DNA typing of cyanoacrylate fumed latent fingerprints using GlobalFiler™ and ForenSeq™ Signature Prep kits.

DNA typing of latent fingerprints is highly desirable to increase chances of individualization. We recovered DNA from Cyanoacrylate (CA) fumed fingerprints and used both GlobalFiler™ and ForenSeq™ DNA Signature Prep kits for DNA typing. For GlobalFiler™, samples were processed using a protocol modified for Low Template (LT)-DNA samples (half-volume reactions, 30 cycles) while for ForenSeq™ DNA Signature Prep, samples were processed using a standard protocol and fluorometer-based library quantitation. We evaluated genotyping success and quality of profiles in terms of completeness, Peak Height Ratio/Allele Coverage Ratio, presence of PCR artifacts and drop-in alleles. With GlobalFiler™, average autosomal STR (aSTR) profile completeness was 44.4% with 2-20 pg, 54.3% with 22-60 pg, and 95% with 64-250 pg DNA input. CODIS uploadable profiles were obtained in 2/10, 3/11, and 11/12 samples in these ranges. With ForenSeq™ DNA Signature Prep, average aSTR profile completeness was 19.7% with 1-20 pg and 45.2% with 22-47 pg but increased to 78.3% with 68-122 pg and 86.7% with 618-1000 pg DNA input. Uploadable profiles were obtained in 0/12, 4/11, 4/7, and 3/3 samples for these ranges. Results show very high sensitivity using both kits. Half-volume reactions and 30 cycles had minimal negative effect on Globalfiler™ profile quality, providing support for wider use after validation experiments to routinely improve results from LT samples. A standard protocol for the ForenSeq™ DNA Signature Prep kit was also highly successful with LT DNA obtained from CA-fumed fingerprints with additional information from isometric STR alleles and other markers.

Population genetic data for 21 autosomal STR loci in the Azerbaijani population using the Globalfiler™ kit.

The Republic of Azerbaijan is located in the southern Caucasus mountains, a region which is linguistically and ethnically diverse. We report allele frequency data for 21 autosomal loci from the Globalfiler™ kit in the Azerbaijani population using 467 individuals from Baku. Exact tests for Hardy Weinberg Equilibrium and Linkage Equilibrium were conducted, and all forensic parameters were estimated. High levels of Expected Heterozygosity HE were seen, with a minimum of 0.637 for TPOX and a maximum of 0.949 for SE33. Polymorphism Information Content (PIC) values for all STR loci were high, ranging from 0.587 for TPOX to 0.947 for SE33. Matching probability (MP) estimates ranged from 0.006 for SE33 to 0.178 for TPOX. Power of Discrimination (PD) values for most of the tested loci (17/21) were ≥ 0.9. The Combined Matching Probability (CMP) and the Combined Power of Discrimination (CPD) for all 21 loci were 7.84 × 10-27 and 1.0 respectively. Exact tests for population differentiation using all available Globalfiler™ datasets from across Europe and Asia reveal a general trend of higher differentiation with increasing geographical separation, but there is a need for additional population data from neighboring regions of Azerbaijan.

Ancient DNA from the Asiatic Wild Dog (Cuon alpinus) from Europe.

The Asiatic wild dog (Cuon alpinus), restricted today largely to South and Southeast Asia, was widespread throughout Eurasia and even reached North America during the Pleistocene. Like many other species, it suffered from a huge range loss towards the end of the Pleistocene and went extinct in most of its former distribution. The fossil record of the dhole is scattered and the identification of fossils can be complicated by an overlap in size and a high morphological similarity between dholes and other canid species. We generated almost complete mitochondrial genomes for six putative dhole fossils from Europe. By using three lines of evidence, i.e., the number of reads mapping to various canid mitochondrial genomes, the evaluation and quantification of the mapping evenness along the reference genomes and phylogenetic analysis, we were able to identify two out of six samples as dhole, whereas four samples represent wolf fossils. This highlights the contribution genetic data can make when trying to identify the species affiliation of fossil specimens. The ancient dhole sequences are highly divergent when compared to modern dhole sequences, but the scarcity of dhole data for comparison impedes a more extensive analysis.

A re-examination of the taxonomic status of Prostoma jenningsi-a Freshwater Nemertean.

