DNA Distribution Research Articles

Assessment of Diamond™ Nucleic Acid Dye for the identification and targeted sampling of latent DNA in operational casework

Recovery and DNA profiling of latent touch DNA deposits is a ubiquitous practice by operational forensic laboratories that provides critical evidence in many criminal investigations. Despite recent improvements in the sensitivity of contemporary DNA profiling kits, the inability to localise and visually quantify touch DNA deposits on an exhibit means that ineffective or unwarranted sampling is often performed leading to poor success. Diamond™ Nucleic Acid Dye (DD) is a fluorescent DNA binding dye which has recently been shown to bind to corneocytes enabling visualisation and targeted sampling of touch DNA deposits under controlled conditions. The ability to translate these findings to operational casework, where a diverse range of substrates is encountered and the amount and distribution of touch DNA is uncontrolled, is currently unknown. Here, we provide the first report on the use of DD in an operational context. Spraying items with DD was shown to have no impact on downstream immunological testing, DNA extraction, or DNA profiling with the GlobalFiler™ PCR amplification kit. DD was shown to effectively locate areas of touch DNA on select exhibits using the Polilight. Issues with background fluorescence, non-specific staining, interference from fingerprint enhancement reagents, or absorbance of the excitation light by black surfaces demonstrated that DD is not compatible with all exhibits. Background fluorescence also prevented the use of DD to screen for the presence of cellular material on IsoHelix swabs post-sampling but it was suitable for screening Lovell DNA tapelifts. A casework trial of 49 plastic bag and tape exhibits showed limited application of DD to triage out negative items as DNA was recovered from items where DD fluorescence was not detected. Where DD fluorescence was detected, its broad distribution prevented targeted sampling and any correlation to be made between the amount observed and DNA yield or profiling outcome. The DD procedure also increased the time taken to search exhibits and risk of inadvertent contamination. Our study suggests that DD is not suited as a generalised screening technique across all touch casework exhibits but further investigation is warranted to determine its applicability to specific exhibit types.

Prevalence of Human Polyomavirus BK Virus in Prostate Cancer Patients and Benign Prostatic Hyperplasia: A Cross-sectional Study on Prostate Patients Referred to Imam Khomeini Hospital in Ahvaz Between 2015 and 2017

Background: Human polyomavirus BK virus (BKV) belongs to the Polyomaviridae family and seems to be a drastic virus in prostate cancer (PCa) etiology. BK virus induces oncogenesis via the expression of large tumor antigen (LTAg) and small tumor antigen (stAg). Also, BKV infection seems to play an essential role in prostate cancer development. Objectives: In this study was aimed to study the prevalence of BKV in benign and cancerous prostate tissues. Methods: In this study, 100 formalin-fixed paraffin-embedded tissues of PCa specimens and benign prostatic hyperplasia (BPH) were collected. The DNA was extracted from tissue samples, and the BKV DNA was investigated using a semi-nested polymerase chain reaction (PCR). The MEGA 6.0 software was used for phylogenetic analysis to assemble the viral genome. A phylogenetic tree was constructed by neighbor-joining analysis with 1,000 replicates of the bootstrap resampling test. Results: The BKV DNA was found in 66% (33/50) of patients with PCa and 36% (18/50) of patients with benign prostatic hyperplasia (BPH) (P = 0.003). The frequency of BKV DNA in different classes of Gleason score (5 - 10) was not significant (0.094). The distribution of BKV DNA among different age groups was not significant (P = 0.086). Conclusions: High frequency of BKV infection was detected in patients with PCa compared to patients with BPH (P = 0.003), and the coexistence of BKV DNA was confirmed in 51% (51/100) of tissue samples, which were confirmed to be subtype 1 of BKV infection.

The InBIO Barcoding Initiative Database: DNA barcodes of Portuguese Hemiptera 01

BackgroundThe InBIO Barcoding Initiative (IBI) Hemiptera 01 dataset contains records of 131 specimens of Hemiptera. Most specimens have been morphologically identified to species or subspecies level and represent 88 species in total. The species of this dataset correspond to about 7.3% of continental Portuguese hemipteran species diversity. All specimens were collected in continental Portugal. Sampling took place from 2015 to 2019 and specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources.New informationThis dataset increases the knowledge on the DNA barcodes and distribution of 88 species of Hemiptera from Portugal. Six species, from five different families, were new additions to the Barcode of Life Data System (BOLD), with another twenty five species barcodes' added from under-represented taxa in BOLD. All specimens have their DNA barcodes publicly accessible through BOLD online database and the distribution data can be accessed through the Global Biodiversity Information Facility (GBIF). Eutettix variabilis and Fieberiella florii are recorded for the first time for Portugal and Siphanta acuta, an invasive species, previously reported from the Portuguese Azores archipelago, is recorded for the first time for continental Portugal.

