Characterization of integrated hepatitis B virus DNA harboring pre-S mutations in hepatocellular carcinoma patients with ground glass hepatocytes.

Ground glass hepatocytes (GGHs) have been associated with hepatocellular carcinoma (HCC) recurrence and poor prognosis. We previously demonstrated that pre-S expression in some GGHs is resistant to current hepatitis B virus (HBV) antiviral therapies. This study aimed to investigate whether integrated HBV DNA (iDNA) is the primary HBV DNA species responsible for sustained pre-S expression in GGH after effective antiviral therapy. We characterized 10 sets of micro-dissected, formalin-fixed-paraffin-embedded, and frozen GGH, HCC, and adjacent hepatitis B surface antigen-negative stained tissues for iDNA, pre-S deletions, and the quantity of covalently closed circular DNA. Eight patients had detectable pre-S deletions, and nine had detectable iDNA. Interestingly, eight patients had integrations within the TERT and CCNE1 genes, which are known recurrent integration sites associated with HCC. Furthermore, we observed a recurrent integration in the ABCC13 gene. Additionally, we identified variations in the type and quantity of pre-S deletions within individual sets of tissues by junction-specific PacBio long-read sequencing. The data from long-read sequencing indicate that some pre-S deletions were acquired following the integration events. Our findings demonstrate that iDNA exists in GGH and can be responsible for sustained pre-S expression in GGH after effective antiviral therapy.

Network analysis with either Illumina or MinION reveals that detecting vertebrate species requires metabarcoding of iDNA from a diverse fly community.

DNA obtained from invertebrates (iDNA) can be metabarcoded in order to survey vertebrate communities. However, little attention has been paid to the interaction between the invertebrate and vertebrate species. Here, we tested for specialization by sampling the dung and carrion fly community of a swamp forest remnant along a disturbance gradient (10 sites: 80-310 m from a road). Approximately, 60% of the baited 407 flies yielded 294 vertebrate identifications based on two COI fragments and 16S. A bipartite network analysis found no statistically significant specialization in the interactions between fly and vertebrate species, but uncommon fly species can carry the signal for vertebrate species that are otherwise difficult to detect with iDNA. A spatial analysis revealed that most of the 20 vertebrate species reported in this study could be detected within 150 m of the road (18 spp.) and that the fly community sourced for iDNA was unexpectedly rich (24 species, 3 families). They carried DNA for rare and common species inhabiting different layers of the forest (ground-dwelling: wild boar, Sunda pangolin, skinks, rats; arboreal: long-tailed macaque, Raffles' banded langur; flying: pin-striped tit-babbler, olive-winged bulbul). All our results were obtained with a new, greatly simplified iDNA protocol that eliminates DNA extraction by obtaining template directly through dissolving fly faeces and regurgitates with water. Lastly, we show that MinION- and Illumina-based metabarcoding yield similar results. We conclude by urging more studies that use different baits and involve experiments that are capable of revealing the dispersal capabilities of the flies carrying the iDNA.

Biodiversity and vector-borne diseases: Host dilution and vector amplification occur simultaneously for Amazonian leishmaniases.

Changes in biodiversity may impact infectious disease transmission through multiple mechanisms. We explored the impact of biodiversity changes on the transmission of Amazonian leishmaniases, a group of wild zoonoses transmitted by phlebotomine sand flies (Psychodidae), which represent an important health burden in a region where biodiversity is both rich and threatened. Using molecular analyses of sand fly pools and blood-fed dipterans, we characterized the disease system in forest sites in French Guiana undergoing different levels of human-induced disturbance. We show that the prevalence of Leishmania parasites in sand flies correlates positively with the relative abundance of mammal species known as Leishmania reservoirs. In addition, Leishmania reservoirs tend to dominate in less diverse mammal communities, in accordance with the dilution effect hypothesis. This results in a negative relationship between Leishmania prevalence and mammal diversity. On the other hand, higher mammal diversity is associated with higher sand fly density, possibly because more diverse mammal communities harbor higher biomass and more abundant feeding resources for sand flies, although more research is needed to identify the factors that shape sand fly communities. As a consequence of these antagonistic effects, decreased mammal diversity comes with an increase of parasite prevalence in sand flies, but has no detectable impact on the density of infected sand flies. These results represent additional evidence that biodiversity changes may simultaneously dilute and amplify vector-borne disease transmission through different mechanisms that need to be better understood before drawing generalities on the biodiversity-disease relationship.

