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Criminals often attempt to conceal blood-stained weapons used in violent crimes, making forensic evidence crucial in solving cases. This study explores the recovery and extraction of trace DNA from sports equipment, including cricket bats, table tennis racquets, and hockey sticks, which are frequently implicated in such incidents. Our research evaluates various double swab collection methods for retrieving trace DNA from these sports items, emphasizing those associated with blunt force trauma. We also compare presumptive and confirmatory tests to establish a direct correlation. This research consistently demonstrated robust DNA recovery, surpassing a 50 % threshold across all tests. Specifically, DNA recovery from buried samples reached an impressive 87 %, while washed samples still yielded a substantial 80 % efficiency. We conducted a comparative analysis between presumptive and confirmatory testing methods, establishing a direct correlation between the two. Variability in DNA recovery efficiency was observed and attributed to factors like the type of surface the items contacted, and ambient humidity levels. In addition to presenting robust DNA recovery rates, statistical analyses were employed to compare methods, establishing correlations and highlighting the influence of environmental factors on DNA recovery efficiency. These findings have significant implications for forensic investigations involving silent weapons crafted from sports equipment, emphasizing the need for standardized protocols and consideration of environmental factors in DNA analysis., Competing Interests: Conflict of interest All authors declare there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The main aim of this work was to validate the detection of saliva samples from denim, cotton, and polyester fabrics aged up to six months, applying rapid immunochromatographic tests resulting in the analysis of nuclear and mitochondrial DNA recovered. A comparison was also carried out between two saliva detection tests, Laboratory and Crime Scene. 50 μl saliva samples (three per time and test) were deposited on denim, cotton, and polyester fabrics. After 1, 3, 7, 14, 21, 30, 60, 90, 150, and 180 days of storage at room temperature, the samples were recovered by swabbing and detected by SERATEC ® Amylase (Laboratory) test and SERATEC ® SALIVA CS (Crime Scene) test (SERATEC ® , Göttingen, Germany). DNA was isolated from the swab extraction buffer applying a silica-based methodology, and quantified based on fluorescent and human-specific quantifications. Then, it was submitted to STR profiling and mtDNA sequencing. According to our results, saliva stains up to six months after deposition remain valid specimens. The intensity of the bands varied among fabric type and time. Total DNA was successfully recovered from all tested samples, though with the limitations of obtaining partial nuclear DNA profiles from the oldest samples. In contrast, complete characterization of mtDNA was achieved from all samples. Lab and CS tests performed similar on the detection of saliva, as well as, DNA yield and profiling. Future research will be able to expand these results, analyzing the stability of other body fluids and the sensitivity of rapid immunochromatographic tests to detect them., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mitochondria contain proteins from two genetic origins. Most mitochondrial proteins are encoded in the nuclear genome, translated in the cytosol, and subsequently imported into the different mitochondrial sub-compartments. A small number is encoded in the mitochondrial DNA (mtDNA). The manipulation of the mtDNA gene expression represents a challenge. Here, we present an in vitro approach using morpholinos chemically linked to a precursor protein to silence gene expression in purified human mitochondria. The protocol is demonstrated with a Jac1-morpholino chimera specifically targeting COX1 mRNA. The chimera import and mitochondrial translation requirements are described in a step-by-step procedure, where the dose-dependent effect of reducing COX1 translation is observed. The affinity and specificity of chimera-mRNA binding also show great applicability to purify transcript-associated proteins by using the imported chimera construct as bait for immunoprecipitation. This new strategy opens up the possibility to address mechanistic questions about gene expression and physiology in mitochondria., (Copyright © 2024. Published by Elsevier Inc.)

Artefacts made from stones, bones and teeth are fundamental to our understanding of human subsistence strategies, behaviour and culture in the Pleistocene. Although these resources are plentiful, it is impossible to associate artefacts to specific human individuals 1 who can be morphologically or genetically characterized, unless they are found within burials, which are rare in this time period. Thus, our ability to discern the societal roles of Pleistocene individuals based on their biological sex or genetic ancestry is limited 2-5 . Here we report the development of a non-destructive method for the gradual release of DNA trapped in ancient bone and tooth artefacts. Application of the method to an Upper Palaeolithic deer tooth pendant from Denisova Cave, Russia, resulted in the recovery of ancient human and deer mitochondrial genomes, which allowed us to estimate the age of the pendant at approximately 19,000-25,000 years. Nuclear DNA analysis identifies the presumed maker or wearer of the pendant as a female individual with strong genetic affinities to a group of Ancient North Eurasian individuals who lived around the same time but were previously found only further east in Siberia. Our work redefines how cultural and genetic records can be linked in prehistoric archaeology., (© 2023. The Author(s).)

Next-generation sequencing has dramatically fostered insect mitogenomic research in recent years. However, studies on the insect mitochondrial genome (mitogenome) assembly mainly rely on the sequencing data from total DNA, which is not cost-effective as a huge data from nuclear DNA are wasted. Besides, many mitogenomic studies require genomic information from individual organisms, whereas the DNA yield from small individual insects is too low to meet the sequencing requirements. Here, we describe a strategy for a high enrichment of insect mitochondrial DNA (mtDNA) using rolling circle amplification (RCA) technique. This strategy consists of standard DNA extraction, RCA enrichment, next-generation sequencing and mitogenome assembly. We have evaluated the performance of this strategy on nine insect species representing eight families of insecta, three other invertebrates, and even two vertebrate specimens. Results show that our strategy is especially suitable for insects, which allows almost all tested insect mtDNA contents to reach 80% and above. A further examination of enrichment efficiency of our strategy among different taxa shows that it is also applicable to other invertebrates and even some vertebrates such as Rhacophorus and ptyas species, although its enrichment efficiency in these groups is lower than that of insects. After treatment with our strategy, small flux sequencing data can realize the assembly of mitogenome with deep coverage, providing a solid base for subsequent mitogenome-based studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The sambar (Rusa unicolor) is one of the largest deer distributed across diverse habitats of India and threatened due to habitat loss, changes in the land-use patterns, illegal poaching, and anthropogenic disturbances. Here, we report the first population genetics account of sambar deer from the Western Himalayas., Methods and Results: We observed relatively compromised genetic diversity (π = 0.0008 ± 0.0006 at mtDNA and Ho = 0.499 ± 0.056 at nDNA). We identified 60 unique individuals using a select panel of seven loci (P ID sib cum 1.60E-03). Bayesian skyline plot showed a stable demographic history since the past 8 kyr with a decline in recent years. The population lacked genetic structuring, likely due to the contiguous distribution and large dispersal patterns of sambar., Conclusion: The preliminary findings are valuable in exploring the utility of genetic diversity in monitoring the sambar population, estimating density following capture-recapture analysis, and aid to the conservation planning of sambar in large landscapes., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Research Question: Which is the optimal extraction method for isolating and quantifying circulating cell-free DNA (ccfDNA) from patients with endometriosis? Endometriosis is a common benign disease, associated with pain, infertility and reduced quality of life. Endometriosis is also a known risk factor for various cancers. Robust biomarkers for early detection and prediction of prognosis, however, are lacking. CcfDNA is an easy to obtain biomarker associated with prognosis of cancer patients and enables non-invasive analysis of somatic mutations. Recently, elevated levels of ccfDNA were detected in patients with endometriosis., Design: Two different ccfDNA extraction methods were compared: Maxwell RSC ccfDNA plasma kit (Maxwell) and QiAamp minElute ccfDNA mini kit (QIAamp). The ccfDNA and circulating mitochondrial DNA (mtDNA) quantities from 34 patients diagnosed with endometriosis were analysed. Fluorometric measurement and quantitative reverse transcription polymerase chain reaction (qRT-PCR) of short and long ALU and mtDNA fragments were used to quantiy ccfDNA., Results: The yield of ccfDNA isolated with the Maxwell method was significantly higher compared with the QIAamp method (P < 0.0001). Integrity of ccfDNA was significantly higher in the QIAamp isolate (P < 0.0001). Recovered mtDNA was not significantly different between both extraction methods used., Conclusions: The choice of extraction method can significantly influence the ccfDNA output and integrity. Both methods, however, enabled isolation of sufficient ccfDNA for further downstream applications. With this approach, isolation of ccfDNA could enable the non-invasive detection and analysis of somatic mutation within endometriosis tissue., (Copyright © 2021 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The origins and taxonomy of the introduced vervet monkey population in Dania Beach, Florida has been unconfirmed due to a lack of documentation and genetic research. Our goal was to determine the introduction history, species identification, and geographic origins of the monkeys. Through interviews, historical archives, and popular media, we traced the monkeys to an escape from the Dania Chimpanzee Farm in 1948. The facility imported primates from Africa for medical research purposes. Historical archives suggest the monkeys were caught in Sierra Leone. We tested the hypothesis of West African origins using three genetic markers: one mitochondrial DNA gene (cytochrome b) and two fragments from the Y-chromosome, the sex-determining gene and the zinc-finger gene. We ran Bayesian and maximum-likelihood analyses to reconstruct phylogenetic trees. Results from all loci confirmed the species identification is Chlorocebus sabaeus. We found no variation among the sampled individuals and found the cytochrome b haplotype to be a complete match to a C. sabaeus sample from Senegal. Phylogenetic analyses showed strong support for the Dania Beach mitochondrial and Y-chromosome lineages to group within a monophyletic C. sabaeus clade endemic to West Africa. Our study provides critical baseline information to the scientific community about a little-known population of Chlorocebus monkeys that have adapted to a novel environment in the southeastern United States.