Prostoma jenningsi was first recorded at the Clay 'Ole pond in Lancashire, UK, in 1969 and was distinguished histologically from other Prostoma by the presence of 11 proboscidial nerves (with all other Prostoma species thought to have 9-10). P. jenningsi was considered to be the only species endemic to Lancashire and listed in the British Red Data Book as 'Insufficiently Known' as well as a 'Species of Principal Importance' under the UK Natural Environment and Rural Communities Act (2006).                A limited number of Prostoma spp were recovered from the Clay 'Ole in 2011 (the first confirmation of the presence of Prostoma spp. since 1999). In 2015, further sampling was undertaken and expanded to other ponds in Lancashire resulting in the discovery of Prostoma spp. at a further 3 locations. Thereafter, DNA sequencing of nuclear 18S ribosomal RNA and mitochondrial Cytochrome Oxidase I (COI) genes were undertaken and phylogenetic analyses performed to establish the taxonomic status of recovered specimens. All available Prostoma sequences (Prostoma eilhardi and Prostoma graecense) were downloaded from GenBank® and Barcode of Life Data System (BOLD) databases for comparison.                All 18S sequences from samples in Lancashire were identical to each other and to all downloaded Prostoma sequences, allowing no further analyses. With COI, 50 individuals were collected from 4 locations across Lancashire and sequenced, comparing a total of 480 base pairs. Average uncorrected p-distances between UK and European samples were low, although some more geographically distant samples from California, USA, displayed higher uncorrected p-distance values.                Results suggest that the Prostoma recovered from the Clay 'Ole (and all other sampled locations in Lancashire) are not distinct from P. eilhardi and P. graecense (as downloaded from GenBank® and BOLD) suggesting that there is a strong case for the species status of P. jenningsi to be revoked. Further regional and national sampling is required to obtain a clearer evaluation of the distribution of Prostoma and the levels of genetic diversity present in the UK. In addition, results from this study indicate that thorough taxonomical re-evaluation of species within the Prostoma genus is required.

Sequence data of six unusual alleles at SE33 and D1S1656 STR Loci.

When profiling a reference dataset of 500 DNA samples for the population of Saudi Arabia, using the GlobalFiler® PCR amplification kit, six unusual alleles were detected. At the SE33 locus, four novel alleles were found: 2, 14.3, 20.3, and 38; two alleles at the D1S1656 locus: 7 and 8 had been previously reported, but no published sequence data was available. The D1S1656 alleles were sequenced using ForenSeq™ DNA Signature Prep with the MiSeq FGx System (Illumina, USA). As the SE33 is not reported by available Massively Parallel Sequencing (MPS) systems, samples that exhibited the unreported alleles were sequenced using BigDye™ Terminator v3.1 Cycle Sequencing Kit. Here we present the sequence and structure of the previously uncharacterized alleles.

SkydancerPlex: A novel STR multiplex validated for forensic use in the hen harrier (Circus cyaneus).

The hen harrier (Circus cyaneus) is a bird of prey which is heavily persecuted in the UK because it preys on the game bird red grouse (Lagopus lagopus scoticus). To help investigations into illegal killings of hen harrier, a STR multiplex kit containing eight short tandem repeat (STR) markers and a chromohelicase DNA binding protein 1 (CHD 1) sexing marker was developed. The multiplex kit was tested for species specificity, sensitivity, robustness, precision, accuracy and stability. Full profiles were obtained with as little as 0.25 ng of template DNA. Concurrent development of an allelic ladder to ensure reliable and accurate allele designation across laboratories makes the SkydancerPlex the first forensic DNA profiling system in a species of wildlife to be fully validated according to SWGDAM and ISFG recommendations. An average profile frequency of 3.67 × 10(-8), a PID estimate of 5.3 × 10(-9) and a PID-SIB estimate of 9.7 × 10(-4) make the SkydancerPlex an extremely powerful kit for individualisation.

Phylogenetic evidence for a case of misleading rather than mislabeling in caviar in the United Kingdom.

Sturgeons and paddlefish are freshwater fish which are highly valued for their caviar. Despite the fact that every single species of sturgeon and paddlefish is listed under CITES, there are reports of illegal trade in caviar where products are deliberately mislabeled. Three samples of caviar purchased in the United Kingdom were investigated for accurate CITES labeling using COI and cyt b sequencing. Initial species identification was carried out using BLAST followed by phylogenetic analyses using both maximum parsimony and maximum likelihood methods. Results showed no evidence for mislabeling with respect to CITES labels in any of the three samples, but we observed clear evidence for a case of misleading the customer in one sample.

FMLP-, thapsigargin-, and H2O2-evoked changes in intracellular free calcium concentration in lymphocytes and neutrophils of type 2 diabetic patients.

Type 2 diabetic (T2DM) patients are immune-compromised having a higher susceptibility to infections and long-term complications in different parts of the body contributing to increased morbidity and mortality. A derangement in the homeostasis of intracellular free calcium concentration [Ca²âº](i) is known to be associated with several diseases in the body including T2DM. Both neutrophils and lymphocytes play active protective roles in host immune response to infection showing impairment in microbicidal functions including phagocytosis and chemotaxis which are calcium-dependent processes. This study evaluated the process of [Ca²âº]i mobilization from both neutrophils and lymphocytes taken from blood of both T2DM patients and healthy age-matched control subjects investigating the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP), thapsigargin (TG), and hydrogen peroxide (H2O2) on [Ca²âº](i) homeostasis. This study employed isolated peripheral blood neutrophils and lymphocytes from 24 T2DM patients and 24 healthy volunteers. Either neutrophils or lymphocytes were stimulated separately with fMLP, TG, or H2O2. Induced changes in [Ca²âº] in both neutrophils and lymphocytes were evaluated using spectrofluorometric methods. Stimulation of human neutrophils and lymphocytes with fMLP, TG, or H2O2 in the presence of [Ca²âº]o resulted in significant decreases in [Ca²âº](i) mobilization from T2DM patients compared with healthy controls. These data indicate that neutrophils and lymphocytes from T2DM patients are less responsive to calcium mobilizing agents compared with granulocytes from healthy controls and this is possibly due to the hyperglycemia. The results suggest that agonist-evoked decrease in [Ca²âº](i) in immune cells might be one of the possible mechanisms of impaired immunity in diabetic patients.