The particle size distribution of environmental DNA varies with species and degradation

Environmental DNA (eDNA) analysis is frequently used as a non-invasive method to investigate species and biodiversity in ecosystems. However, such eDNA may represent both organisms currently present as well as species that released their DNA some point in the past, thereby representing a mix of current and historic biodiversity. This may lead to a false-positive detection of organisms' presence. As the eDNA particle size distribution (PSD) changes along with the decay process, it may facilitate solving the above problem. Here, we set up tank experiments with snails, zebrafish and daphnids, respectively, to monitor the change in eDNA PSD and eDNA degradation through time after removing organisms. We found that zebrafish eDNA decays more slowly for larger particle sizes. Across all species tested, the percentage of large size ranges tended to increase over time while the smaller sizes showed relatively fast decay rates. As a result, PSD changed consistently with eDNA decay, although initial PSD varied between species. In combination, we propose that eDNA PSD can be used to assess the current prevalence of organisms at an eDNA sampling location while avoiding false-positives on the presence of species. Our findings expand the applicability of eDNA for monitoring target species in freshwater ecosystems.

Ion-dependent DNA configuration in bacteriophage capsids

Bacteriophages densely pack their long double-stranded DNA genome inside a protein capsid. The conformation of the viral genome inside the capsid is consistent with a hexagonal liquid crystalline structure. Experiments have confirmed that the details of the hexagonal packing depend on the electrochemistry of the capsid and its environment. In this work, we propose a biophysical model that quantifies the relationship between DNA configurations inside bacteriophage capsids and the types and concentrations of ions present in a biological system. We introduce an expression for the free energy that combines the electrostatic energy with contributions from bending of individual segments of DNA and Lennard-Jones-type interactions between these segments. The equilibrium points of this energy solve a partial differential equation that defines the distributions of DNA and the ions inside the capsid. We develop a computational approach that allows us to simulate much larger systems than what is possible using the existing molecular-level methods. In particular, we are able to estimate bending and repulsion between the DNA segments as well as the full electrochemistry of the solution, both inside and outside of the capsid. The numerical results show good agreement with existing experiments and with molecular dynamics simulations for small capsids.

Bayesian Skyline Plots disagree with range size changes based on Species Distribution Models for Holarctic birds.

During the Quaternary, large climate oscillations impacted the distribution and demography of species globally. Two approaches have played a major role in reconstructing changes through time: Bayesian Skyline Plots (BSPs), which reconstruct population fluctuations based on genetic data, and Species Distribution Models (SDMs), which allow us to back-cast the range occupied by a species based on its climatic preferences. In this paper, we contrast these two approaches by applying them to a large data set of 102 Holarctic bird species, for which both mitochondrial DNA sequences and distribution maps are available, to reconstruct their dynamics since the Last Glacial Maximum (LGM). Most species experienced an increase in effective population size (Ne , as estimated by BSPs) as well as an increase in geographical range (as reconstructed by SDMs) since the LGM; however, we found no correlation between the magnitude of changes in Ne and range size. The only clear signal we could detect was a later and greater increase in Ne for wetland birds compared to species that live in other habitats, a probable consequence of a delayed and more extensive increase in the extent of this habitat type after the LGM. The lack of correlation between SDM and BSP reconstructions could not be reconciled even when range shifts were considered. We suggest that this pattern might be linked to changes in population densities, which can be independent of range changes, and caution that interpreting either SDMs or BSPs independently is problematic and potentially misleading.