The habitats of Galago moholi are suspected to be largely fragmented, while the species is thought to be expanding further into the southernmost fringe of its range, as well as into human settlements. To date, no intraspecific molecular genetic studies have been published on G. moholi. Here we estimate the genetic diversity and connectivity of populations of G. moholi using two mitochondrial gene regions, the cytochrome C oxidase subunit I gene (COI) and the displacement loop of the control region (D-loop). Samples from five localities in northern South Africa were obtained from archived collections. The two mitochondrial DNA gene regions were amplified and sequenced to provide population summary statistics, differentiation [proportion of the total genetic variation in a population relative to the total genetic variance of all the populations (F ST ), differentiation within populations among regions (Φ ST )], genetic distance and structure. There was discernible genetic structure among the individuals, with two COI and six D-loop haplotypes belonging to two genetically different groups. There was population differentiation among regions (F ST  = 0.670; Φ ST  = 0.783; P < 0.01). However, there were low levels of differentiation among populations, as haplotypes were shared between distant populations. Adjacent populations were as divergent from each other as from distant populations. The results suggest that genetic introgression, most likely due to past migrations or recent unintentional translocations that include the animal trade, may have led to connectivity among populations.

Bones and teeth are important sources of Pleistocene hominin DNA, but are rarely recovered at archaeological sites. Mitochondrial DNA (mtDNA) has been retrieved from cave sediments but provides limited value for studying population relationships. We therefore developed methods for the enrichment and analysis of nuclear DNA from sediments and applied them to cave deposits in western Europe and southern Siberia dated to between 200,000 and 50,000 years ago. We detected a population replacement in northern Spain about 100,000 years ago, which was accompanied by a turnover of mtDNA. We also identified two radiation events in Neanderthal history during the early part of the Late Pleistocene. Our work lays the ground for studying the population history of ancient hominins from trace amounts of nuclear DNA in sediments., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

In the event of fires, natural disasters, and other events associated with high temperature, bones and teeth are the only source of genetic material for identifying human or animal carcasses. To obtain reliable final results of identification tests, the use of appropriate nucleic acid extraction methods is crucial. Therefore, the main objective of this research was to evaluate the effectiveness of selected methods of DNA isolation from animal burnt bones and teeth. In addition, the effect of the duration of high temperature on the stability of nuclear and mitochondrial DNA in these tissues was determined, as well as the possibility of using the genetic material obtained for species identification of remains of unknown origin. Bones and teeth collected during necropsy of dogs were burnt in a laboratory oven at 400 °C (752 °F; 673.15 K) for 5, 10, 15, 30, 45 and 60 min. DNA was isolated according to four different protocols, using three commercial kits, i.e. the PrepFiler® Forensic DNA Extraction Kit from Applied Biosystems, the QIAamp® DNA Investigator Kit from QIAGEN, and the DNA Mini Kit from Syngen, as well as a classic organic method. The effectiveness of these methods was compared by assessing the amount of isolated DNA using Real-Time PCR and its purity using a NanoDrop™ spectrophotometer. Each isolate was also subjected to PCR with primers designed to amplify fragments of dog mitochondrial DNA. The effectiveness of species identification was assessed for the method showing the best DNA recovery and for the organic method, considered the gold standard for analysis of difficult material. The QIAamp® DNA Investigator Kit showed the highest efficiency of DNA isolation from bones and teeth burnt for 15 min (the longest burning time for which DNA could still be recovered from bones and teeth). The results of the experiment clearly indicate that DNA stability in hard tissues depends on how long they burn. In the case of exposure to 400 °C, reliable genetic testing, including species identification, is possible when the burning time does not exceed 15 min. Among the hard tissues examined, bones proved more suitable than teeth for identification purposes. It was also concluded that identification of bone remains with extreme heat damage should be based on mitochondrial DNA analysis., (Copyright © 2021 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Mitochondrial DNA (mtDNA) has been demonstrated to be a reliable biomarker of UV-induced genetic damage in both animal and human skin. Properties of the mitochondrial genome which allow for its use as a biomarker of damage include its presence in multiple copies within a cell, its limited repair mechanisms, and its lack of protective histones. To measure UV-induced mtDNA damage (particularly in the form of strand breaks), real-time quantitative PCR (qPCR) is used, based on the observation that PCR amplification efficiency is decreased in the presence of high levels of damage. Here, we describe the measurement of UV-induced mtDNA damage which includes the extraction of cellular DNA, qPCR to determine the relative amount of mtDNA, qPCR to determine UV-induced damage within a long strand of mtDNA, and the verification of the amplification process using gel electrophoresis.

