, Zn, Sr and Y in the Martian sample. The results obtained are effective for the characterization of meteorites, proving thus that it is possible to obtain important information about the chemical composition, as well as about the distribution and the internal structure of these materials, evaluating aspects such as density and porosity.

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: results of a fourth and fifth collaborative EDNAP exercise.

The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were used for the identification of vaginal secretion: Vag triplex (MYOZ1, CYP2B7P1 and MUC4) and a Lactobacillus-specific Lacto triplex (Ljen, Lcris, Lgas). The laboratories used different chemistries and instrumentation and all were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA allowed for positive identification of the tissue/fluid source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling, also from old and compromised casework samples. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid identification method that can easily be combined with current STR typing technology.

RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise.

A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10-0.01 µl saliva, 5-0.01 µl semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 µl saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies.

GHEP-ISFG proficiency test 2011: Paper challenge on evaluation of mitochondrial DNA results.

The GHEP-ISFG Working Group performed a collaborative exercise to monitor the current practice of mitochondrial (mt)DNA reporting. The participating laboratories were invited to evaluate a hypothetical case example and assess the statistical significance of a match between the haplotypes of a case (hair) sample and a suspect. A total of 31 forensic laboratories participated of which all but one used the EMPOP database. Nevertheless, we observed a tenfold range of reported LR values (32-333.4), which was mainly due to the selection of different reference datasets in EMPOP but also due to different applied formulae. The results suggest the need for more standardization as well as additional research to harmonize the reporting of mtDNA evidence.

The tooth for molecular analysis and identification : a forensic approach.

The aim of this study is to optimize laboratory preparation of teeth for DNA identification. By sectioning the tooth topographically into two different radicular portions, it was analyzed whether these portions of mineralized tissue differ in the quantity and quality of DNA they contain. 25 teeth were subject to different experimental conditions and total DNA was quantified for each individual tooth's radicular portion: apical and remaining root, according to a 2003 study by Gaytemenn and Sweet. We verified, with statistically significant figures, that the apical portion of the tooth is that which contains the greatest quantity of DNA. Different analytical procedures were studied for various polymorphic markers to evaluate the quality of the DNA. We concluded that the tooth is topographically distinct in both DNA quantity and quality. The tooth's apical portion is the preferential choice in sample preparation of dental mineralized tissue for molecular analysis and identification.

Determination of Cu/Zn and Fe in human serum of patients with sickle cell anemia using radiation synchrotron.

In this work we analyzed serum samples from patients with Sickle Cell Anemia (SCA) using Total Reflection X-Ray Fluorescence using Synchrotron Radiation (SRTXRF). The SRTXRF measurements were performed at the X-Ray Fluorescence Beamline at the Brazilian National Synchrotron Light Laboratory (LNLS). We studied forty-three patients aged 18-50 suffering from SCA and sixty healthy volunteers aged 18-60. It was possible to determine the concentrations of the following elements: P, S, Cl, K, Ca, Fe, Cu, Zn, Br and Rb. Moreover, there are evidences of an association among Fe, Cu, Zn and Cu/Zn in the SCA pathogenesis process. The concentrations of Fe and Cu in the serum samples of patients with SCA were larger, 120% and 20%, respectively, when compared with the CG. The serum level Cu/Zn ratio was significantly higher (60%) in the serum samples from patients suffering from SCA than from the CG. Therefore, the Cu/Zn ratio can be used as an adjuvant index in enhancement for diagnosis of SCA.

Elemental distribution images in prostate samples by X-ray fluorescence microtomography.

An X-ray transmission microtomography (CT) system combined with an X-ray fluorescence microtomography (XRFµCT) system was implemented in the Brazilian Synchrotron Light Laboratory (LNLS), in order to determine the elemental distribution in prostate samples aiming at establishing a correlation between the concentration of some elements and the characteristics and pathology of the tissues. The CT images were reconstructed using a filtered-back projection algorithm and the XRFµCT images were reconstructed using a filtered-back projection algorithm with absorption corrections.

RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise.

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 µl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.

The GHEP-EMPOP collaboration on mtDNA population data--A new resource for forensic casework.

Mitochondrial DNA (mtDNA) population data for forensic purposes are still scarce for some populations, which may limit the evaluation of forensic evidence especially when the rarity of a haplotype needs to be determined in a database search. In order to improve the collection of mtDNA lineages from the Iberian and South American subcontinents, we here report the results of a collaborative study involving nine laboratories from the Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) and EMPOP. The individual laboratories contributed population data that were generated throughout the past 10 years, but in the majority of cases have not been made available to the scientific community. A total of 1019 haplotypes from Iberia (Basque Country, 2 general Spanish populations, 2 North and 1 Central Portugal populations), and Latin America (3 populations from São Paulo) were collected, reviewed and harmonized according to defined EMPOP criteria. The majority of data ambiguities that were found during the reviewing process (41 in total) were transcription errors confirming that the documentation process is still the most error-prone stage in reporting mtDNA population data, especially when performed manually. This GHEP-EMPOP collaboration has significantly improved the quality of the individual mtDNA datasets and adds mtDNA population data as valuable resource to the EMPOP database (www.empop.org).