Systematic Assessment of GATA2 Genetic Variation Reveals the Presence of Novel Disease-Causing Synonymous Exonic Mutations

Among its different clinical presentations, GATA2 deficiency had been recognized as the most frequent hereditary predisposition to pediatric myelodysplastic syndromes (MDS), accounting for 15% of MDS with excess of blasts (MDS-EB), with a particularly high prevalence among children with monosomy 7 (37%). A pattern of three major classes of germline mutations can be recognized in the roughly 350 reported cases with GATA2 deficiency: I) protein truncating (stop-gain, frameshift, splice-site), II) missense within or adjacent to zinc finger 2 (ZnF2), and, III) noncoding substitutions in the conserved EBOX-GATA-ETS regulatory region in intron 4, (NM_032638.4). The rare aberrations (class IV) include in frame indels and whole gene deletions. For the purpose of this study we introduced the above classification to systematically categorize various types of GATA2 variations and to assess the prevalence and pathogenic significance of silent (synonymous) substitutions, referred to as class V.Our study cohort consisted of 723 children and adolescents with primary MDS enrolled in prospective registry of the European Working Group of childhood MDS (EWOG-MDS). Typical GATA2 germline mutations were found in 69/723 cases. Additional 28 GATA2-deficient individuals with heterogeneous phenotypes diagnosed at our institutions were added to the analysis. We excluded patients with secondary MDS, in which GATA2 is not etiologically involved as previously reported by our group. Results were compared to those of 138632 individuals in the Genome Aggregation Database (GnomAD). Various in-silico prediction tools to evaluate deleteriousness were employed, and selected mutations were investigated in a GATA reporter system.First, we categorized the “typical” germline GATA2 mutations identified in 96 patients. Class I = truncating mutations were present in 51.5% (49/96), class II = missense in ZnF2 in 37.5% (36/96), class III = deep intron 4 in 8.3% (8/96) and class IV = other in 3.15% (3/96) of patients. Genotype-phenotype analysis failed to establish a significant correlation. We next systematically assessed the prevalence of other potentially pathogenic coding variants. Missense alterations p.Ala164Thr, p.Pro161Ala, and p.Pro250Ala (present in 34.8%, 1.6% and 0.9% GnomAD individuals) were neither significantly enriched, nor they displayed “synergistic” co-occurrence in pediatric MDS. This essentially depicts an unlikely role in MDS predisposition. We further performed transactivation activity in a GATA reporter system for p.Pro161Ala and observed no significant alteration.Finally, we focused on the evaluation of synonymous exonic substitutions resulting in nucleotide exchange without alteration of protein sequence. Recent cancer studies proposed that synonymous mutations might alter protein folding and stability, also such variants can introduce novel splice sites leading to premature RNA degradation. The synonymous substitutions that are common according to GnomAD: p.Pro5Pro (88.3% GnomAD), p.Thr188Thr (8.0%) and p.Ala411Ala (4.3%) showed similar frequencies in our patient cohort. Ultimately, we identified 6 patients with 5 novel or very rare synonymous exonic substitutions, referred to as “class V” variants (Fig. 1): p.Thr117Thr found in 2 unrelated pedigrees, p.Gly327Gly, p.Leu217Leu, p.Ala341Ala, and p.Pro472Pro (detected in 0, 0, 2, 6, and 70 out of >130 Tsd. GnomAD individuals, respectively). Median age at MDS diagnosis was 12 (4-15) years and 4/6 patients have acquired monosomy 7.In silico prediction assigned a high chance of splice defects to p.Thr117Thr, p.Leu217Leu, and p.Ala341Ala variants. Sequencing from blood of 2 unrelated patients with p.Thr117Thr confirmed complete loss of mutated allele in mRNA as compared to 50:50% distribution in DNA; while the degradation of p.Leu217Leu mutant mRNA was less prominent. Expectedly, GATA reporter assays performed for synonymous variants did not reveal altered transactivation. Further ongoing studies aim at detailed examination of altered splicing and characterization of protein stability.In sum, we establish that noncoding substitutions in GATA2, believed thus far to be silent, constitute a novel disease-causing mutational class. In contrast, rare SNPs (p.Pro161Ala and p.Pro250Ala) have a likely neutral effect towards MDS predisposition. DisclosuresLübbert:Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding.

A Compositional Model to Predict the Aggregated Isotope Distribution for Average DNA and RNA Oligonucleotides.