A female Bornean orangutan (Pongo pygmaeus) aged 11 years and 6 months was examined by veterinarians after caretakers observed lethargy and facial grimacing. Within 72 h the primate had left-sided hemiparesis that worsened over the next week. An MRI revealed a focal right-sided cerebral mass suspected to be a neoplasm. Ten days after onset of clinical signs, the orangutan died. On postmortem exam, the medial right parietal lobe was replaced by a 7 × 4 × 3.5 cm focus of neuromalacia and hemorrhage that displaced the lateral ventricle and abutted the corpus callosum. Histopathology of the cerebral lesion revealed pyogranulomatous meningoencephalitis with intralesional amoeba trophozoites and rare cysts. Fresh parietal lobe was submitted to the Centers for Disease Control and Prevention lab for multiplex free-living amoebae real-time PCR and detected Balamuthia mandrillaris DNA at a high burden. Mitochondrial DNA was sequenced, and a 760-bp locus 19443F/20251R was compared to several human infections of B. mandrillaris and shown to be identical to the isolates from four human cases of encephalitis: 1998 in Australia, 1999 in California, 2000 in New York, and 2010 in Arizona. Indirect immunofluorescent antibody testing of stored serum samples indicated exposure to B. mandrillaris for at least 2 years prior to death. Within 1 week of the orangutan's death, water from the exhibit was analyzed and identified the presence of B. mandrillaris DNA, elucidating a possible source of exposure. B. mandrillaris, first reported in a mandrill in 1986, has since occurred in humans and animals and is now considered an important emerging pathogen.

In recent years, next-generation sequencing (NGS) has become a powerful tool for studying both inherited and somatic heteroplasmic mitochondrial DNA (mtDNA) variation. NGS has proved particularly powerful when combined with single-cell isolation techniques, allowing the investigation of low-level heteroplasmic variants both between cells and within tissues. Nevertheless, there remain significant challenges, especially around the selective enrichment of mtDNA from total cellular DNA and the avoidance of nuclear pseudogenes. This chapter summarizes the techniques needed to enrich, amplify, sequence, and analyse mtDNA using NGS .

Changes in circulating mitochondrial DNA (mtDNA) are widely used to indicate mitochondrial dysfunction in common non-genetic diseases where mitochondrial dysfunction may play a role. However, the methodology being used is not always specific and reproducible, and most studies use whole blood rather than evaluating cellular and cell-free mtDNA separately. Cellular mtDNA is contained within the mitochondrion and encodes vital subunits of the OXPHOS machinery. Conversely, cell-free mtDNA can have harmful effects, triggering inflammatory responses and potentially contributing to pathogenic processes. In this chapter, we describe a protocol to accurately measure the amount of cellular and cell-free human mtDNA in peripheral blood. Absolute quantification is carried out using real-time quantitative PCR (qPCR) to quantify cellular mtDNA, measured as the mitochondrial genome to nuclear genome ratio (designated the Mt/N ratio) in whole blood and peripheral blood mononuclear cells (PBMCs) and the number of mtDNA copies per μL in plasma and serum. We describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions, (2) prepare DNA from each of these fractions, (3) prepare dilution standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Chondrocytes, the only cells in articular cartilage, are metabolically active and responsible for the turnover of extracellular matrix and maintenance of the tissue homeostasis. Changes in chondrocyte function can cause degradation of the matrix and loss of articular cartilage integrity, leading to development and progression of osteoarthritis (OA). These changes are exemplified by accumulated mitochondrial damage and dysfunction. Because mitochondria are the critical organelles to produce energy and play a key role in cellular processes, the approaches to assess mitochondrial function under both physiological and pathological conditions enable us to uncover the mechanisms on how dysfunction of mitochondria in chondrocytes mediates signaling pathways that are involved in disturbance of cartilage homeostasis. In this chapter, we describe the methods to evaluate mitochondrial biogenesis, activity and mitochondrial DNA (mtDNA) integrity in chondrocytes.

The aim of this study was to assess two protocols for their capacities to simultaneously isolate RNA, mtDNA and ncDNA from mammalian cells. We compared the Invitrogen TRIzol-based method and Qiagen DNeasy columns, using the HepG2 cell line and human primary glioblastoma stem cells. Both methods allowed the isolation of all three types of nucleic acids and provided similar yields in mtDNA. However, the yield in ncDNA was more than tenfold higher on columns, as observed for both cell types. Conversely, the TRIzol method proved more reproducible and was the method of choice for isolating RNA from glioblastoma cells, as demonstrated for the housekeeping genes RPLP0 and RPS9 .

Accurate identification of species is a prerequisite for successful biodiversity management and further genetic studies. Species identification techniques often require both morphological diagnostics and molecular tools, such as DNA barcoding, for correct identification. In particular, the use of the subunit I of the mitochondrial cytochrome c oxidase (COI) gene for DNA barcoding has proven useful in species identification for insects. However, to date, no studies have been carried out on the DNA barcoding of Nigerian butterflies. We evaluated the utility of DNA barcoding applied for the first time to 735 butterfly specimens from southern Nigeria. In total, 699 DNA barcodes, resulting in a record of 116 species belonging to 57 genera, were generated. Our study sample comprised 807 DNA barcodes based on sequences generated from our current study and 108 others retrieved from BOLD. Different molecular analyses, including genetic distance-based evaluation (Neighbor-Joining, Maximum Likelihood and Bayesian trees) and species delimitation tests (TaxonDNA, Automated Barcode Gap Discovery, General Mixed Yule-Coalescent, and Bayesian Poisson Tree Processes) were performed to accurately identify and delineate species. The genetic distance-based analyses resulted in 163 well-separated clusters consisting of 147 described and 16 unidentified species. Our findings indicate that about 90.20% of the butterfly species were explicitly discriminated using DNA barcodes. Also, our field collections reported the first country records of ten butterfly species-Acraea serena, Amauris cf. dannfelti, Aterica galena extensa, Axione tjoane rubescens, Charaxes galleyanus, Papilio lormieri lormeri, Pentila alba, Precis actia, Precis tugela, and Tagiades flesus. Further, DNA barcodes revealed a high mitochondrial intraspecific divergence of more than 3% in Bicyclus vulgaris vulgaris and Colotis evagore. Furthermore, our result revealed an overall high haplotype (gene) diversity (0.9764), suggesting that DNA barcoding can provide information at a population level for Nigerian butterflies. The present study confirms the efficiency of DNA barcoding for identifying butterflies from Nigeria. To gain a better understanding of regional variation in DNA barcodes of this biogeographically complex area, future work should expand the DNA barcode reference library to include all butterfly species from Nigeria as well as surrounding countries. Also, further studies, involving relevant genetic and eco-morphological datasets, are required to understand processes governing mitochondrial intraspecific divergences reported in some species complexes.

In pair-living mammals, genetic monogamy is extremely rare. One possible reason is that in socially monogamous animals, mate choice can be severely constrained, increasing the risk of inbreeding or pairing with an incompatible or low-quality partner. To escape these constraints, individuals might engage in extra-pair copulations. Alternatively, inbreeding can be avoided by dispersal. However, little is known about the interactions between mating system, mate choice, and dispersal in pair-living mammals. Here we genotyped 41 wild individuals from 14 groups of coppery titi monkeys (Plecturocebus cupreus) in Peruvian Amazon using 18 microsatellite loci. Parentage analyses of 18 young revealed no cases of extra-pair paternity, indicating that the study population is mostly genetically monogamous. We did not find evidence for relatedness- or heterozygosity-based mate choice. Despite the lack of evidence for active inbreeding avoidance via mate choice, mating partners were on average not related. We further found that dispersal was not sex-biased, with both sexes dispersing opportunistically over varying distances. Our findings suggest that even opportunistic dispersal, as long as it is not constrained, can generate sufficient genetic diversity to prevent inbreeding. This, in turn, can render active inbreeding avoidance via mate choice and extra-pair copulations less necessary, helping to maintain genetic monogamy.

Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of ∼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Ware et al.)

Nuclear mitochondrial DNA segments (NUMTs) are generated via transfer of portions of the mitochondrial genome into the nuclear genome. Given their common origin, there is the possibility that both the mitochondrial and NUMT segments may co-amplify using the same set of primers. Thus, analysis of the variation of the mitochondrial genome must take into account this co-amplification of mitochondrial and NUMT sequences. The study herein builds on data from the study by Strobl et al. (Strobl et al., 2019), in which multiple point heteroplasmies were called with an "N" to prevent labeling NUMT sequences mimicking mitochondrial heteroplasmy and being interpreted as true mitochondrial in origin sequence variants. Each of these point heteroplasmies was studied in greater detail, both molecularly and bioinformatically, to determine whether NUMT or true mitochondrial DNA variation was present. The bioinformatic and molecular tools available to help distinguish between NUMT and mitochondrial DNA and the effect of NUMT sequences on interpretation were discussed., (Copyright © 2020 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Pleistocene glacial-interglacial cycles are correlated with dramatic temperature oscillations. Examining how species responded to these natural fluctuations can provide valuable insights into the impacts of present-day anthropogenic climate change. Here we present a phylogeographic study of the extinct American mastodon (Mammut americanum), based on 35 complete mitochondrial genomes. These data reveal the presence of multiple lineages within this species, including two distinct clades from eastern Beringia. Our molecular date estimates suggest that these clades arose at different times, supporting a pattern of repeated northern expansion and local extirpation in response to glacial cycling. Consistent with this hypothesis, we also note lower levels of genetic diversity among northern mastodons than in endemic clades south of the continental ice sheets. The results of our study highlight the complex relationships between population dispersals and climate change, and can provide testable hypotheses for extant species expected to experience substantial biogeographic impacts from rising temperatures.

Detection of food adulteration is a challenge. However, the identification of adulterated meat in processed products is important for health and personal preference. Mitochondrial genomic DNA (mtDNA) is a good candidate for reliable identification of meat ingredients; however, the extraction of mtDNA from processed products is a bottleneck for development of detection strategies. Therefore, we constructed a rapid (~5 min) mtDNA extraction device. mtDNAs from different meat samples, such as pork (Sus scrofa), chicken (Gallus gallus), and beef (Bos taurus), were successfully detected in up to 0.1% adulterated animal species. We believe that the proposed strategy could be applied to detect animal species from processed meat products to reduce fraudulent practices., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cytosine methylation is the leading epigenetic modification on DNA playing a role in gene regulation. Methylation can occur in cytosines of any nucleic acids in cytosol (as mitochondrial DNA, mtDNA) and in nuclear DNA (ncDNA). mtDNA exists as multiple copies within numerous mitochondria. This suggests that the number of mitochondria and mtDNA copy number can indicate the presence of a significant amount of DNA methylation within total DNA methylation detected. However, immunofluorescence method does not have a step to discriminate the staining between ncDNA and mtDNA. Antibodies used in immunological methods are methylation-specific but not selective for DNA type and they can bind to methylated cytosines in any DNA within the specimen. Current study aimed to understand whether mtDNA methylation interferes with the detection of nuclear DNA methylation by immunofluorescence and affinity enrichment (ELISA) in different mammalian cells. Experiments were performed to distinguish methylation between mtDNA and ncDNA. Immunofluorescence showed that there was no significant difference in the detected amount of methylation between mitochondrial and nuclear DNA. But ELISA revealed that up to 25% of cellular methylation was derived from mitochondria. This suggests that significant contamination of mtDNA methylation with ncDNA methylation can result in overestimation of the quantitative level of nuclear methylation., Competing Interests: Declaration of competing interest The author declares that no conflict of interest exists., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The genus Brassica mainly comprises three diploid and three recently derived allotetraploid species, most of which are highly important vegetable, oil or ornamental crops cultivated worldwide. Despite being extensively studied, the origination of B. napus and certain detailed interspecific relationships within Brassica genus remains undetermined and somewhere confused. In the current high-throughput sequencing era, a systemic comparative genomic study based on a large population is necessary and would be crucial to resolve these questions., Results: The chloroplast DNA and mitochondrial DNA were synchronously resequenced in a selected set of Brassica materials, which contain 72 accessions and maximally integrated the known Brassica species. The Brassica genomewide cpDNA and mtDNA variations have been identified. Detailed phylogenetic relationships inside and around Brassica genus have been delineated by the cpDNA- and mtDNA- variation derived phylogenies. Different from B. juncea and B. carinata, the natural B. napus contains three major cytoplasmic haplotypes: the cam-type which directly inherited from B. rapa, polima-type which is close to cam-type as a sister, and the mysterious but predominant nap-type. Certain sparse C-genome wild species might have primarily contributed the nap-type cytoplasm and the corresponding C subgenome to B. napus, implied by their con-clustering in both phylogenies. The strictly concurrent inheritance of mtDNA and cpDNA were dramatically disturbed in the B. napus cytoplasmic male sterile lines (e.g., mori and nsa). The genera Raphanus, Sinapis, Eruca, Moricandia show a strong parallel evolutional relationships with Brassica., Conclusions: The overall variation data and elaborated phylogenetic relationships provide further insights into genetic understanding of Brassica, which can substantially facilitate the development of novel Brassica germplasms.

Lyme borreliosis (LB) is the archetypal emerging zoonosis and is dependent on transmission by ticks in the genus Ixodes. Understanding the origin, maintenance, and spread of these ticks contributes much to our understanding of the spread of LB and other disease agents borne by these ticks. We collected 1232 Ixodes scapularis ticks from 17 east coast sites ranging from New Hampshire to Florida and used mtDNA, three nuclear genetic loci, and incorporated Bayesian analyses to resolve geographically distinct tick populations and compare their demographic histories. A sparse, stable, and genetically diverse population of ticks in the Southeastern US, that is rarely infected with the agent of LB is genetically distinct from an abundant, expanding, and comparatively uniform population in the Northeast, where epidemic LB now constitutes the most important vector borne disease in the United States. The contrasting geography and demography of tick populations, interpreted in the context of the geological history of the region, suggests that during the last glacial period such ticks occupied distinct refugia, with only the northern-most site of refuge giving rise to those ticks and pathogens now fueling the epidemic.