The masking effect of extracellular DNA and robustness of intracellular DNA in anaerobic digester NGS studies: A discriminatory study of the total DNA pool.

Most commonly, next generation sequencing-based microbiome studies are performed on the total DNA (totDNA) pool; however, this consists of extracellular- (exDNA) and intracellular (iDNA) DNA fractions. By investigating the microbiomes of different anaerobic digesters over time, we found that totDNA suggested lower species richness considering all and/or only common species and yielded fewer unique reads as compared to iDNA. Additionally, exDNA-derived sequences were more similar to those from totDNA than from iDNA and, finally, iDNA showed the best performance in tracking temporal changes in microbial communities. We postulate that abundant sequences present within the exDNA fraction mask the overall results of totDNA and provide evidence that exDNA has the potential to qualitatively bias microbiome studies at least in the anaerobic digester environment as it contains information about cells that were lysed hours or days ago. iDNA, however, was found to be more appropriate in providing reliable genetic information about potentially alive as well as rare microbes within the target habitat.

Iron status and associations with physical performance during basic combat training in female New Zealand Army recruits.

Decreases in Fe status have been reported in military women during initial training periods of 8-10 weeks. The present study aimed to characterise Fe status and associations with physical performance in female New Zealand Army recruits during a 16-week basic combat training (BCT) course. Fe status indicators - Hb, serum ferritin (sFer), soluble transferrin receptor (sTfR), transferrin saturation (TS) and erythrocyte distribution width (RDW) - were assessed at the beginning (baseline) and end of BCT in seventy-six volunteers without Fe-deficiency non-anaemia (sFer 10 mg/l at baseline or end. A timed 2·4 km run followed by maximum press-ups were performed at baseline and midpoint (week 8) to assess physical performance. Changes in Fe status were investigated using paired t tests and associations between Fe status and physical performance evaluated using Pearson correlation coefficients. sFer (56·6 (sd 33·7) v. 38·4 (sd 23·8) µg/l) and TS (38·8 (sd 13·9) v. 34·4 (sd 11·5) %) decreased (P<0·001 and P=0·014, respectively), while sTfR (1·21 (sd 0·27) v. 1·39 (sd 0·35) mg/l) and RDW (12·8 (sd 0·6) v. 13·2 (sd 0·7) %) increased (P<0·001) from baseline to end. Hb (140·6 (sd 7·5) v. 142·9 (sd 7·9) g/l) increased (P=0·009) during BCT. At end, sTfR was positively (r 0·29, P=0·012) and TS inversely associated (r -0·32, P=0·005) with midpoint run time. There were no significant correlations between Fe status and press-ups. Storage and functional Fe parameters indicated a decline in Fe status in female recruits during BCT. Correlations between tissue-Fe indicators and run times suggest impaired aerobic fitness. Optimal Fe status appears paramount for enabling success in female recruits during military training.

The use of extracellular DNA as a proxy for specific microbial activity.

The ubiquity and relevance of extracellular DNA (exDNA) are well-known and increasingly gaining importance in many fields of application such as medicine and environmental microbiology. Although sources and types of exDNA are manifold, ratios of specific DNA-molecules inside and outside of living cells can give reliable information about the activity of entire systems and of specific microbial groups or species. Here, we introduce a method to discriminate between internal (iDNA), as well as bound and free exDNA, and evaluate various DNA fractions and related ratios (ex:iDNA) regarding their applicability to be used as a fast, convenient, and reliable alternative to more tedious RNA-based activity measurements. In order to deal with microbial consortia that can be regulated regarding their activity, we tested and evaluated the proposed method in comparison to sophisticated dehydrogenase- and RNA-based activity measurements with two anaerobic microbial consortia (anaerobic fungi and syntrophic archaea and a microbial rumen consortium) and three levels of resolution (overall activity, total bacteria, methanogenic archaea). Furthermore, we introduce a 28S rRNA gene-specific primer set and qPCR protocol, targeting anaerobic fungi (Neocallimastigomycota). Our findings show that the amount of actively released free exDNA (fDNA) strongly correlates with different activity measurements and is thus suggested to serve as a proxy for microbial activity.