Structural modifications of DNA and RNA molecules play a pivotal role in epigenetic and posttranscriptional regulation. To characterise these modifications, more and more MS and MS/MS- based tools for the analysis of nucleic acids are being developed. To identify an oligonucleotide in a mass spectrum, it is useful to compare the obtained isotope pattern of the molecule of interest to the one that is theoretically expected based on its elemental composition. However, this is not straightforward when the identity of the molecule under investigation is unknown. Here, we present a modelling approach for the prediction of the aggregated isotope distribution of an average DNA or RNA molecule when a particular (monoisotopic) mass is available. For this purpose, a theoretical database of all possible DNA/RNA oligonucleotides up to a mass of 25 kDa is created, and the aggregated isotope distribution for the entire database of oligonucleotides is generated using the BRAIN algorithm. Since this isotope information is compositional in nature, the modelling method is based on the additive log-ratio analysis of Aitchison. As a result, a univariate weighted polynomial regression model of order 10 is fitted to predict the first 20 isotope peaks for DNA and RNA molecules. The performance of the prediction model is assessed by using a mean squared error approach and a modified Pearson’s χ2 goodness-of-fit measure on experimental data. Our analysis has indicated that the variability in spectral accuracy contributed more to the errors than the approximation of the theoretical isotope distribution by our proposed average DNA/RNA model. The prediction model is implemented as an online tool. An R function can be downloaded to incorporate the method in custom analysis workflows to process mass spectral data.

Assessment of the Oral Delivery of a Myelin Basic Protein Gene Promoter with Antiapoptotic bcl-xL (pMBP-bcl-xL) DNA by Cyclic Peptide Nanotubes with Two Aspect Ratios and Its Biodistribution in the Brain and Spinal Cord.

Cyclo-(D-Trp-Tyr) peptide nanotubes (PNTs) were reported to be potential carriers for oral gene delivery in our previous study; however, the effect of the aspect ratio (AR) of these PNTs on gene delivery in vivo could affect penetration or interception in biological environments. The aim of this study was to assess the feasibility of cyclo-(D-Trp-Tyr) PNTs with two ARs as carriers for oral pMBP-bcl-xL-hRluc delivery to the spinal cord to treat spinal cord injury (SCI). We evaluated the biodistribution of oligodendrocyte (OLG)-specific myelin basic protein gene promoter-driven antiapoptotic DNA (pMBP-bcl-xL) to the brain and spinal cord delivered with cyclo-(D-Trp-Tyr) PNTs with large (L) and small (S) PNTs with two ARs. After complex formation, the length, width, and AR of the L-PNTs/DNA were 77.86 ± 3.30, 6.51 ± 0.28, and 13.75 ± 7.29 μm, respectively, and the length and width of the S-PNTs/DNA were 1.17 ± 0.52 and 0.17 ± 0.05 μm, respectively, giving an AR of 7.12 ± 3.17 as detected by scanning electron microscopy. Each of these three parameters exhibited significant differences (p < 0.05) between L-PNTs/DNA and S-PNTs/DNA. However, there were no significant differences (p > 0.05) between the L-PNTs and S-PNTs for either their DNA encapsulation efficiency (29.72 ± 14.19 and 34.31 ± 16.78%, respectively) or loading efficiency (5.15 ± 2.58 and 5.95 ± 2.91%). The results of the in vitro analysis showed that the S-PNT/DNA complexes had a significantly higher DNA release rate and DNA permeation in the duodenum than the L-PNT/DNA complexes. Using Cy5 and TM-rhodamine to individually and chemically conjugate the PNTs with plasmid DNA, we observed, using laser confocal microscopy, that the PNTs and DNA colocalized in complexes. We further confirmed the complexation between DNA and the PNTs using fluorescence resonance energy transfer (FRET). Data from an in vivo imaging system (IVIS) showed that there was no significant difference (p > 0.05) in PNT distribution between L-PNTs/DNA and S-PNTs/DNA within 4 h. However, the S-PNT/DNA group had a significantly higher DNA distribution (p < 0.05) in several organs, including the ilium, heart, lungs, spleen, kidneys, testes, brain, and spinal cord. Finally, we determined the bcl-xL protein expression levels in the brain and spinal cord regions for the L-PNT/DNA and S-PNT/DNA complex formulations. These results suggested that either L-PNTs or S-PNTs may be used as potential carriers for oral gene delivery to treat SCI.

Equid herpesvirus-1 Distribution in Equine Lymphoid and Neural Tissues 70 Days Post Infection.