Paternal and maternal lineages in Guinea-Bissau population.

The aim of the present work was to study the origin of paternal and maternal lineages in Guinea-Bissau population, inferred by phylogeographic analyses of mtDNA and Y chromosome defined haplogroups. To determine the male lineages present in Guinea-Bissau, 33 unrelated males were typed using a PCR-SNaPshot multiplex based method including 24 Y-SNPs, which characterize the main haplogroups in sub-Saharan Africa and Western Europe. In the same samples, 17 Y-STRs (included in the YFiler kit, Applied Biosystems) were additionally typed. The most frequent lineages observed were E1b1a (xE1b1a4,7)-M2 (68%) and E1a-M33 (15%). The European haplogroup R1b1-P25 was represented with a frequency of 12%. The two hypervariable mtDNA regions were sequenced in 79 unrelated individuals from Guinea-Bissau, and haplogroups were classified based on control region motifs using mtDNA manager. A high diversity of haplogroups was determined in our sample being the most frequent haplogroups characteristic of populations from sub-Saharan Africa, namely L2a1 (15%), L3d (13%), L2c (9%), L3e4 (9%), L0a1 (8%), L1b (6%) and L1c1 (6%). None of the typical European haplogroups (H, J and T) were found in the present sample of Guinea-Bissau. From our results, it is possible to confirm that Guinea-Bissau presents a typically West African profile, marked by a high frequency of the Y chromosome haplogroup E1b1a(xE1b1a4,7)-M2 and a high proportion of mtDNA lineages belonging to the sub-Saharan specific sub-clusters L1 to L3 (89%). A small European influx has been also detected, although restricted to the male lineages.

Mitochondrial DNA sequence analysis of a native Bolivian population.

Mitochondrial DNA analysis is very useful for the interpretation of the history of human migration and to estimate the frequency of a haplotype in the forensic context. From a human settlement perspective, La Paz area is greatly interesting since the first planned city of the region is located there. Samples from 110 individuals from La Paz were studied analysing the polymorphisms in the D-loop, hypervariable region I (HVI) and hypervariable region II (HVII) in order to verify the genetic diversity. The aim of this study was to start the creation of a population database in order to obtain the genetic interpopulation variability and classify haplotypes into characteristic haplogroups of South America. A total of 97 different haplotypes were identified, 90 being unique, expressed by 122 polymorphic nucleotide positions. Nucleotide and sequence diversity were estimated to be 0.015 +/- 0.0075 and 0.996, respectively. Haplogroup distribution in the samples was 57.27% B4, 19.09% C1, 10.00% A2, 3.64% D1, 2.73% D4h3, 1.82% H, and 0.91% for each of the haplogroups A4, B4c1a, CZ, D4J, M7a and M8/N9b. The rate of length heteroplasmy was 36.36% in HVI and 52.73% in HVII. Phylogenetic analysis reveals proximity to the Korean, Chilean aboriginal, Japanese and Australian populations. The estimated genetic variability of the studied population was high, suggesting an early settlement.

Biological tissues analysis by XRF microtomography.

The main of this work is to determine the elemental distribution in breast and prostate tissue samples in order to verify the concentration of some elements correlated with characteristics and pathology of each tissue observed by the X-ray transmission microtomography (microCT). The experiments were performed at the X-ray fluorescence beamline of the Brazilian Synchrotron Light Laboratory. The microCT images were reconstructed using a filtered-back-projection algorithm and the XRF microtomographies were reconstructed using a filtered-back-projection algorithm with absorption corrections.

2006 GEP-ISFG collaborative exercise on mtDNA: reflections about interpretation, artefacts, and DNA mixtures.

We report the results of the seventh edition of the GEP-ISFG mitochondrial DNA (mtDNA) collaborative exercise. The samples submitted to the participant laboratories were blood stains from a maternity case and simulated forensic samples, including a case of mixture. The success rate for the blood stains was moderate ( approximately 77%); even though four inexperienced laboratories concentrated about one-third of the total errors. A similar success was obtained for the analysis of mixed samples (78.8% for a hair-saliva mixture and 69.2% for a saliva-saliva mixture). Two laboratories also dissected the haplotypes contributing to the saliva-saliva mixture. Most of the errors were due to reading problems and misinterpretation of electropherograms, demonstrating once more that the lack of a solid devised experimental approach is the main cause of error in mtDNA testing.

Topographic trace-elemental analysis in the brain of Wistar rats by X-ray microfluorescence with synchrotron radiation.

