Many direct-to-consumer canine genetic tests can identify the breed of purebred dogs.

OBJECTIVE: To compare pedigree documentation and genetic test results to evaluate whether user-provided photographs influence the breed ancestry predictions of direct-to-consumer (DTC) genetic tests for dogs. ANIMALS: 12 registered purebred pet dogs representing 12 different breeds. METHODS: Each dog owner submitted 6 buccal swabs, 1 to each of 6 DTC genetic testing companies. Experimenters registered each sample per manufacturer instructions. For half of the dogs, the registration included a photograph of the DNA donor. For the other half of the dogs, photographs were swapped between dogs. DNA analysis and breed ancestry prediction were conducted by each company. The effect of condition (ie, matching vs shuffled photograph) was evaluated for each company's breed predictions. As a positive control, a convolutional neural network was also used to predict breed based solely on the photograph. RESULTS: Results from 5 of the 6 tests always included the dog's registered breed. One test and the convolutional neural network were unlikely to identify the registered breed and frequently returned results that were more similar to the photograph than the DNA. Additionally, differences in the predictions made across all tests underscored the challenge of identifying breed ancestry, even in purebred dogs. CLINICAL RELEVANCE: Veterinarians are likely to encounter patients who have conducted DTC genetic testing and may be asked to explain the results of genetic tests they did not order. This systematic comparison of commercially available tests provides context for interpreting results from consumer-grade DTC genetic testing kits.

9S1R nullomer peptide induces mitochondrial pathology, metabolic suppression, and enhanced immune cell infiltration, in triple-negative breast cancer mouse model.

Nullomers are the shortest strings of absent amino acid (aa) sequences in a species or group of species. Primes are those nullomers that have not been detected in the genome of any species. 9S1R is a 5-aa peptide prime sequence attached to 5-arginine aa, used to treat triple negative breast cancer (TNBC) in an in vivo mouse model. This unique peptide, administered with a trehalose carrier (9S1R-NulloPT), offers enhanced solubility and exhibits distinct anti-cancer effects against TNBC. In our study, we investigated the effect of 9S1R-NulloPT on tumor growth, metabolism, metastatic burden, tumor immune-microenvironment (TME), and transcriptome of aggressive mouse TNBC tumors. Notably, treated mice had smaller tumors in the initial phase of the treatment, as compared to untreated control, and diminished in vivo and ex vivo bioluminescence at later-stages - indicative of metabolically quiescent, dying tumors. The treatment also caused changes in TME with increased infiltration of immune cells and altered tumor transcriptome, with 365 upregulated genes and 710 downregulated genes. Consistent with in vitro data, downregulated genes were enriched in cellular metabolic processes (179), specifically mitochondrial TCA cycle/oxidative phosphorylation (44), and translation machinery/ribosome biogenesis (45). The upregulated genes were associated with the developmental (13), ECM organization (12) and focal adhesion pathways (7). In conclusion, our study demonstrates that 9S1R-NulloPT effectively reduced tumor growth during its initial phase, altering the TME and tumor transcriptome. The treatment induced mitochondrial pathology which led to a metabolic deceleration in tumors, aligning with in vitro observations.

Nullomer peptide increases immune cell infiltration and reduces tumor metabolism in triple negative breast cancer mouse model.