Equid herpesvirus-1 (EHV-1) causes respiratory disease, abortion and myeloencephalopathy in horses worldwide. As member of the Alphaherpesvirinae, latency is key to EHV-1 epidemiology. EHV-1 latent infection has been detected in the trigeminal ganglion (TG), respiratory associated lymphoid tissue (RALT) and peripheral blood mononuclear cells (PBMC) but additional locations are likely. The aim of this study was to investigate the distribution of viral DNA throughout the equine body. Twenty-five horses divided into three groups were experimentally infected via intranasal instillation with one of three EHV-1 viruses and euthanized on Day 70, post infection. During necropsy, TG, various sympathetic/parasympathetic ganglia of head, neck, thorax and abdomen, spinal cord dorsal root ganglia, RALT, mesenteric lymph nodes, spleen and PBMC of each horse were collected. Genomic viral loads and L-(late) gene transcriptional activity in each tissue and PBMC were measured using qPCR. In addition, immunohistochemistry (IHC) was applied on neural parenchyma tissue sections. EHV-1 DNA was detected in many neural and lymphoid tissue sections, but not in PBMC. L-gene transcriptional activity was not detected in any sample, and translational activity was not apparent on IHC. Tissue tropism differed between the Ab4 wild type and the two mutant viruses.

Abstract P-47: Analysis of Phosphorus Distribution in Giant Bacteriophage Capsid by Electron Energy Loss Spectroscopy

Background: We have recently developed a method to visualize the distribution of DNA in the cytoplasm of bacteria by analytical electron microscopy (EM), using the Phosphorus signal (dsDNA contains two phosphate groups per each nucleotide pair), that was detected and mapped onto the image of the cell (Danilova et al, 2020; Loiko et al, 2020). Here we applied this technique to study much smaller objects – the DNA packing inside bacteriophage heads. We studied phiEL, giant phiKZ-like bacteriophage of the Myoviridae family that infects Pseudomonas aeruginosa (Krylov et al, 2003). We have earlier demonstrated that this phage contains an ‘inner body’ inside its capsid, which is responsible for the specific DNA packing (Sokolova et al, 2014). Methods: The phage propagation was performed as described before (Sokolova et al, 2014). A 3 ul sample of purified bacteriophage phiEL was applied to the glow-discharged carbon-coated copper grid and stained with freshly prepared ammonium Molybdate 2% aquatic solution for 30 sec. Grids were loaded into Gatan cooling holder and the temperature of the specimen was kept at -180°C. EELS spectra and phosphorus elemental maps were obtained on JEOL2100 microscope, operating at 200 kV with the Gatan GIF Quantum ER spectrometer in STEM mode. Pixel size was set to 15-20 nm. STEM drift correction was applied after each 40-50 pixels. Each spectrum was obtained at a 6.0 mrad collection angle, 0.25 eV dispersion, and 132 eV energy shift. The spectra from different pixels were aligned to carbon K-edge. Results: Phosphorus mapping inside and outside the bacteriophage capsid was performed (Fig. 1). Outside the capsid, the phosphorus signal was practically absent, which corresponds to the presence of DNA only inside the capsid. The distribution of phosphorus inside the capsid was uneven: the rectangular area in the middle of the capsid contained a weak signal, while a more intense signal was detected on the periphery. This can be explained by the presence of an ‘inner body’ inside (Fig. 1C). Conclusion: Thus, our results justify the possibility of using the analytical EM technique to study the distribution of DNA by mapping Phosphorus in biological nano-objects at relatively low content of the element.

Multi-scaled Monte Carlo calculation for radon-induced cellular damage in the bronchial airway epithelium

Radon is a leading cause of lung cancer in indoor public and mining workers. Inhaled radon progeny releases alpha particles, which can damage cells in the airway epithelium. The extent and complexity of cellular damage vary depending on the alpha particle’s kinetic energy and cell characteristics. We developed a framework to quantitate the cellular damage on the nanometer and micrometer scales at different intensities of exposure to radon progenies Po-218 and Po-214. Energy depositions along the tracks of alpha particles that were slowing down were simulated on a nanometer scale using the Monte Carlo code Geant4-DNA. The nano-scaled track histories in a 5 μm radius and 1 μm-thick cylindrical volume were integrated into the tracking scheme of alpha trajectories in a micron-scale bronchial epithelium segment in the user-written SNU-CDS program. Damage distribution in cellular DNA was estimated for six cell types in the epithelium. Deep-sited cell nuclei in the epithelium would have less chance of being hit, but DNA damage from a single hit would be more serious, because low-energy alpha particles of high LET would hit the nuclei. The greater damage in deep-sited nuclei was due to the 7.69 MeV alpha particles emitted from Po-214. From daily work under 1 WL of radon concentration, basal cells would respond with the highest portion of complex DSBs among the suspected progenitor cells in the most exposed regions of the lung epithelium.

