Body fluid identification using a targeted mRNA massively parallel sequencing approach - results of a EUROFORGEN/EDNAP collaborative exercise.

In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laboratories, in order to test the efficacy of targeted mRNA sequencing to identify body fluids. The task was to analyse the supplied dried body fluid stains and, optionally, participants' own bona fide or mock casework samples of human origin, according to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. There was some inter-laboratory variability in read counts, but overall the results of the laboratories were comparable in that highly expressed markers showed high read counts and less expressed markers showed lower counts. We performed a partial least squares (PLS) analysis on the data, where blood, menstrual blood, saliva and semen markers and samples clustered well. The results of this collaborative mRNA massively parallel sequencing (MPS) exercise support targeted mRNA sequencing as a reliable body fluid identification method that could be added to the repertoire of forensic MPS panels.

Mitochondrial inheritance is delayed in Saccharomyces cerevisiae cells lacking the serine/threonine phosphatase PTC1.

In wild-type yeast mitochondrial inheritance occurs early in the cell cycle concomitant with bud emergence. Cells lacking the PTC1 gene initially produce buds without a mitochondrial compartment; however, these buds later receive part of the mitochondrial network from the mother cell. Thus, the loss of PTC1 causes a delay, but not a complete block, in mitochondrial transport. PTC1 encodes a serine/threonine phosphatase in the high-osmolarity glycerol response (HOG) pathway. The mitochondrial inheritance delay in the ptc1 mutant is not attributable to changes in intracellular glycerol concentrations or defects in the organization of the actin cytoskeleton. Moreover, epistasis experiments with ptc1delta and mutations in HOG pathway kinases reveal that PTC1 is not acting through the HOG pathway to control the timing of mitochondrial inheritance. Instead, PTC1 may be acting either directly or through a different signaling pathway to affect the mitochondrial transport machinery in the cell. These studies indicate that the timing of mitochondrial transport in wild-type cells is genetically controlled and provide new evidence that mitochondrial inheritance does not depend on a physical link between the mitochondrial network and the incipient bud site.

Vacuole partitioning during meiotic division in yeast.

We have examined the partitioning of the yeast vacuole during meiotic division. In pulse-chase experiments, vacuoles labeled with the lumenal ade2 fluorophore or the membrane-specific dye FM 4-64 were not inherited by haploid spores. Instead, these fluorescent markers were excluded from spores and trapped between the spore cell walls and the ascus. Serial optical sections using a confocal microscope confirmed that spores did not inherit detectable amounts of fluorescently labeled vacuoles. Moreover, indirect immunofluorescence studies established that an endogenous vacuolar membrane protein, alkaline phosphatase, and a soluable vacuolar protease, carboxypeptidase Y. were also detected outside spores after meiotic division. Spores that did not inherit ade2- or FM 4-64-labeled vacuoles did generate an organelle that could be visualized by subsequent staining with vacuole-specific fluorophores. These data contrast with genetic evidence that a soluble vacuolar protease is inherited by spores. When the partitioning of both types of markers was examined in sporulating cultures, the vacuolar protease activity was inherited by spores while fluorescently labeled vacuoles were largely excluded from spores. Our results indicate that the majority of the diploid vacuole, both soluble contents and membrane-bound components, are excluded from spores formed during meiotic division.

RNA/DNA co-analysis from human skin and contact traces--results of a sixth collaborative EDNAP exercise.

The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology.

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.

Male dominance rank, mating and reproductive success in captive bonobos (Pan paniscus).

In the recent past, application of DNA genotyping techniques has enabled researchers to more accurately test relationships between dominance rank (DR), mating success (MS) and reproductive success (RS). Paternity studies often reveal that reproductive outcome does not always correlate with male DR and/or MS and thus open room for discussion and interpretation of alternative reproductive tactics of both sexes. In this study, we analysed male DR, MS and RS in a group of bonobos at Twycross Zoo (UK). Genetic relationships were determined using 8 tetrameric microsatellite loci. Despite clear and asymmetric dominance relationships, analysed using normalised David's scores based on a dyadic index of dominance among the group's 3 mature males, we found that the most dominant male did not sire the most offspring. In fact, both infants conceived during the observation period were found to be sired by the lower-ranking males. Although the alpha male had almost exclusive mating access to one of the females during the time she was showing a maximal anogenital swelling, her infant was sired by the lowest-ranking male who mostly mated with her when outside the maximal swelling period. This result suggests that either sperm competition operates and/or ovulation is decoupled from the phase of maximal anogenital swelling which could allow greater female choice.

Confocal microscopy of F-actin distribution in Xenopus oocytes.

We have used rhodamine-conjugated phalloidin and confocal microscopy to examine the organisation of filamentous actin (F-actin) during oogenesis in Xenopus laevis. F-actin was restricted to a thin shell in the cortex of oogonia and post-mitotic oocytes less than 35 microns in diameter. In oocytes with diameters of 35-50 microns, F-actin was observed in three cellular domains: in the cortex, in the germinal vesicle (GV) and in a network of cytoplasmic cables. Initially, actin cables were sparsely distributed in the cytoplasm, with no evidence of discrete organising centres. In larger stage I oocytes, a dense network of actin cables extended throughout the cytoplasm, linking the GV and mitochondrial mass to the cortical actin shell. Apart from the F-actin associated with the mitochondrial mass, no evidence of a polarised distribution of F-actin was apparent in stage I oocytes. F-actin was observed also in the cortex and the GV of stage VI oocytes, and a network of cytoplasmic cables surrounded the GV. Cytoplasmic actin cables extended from the GV to the animal cortex, and formed a three-dimensional network surrounding clusters of yolk platelets in the vegetal cytoplasm. Finally, disruption of F-actin in stage VI oocytes with cytochalasin resulted in distortion and apparent rotation of the GV in the animal hemisphere, suggesting that actin plays a role in maintaining the polarised organisation of amphibian oocytes.