Background: Nullomers are the shortest strings of absent amino acid (aa) sequences in a species or group of species. Primes are those nullomers that have not been detected in the genome of any species. 9S1R is a 5-aa peptide derived from a prime sequence that is tagged with 5 arginine aa, used to treat triple negative breast cancer (TNBC) in an in vivo TNBC mouse model. 9S1R is administered in trehalose (9S1R-NulloPT), which enhances solubility and exhibits some independent effects against tumor growth and is thus an important component in the drug preparation. Method: We examined the effect of 9S1R-NulloPT on tumor growth, metabolism, metastatic burden, necrosis, tumor immune microenvironment, and the transcriptome of aggressive mouse TNBC tumors. Results: The peptide-treated mice had smaller tumors in the initial phase of the treatment, as compared to the untreated control, and reduced in vivo bioluminescence at later stages, which is indicative of metabolically inactive tumors. A decrease in ex vivo bioluminescence was also observed in the excised tumors of treated mice, but not in the secondary metastasis in the lungs. The treatment also caused changes in tumor immune microenvironment with increased infiltration of immune cells and margin inflammation. The treatment upregulated 365 genes and downregulated 710 genes in tumors compared to the untreated group. Consistent with in vitro findings in breast cancer cell lines, downregulated genes in the treated TNBC tumors include Cellular Metabolic Process Related genes (179), specifically mitochondrial genes associated with TCA cycle/oxidative phosphorylation (44), and translation machinery/ribosome biogenesis genes (45). Among upregulated genes, the Developmental Pathway (13), ECM Organization (12) and Focal Adhesion Related Pathways (7) were noteworthy. We also present data from a pilot study using a bilateral BC mouse model, which supports our findings. Conclusion: In conclusion, although 9S1R-NulloPT was moderate at reducing the tumor volume, it altered the tumor immune microenvironment as well as the tumor transcriptome, rendering tumors metabolically less active by downregulating the mitochondrial function and ribosome biogenesis. This corroborates previously published in vitro findings.

Correcting forensic DNA errors.

DNA mixture interpretation can produce opposing conclusions by qualified forensic analysts, even within the same laboratory. The long-delayed publication of the National Institutes of Standards and Technology (NIST) study of 109 North American crime laboratories in this journal demonstrates this most clearly. This latest study supports earlier work that shows common methods such as the Combined Probability of Inclusion (CPI) have wrongly included innocent people as contributors to DNA mixtures. The 2016 President's Council of Advisors on Science and Technology report concluded, "In summary, the interpretation of complex DNA mixtures with the CPI statistic has been an inadequately specified-and thus inappropriately subjective-method. As such, the method is clearly not foundationally valid" [7]. The adoption of probabilistic genotyping by many laboratories will certainly prevent some of these errors from occurring in the future, but the same laboratories that produced past errors can also now review old cases with their new software-without additional bench work. It is critical that laboratories adopt procedures and policies to do this.

Allele frequencies of 15 STR loci (Identifiler™ kit) in Basque-Americans.

Individuals with Basque ancestry form a historically and culturally important minority of the population of the western United States. Allele frequencies for the 15 autosomal STRs in the AmpFlSTR® Identifiler® PCR Amplification Kit (Applied Biosystems) from 156 unrelated self-identified Basque individuals born in the United States are presented. Allele frequencies were used to calculate parameters commonly used in genetics and forensics including power of discrimination (PD), power of exclusion (PE), polymorphic information content (PIC), and expected heterozygosity (He). The sample population was also compared with the European Basque population and the major American ethnicities.

The effect of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel and normal cell lines.

BACKGROUND: Nullomer peptides are the smallest sequences absent from databases of natural proteins. We first began compiling a list of absent 5-amino acid strings in 2006 (1). We report here the effects of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel, derived from human cancers of 9 organs (kidney, ovary, skin melanoma, lung, brain, lung, colon, prostate and the hematopoietic system), and four normal cell lines (endothelial HUVEC, skin fibroblasts BJ, colon epithelial FHC and normal prostate RWPE-1). METHODS: NCI-60 cancer cell panel and four normal cell lines were cultured in vitro in RPMI1640 supplemented with 10% Hyclone fetal bovine serum and exposed for 48 h to 5 µM, 25 µM and 50 µM of peptides 9R, 9S1R and 124R. Viability was assessed by CCK-8 assay. For peptide ATP depletion effects, one cell line representing each organ in the NCI-60 panel, and four normal cell lines were exposed to 50 µM of peptides 9R, 9S1R and 124R for 3 h. The ATP content was assessed in whole cells, and their supernatants. RESULTS: Peptides 9S1R and 9R are respectively lethal to 95 and 81.6% of the 60 cancer cell lines tested. Control peptide 124R has no effect on the growth of these cells. Especially interesting the fact that peptides 9R and 9S1R are capable of killing drug-resistant and hormone-resistant cell lines, and even cancer stem cells. Peptides 9R and 9S1R have a broader activity spectrum than many cancer drugs in current use, can completely deplete cellular ATP within 3 h, and are less toxic to 3 of the 4 normal cell lines tested than they are to several cancers. CONCLUSIONS: Nullomer peptides 9R and 9S1R have a large broad lethal effect on cancer cell lines derived from nine organs represented in the NCI-60 panel. This broad activity crosses many of the categorical divisions used in the general classification of cancers: solid vs liquid cancers, drug sensitive vs drug resistant, hormone sensitive vs hormone resistant, cytokine sensitive vs cytokine non sensitive, slow growing vs rapid growing, differentiated vs dedifferentiated cancers. Furthermore peptides 9R and 9S1R are lethal to cancer stem cells and breast canrcinosarcoma.