Preparation, Description and Evaluation of the Lethality Acid Loaded Liposomal Nanoparticles Against in Vitro Colon and Liver Cancer

Today, the use of nanoparticles as carriers in the science of drug delivery is of particular importance. Improving drug performance and reducing side effects due to changes in the pharmacokinetic properties of the drug are special benefits of drug Nano-carriers. Monolayer and bilayer Nano -liposomes were synthesized by reverse phase evaporation and thin film hydration with HEPES buffer and Snoike operation, respectively. Scanning electron microscopy and transmission electron microscopy examinations show that Pegylated Nano-Phytosomes have a spherical shape and much less aggregation is observed compared to non-Pegylated Nano-phytosomes. In order to evaluate the rate of drug release and the effect of peeling on the quality of drug release, dialysis bag method was used. The results of drug release indicate the rapid release of the free drug compared to the release of the drug from Glycyrrhizic acid-loaded Nano-Phytosomes. The results of apoptosis test also showed equal distribution of DNA of healthy cells and sphericity of cell nucleus. In order to investigate the effect of cell mortality in the monolayer nano-liposomes section, two HepG2 and KATO III cell lines were used and in the bilayer Nano-Phytosomes section, two cell lines DLD-1 and LIM-2405 related to colon cancer were used.

Temporal-dependent effects of DNA degradation on frozen tissues archived at −80°C

Abstract Frozen tissues, associated with natural history and biological collections, historically have been archived at temperatures between −20°C and −80°C. More recently, the availability of liquid nitrogen systems has enabled the storage of tissue samples (biobanking) at temperatures as low as −196°C. Currently, it is not known how the degree of coldness (e.g., −80°C or −196°C) or longevity (time in storage) impacts preservation of tissue samples. To examine the effects of long-term storage (−80°C and −196°C) on DNA degradation, tissue samples (muscle and liver) archived for 30, 20, 10, or 1 years were obtained from the Natural Science Research Laboratory at Texas Tech University. The integrity of DNA (measured as molecular weight and fragment length) extracted from samples was determined using automated DNA isolation methods followed by microfluidic distribution measurement. DNA distributions were compared using measures of central tendency, a regression-based molecular mass profile, and as a latent variable in a structural equation model. Muscle samples consistently outperformed liver samples in terms of quality of DNA yield. Also, muscle samples exhibited a significant linear relationship with time in which older samples were more degraded than were recent samples. The signal for a temporal effect on DNA was strongest when considering a latent variable of DNA quality based on mode and kurtosis; 37% of the variation in the latent variable was explained by variation in units of time. More recent time points tended to be more similar, but the temporal effect on the latent variable remained strong even when the oldest samples were removed from the analysis. In contrast, integrity of DNA from liver samples did not have a significant linear relationship with time; however, in some years they exhibited non-normally distributed DNA quality metrics that may have reflected sensitivity of liver tissue to degradation during specimen preparation, DNA extraction, or archive parameters. Results indicated that tissue type and temporal effects influenced rates of DNA degradation, with the latter emphasizing the long-term value of biobanking at the coldest temperatures possible (liquid nitrogen storage) to mitigate degradation of biological samples of ever-increasing scientific value.

Comparative Distribution of Repetitive Sequences in the Karyotypes of Xenopus tropicalis and Xenopus laevis (Anura, Pipidae).