We generated a phylogeny for Caucasian rock lizards (Darevskia), and included six other families of true lizards (Lacertini), based on complete mitochondrial genome analysis. Next-generation sequencing (NGS) of genomic DNA was used to obtain 16 new mitogenomes of Darevskia. These, along with 35 sequences downloaded from GenBank: genera Darevskia, Zootoca, Podarcis, Phoenicolacerta, Takydromus, Lacerta, and Eremias-were used in the analysis. All four analytical methods (Bayesian Inference, BI; Maximum Likelihood, ML; Maximum Parsimony, MP; and Neighbor-Joining, NJ) showed almost congruent intra-generic topologies for Darevskia and other lizard genera. However, ML and NJ methods on one side, and BI and MP methods on the other harvested conflicting phylogenies. The ML/NJ topology supports earlier published separation of Darevskia into three mitochondrial clades (Murphy, Fu, Macculloch, Darevsky, and Kupinova, 2000), but BI and MP topologies support that the basal branching occurred between D. parvula from the western Lesser Caucasus and the rest of Darevskia. All topologies altered the phylogenetic position of some individual species, including D. daghestanica, D. derjugini, and D. chlorogaster. Reanalysis after excluding four saturated genes from the data set, and excluding genus Eremias gives fully convergent topologies. The most basal branching for true lizards was between Far Eastern Takydromus and the Western Eurasian genera (BI). Comparing phylogenetic performance of individual genes relative to whole mitogenome data, concatenated 16S RNA (the least saturated gene in our analyses) and Cytochrome b genes generate a robust phylogeny that is fully congruent with that based on the complete mitogenome., Competing Interests: The authors have declared that no competing interests exist.

The Indo-Pacific nassariids (genus Nassarius ) possesses the highest diversity within the family Nassariidae. However, the previous shell or radula-based classification of Nassarius is quite confusing due to the homoplasy of certain morphological characteristics. The toxin accumulators Nassarius glans and Nassarius siquijorensis are widely distributed in the subtidal regions of the Indo-Pacific Ocean. In spite of their biological significance, the phylogenetic positions of N . glans and N . siquijorensis are still undetermined. In the present study, the complete mitochondrial genomes of N . glans and N . siquijorensis were sequenced. The present mitochondrial genomes were 15,296 and 15,337 bp in length, respectively, showing negative AT skews and positive GC skews as well as a bias of AT rich on the heavy strand. They contained 13 protein coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and several noncoding regions, and their gene order was identical to most caenogastropods. Based on the nucleotide sequences combining 13 protein coding genes and two rRNA genes, a well-supported phylogeny of Nassarius was reconstructed, and several morphological synapomorphies were observed corresponding to the phylogenetic framework. In addition, the sister group relationship between N. variciferus and the remaining toxin-accumulated nassariids was determined, suggesting that the phylogeny might be related to their diet. The divergence time estimation analysis revealed a correlation between speciation events of nassariids and glacial cycles during the Pliocene-Pleistocene epoch.

Colletotrichum species form one of the most economically significant groups of pathogenic fungi and lead to significant losses in the production of major crops-in particular, fruits, vegetables, ornamental plants, shrubs, and trees. Members of the genus Colletotrichum cause anthracnose disease in many plants. Due to their considerable variation, these fungi have been widely investigated in genetic studies as model organisms. Here, we report the complete mitochondrial genome sequences of four Colletotrichum species ( C. fioriniae, C. lupini, C. salicis, and C. tamarilloi ). The reported circular mitogenomes range from 30,020 ( C. fioriniae ) to 36,554 bp ( C. lupini ) in size and have identical sets of genes, including 15 protein-coding genes, two ribosomal RNA genes, and 29 tRNA genes. All four mitogenomes are characterized by a rather poor repetitive sequence content with only forward repeat representatives and a low number of microsatellites. The topology of the phylogenetic tree reflects the systematic positions of the studied species, with representatives of each Colletotrichum species complex gathered in one clade. A comparative analysis reveals consistency in the gene composition and order of Colletotrichum mitogenomes, although some highly divergent regions are also identified, like the rps3 gene which appears as a source of potential diagnostic markers for all studied Colletotrichum species.

The Middle to Upper Palaeolithic transition in Europe witnessed the replacement and partial absorption of local Neanderthal populations by Homo sapiens populations of African origin 1 . However, this process probably varied across regions and its details remain largely unknown. In particular, the duration of chronological overlap between the two groups is much debated, as are the implications of this overlap for the nature of the biological and cultural interactions between Neanderthals and H. sapiens. Here we report the discovery and direct dating of human remains found in association with Initial Upper Palaeolithic artefacts 2 , from excavations at Bacho Kiro Cave (Bulgaria). Morphological analysis of a tooth and mitochondrial DNA from several hominin bone fragments, identified through proteomic screening, assign these finds to H. sapiens and link the expansion of Initial Upper Palaeolithic technologies with the spread of H. sapiens into the mid-latitudes of Eurasia before 45 thousand years ago 3 . The excavations yielded a wealth of bone artefacts, including pendants manufactured from cave bear teeth that are reminiscent of those later produced by the last Neanderthals of western Europe 4-6 . These finds are consistent with models based on the arrival of multiple waves of H. sapiens into Europe coming into contact with declining Neanderthal populations 7,8 .

Population monitoring is critical to effective conservation, but forest living taxa can be difficult to directly observe. This has been true of African forest elephants (Loxodonta cyclotis), for which we have limited information regarding population size and social behavior despite their threatened conservation status. In this study, we estimated demographic parameters using genetic capture-recapture of forest elephants in the southern Industrial Corridor of the Gamba Complex of Protected Areas in southwestern Gabon, which is considered a global stronghold for forest elephants. Additionally, we examined social networks, predicting that we would find matrilineal structure seen in both savanna and forest elephants. Given 95% confidence intervals, we estimate population size in the sampled area to be between 754 and 1,502 individuals and our best density estimate ranges from 0.47 to 0.80 elephants per km2. When extrapolated across the entire Industrial Corridor, this estimate suggests an elephant population size of 3,033 to 6,043 based on abundance or 1,684 to 2,832 based on density, approximately 40-80% smaller than previously suggested. Our social network analysis revealed approximately half of network components included females with different mitochondrial haplotypes suggesting a wider range of variation in forest elephant sociality than previously thought. This study emphasizes the threatened status of forest elephants and demonstrates the need to further refine baseline estimates of population size and knowledge on social behavior in this taxon, both of which will aid in determining how population dynamics in this keystone species may be changing through time in relation to increasing conservation threats., Competing Interests: The authors have declared that no competing interests exist.

The mitochondrial theory of aging attributes much of the aging process to mitochondrial DNA damage. The polymerase gamma (PolG) mutant mouse was designed to evaluate this theory and thus carries a mutated proofreading region of polymerase gamma (D257A) that exclusively transcribes the mitochondrial genome. As a result, PolGD257A mice accumulate mitochondrial DNA (mtDNA) mutations that lead to premature aging, as evidenced by hair loss, weight loss, kyphosis, increased rates of apoptosis, organ damage, and an early death, occurring around 12 months of age. Research has shown that exercise decreases skeletal muscle mtDNA mutations and normalizes protein levels in PolG mice. However, brain mtDNA changes with exercise in PolG mice have not been studied. We found no effects of exercise on mtDNA mutations or copy number in either the brain or liver of PolG mice, despite changes to body mass. Our results suggest that mitochondrial mutations play little role in exercise-brain interactions in the PolG model of accelerated aging. In addition to evaluating the effect of exercise on mtDNA outcomes, we also implemented novel methods for both extracting mtDNA and measuring mtDNA mutations, with aims for improving the efficiency and accuracy of these methods., Competing Interests: The authors have declared that no competing interests exist.