Case report: Coincidental inclusion in a 17-locus Y-STR mixture, wrongful conviction and exoneration.

We report the case of a suspect (Suspect-3) who was convicted (and later exonerated) of participating in the multiple-attacker rape of two women. The forensic evidence against him was his inclusion in a 17-marker Y-STR mixture isolated from semen on one victim's clothing. The DNA inclusion produced a match statistic with a combined probability of inclusion of 1 in 741, and a Likelihood Ratio of 3296. While the defense team was told that Suspect-3 was included in the semen DNA mixture, they were not told that all of the Y-STR alleles could also be explained by just the other two accused attackers' haplotypes. Suspect-3 was subsequently freed after the Taiwan Association for Innocence requested re-examination of the incriminating mixed DNA sample. The Criminal Investigation Bureau was then able to exclude him using an extended set of Y-STR markers (23 loci), leading to his exoneration.

Morphologic and molecular description of Metopus fuscus Kahl from North America and new rDNA sequences from seven metopids (Armophorea, Metopidae).

Most species in the large ciliate genus Metopus Claparède & Lachmann, 1858 lack detailed descriptions based on modern morphologic and molecular methods. This lack of data for the vast majority of species hampers application of a morphospecies approach to the taxonomy of Metopus and other armophorids. In this report we redescribe the large species, Metopus fuscusKahl, 1927 based on in vivo observation, silver impregnation, scanning electron microscopy, and single-cell 18S rDNA sequencing of a freshwater North American (Idaho) population. Metopus fuscus invariably has a perinuclear envelope of endosymbiotic bacteria not found in other species. Unlike the original description of a single row of coarse granules between ciliary rows, the Idaho population has five loose rows of small interkinetal granules. We discuss the possible importance of this character in metopids. We also provide a phylogenetic analysis including seven other new metopid 18S rDNA sequences: Brachonella spiralis, B. galeata, Metopus laminarius, M. setosus, M. striatus, M. violaceus, Palmarella lata. Metopus fuscus and M. setosus form a fully supported clade, challenging previous morphospecies groupings. We discuss some ambiguities of armophorid morphologic terminology in the earlier literature. Our phylogenetic analysis of Idaho metopids indicates that the genera Metopus and Brachonella are both nonmonophyletic.

The first successful use of a low stringency familial match in a French criminal investigation.

We describe how a very simple application of familial searching resolved a decade-old, high-profile rape/murder in France. This was the first use of familial searching in a criminal case using the French STR DNA database, which contains approximately 1,800,000 profiles. When an unknown forensic profile (18 loci) was searched against the French arrestee/offender database using CODIS configured for a low stringency search, a single low stringency match was identified. This profile was attributed to the father of the man suspected to be the source of the semen recovered from the murder victim Elodie Kulik. The identification was confirmed using Y-chromosome DNA from the putative father, an STR profile from the mother, and finally a tissue sample from the exhumed body of the man who left the semen. Because of this identification, the investigators are now pursuing possible co-conspirators.

Morphology and phylogeny of Bryophryoides ocellatus n. g., n. sp. (Ciliophora, Colpodea) from in situ soil percolates of Idaho, U.S.A.