Xenopus laevis and its diploid relative, Xenopus tropicalis, are the most used amphibian models. Their genomes have been sequenced, and they are emerging as model organisms for research into disease mechanisms. Despite the growing knowledge on their genomes based on data obtained from massive genome sequencing, basic research on repetitive sequences in these species is lacking. This study conducted a comparative analysis of repetitive sequences in X. laevis and X. tropicalis. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) with Cot DNA of both species revealed a conserved enrichment of repetitive sequences at the ends of the chromosomes in these Xenopus species. The repeated sequences located on the short arm of chromosome 3 from X. tropicalis were not related to the sequences on the short arm of chromosomes 3L and 3S from X. laevis, although these chromosomes were homoeologous, indicating that these regions evolved independently in these species. Furthermore, all the other repetitive sequences in X. tropicalis and X. laevis may be species-specific, as they were not revealed in cross-species hybridizations. Painting experiments in X. laevis with chromosome 7 from X. tropicalis revealed shared sequences with the short arm of chromosome 3L. These regions could be related by the presence of the nucleolus organizer region (NOR) in both chromosomes, although the region revealed by chromosome painting in the short arm of chromosome 3L in X. laevis did not correspond to 18S + 28S rDNA sequences, as they did not colocalize. The identification of these repeated sequences is of interest as they provide an explanation to some problems already described in the genome assemblies of these species. Furthermore, the distribution of repetitive DNA in the genomes of X. laevis and X. tropicalis might be a valuable marker to assist us in understanding the genome evolution in a group characterized by numerous polyploidization events coupled with hybridizations.

The interplay between DNA and histone methylation: molecular mechanisms and disease implications.

Methylation of cytosine in CpG dinucleotides and histone lysine and arginine residues is a chromatin modification that critically contributes to the regulation of genome integrity, replication, and accessibility. A strong correlation exists between the genome-wide distribution of DNA and histone methylation, suggesting an intimate relationship between these epigenetic marks. Indeed, accumulating literature reveals complex mechanisms underlying the molecular crosstalk between DNA and histone methylation. These in vitro and in vivo discoveries are further supported by the finding that genes encoding DNA- and histone-modifying enzymes are often mutated in overlapping human diseases. Here, we summarize recent advances in understanding how DNA and histone methylation cooperate to maintain the cellular epigenomic landscape. We will also discuss the potential implication of these insights for understanding the etiology of, and developing biomarkers and therapies for, human congenital disorders and cancers that are driven by chromatin abnormalities.

Mitochondrial dynamics in health and disease.

In animals, mitochondria are mainly organised into an interconnected tubular network extending across the cell along a cytoskeletal scaffold. Mitochondrial fission and fusion, as well as distribution along cytoskeletal tracks, are counterbalancing mechanisms acting in concert to maintain a mitochondrial network tuned to cellular function. Balanced mitochondrial dynamics permits quality control of the network including biogenesis and turnover, and distribution of mitochondrial DNA, and is linked to metabolic status. Cellular and organismal health relies on a delicate balance between fission and fusion, and large rearrangements in the mitochondrial network can be seen in response to cellular insults and disease. Indeed, dysfunction in the major components of the fission and fusion machineries including dynamin-related protein 1 (DRP1), mitofusins 1 and 2 (MFN1, MFN2) and optic atrophy protein 1 (OPA1) and ensuing imbalance of mitochondrial dynamics can lead to neurodegenerative disease. Altered mitochondrial dynamics is also seen in more common diseases. In this review, the machinery involved in mitochondrial dynamics and their dysfunction in disease will be discussed.

Circumglobal distribution of fish environmental DNA in coral reefs

Coral reefs host the highest fish diversity on Earth despite covering less than 0.1% of the ocean’s seafloor. At the same time they are also extremely threatened. Data syntheses over decades of surveys estimate the total number of coral reef fishes to vary from 2,400 to 8,000 species distributed among roughly 100 families. But this diversity remains largely unknown. Here, we investigated how environmental DNA (eDNA) could describe the distribution of fish diversity in coral reefs. We generated 504,457,267 raw 12S ribosomal DNA (rDNA) sequence reads from 251 samples (2,693 PCR replicates) collected at 25 sites in 145 stations covering five regions across the Indian, Pacific and Atlantic Oceans. Bioinformatic analysis clustered these sequences into 2,160 molecular operational taxonomic units (MOTUs) corresponding to distinct species (Marques 2020) We compared our results, with visual census surveys from Reef Life Survey, on 2,813 transects in tropical regions. Our outcomes demonstrate the capacity of eDNA metabarcoding from water samples to reconstruct well-known biogeographic patterns of fish diversity on coral reefs, such as species richness gradients towards the coral triangle, and family proportion stability across sites (Bellwood and Hughes 2001). Additionally, eDNA survey data documented a higher fish species (16%) and family (50%) diversity than estimates obtained with underwater visual surveys carried out on 20 times more sites. MOTU richness per family retrieved with eDNA closely matched fish species richness within families recorded in visual census data. However, eDNA revealed higher richness of reef-associated species and species from adjacent habitats, of cryptobenthic or nocturnal species, but also of pelagic and wide-ranging species. eDNA survey data showed that fish diversity is characterized by spatially heterogeneous species assemblages among regions, with more dissimilarity among adjacent coral reefs than detected with visual survey data. Unlike visual surveys, eDNA metabarcoding revealed the same prevalence of rarity as expected under the neutral theory of biodiversity, suggesting the predominance of random processes and ecological equivalence within trophic groups at large scale to explain fish biodiversity patterns on coral reefs. Our study demonstrates how sequencing eDNA from water provides a rapid and effective approach to characterize and assess coral reef diversity across large spatial scales, thereby also uncovering hidden biodiversity patterns.