Background: Mitochondrial DNA (mtDNA) copy number in peripheral blood has been found to be associated with risk of developing several cancers. However, data on pancreatic ductal adenocarcinoma (PDAC) are very limited., Methods: To further our knowledge on this topic, we measured relative mtDNA copy number by a quantitative real-time PCR assay in peripheral leukocyte samples of 476 PDAC cases and 357 controls nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort., Results: We observed lower mtDNA copy number with advancing age ( P = 6.54 × 10 -5 ) and with a high body mass index (BMI) level ( P = 0.004) and no association with sex, smoking behavior, and alcohol consumption. We found an association between increased mtDNA copy number and decreased risk of developing PDAC with an odds ratios (OR) of 0.35 [95% confidence interval (CI), 0.16-0.79; P = 0.01] when comparing the fifth quintile with the first using an unconditional logistic regression and an OR of 0.19 (95% CI, 0.07-0.52; P = 0.001) with a conditional analysis. Analyses stratified by BMI showed an association between high mtDNA copy number and decreased risk in the stratum of normal weight, consistent with the main analyses., Conclusions: Our results suggest a protective effect of a higher number of mitochondria, measured in peripheral blood leukocytes, on PDAC risk., Impact: Our findings highlight the importance of understanding the mitochondrial biology in pancreatic cancer., (©2020 American Association for Cancer Research.)

Our study purpose was to evaluate mitochondrial (mt)DNA and RNA in peripheral blood mononuclear cells (PBMCs) and body shape changes (BSC) in HBV-infected patients. mtDNA and mtRNA were measured in PBMCs. The presence of BSC was evaluated through a questionnaire and clinical evaluation. A total of 157 subjects were enrolled, of these 107 were HBV-infected patients, 54 receiving nucleoside analogues (NAs, Group A), 53 naive to antivirals (Group B) and 50 age-sex matched controls (Group C). All HBV-treated patients had negative HBV-DNA. Twenty (37,0%) received lamivudine + adefovir, 20 (37.0%) tenofovir, 2 (3.7%) lamivudine and 12 (22.2%) entecavir. Therapy median duration was 38 months (IQR 20-60) in NA-treated patients. Group A showed significantly higher mtDNA/nuclear (n) DNA ratio (p = 0.000008) compared to Group C and Group B (p = 0.002). Group B showed significantly higher mtDNA/nDNA ratio compared to Group C (p = 0.017). Group A and B had significantly lower mtRNA/nRNA ratio compared to Group C (p = 0.00003 and p = 0.00006, respectively). Tenofovir and entecavir showed less impact compared to lamivudine + adefovir. mtDNA/nDNA ratio positively (Rho = 0.34, p < 0.05) and mtRNA/nRNA ratio negatively (Rho = -0.34, p < 0.05) correlated with therapy duration. BSC were significantly more frequent in Group A [10/54 (18.5%)] compared to Group B [3/53 (5.6%, p = 0.04)] and Group C [0/50, (p = 0.0009)]. In conclusion, long-term NA therapy was associated both to mitochondrial toxicity and BSC, showing significant differences in mtDNA and mtRNA levels. Tenofovir and entecavir showed lower impact on alterations, compared to 1 st generation NA.

Male infertility is commonly associated with sperm abnormalities including asthenozoospermia. The molecular basis of asthenozoospermia was linked to mitochondrial DNA (mtDNA) mutations. The 4,977-bp human mtDNA deletion is one of the most common mutations of spermatozoa and results in loss of about 33% of the mitochondrial genome. In this preliminary study, we aimed to investigate the presence of 4,977-bp mtDNA deletion in asthenozoospermic infertile men in Jordan. Semen specimens of 120 asthenozoospermic infertile men and 80 normozoospermic individuals were collected at the in vitro fertilization unit. MtDNA was extracted after the enrichment of spermatozoa; then, polymerase chain reaction was performed using 4,977-bp mtDNA deletion-specific primers. The deletion of 4,977-bp mtDNA was detected in 79.2% of asthenozoospermic patients compared to 10% in normozoospermic controls. The results showed a significant association between the presence of 4,977-bp mtDNA deletion and the asthenozoospermia and infertility (OR = 34.2000, 95% CI = 14.57-80.26, p-value < .001). In conclusion, our findings underscored a strong association between 4,977-bp mtDNA deletion and asthenozoospermia in the Jordanian population., (© 2019 Blackwell Verlag GmbH.)

Over the centuries, the geographical distribution of brown bear (Ursus arctos) across the Iberian Peninsula has been decreasing, with the species currently confined to North Iberia. The Cantabrian brown bear population is one of the smallest in Europe and is structured into two subpopulations, positioned along an east-west axis. Given the current critically endangered status of this population, it is essential to have a clear picture of its within-population genetic patterns and processes. We use a set of three molecular markers (mitochondrial DNA, autosomal microsatellites and sex markers) to clarify the genetic origins and assess the migration patterns and gene flow of the Cantabrian brown bear population. Our results reveal the presence of two different mitochondrial (matrilineal) haplotypes in the Cantabrian population, both belonging to European brown bear clade 1a. The two haplotypes are geographically structured between Eastern (haplotype CanE) and Western Cantabrian (haplotype CanW) subpopulations, which is consistent with the genetic structure previously identified using nuclear markers. Additionally, we show that CanE is closer to the historical Pyrenean (Pyr) haplotype than to CanW. Despite strong structuring at the levels of mtDNA and nuclear loci, there is evidence of bidirectional gene flow and admixture among subpopulations. Gene flow is asymmetrical and significantly more intense from the Eastern to the Western Cantabrian subpopulation. In fact, we only detected first generation male migrants from the Eastern to the Western Cantabrian subpopulation. These results suggest more intense migration from the smaller and more vulnerable Eastern Cantabrian subpopulation towards the larger and more stable Western Cantabrian subpopulation. These new insights are relevant for assessments of on-going conservation measures, namely the role of dispersal corridors and enhanced connectivity. We discuss the importance of complementary conservation measures, such as human-wildlife conflict mitigation and habitat improvement, for the conservation of a viable Cantabrian brown bear population., Competing Interests: The authors have declared that no competing interests exist.