We describe the morphology and 18S rDNA phylogeny of Bryophryoides ocellatus n. g., n. sp., a bryophryid ciliate inhabiting in situ soil percolates from Idaho, U.S.A. The new genus is distinguished from other bryophryid genera by a combination of the following features: (1) kreyellid (irregularly meshed) silverline pattern, (2) polymorphic adoral organelles in the preoral suture, (3) absence of vestibular kineties. In phylogenetic analyses, Bryophryoides ocellatus is most closely related to Bryophrya gemmea. The 18S rDNA sequence pairwise distance of 2% between these genera, while similar to that between many colpodidan species, exceeds that between some colpodidan genera (e.g. Mykophagophrys and Pseudoplatyophrya, 1.1%), further supporting establishment of the new genus. Topology hypothesis testing strongly supports the monophyly of the Colpodida including the bryophryids. Despite weak nodal support, tests of topology constraints narrowly reject the non-monophyly of the sequenced Bryophryidae (Bryophrya+Bryophryoides+Notoxoma). Likewise, the monophyletic origin of the sequenced Bryophryidae is indicated in the phylogenetic networks though with low support.

Safeguarding forensic DNA reference samples with nullomer barcodes.

Unintended transfer of biological material containing DNA is a concern to all laboratories conducting PCR analysis. While forensic laboratories have protocols in place to reduce the possibility of contaminating casework samples, there is no way to detect when a reference sample is mislabeled as evidence, or contaminates a forensic sample. Thus there is public concern regarding the safeguarding of DNA submitted to crime labs. We demonstrate a method of introducing an internal amplification control to reference samples, in the form of a nullomer barcode which is based upon sequences absent or rare from publically accessible DNA databases. The detection of this barcode would indicate that the source of analyzed DNA was from a reference sample provided by an individual, and not from an evidence sample. We demonstrate that the nullomers can be added directly to collection devices (FTA paper) to allow tagging during the process of sample collection. We show that such nullomer oligonucleotides can be added to existing forensic typing and quantification kits, without affecting genotyping or quantification results. Finally, we show that even when diluted a million-fold and spilled on a knife, the nullomer tags can be clearly detected. These tags support the National Research Council of the National Academy recommendation that "Quality control procedures should be designed to identify mistakes, fraud, and bias" in forensic science (National Academy of Sciences, 2009).

Nullomer derived anticancer peptides (NulloPs): differential lethal effects on normal and cancer cells in vitro.

We demonstrate the first use of the nullomer (absent sequences) approach to drug discovery and development. Nullomers are the shortest absent sequences determined in a species, or group of species. By identifying the shortest absent peptide sequences from the NCBI databases, we screened several potential anti-cancer peptides. In order to improve cell penetration and solubility we added short poly arginine tails (5Rs), and initially solubilized the peptides in 1M trehalose. The results for one of the absent sequences 9R (RRRRRNWMWC), and its scrambled version 9S1R (RRRRRWCMNW) are reported here. We refer to these peptides derived from nullomers as PolyArgNulloPs. A control PolyArgNulloP, 124R (RRRRRWFMHW), was also included. The lethal effects of 9R and 9S1R are mediated by mitochondrial impairment as demonstrated by increased ROS production, ATP depletion, cell growth inhibition, and ultimately cell death. These effects increase over time for cancer cells with a concomitant drop in IC-50 for breast and prostate cancer cells. This is in sharp contrast to the effects in normal cells, which show a decreased sensitivity to the NulloPs over time.

Y-STR variation in the Basque diaspora in the Western USA: evolutionary and forensic perspectives.

Individuals of Basque origin migrated in large numbers to the Western USA in the second half of the nineteenth century, and the flow continued with less intensity during the last century. The European source population, that of the Basque Country, has long been a cultural and geographical isolate. Previous studies have demonstrated that Y-STR frequencies of Basques are different from those of other Spanish and European populations [1]. The Basque diaspora in the Western USA is a recent migration, but the founder effect and the incorporation of new American Y chromosomes into the paternal genetic pool of the Basque diaspora could have influenced its genetic structure and could thus have practical implications for forensic genetics. To check for genetic substructure among the European source and Basque diaspora populations and determine the most suitable population database for the Basque diaspora in the Western USA, we have analysed the haplotype distribution of 17 Y-STRs in both populations. We have found that the Basque diaspora in the Western USA largely conserve the Y chromosome lineage characteristic of the autochthonous European Basque population with no statistically significant differences. This implies that a common 17 Y-STR Basque population database could be used to calculate identification or kinship parameters regardless of whether the Basque individuals are from the European Basque Country or from the Basque diaspora in the Western USA.