LifeDNAquatic: A priori settings and refined pipelines of eDNA metabarcoding to generate "big data" for solid fish, mussel and amphibian SDMs

Sound environmental management decisions - in accordance with the EU WFD for aquatic ecosystems – mainly depend on reliable species presence- and distribution- data. Here we present a workflow from sampling strategies to results and decision making using eDNA metabarcoding analyses for fish, amphibians, and mussels from habitat to landscape scales with focus on sampling strategies for "big data" in marine and freshwater ecosystems in Sweden. The project LifeDNAquatic highlights a solid eDNA pipeline and comparison of methods, which cover field planning and the entire pipeline generating data for Species Distribution Models (SDMs). Intense sampling over a large river catchment highlights previoulsy unanswered questionsand and provides insights to a priori settings for sampling strategies to retrieve "big data". The results provide novel insights to DNA distribution in the environment, seasonal and spatial changes in eDNA composition, and validation of data.

Composition and distribution of fish environmental DNA in an Adirondack watershed.

BackgroundEnvironmental DNA (eDNA) surveys are appealing options for monitoring aquatic biodiversity. While factors affecting eDNA persistence, capture and amplification have been heavily studied, watershed-scale surveys of fish communities and our confidence in such need further exploration.MethodsWe characterized fish eDNA compositions using rapid, low-volume filtering with replicate and control samples scaled for a single Illumina MiSeq flow cell, using the mitochondrial 12S ribosomal RNA locus for taxonomic profiling. Our goals were to determine: (1) spatiotemporal variation in eDNA abundance, (2) the filtrate needed to achieve strong sequencing libraries, (3) the taxonomic resolution of 12S ribosomal sequences in the study environment, (4) the portion of the expected fish community detectable by 12S sequencing, (5) biases in species recovery, (6) correlations between eDNA compositions and catch per unit effort (CPUE) and (7) the extent that eDNA profiles reflect major watershed features. Our bioinformatic approach included (1) estimation of sequencing error from unambiguous mappings and simulation of taxonomic assignment error under various mapping criteria; (2) binning of species based on inferred assignment error rather than by taxonomic rank; and (3) visualization of mismatch distributions to facilitate discovery of distinct haplotypes attributed to the same reference. Our approach was implemented within the St. Regis River, NY, USA, which supports tribal and recreational fisheries and has been a target of restoration activities. We used a large record of St. Regis-specific observations to validate our assignments.ResultsWe found that 300 mL drawn through 25-mm cellulose nitrate filters yielded greater than 5 ng/µL DNA at most sites in summer, which was an approximate threshold for generating strong sequencing libraries in our hands. Using inferred sequence error rates, we binned 12S references for 110 species on a state checklist into 85 single-species bins and seven multispecies bins. Of 48 bins observed by capture survey in the St. Regis, we detected eDNA consistent with 40, with an additional four detections flagged as potential contaminants. Sixteen unobserved species detected by eDNA ranged from plausible to implausible based on distributional data, whereas six observed species had no 12S reference sequence. Summed log-ratio compositions of eDNA-detected taxa correlated with log(CPUE) (Pearson’s R = 0.655, P < 0.001). Shifts in eDNA composition of several taxa and a genotypic shift in channel catfish (Ictalurus punctatus) coincided with the Hogansburg Dam, NY, USA. In summary, a simple filtering apparatus operated by field crews without prior expertise gave useful summaries of eDNA composition with minimal evidence of field contamination. 12S sequencing achieved useful taxonomic resolution despite the short marker length, and data exploration with standard bioinformatic tools clarified taxonomic uncertainty and sources of error.