Mitochondrial DNA copy number (mtDNA-CN), a measure of the number of mitochondrial genomes per cell, is a minimally invasive proxy measure for mitochondrial function and has been associated with several aging-related diseases. Although quantitative real-time PCR (qPCR) is the current gold standard method for measuring mtDNA-CN, mtDNA-CN can also be measured from genotyping microarray probe intensities and DNA sequencing read counts. To conduct a comprehensive examination on the performance of these methods, we use known mtDNA-CN correlates (age, sex, white blood cell count, Duffy locus genotype, incident cardiovascular disease) to evaluate mtDNA-CN calculated from qPCR, two microarray platforms, as well as whole genome (WGS) and whole exome sequence (WES) data across 1,085 participants from the Atherosclerosis Risk in Communities (ARIC) study and 3,489 participants from the Multi-Ethnic Study of Atherosclerosis (MESA). We observe mtDNA-CN derived from WGS data is significantly more associated with known correlates compared to all other methods (p < 0.001). Additionally, mtDNA-CN measured from WGS is on average more significantly associated with traits by 5.6 orders of magnitude and has effect size estimates 5.8 times more extreme than the current gold standard of qPCR. We further investigated the role of DNA extraction method on mtDNA-CN estimate reproducibility and found mtDNA-CN estimated from cell lysate is significantly less variable than traditional phenol-chloroform-isoamyl alcohol (p = 5.44x10-4) and silica-based column selection (p = 2.82x10-7). In conclusion, we recommend the field moves towards more accurate methods for mtDNA-CN, as well as re-analyze trait associations as more WGS data becomes available from larger initiatives such as TOPMed., Competing Interests: The authors have declared that no competing interests exist.

Several starfish (Echinodermata, Asteroidea) are keystone species of marine ecosystems, but some of the species are difficult to identify using morphological criteria only. The common sunstar, Crossaster papposus (Linnaeus, 1767), is a conspicuous species with a wide circumboreal distribution. In 1900, a closely similar species, C. squamatus (Döderlein, 1900) was described from the NE Atlantic Ocean, but subsequent authors have differed in their views on whether this is a valid taxon or rather an ecotype associated with temperature variations. We assessed the differentiating morphological characters of specimens from Norwegian and Greenland waters identified as C. papposus and C. squamatus and compared their distributions in the NE Atlantic as inferred from research cruises. The field data show that C. papposus is found mainly in temperate and shallow waters, whereas C. squamatus resides on the shelf-break in colder, mixed water masses. Intraspecific diversity and interspecific genetic differentiation of the two putative species, and their phylogenetic relationships to several Crossaster congeners worldwide, were explored using mitochondrial and nuclear DNA sequences. The molecular evidence suggests that C. papposus is the more diverse and geographically structured taxon, in line with its wide distribution. C. papposus and C. squamatus are closely related, yet clearly distinct taxa, while C. papposus and C. multispinus H.L. Clark, 1916, the latter from the South Pacific Ocean, are closely related, possibly sister taxa., Competing Interests: The Authors have declared that no competing interests exist.

Isolation of mitochondrial DNA from root tissues of mung bean (Vigna radiata) is quite tedious, complex, and often results in low yield. Hence here we show a simple, rapid, and improved protocol for isolation of mitochondrial DNA from root tissues of hydroponically grown mung bean plants. This method involves purification of mitochondria and subsequent isolation of DNA from obtained purified mitochondria. For this purpose, mitochondria were isolated using a discontinuous Percoll gradient centrifugation followed by RNase I treatment to the isolated DNA to remove any traces of RNA contamination. The mitochondrial DNA was isolated from mitochondrial samples by commonly used CTAB method. The specificity of isolated mitochondrial DNA was confirmed using mtDNA-specific genes (NAD1 and COX3). β-Actin primer was used to check the nuclear DNA contamination. PCR amplification was observed in mtDNA specific genes NAD1 and COX3 except nuclear encoded β-actin gene suggesting that mitochondrial DNA was not contaminated by nuclear DNA.

DNA combing technology is a powerful methodology for the study of DNA replication in vivo. This tool can be used to identify origins of replication, assess of directionality of forks, and measure fork speed. Over the years, the method has been used extensively to study nuclear DNA replication. The first step involves the incorporation of thymidine analogs (CldU and IdU) into nascent DNA chains and followed by their visualization with immunofluorescence using antibodies that can distinguish the two analogs. Recently, we adapted and fine-tuned DNA combing technology to the specifics of mitochondrial DNA (Phillips et al., 2017, p. 155). The protocol, which we termed mito-SMARD (mitochondrial single molecule analysis of replication DNA), provides in vivo insight into mitochondrial DNA (mtDNA) replication with high resolution., Competing Interests: Competing interests Agnel Sfeir is a cofounder and shareholder in Repare Therapeutics., (© 2020 Elsevier Inc. All rights reserved.)

A set of historic murders, known as the "Jack the Ripper murders," started in London in August 1888. The killer's identity has remained a mystery to date. Here, we describe the investigation of, to our knowledge, the only remaining physical evidence linked to these murders, recovered from one of the victims at the scene of the crime. We applied novel, minimally destructive techniques for sample recovery from forensically relevant stains on the evidence and separated single cells linked to the suspect, followed by phenotypic analysis. The mtDNA profiles of both the victim and the suspect matched the corresponding reference samples, fortifying the link of the evidence to the crime scene. Genomic DNA from single cells recovered from the evidence was amplified, and the phenotypic information acquired matched the only witness statement regarded as reliable. To our knowledge, this is the most advanced study to date regarding this case., (© 2019 American Academy of Forensic Sciences.)

The novel dipstick DNA extraction method was tested for its reliability and usability for biological barcoding in comparison to a commercial kit and to a simplified isopropanol precipitation method using crayfish gill tissue. Following DNA extraction, the mitochondrial COI-gene was amplified in a PCR-reaction using a standard set of universal invertebrate primers. All three extraction techniques resulted in successful amplifications. With the dipstick method, PCR immediately follows the very brief DNA extraction technique. We suggest that the dipstick method is an affordable, efficient, and reliable DNA extraction method uniquely suited for biological barcoding that results in reliable and reproducible amplification for downstream applications such as sequencing. Additional tests on crayfish with primers for different parts of the mitochondrial genome and on fish using specific fish COI-primers confirmed these findings. Due to the few steps involved in the DNA extraction procedure the dipstick technique is also highly recommended for high school and university biology courses.

Traditional approaches for interrogating the mitochondrial genome often involve laborious extraction and enrichment protocols followed by Sanger sequencing. Although preparation techniques are still demanding, the advent of next-generation or massively parallel sequencing has made it possible to routinely obtain nucleotide-level data with relative ease. These short-read sequencing platforms offer deep coverage with unparalleled read accuracy in high-complexity genomic regions but encounter numerous difficulties in the low-complexity homopolymeric sequences characteristic of the mitochondrial genome. The inability to discern identical units within monomeric repeats and resolve copy-number variations for heteroplasmy detection results in suboptimal genome assemblies that ultimately complicate downstream data analysis and interpretation of biological significance. Oxford Nanopore Technologies offers the ability to generate long-read sequencing data on a pocket-sized device known as the MinION. Nanopore-based sequencing is scalable, portable, and theoretically capable of sequencing the entire mitochondrial genome in a single contig. Furthermore, the recent development of a nanopore protein with dual reader heads allows for clear identification of nucleotides within homopolymeric stretches, significantly increasing resolution throughout these regions. The unrestricted read lengths, superior homopolymeric resolution, and affordability of the MinION device make it an attractive alternative to the labor-intensive, time-consuming, and costly mainstay deep-sequencing platforms. This article describes three approaches to extract, prepare, and sequence mitochondrial DNA on the Oxford Nanopore MinION device. Two of the workflows include enrichment of mitochondrial DNA prior to sequencing, whereas the other relies on direct sequencing of native genomic DNA to allow for simultaneous assessment of the nuclear and mitochondrial genomes. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Enrichment-free mitochondrial DNA sequencing Alternate Protocol 1: Mitochondrial DNA sequencing following enrichment with polymerase chain reaction (PCR) Alternate Protocol 2: Mitochondrial DNA sequencing following enrichment with PCR-free hybridization capture Support Protocol 1: DNA quantification and quality assessment using the Agilent 4200 TapeStation System Support Protocol 2: AMPure XP bead clean-up Support Protocol 3: Suggested data analysis pipeline., (© 2019 John Wiley & Sons, Inc.)