Subjectivity and bias in forensic DNA mixture interpretation.

The objectivity of forensic science decision making has received increased attention and scrutiny. However, there are only a few published studies experimentally addressing the potential for contextual bias. Because of the esteem of DNA evidence, it is important to study and assess the impact of subjectivity and bias on DNA mixture interpretation. The study reported here presents empirical data suggesting that DNA mixture interpretation is subjective. When 17 North American expert DNA examiners were asked for their interpretation of data from an adjudicated criminal case in that jurisdiction, they produced inconsistent interpretations. Furthermore, the majority of 'context free' experts disagreed with the laboratory's pre-trial conclusions, suggesting that the extraneous context of the criminal case may have influenced the interpretation of the DNA evidence, thereby showing a biasing effect of contextual information in DNA mixture interpretation.

The genetics of innocence: analysis of 194 U.S. DNA exonerations.

This new analysis of 194 DNA exonerations, representing 171 criminal events, examines the types of evidence and DNA testing that have been used to free the victims of wrongful conviction. The types of DNA testing used to free the innocent parallels the growth of these techniques in forensic science. Short tandem repeat (STR) analysis now prevails (70%), though Y-STR analysis (16%) and mitochondrial testing (10%) are still used when STR analysis is not feasible, and the recently developed mini-STRs have been used for exonerations since 2008 (2.6%). The types of exculpatory evidence included intimate swabs (65%), clothing (53%), hair (13%), fingernail evidence (5%), cigarettes (3%), and other evidence. The most common factor associated with wrongful convictions was misidentification (75%), including misidentification by the victim (65%). False confessions (including admissions and pleas) were obtained in 30% of the cases, and informant testimony (including jailhouse and government informants) was used in 22% of the false convictions. Several types of invalid forensic science testimony were used to wrongfully convict in the 146 trials where transcripts or reliable forensic science data were available for analysis. Invalid testimony included serology (38%), hair comparison (22%), fingerprint comparison (2%), and bite mark comparison (3%). In 43% of the exonerations, the true perpetrator of the crime was identified through postconviction testing.

Mitochondrial DNA analysis of an immigrant Basque population: loss of diversity due to founder effects.

The Basques have a well-documented history of migration and settlement in the Americas, and they often retain cultural identity across generations. Numerous genetic studies have been carried out on European Basques; thus, immigrant Basques are an ideal population for investigating the genetic consequences of a recent human migration event. We have sampled 53 unrelated individuals with Basque ancestry in Boise, Idaho and determined the mitochondrial DNA (mtDNA) sequence variation of the first and second hypervariable regions. Thirty-six mtDNA haplotypes were detected in our sample. We found evidence of genetic changes consistent with founder effects, which is compatible with the known history of migration. Compared with the European Basque population, the immigrant Basques are significantly different in terms of haplogroup frequency distribution and diversity. They have a lower measure of weighted intralineage mean pairwise diversity (WIMP) and greater genetic distance from other European populations. These data indicate that this immigrant Basque population has experienced a reduction in genetic diversity compared with the putative source population. However, this loss of diversity is not detectable using indices of demographic history such as Tajima's D and Fu's F. This study represents the first description of mtDNA diversity in an immigrant Basque population, and our findings indicate that founder effects accompanying this relatively recent migration event have shaped the genetic diversity of this population.

Morphology, morphometrics, and molecular characterization of Bryophrya gemmea n. sp. (Ciliophora, Colpodea): implications for the phylogeny and evolutionary scenario for the formation of oral ciliature in the order Colpodida.