Iron deficiency without anaemia is a potential cause of fatigue: meta-analyses of randomised controlled trials and cross-sectional studies.

Fe deficiency is a prevalent nutritional disease, and fatigue is a common complaint in the general and patient population. The association between Fe deficiency without anaemia (IDNA) and fatigue is unclear. Here, we performed a meta-analysis to evaluate the therapeutic effect of Fe on fatigue in patients with IDNA and the association between IDNA and fatigue in the population. Articles from the PubMed database up to 19 January 2016 were systematically searched. A total of six relevant randomised controlled trials (RCT) and six relevant cross-sectional studies were identified. All outcomes were converted into effect sizes. In the meta-analysis of the six RCT, we identified a significant therapeutic effect of Fe in fatigue patients with IDNA (pooled effect size 0·33; 95 % CI 0·17, 0·48; I 2=0·0 %; P<0·0001). A sensitivity analysis found that the overall results (i.e. significant association) were robust. In the meta-analysis of the six cross-sectional studies, the association between IDNA and fatigue was not significant (pooled effect size 0·10; 95 % CI -0·11, 0·31; I 2=57·4 %; P=0·362). A sensitivity analysis found that the overall results (i.e. no significant association) were not robust; removal of one study made the outcomes significant. These meta-analyses suggest that improving Fe status may decrease fatigue. Further research is necessary to identify diagnostic criteria for selecting fatigue patients who might benefit from Fe therapy and to assess the prevalence of IDNA with fatigue in the general population.

An invertebrate stomach's view on vertebrate ecology: certain invertebrates could be used as "vertebrate samplers" and deliver DNA-based information on many aspects of vertebrate ecology.

Recent studies suggest that vertebrate genetic material ingested by invertebrates (iDNA) can be used to investigate vertebrate ecology. Given the ubiquity of invertebrates that feed on vertebrates across the globe, iDNA might qualify as a very powerful tool for 21st century population and conservation biologists. Here, we identify some invertebrate characteristics that will likely influence iDNA retrieval and elaborate on the potential uses of invertebrate-derived information. We hypothesize that beyond inventorying local faunal diversity, iDNA should allow for more profound insights into wildlife population density, size, mortality, and infectious agents. Based on the similarities of iDNA with other low-quality sources of DNA, a general technical framework for iDNA analyses is proposed. As it is likely that no such thing as a single ideal iDNA sampler exists, forthcoming research efforts should aim at cataloguing invertebrate properties relevant to iDNA retrieval so as to guide future usage of the invertebrate tool box.

Single-Dose Immunogenic DNA Vaccines Coding for Live-Attenuated Alpha- and Flaviviruses.

Single-dose, immunogenic DNA (iDNA) vaccines coding for whole live-attenuated viruses are reviewed. This platform, sometimes called immunization DNA, has been used for vaccine development for flavi- and alphaviruses. An iDNA vaccine uses plasmid DNA to launch live-attenuated virus vaccines in vitro or in vivo. When iDNA is injected into mammalian cells in vitro or in vivo, the RNA genome of an attenuated virus is transcribed, which starts replication of a defined, live-attenuated vaccine virus in cell culture or the cells of a vaccine recipient. In the latter case, an immune response to the live virus vaccine is elicited, which protects against the pathogenic virus. Unlike other nucleic acid vaccines, such as mRNA and standard DNA vaccines, iDNA vaccines elicit protection with a single dose, thus providing major improvement to epidemic preparedness. Still, iDNA vaccines retain the advantages of other nucleic acid vaccines. In summary, the iDNA platform combines the advantages of reverse genetics and DNA immunization with the high immunogenicity of live-attenuated vaccines, resulting in enhanced safety and immunogenicity. This vaccine platform has expanded the field of genetic DNA and RNA vaccines with a novel type of immunogenic DNA vaccines that encode entire live-attenuated viruses.

The effects of climate and soil depth on living and dead bacterial communities along a longitudinal gradient in Chile.