Pneumocystis jirovecii pneumonia (PJP) is an important cause of pneumonia in the HIV-negative immunocompromised population, for whom the fungal load is low, the differential diagnosis is difficult, and a bronchoalveolar lavage (BAL) sample is often not readily available. Molecular techniques have improved the microbiological diagnosis in this scenario. The usefulness of two real-time PCR techniques targeting nuclear single-copy and mitochondrial multicopy genes, respectively, applied to oral wash specimens (OW) for PJP diagnosis was assessed, and its accuracy was compared to a BAL fluid-based diagnosis. Immunocompromised patients having PJP in the differential diagnosis of an acute respiratory episode, and from whom OW and BAL or lung biopsy specimens were obtained ≤48 h apart, were retrospectively included. PCRs targeting the dihydropteroate synthase gene (DHPS) and the mitochondrial small-subunit (mtSSU) rRNA gene were performed in paired OW-BAL specimens. Thirty-six patients were included (88.6% HIV negative). Fifteen patients (41.7%) were classified as PJP, and a further 8 were considered P. jirovecii colonized. Quantification of DHPS and mtSSU in BAL fluid showed an accuracy of 96.9% and 93.0%, respectively, for PJP diagnosis, whereas a qualitative approach performed better when applied to OW (accuracy, 91.7%) irrespective of the PCR target studied (kappa = 1). Qualitative molecular diagnosis applied to OW showed an excellent performance for PJP diagnosis regardless of the target studied, being easier to interpret than the quantitative approach needed for BAL fluid., (Copyright © 2019 American Society for Microbiology.)

Between 1990 and 2018, the Defense POW/MIA Accounting Agency submitted 2177 cranial elements and 1565 teeth to the Armed Forces Medical Examiner System-Armed Forces DNA Identification Laboratory for DNA testing. In an effort to identify missing United States service members, materials were recovered from wartime losses inclusive of World War II, the Korean War, and Southeast Asia. Using four different DNA extraction protocols, DNA testing was performed using mitochondrial DNA Sanger sequencing, modified AmpFlSTR ® Yfiler™, AmpFlSTR ® MiniFiler™, PowerPlex ® Fusion, or Next Generation Sequencing. This paper aims to provide optimal strategies for the DNA testing of skeletonized cranial materials. Cranial elements produced the most consistent results in Sanger sequencing using an organic purification; however, teeth were most successful for the same platform with an inorganic purification. The inverse is true for STR testing of cranial bones. Of the cranial elements, the temporal provided the most consistent results., (© 2019 American Academy of Forensic Sciences.)

This study reports the molecular differentiation of females of Anopheles maculatus s.l. collected in eight localities on five islands in the Indonesian Archipelago: Hargowilis and Hargotirto villages of Central Java Province, North Kalimantan Province, Sabang off the northern tip of Sumatra Province, Sumba Island of East Nusa Tenggara Province and Sulawesi Province. Analyses based on rDNA (ITS2 and D3) and mtDNA (COII) sequences revealed the presence of An. greeni for the first time in North Kalimantan, and at least one novel (previously unrecognized) species of the Maculatus Group in Central Java (Hargowilis). Despite the similarity of rDNA markers of specimens of An. maculatus s.l. from Central Java and Sulawesi, their COII sequences are highly divergent (3.3%), which might indicate the presence of a further new species. Specimens of An. maculatus s.l. from the other localities had identical rDNA sequences to most An. maculatus s.s. from mainland Southeast Asia, but moderate divergence in their COII sequences (1.2-2.1%). The latter might indicate there are further novel species within the Maculatus Complex. However, as the divergence at COII may be the result of geographical structuring within species related to the historical biogeography of the region, further studies are needed to shed light on this possibility., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Adolescent idiopathic scoliosis (AIS) which characterized by complex three-dimensional deformity of spine has been difficult to cure because of the unknown etiopathology and uncertainty of progression. Nowadays, circulating cell-free (ccf) DNA was found to be a potential biomarker for several benign and malignant diseases. However, whether ccf DNA can be a biomarker for AIS has not been reported yet. In this study, we investigate the circulating cell-free nuclear DNA (ccf n-DNA) and mitochondrial DNA (ccf mt-DNA) concentrations in the plasma of patients with AIS and controls (CT), and the changed plasma ccf n-DNA and ccf mt-DNA levels and their association with clinical parameters were assessed., Methods: The plasma of peripheral blood from 69 AIS patients and 21 age-matched CT was collected for ccf DNA analysis. Quantitative PCR was used to detect ccf n-DNA and ccf mt-DNA levels, and correlation analyses between the ccf n-DNA and ccf mt-DNA levels and clinical characteristics were conducted. Receiver operator curves (ROC) were used to analyze the sensitivity and specificity of ccf n-DNA and ccf mt-DNA levels to different characteristics., Results: The plasma ccf n-DNA levels of both GAPDH and ACTB were significantly decreased in AIS patients compared with those in controls, while the plasma ccf mt-DNA levels did not changed. According to sex-related analyses, the ccf n-DNA levels in male CT-M was higher than that in female CT and male AIS, but the ccf n-DNA levels in female AIS was not significantly changed when compared with male AIS or female CT. However, the concentration of ccf mt-DNA in female AIS increased significantly when compared with male AIS. Surprisingly, Lenke type-related analyses suggested that Lenke type 1 patients had lower ccf n-DNA levels, whereas Lenke type 5 patients had higher ccf mt-DNA levels compared with those of controls. However, a lower sensitivity and specificity of AIS predicted by ccf n-DNA or ccf mt-DNA levels was observed, whether in total, by sex, or by Lenke type., Conclusion: Although with no/little predictive accuracy of AIS/progressed AIS by ccf DNA levels, significantly changed plasma ccf DNA levels were observed in AIS patients compared with those in controls.

A 75-year-old man presented with a 1-cm large elastic soft subcutaneous nodule on the left side of the umbilicus, which when excised showed presence of a helminthic form within the granulomatous lesions. Morphologically, the helminth was considered to be of the genus Dirofilaria, and the patient showed increased serum antibody titer against canine filaria. The partial DNA sequence of the mitochondrial 12S rRNA gene locus of this clinical isolate showed the highest nucleotide identity (89.6%) with Dirofilaria repens; however, the phylogenetic analysis addressed the haplotype and Dirofilaria ursi as outgroups of the clusters of D. repens and Dirofilaria immitis, which are the causal agents of most human dirofilariasis. As like bear filaria D. ursi, a wide variety of other carnivore-parasitizing filaria species have rarely been reported in humans. The newly detected genetic haplotype in this case may correspond to one of these species of Dirofilaria, though the genetic references are not available thus far., (© 2019 Japanese Dermatological Association.)