F-actin is required for spindle anchoring and rotation in Xenopus oocytes: a re-examination of the effects of cytochalasin B on oocyte maturation.

We used confocal immunofluorescence microscopy to examine spindle migration, morphology and orientation during the maturation of Xenopus oocytes, in the presence or absence of cytochalasin B (CB), an inhibitor of actin assembly. Treatment with CB during maturation (10-50 micrograms/ml beginning 0-3 h prior to addition of progesterone) disrupted the normal organisation of the novel MTOC and transient microtubule array (MTOC-TMA complex) that serves as the immediate precursor of the first meiotic spindle, suggesting that F-actin plays an important role in the assembly or maintenance of this complex. However, CB treatment did not block translocation of the MTOC-TMA complex to the oocyte cortex, suggesting that MTOC-TMA translocation is not dependent on an actin-based mechanism. Bipolar spindles were observed in CB-treated oocytes fixed during both M1 and M2. However, rotation of the M1 and M2 spindles into an orientation orthogonal to the oocyte surface was inhibited by CB. Rhodamine-phalloidin revealed a concentration of F-actin at the site of M1 spindle attachment, further suggesting that cortical actin is required for anchoring and rotation of the meiotic spindles. Finally, the incidence of M1 monasters was significantly increased in CB-treated oocytes, suggesting that interactions between the nascent M1 spindle and cortex are dependent on F-actin.

Factors affecting the amount of genomic DNA extracted from ape faeces and the identification of an improved sample storage method.

Abstract Genetic analysis using noninvasively collected samples such as faeces continues to pose a formidable challenge because of unpredictable variation in the extent to which usable DNA is obtained. We investigated the influence of multiple variables on the quantity of DNA extracted from faecal samples from wild mountain gorillas and chimpanzees. There was a small negative correlation between temperature at time of collection and the amount of DNA obtained. Storage of samples either in RNAlater solution or dried using silica gel beads produced similar results, but significantly higher amounts of DNA were obtained using a novel protocol that combines a short period of storage in ethanol with subsequent desiccation using silica.

Reconstructing the history of human limb development: lessons from birth defects.

A major goal of biology has been to understand the developmental mechanisms behind evolutionary trends. This has led to a growing interest in studying the molecular basis of the evolution of developmental programs such as those mediating the diversification of tetrapod limbs. Over the last 10 y, it has become clear that the genes and general developmental programs used to build a limb are strongly conserved among widely disparate species. This finding suggests that altered regulation of the timing and locations of developmental events may be responsible for the morphologic variation observed among some species. However, genetic analyses of the regulatory regions of genes controlling vertebrate developmental programs are very limited. Characterization of the genetic basis of human birth defects of the limb provides an opportunity to dissect the developmental programs used to modify the architecture of the hominoid limb. This may allow us to assess the relative contributions of altered gene regulation to morphologic variation among species and reconstruct the evolutionary history of the hominid limb. Such insight is also important because morphologic differences in the hominid upper limb have been correlated with the use of tools, and tool making is often regarded as the milestone that marked the emergence of the genus Homo.

Gene flow on the ice: genetic differentiation among Adélie penguin colonies around Antarctica.

Each summer Adélie penguins breed in large disjunct colonies on ice-free areas around the Antarctic continent. Comprising > 10 million birds, this species represents a dominant feature of the Antarctic ecosystem. The patchy distribution within a large geographical range, natal philopatry and a probable history of refugia, suggest that this species is likely to exhibit significant genetic differentiation within and among colonies. We present data from seven microsatellite DNA loci for 442 individuals from 13 locations around the Antarctic continent. With the exception of one locus, there was no significant genic or genotypic heterogeneity across populations. Pairwise FST values were low with no value > 0.02. When all colonies were compared in a single analysis, the overall FST value was 0.0007. Moreover, assignment tests were relatively ineffective at correctly placing individuals into their respective collection sites. These data reveal a lack of genetic differentiation between Adélie penguin colonies around the Antarctic continent, despite substantial levels of genetic variation. We consider this homogeneity in terms of the dispersal of individuals among colonies and the size of breeding groups and discuss our results in terms of the glacial history of Antarctica.

The spectrum of mutations in TBX3: Genotype/Phenotype relationship in ulnar-mammary syndrome.

Ulnar-mammary syndrome (UMS) is a pleiotropic disorder affecting limb, apocrine-gland, tooth, hair, and genital development. Mutations that disrupt the DNA-binding domain of the T-box gene, TBX3, have been demonstrated to cause UMS. However, the 3' terminus of the open reading frame (ORF) of TBX3 was not identified, and mutations were detected in only two families with UMS. Furthermore, no substantial homology outside the T-box was found among TBX3 and its orthologues. The subsequent cloning of new TBX3 cDNAs allowed us to complete the characterization of TBX3 and to identify alternatively transcribed TBX3 transcripts, including one that interrupts the T-box. The complete ORF of TBX3 is predicted to encode a 723-residue protein, of which 255 amino acids are encoded by newly identified exons. Comparison of other T-box genes to TBX3 indicates regions of substantial homology outside the DNA-binding domain. Novel mutations have been found in all of eight newly reported families with UMS, including five mutations downstream of the region encoding the T-box. This suggests that a domain(s) outside the T-box is highly conserved and important for the function of TBX3. We found no obvious phenotypic differences between those who have missense mutations and those who have deletions or frameshifts.