Soil bacterial communities play a critical role in shaping soil stability and formation, exhibiting a dynamic interaction with local climate and soil depth. We employed an innovative DNA separation method to characterize microbial assemblages in low-biomass environments such as deserts and distinguish between intracellular DNA (iDNA) and extracellular DNA (eDNA) in soils. This approach, combined with analyses of physicochemical properties and co-occurrence networks, investigated soil bacterial communities across four sites representing diverse climatic gradients (i.e., arid, semi-arid, Mediterranean, and humid) along the Chilean Coastal Cordillera. The separation method yielded a distinctive unimodal pattern in the iDNA pool alpha diversity, increasing from arid to semi-arid climates and decreasing in humid environments, highlighting the rapid feedback of the iDNA community to increasing soil moisture. In the arid region, harsh surface conditions restrict bacterial growth, leading to peak iDNA abundance and diversity occurring in slightly deeper layers than the other sites. Our findings confirmed the association between specialist bacteria and ecosystem-functional traits. We observed transitions from Halomonas and Delftia, resistant to extreme arid environments, to Class AD3 and the genus Bradyrhizobium, associated with plants and organic matter in humid environments. The distance-based redundancy analysis (dbRDA) analysis revealed that soil pH and moisture were the key parameters that influenced bacterial community variation. The eDNA community correlated slightly better with the environment than the iDNA community. Soil depth was found to influence the iDNA community significantly but not the eDNA community, which might be related to depth-related metabolic activity. Our investigation into iDNA communities uncovered deterministic community assembly and distinct co-occurrence modules correlated with unique bacterial taxa, thereby showing connections with sites and key environmental factors. The study additionally revealed the effects of climatic gradients and soil depth on living and dead bacterial communities, emphasizing the need to distinguish between iDNA and eDNA pools.

Mammal dung-dung beetle trophic networks: an improved method based on gut-content DNA.

Background: Dung beetles provide many important ecosystem services, including dung decomposition, pathogen control, soil aeration, and secondary seed dispersal. Yet, the biology of most dung beetles remains unknown. Natural diets are poorly studied, partly because previous research has focused on choice or attraction experiments using few, easily accessible dung types from zoo animals, farm animals, or humans. This way, many links within natural food webs have certainly been missed. In this work, we aimed to establish a protocol to analyze the natural diets of dung beetles using DNA gut barcoding. Methods: First, the feasibility of gut-content DNA extraction and amplification of 12s rDNA from six different mammal dung types was tested in the laboratory. We then applied the method to beetles caught in pitfall traps in Ecuador and Germany by using 12s rDNA primers. For a subset of the dung beetles caught in the Ecuador sampling, we also used 16s rDNA primers to see if these would improve the number of species we could identify. We predicted the likelihood of amplifying DNA using gut fullness, DNA concentration, PCR primer, collection method, and beetle species as predictor variables in a dominance analysis. Based on the gut barcodes, we generated a dung beetle-mammal network for both field sites (Ecuador and Germany) and analyzed the levels of network specificity. Results: We successfully amplified mammal DNA from dung beetle gut contents for 128 specimens, which included such prominent species as Panthera onca (jaguar) and Puma concolor (puma). The overall success rate of DNA amplification was 53%. The best predictors for amplification success were gut fullness and DNA concentration, suggesting the success rate can be increased by focusing on beetles with a full gut. The mammal dung-dung beetle networks differed from purely random network models and showed a moderate degree of network specialization (H2': Ecuador = 0.49; Germany = 0.41). Conclusion: We here present a reliable method of extracting and amplifying gut-content DNA from dung beetles. Identifying mammal dung via DNA reference libraries, we created mammal dung-dung beetle trophic networks. This has benefits over previous methods because we inventoried the natural mammal dung resources of dung beetles instead of using artificial mammal baits. Our results revealed higher levels of specialization than expected and more rodent DNA than expected in Germany, suggesting that the presented method provides more detailed insights into mammal dung-dung beetle networks. In addition, the method could have applications for mammal monitoring in many ecosystems.

Mitochondrial Genomes Assembled from Non-Invasive eDNA Metagenomic Scat Samples in Critically Endangered Mammals.