We studied the morphology, morphometry, resting, and reproductive cysts, as well as the molecular phylogeny of Bryophrya gemmea n. sp., a colpodid ciliate that was discovered in ephemeral puddles in Idaho, northwest United States. This new species is distinguished from congeners by the irregularly pentagonal adoral organelles, four to five vestibular kineties, the single micronucleus, and one to three rows of brightly refractive protuberant interkinetal cortical granules to the right of the preoral suture. Resting cysts have two distinct membranes and an outer mucous coat. As typical for most colpodids, reproduction occurs in division cysts but details of ontogenesis are unknown. The 18S rRNA gene sequence shows only weak support for the phylogenetic relationship between Bryophrya and the bryophryid genus Notoxoma previously inferred from morphologic characters. Further, our molecular phylogenies classify bryophryids rather basal within the order Colpodida, not supporting ordinal status suggested by morphologists. Based on molecular data and morphologic characters, the colpodid genus Ilsiella is removed from the family Marynidae and placed in a new family, Ilsiellidae. Considering the molecular data, an evolutionary scenario for the formation of colpodid oral structures from a cyrtolophosidid ancestor through a bryophryid intermediate is proposed.

The Y-STR genetic diversity of an Idaho Basque population, with comparison to European Basques and US Caucasians.

Fifty unrelated Basque males from southwest Idaho were typed for the 17 Y-STR loci in the Yfiler multiplex kit (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, YGATA H4.1 and DYS385a/b). In total, 42 haplotypes were identified, with no more than two individuals sharing a single haplotype. The haplotype diversity (HD) was 0.9935, and gene diversity (D) over loci was 0.457 ± 0.137. The Idaho Basque population was compared to the source population from the Basque autonomous region of Northern Spain and Southern France, as well as a United States Caucasian population. The haplotype diversity for the immigrant Basque sample is within 0.4% of the haplotype diversity of the European Basques (0.9903); thus the power of discrimination is similar for each population. The Idaho Basque population has less diversity in 9 out of 16 loci (considering DYS385a/b together) and 3% less diversity across all loci, compared to the European Basque population. A multidimensional scaling analysis (MDS) was created using pairwise R(ST) values to compare the Idaho Basques to other populations. Based upon R(ST) and F(ST) measures, no significant differentiation was found between the Idaho and source European Basque population.

Dockomatic - automated ligand creation and docking.

BACKGROUND: The application of computational modeling to rationally design drugs and characterize macro biomolecular receptors has proven increasingly useful due to the accessibility of computing clusters and clouds. AutoDock is a well-known and powerful software program used to model ligand to receptor binding interactions. In its current version, AutoDock requires significant amounts of user time to setup and run jobs, and collect results. This paper presents DockoMatic, a user friendly Graphical User Interface (GUI) application that eases and automates the creation and management of AutoDock jobs for high throughput screening of ligand to receptor interactions. RESULTS: DockoMatic allows the user to invoke and manage AutoDock jobs on a single computer or cluster, including jobs for evaluating secondary ligand interactions. It also automates the process of collecting, summarizing, and viewing results. In addition, DockoMatic automates creation of peptide ligand .pdb files from strings of single-letter amino acid abbreviations. CONCLUSIONS: DockoMatic significantly reduces the complexity of managing multiple AutoDock jobs by facilitating ligand and AutoDock job creation and management.

Ribonucleotide and ribonucleoside determination by ambient pressure ion mobility spectrometry.

Detection limits and reduced mobilities for 12 ribonucleotides and 4 ribonucleosides were measured by ambient pressure electrospray ionization-ion mobility spectrometry (ESI-IMS). With the instrument used in this study it was possible to separate some of these compounds within mixtures. Detection limits reported for ribonucleotides and ribonucleosides ranged from 15 to 300 pmol and the reduced mobilities ranged from 41 to 56 suggesting that ambient pressure ESI-IMS may be used for their rapid and sensitive separation and detection. This report demonstrates that it was possible to use ion mobility spectrometry (IMS) to obtain a spectrum for the separation of nucleotides and nucleosides in less than 1 min. The application holds great promise for nucleotide analysis in the area of separating DNA fragments in genome sequencing and also for forensics DNA typing examinations used for the identification of blood stains in crime scenes and paternity testing.