Use of non-human DNA analysis in forensic science: a mini review.

Analysis of non-human DNA in forensic science, first reported about two decades ago, is now commonplace. Results have been used as evidence in court in a variety of cases ranging from abduction and murder to patent infringement and dog attack. DNA from diverse species, including commonly encountered pets such as dogs and cats, to plants, viruses and bacteria has been used and the sheer potential offered by such analyses has been proven. In this review, using case examples throughout, we detail the considerable literature in this field.

Reliable and robust molecular sexing of the hen harrier (Circus cyaneus) using PCR-RFLP analysis of the CHD 1 gene.

The hen harrier (Circus cyaneus) is a bird of prey that is persecuted in the United Kingdom, and there is a need for a DNA-based individual identification and sexing system for the use in forensic investigations. This study reports a new set of PCR primers for the chromo-helicase-DNA-binding protein 1 gene, which allows sexing using PCR-RFLP. Instead of exonic primers that amplify across a large intron, this set consists of a primer within the intron, enabling reduction in amplicon sizes from 356 to 212 bp and 565 to 219 bp in W and Z chromosomes. DNA degradation and dilution experiments demonstrate that this set is significantly more robust than one that amplifies across the intron, and sequencing of the intronic primer-binding region across several individuals shows that it is highly conserved. While our objective is to incorporate this primer set into an STR-based individualization kit, it may in the meantime prove useful in forensic or conservation studies.

Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit.

DNA extracted from forensic samples can be degraded and also contain co-extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co-amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC90 and IAC410) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX-labelled primers. Co-amplification of IAC90 and IAC410 was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC90 and IAC410 were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC90 signal was still present after all human DNA loci fail to amplify; in contrast, the IAC410 signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles.

STR data for the AmpFlSTR SGM Plus loci from two South Asian populations.

Using the Applied Biosystems' AmpFlSTR SGM Plus PCR amplification kit, we studied the allele frequency distribution of 10 STR loci in two south Asian populations: one from the Gujarat region of India represented by 172 unrelated Gujaratis, now resident in England; and a Pakistani population, represented by 155 unrelated individuals. Gujarat borders southeast Pakistan. There were no significant deviations from Hardy-Weinberg equilibrium in either population after Bonferroni correction. The combined power of discrimination and exclusion for the Indian population were 0.999999999999544 and 0.9999785, respectively; for the Pakistani population, they were 0.999999999999865 and 0.9998975, respectively. F(ST) (or theta) between these two populations was estimated as 0.00146.

Application of a double-enrichment procedure for microsatellite isolation and the use of tailed primers for high throughput genotyping

The number of microsatellite loci and their allelic diversity contribute to increase accuracy and informativity of genetic estimates, however, the isolation of microsatellite loci is not only laborious but also quite expensive. We used (GATA)n and (GACA)n tetranucleotide probes and single- and double-enrichment hybridization to construct and screen a genomic library with an increased proportion of DNA fragments containing repeat motifs. Repeats were found using both types of hybridization but the double-enrichment procedure recovered sequences of which 100 percent contained (GATA)n and (GACA)n motifs. Microsatellite loci primers were then designed with an M13R-tail or CAG-tag to produce scorable PCR products with minimal stutter. The approach used in this study suggests that double-enrichment is a worthwhile strategy when isolating repeat motifs from eukaryotic genomes. Moreover, the use of tailed microsatellite primers provides increased resolution for compound microsatellite loci, with a significant decrease in costs.

Simultaneous overexpression of GH and STAT5b genes inhibits the STAT5 signalling pathway in tilapia (Oreochromis niloticus) embryos

In this study, we describe the use of a STAT5 responsive element (LHRE) reporter gene to monitor the activity of the growth hormone (GH) transduction pathway following expression of heterologous fish GH and rat STAT5b in tilapia embryos and fish fibroblast cells. Our results indicate that both GH and STAT5b are able to activate the LHRE at high levels when transferred separately, demonstrating the substantial level of conservation of the GH signal transduction pathways between fish and mammals. Unexpectedly, co-expression experiments show a strong inhibition of the GH-dependent activation, suggesting that simultaneous GH and STAT5b overexpression can counteract effects of GH expression in tilapia embryos