The abundance of many large-bodied vertebrates, both in marine and terrestrial environments, has declined substantially due to global and regional climate stressors that define the Anthropocene. The development of genetic tools that can serve to monitor population's health non-intrusively and inform strategies for the recovery of these species is crucial. In this study, we formally evaluate whether whole mitochondrial genomes can be assembled from environmental DNA (eDNA) metagenomics scat samples. Mitogenomes of four different large vertebrates, the panda bear (Ailuropoda melanoleuca), the moon bear (Ursus thibetanus), the Java pangolin (Manis javanica), and the the North Atlantic right whale (Eubalaena glacialis) were assembled and circularized using the pipeline GetOrganelle with a coverage ranging from 12x to 480x in 14 out of 18 different eDNA samples. Partial mitochondrial genomes were retrieved from three other eDNA samples. The complete mitochondrial genomes of the studied species were AT-rich and comprised 13 protein coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a putative D-loop/control region. Synteny observed in all assembled mitogenomes was identical to that reported for specimens of the same and other closely related species. This study demonstrates that it is possible to assemble accurate whole mitochondrial chromosomes from eDNA samples (scats) using forthright bench and bioinformatics workflows. The retrieval of mitochondrial genomes from eDNA samples represents a tool to support bioprospecting, bio-monitoring, and other non-intrusive conservation strategies in species considered 'vulnerable', 'endangered', and/or 'critically endangered' by the IUCN Red List of Threatened Species.

Blowfly-derived mammal DNA as mammal diversity assessment tool: Determination of dispersal activity and flight range of tropical blowflies.

Mammalian DNA extracted from the invertebrates, especially blowfly-derived DNA, has been suggested as a useful tool to complement traditional field methods for terrestrial mammal monitoring. However, the accuracy of the estimated location of the target mammal detected from blowfly-derived DNA is largely dependent on the knowledge of blowflies' dispersal range. Presently, published data on adult blowfly dispersal capabilities remain scarce and mostly limited to temperate and subtropical regions, with no published report on the adult blowfly dispersal range in the Tropics. We seek to determine the blowfly flight range and dispersal activity in a tropical plantation in Malaysia by mark-release-recapture of approximately 3000 wild blowflies by use of rotten fish-baited traps for nine consecutive days. Out of the 3000 marked Chrysomya spp., only 1.5% (43) were recaptured during the 9-day sampling period. The majority of the blowflies (79%) were recaptured 1 km from the release point, while 20.9% were caught about 2-3 km from the release point. One individual blowfly travelled as far as 3 km and before being recaptured, which was the maximum dispersal distance recorded in this study. This result suggests that the estimated locations of the mammals detected from blowfly-derived iDNA is likely to be within 1-2 km radius from the origin of the blowfly sampling location. However, a more accurate estimated distance between the target mammal and the blowfly sampling location requires further investigation due to various factors, such as blowfly species, wind speed and direction that may potentially affect the blowfly dispersal activities. This study contributes further understanding on the development of a blowfly-derived DNA method as a mammalian monitoring tool in the tropical forests.

Using eDNA for mammal inventories still needs naturalist expertise, a meta-analysis.

DNA from the environment (eDNA) has been increasingly used as a new tool to conduct biodiversity assessment. Because of its noninvasive and less time-consuming nature, many studies of recent years solely rely on this information to establish a species inventory. eDNA metabarcoding has been shown to be an efficient method in aquatic ecosystems, especially for fish. However, detection efficiency is not clear for mammals. Using the existing literature, we conducted a meta-analysis to investigate if eDNA metabarcoding allows greater detection success compared to conventional surveys (such as field surveys, camera traps, etc.). Although only 28 articles were retrieved, showing the lack of comparative studies, still representing more than 900 taxa detected, we found that detection success was method dependent, but most importantly varies on the taxonomy of the targeted taxa. eDNA metabarcoding performed poorly for bats compared to the traditional mist nests. However, strong detection overlaps were found between conventional surveys and eDNA for large-bodied mammals such as ungulates, primates, and carnivores. Overall, we argue that using both molecular and field approaches can complement each other and can maximize the most accurate biodiversity assessment and there is much room for metabarcoding optimization to reach their full potential compared to traditional surveys.

Narciclasine, a novel topoisomerase I inhibitor, exhibited potent anti-cancer activity against cancer cells.