Knowledge about the spatial distribution and the local concentration of trace elements in tissues is of great importance, since trace elements are involved in many biological functions of living organisms. However, there are few methods available to measure the spatial (two (three)-dimensional) elemental distribution in animal brain. X-ray microfluorescence with synchrotron radiation is a multielemental mapping technique, which was used in this work to determine the topographic of iron, zinc and copper in coronal sections of female Wistar rats of different ages. Young (14 days old) and middle-aged (20 months old) rats (n = 8) were analyzed. The measurements were carried out at the XRF beam line at the Synchrotron Light National Laboratory (Campinas, Brazil). Two-dimensional scanning was performed in order to study the tendency of elemental concentration variation. The acquisition time for each pixel was 10 s/step and the step size was 300 microm/step in both directions. It was observed that the iron distribution was more conspicuous in the cortical area, thalamus and bellow the thalamus. On the other hand, the zinc distribution was more pronounced in the hippocampus. The iron, copper and zinc levels increased with advancing age. Therefore, this study reinforces the idea that these elements are involved in the chemical mechanisms of the brain that induce some neurological diseases, since they are also present in high levels in specific areas of the brain, such as the hippocampus and the substantia nigra of patients with these disorders.

Evaluation of osteoporotic bone structure through synchrotron radiation X-ray microfluorescence images.

The abnormal accumulation or deficiency of trace elements may theoretically impair the formation of bone and contribute to osteoporosis. In this context, the knowledge of major and trace elements is very important in order to clarify many issues regarding diseases of the bone, such as osteoporosis, that remain unresolved. Several kinds of imaging techniques can be useful to access morphology and the minerals present in osteoporotic bones. In this work, synchrotron radiation X-ray microfluorescence was used as an X-ray imaging technique to investigate bone structures. Therefore, this research aims to improve the knowledge about some aspects of bone quality. The measurements were carried out at the Brazilian Synchrotron Laboratory Light Laboratory, in Brazil. A white beam with an energy range of 4-23 keV, a 45 degrees /45 degrees geometry and a capillary optics were used. It was demonstrated that bone quality can and must be evaluated not only by considering the architecture of bones but also by taking into account the concentration and the distribution of minerals. Our results showed that the elemental distributions in bone zones on a micron scale were very helpful to understand functions in those structures.

Elemental distribution mapping on breast tissue samples.

The main of this work is to determine the elemental and absorption distribution map in breast tissue samples by X-ray fluorescence and X-ray transmission microtomography. The experiments were performed at the X-ray fluorescence beamline (D09B-XRF) in the Brazilian Synchrotron Light Source (LNLS), Campinas, Brazil. A quasi-monochromatic beam produced by a multilayer monochromator was used as an incident beam. The sample was placed on a high precision goniometer and translation stages that allow rotating as well as translating it perpendicularly to the beam. The fluorescence photons were collected with an energy dispersive HPGe detector placed at 90 degrees to the incident beam, while transmitted photons were detected with a fast Na(Tl) scintillation counter () placed behind the sample on the beam direction. All the tomographic images were reconstructed using a filtered-back projection algorithm. In the breast tissue samples, it was possible to determine the distribution of Zn, Cu and Fe in healthy tissue and tumor.

Bone diagnosis by X-ray techniques.

In this work, two X-ray techniques used were 3D microcomputed tomography (micro-CT) and X-ray microfluorescence (micro-XRF) in order to investigate the internal structure of the bone samples. Those two techniques work together, e.g. as a complement to each other, to characterize bones structure and composition. Initially, the specimens were used to do the scan procedure in the microcomputer tomography system and the second step consists of doing the X-ray microfluorescence analysis. The results show that both techniques are powerful methods for analyzing, inspecting and characterizing bone samples: they are alternative procedures for examining bone structures and compositions and they are complementary.

Multi-elemental analysis of produced water by synchrotron radiation total reflection X-ray fluorescence.

The synchrotron radiation total reflection X-ray fluorescence (SRTXRF) technique was used for the analysis of heavy metals in produced water samples from oil field in Rio Grande do Norte, in order to determine potential sources of pollution. Since the inorganic components in produced water generally resembling sea water, pre-concentration procedures have been applied to increase the concentration of the analyte of interest and to minimize the salt matrix effects. This technique allows us to determine the contents of V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Hg and Pb in 20 produced water samples. The great majority of the sampling points presented low elemental concentration value. However, in some sample, the Fe, Ni, Cu, Zn and Hg concentration were higher than the established limits by the Brazilian legislation.

Individual genetic identification of biological samples: a case of an aircraft accident.

In 2003, an aircraft accident occurred in the Madeira island north coast, in which 10 persons (4 female and 6 male) have died. STRs (autosomic and Y-chromosome) and mtDNA were made in order to identify the recovered human body remains and results compared with relatives.

Histologic evaluation of the osteoinductive property of autogenous demineralized dentin matrix on surgical bone defects in rabbit skulls using human amniotic membrane for guided bone regeneration.

The aim of this investigation was to evaluate the osteoinductive property of autogenous demineralized dentin matrix (ADDM) on experimental surgical bone defects in the parietal bone of rabbits using the guided bone regeneration (GBR) technique incorporating human amniotic membrane (HAM). Thirty-six rabbits were divided into 2 groups, HAM and ADDM+HAM. It was possible to conclude that HAM did not interfere with bone repair and was resorbed. Slices of ADDM induced direct bone formation and were incorporated by the newly formed bone tissue and remodeled. The bone defects healed faster in the ADDM+HAM group than in the group with HAM only.