DNA topoisomerases are essential nuclear enzymes in correcting topological DNA errors and maintaining DNA integrity. Topoisomerase inhibitors are a significant class of cancer chemotherapeutics with a definite curative effect. Natural products are a rich source of lead compounds for drug discovery, including anti-tumor drugs. In this study, we found that narciclasine (NCS), an amaryllidaceae alkaloid, is a novel inhibitor of topoisomerase I (topo I). Our data demonstrated that NCS inhibited topo I activity and reversed its unwinding effect on p-HOT DNA substrate. However, it had no obvious effect on topo II activity. The molecular mechanism of NCS inhibited topo I showed that NCS did not stabilize topo-DNA covalent complexes in cells, indicating that NCS is not a topo I poison. A blind docking result showed that NCS could bind to topo I, suggesting that NCS might be a topo I suppressor. Additionally, NCS exhibited a potent anti-proliferation effect in various cancer cells. NCS arrested the cell cycle at G2/M phase and induced cell apoptosis. Our study reveals the antitumor mechanisms of NCS and provides a good foundation for the development of anti-cancer drugs based on topo I inhibition.

The potential of aquatic bloodfeeding and nonbloodfeeding leeches as a tool for iDNA characterisation.

Leeches play important roles in food webs due to their abundance, diversity and feeding habits. Studies using invertebrate-derived DNA (iDNA) extracted from leech gut contents to target vertebrate DNA have focused on the Indo-Pacific region and mainly leveraged the leech family Haemadipsidae, composed of bloodfeeding terrestrial leeches, while predatory, fluid/tissue-feeding and aquatic bloodfeeding species have been largely disregarded. While there is some general knowledge regarding the taxonomic groups that leeches prefer to feed on, detailed taxonomic resolution is missing and, therefore, their potential use for monitoring animals is unknown. In this study, 116 leeches from 12 species (six families) and spanning the three feeding habits were collected in Mexico and Canada. We used DNA metabarcoding to investigate their diet and assess their potential use for biodiversity monitoring. We detected vertebrates from five orders including fish, turtles and birds in the diet of aquatic bloodfeeding leeches; eight invertebrate orders of annelids, arthropods and molluscs in leeches that feed on body fluids and tissues; and 10 orders of invertebrates belonging to Arthropoda and Annelida, as well as one vertebrate and one parasitic nematode, in predatory leeches. These results show the potential use of iDNA from aquatic bloodfeeding leeches for retrieving vertebrate taxa, and from predatory and fluid-feeding leeches for invertebrates. Our study provides information about the dietary range of freshwater leeches and one terrestrial leech and contributes proof-of-concept for the use of these leeches for animal monitoring, expanding our knowledge of the use of iDNA from leech gut contents to North America.

Invertebrates for vertebrate biodiversity monitoring: Comparisons using three insect taxa as iDNA samplers.

Metabarcoding of environmental DNA (eDNA) is now widely used to build diversity profiles from DNA that has been shed by species into the environment. There is substantial interest in the expansion of eDNA approaches for improved detection of terrestrial vertebrates using invertebrate-derived DNA (iDNA) in which hematophagous, sarcophagous, and coprophagous invertebrates sample vertebrate blood, carrion, or faeces. Here, we used metabarcoding and multiple iDNA samplers (carrion flies, sandflies, and mosquitos) collected from 39 forested sites in the southern Amazon to profile gamma and alpha diversity. Our main objectives were to (1) compare diversity found with iDNA to camera trapping, which is the conventional method of vertebrate diversity surveillance; and (2) compare each of the iDNA samplers to assess the effectiveness, efficiency, and potential biases associated with each sampler. In total, we collected and analysed 1759 carrion flies, 48,686 sandflies, and 4776 mosquitos. Carrion flies revealed the greatest total vertebrate species richness at the landscape level, despite the least amount of sampling effort and the fewest number of individuals captured for metabarcoding, followed by sandflies. Camera traps had the highest median species richness at the site-level but showed strong bias towards carnivore and ungulate species and missed much of the diversity described by iDNA methods. Mosquitos showed a strong feeding preference for humans as did sandflies for armadillos, thus presenting potential utility to further study related to host-vector interactions.

Environmental DNA in human and veterinary parasitology - Current applications and future prospects for monitoring and control.

Parasites are important pathogens with significant global economic, public and animal health impacts. Successful control or elimination of many parasitic diseases, not least neglected tropical parasites, will require scalable, sensitive and cost-effective monitoring tools. Environmental DNA (eDNA) methods, used extensively in ecology for biomonitoring in natural ecosystems, offer promising advantages such reduced costs and labor requirements for species monitoring. Yet, the use of eDNA-based methods in parasitology and disease surveillance, has only recently begun to be explored. With this review, we wish to give an up-to-date overview of current uses and limitations of eDNA in human and veterinary parasitology, and how existing challenges can be overcome to fully utilize the potential of eDNA for monitoring and control of parasitic diseases. We begin by systematically searching published literature to identify studies that apply eDNA methods in parasitology and synthesize the main findings from these studies. We find that eDNA applications in parasitology only account for a small proportion (73/1960) of all eDNA publications up to now, and even fewer (27/73) studies, that apply eDNA methods specifically for parasites of human or veterinary importance. The majority of studies concern snail-borne trematodes and their intermediate host snails, while a few apply eDNA for mosquito vector species detection. A strong geographical bias, with only very few studies undertaken on the African continent, where parasites are of the biggest public health concern, is also noted. Current obstacles hindering further advances of eDNA methods in parasitology include incomplete reference databases, and challenges related to real-time monitoring in remote areas, and in certain LMIC settings. Finally, we point to future opportunities for eDNA-based research in parasitology and highlight recent innovations in eDNA research, which could further develop its application for monitoring and control of parasitic diseases and vectors in the future.

Abundance, diversity, and host assignment of total, intracellular, and extracellular antibiotic resistance genes in riverbed sediments.

Human health risk assessment for environmental antibiotic resistant microbes requires not only quantifying the abundance of antibiotic resistance genes (ARGs) in environmental matrices, but also understanding their hosts and genetic context. Further, differentiating ARGs in intracellular and extracellular DNA (iDNA and eDNA) fractions may help refine our understanding of ARG transferability. The objectives of this study were to understand the (O1) abundance and diversity of extracellular, intracellular, and total ARGs along a land use gradient and (O2) impact of bioinformatics pipeline on the assignment of putative hosts for the ARGs observed in the different DNA fractions. Sediment samples were collected along a land use gradient in the Raritan River, New Jersey, USA. DNA was extracted to separate eDNA and iDNA and qPCR was performed for select ARGs and the 16S rRNA gene. Shotgun metagenomic sequencing was performed on DNA extracts for the different DNA fractions. ARG hosts were assigned via two different bioinformatic pipelines: network analysis of raw reads versus assembly. Results of the two pipelines were compared to evaluate their performance in terms of number and diversity of linkages and accuracy of in silico matrix spike host assignments. No differences were observed in the 16S rRNA gene normalized sul1 concentrations between the DNA fractions. The overall microbial community structure was more similar for iDNA and total DNA compared to eDNA and generally clustered by sampling site. ARGs associated with mobile genetic elements increased in iDNA for the downstream sites. Regarding host assignment, the raw reads pipeline via network analysis identified 247 ARG hosts as compared to 53 hosts identified by assembly pipeline. Other comparisons between the pipelines were made including ARG assignment to taxa containing waterborne pathogens and practical considerations regarding processing time.

Live-Attenuated VEEV Vaccine Delivered by iDNA Using Microneedles Is Immunogenic in Rabbits.

Effective and simple delivery of DNA vaccines remains a key to successful clinical applications. Previously, we developed a novel class of DNA vaccines, sometimes called iDNA, which encodes the whole live-attenuated vaccine viruses. Compared to a standard DNA vaccine, an iDNA vaccine required a low dose to launch a live-attenuated vaccine in vitro or in vivo. The goal of this pilot study was to investigate if iDNA vaccine encoding live-attenuated Venezuelan equine encephalitis virus (VEEV) can be efficiently delivered in vivo by a microneedle device using a single-dose vaccination with naked iDNA plasmid. For this purpose, we used pMG4020 plasmid encoding live-attenuated V4020 vaccine of VEE virus. The V4020 virus contains structural gene rearrangement, as well as attenuating mutations genetically engineered to prevent reversion mutations. The pMG4020 was administered to experimental rabbits by using a hollow microstructured transdermal system (hMTS) microneedle device. No adverse events to vaccination were noted. Animals that received pMG4020 plasmid have successfully seroconverted, with high plaque reduction neutralization test (PRNT) antibody titers, similar to those observed in animals that received V4020 virus in place of the pMG4020 iDNA plasmid. We conclude that naked iDNA vaccine can be successfully delivered in vivo by using a single-dose vaccination with a microneedle device.