Tool For Molecular Monitoring Research Articles

Environmental DNA as a tool to reconstruct catch composition for longline fisheries vessels

Global wild-capture fisheries are a large and diverse sector requiring various tools for fisheries-dependant data collection and effective Monitoring, Control and Surveillance (MCS). Here we present a novel protocol to collect eDNA from brine tanks onboard commercial longline vessels to reconstruct catch composition. We collected samples from nine vessels operating out of the Eastern Tuna Billfish Fishery, Australia, validating eDNA results with reliable catch data consisting of seven target and bycatch species. Environmental DNA was highly effective for detecting species retained on vessels without contamination or false positives. For four vessels, logbook data and eDNA were consistent with detections of all species. The remaining vessels detected all species except for rare catches of short-billed spearfish (Tetrapturus angustirostris). Similarities between rank abundance distributions of catch and eDNA reads were observed with logbook data mirrored when eDNA sequences were organised into rank order abundance. The method was effective at identifying highly abundant taxa retained in brine tanks- tuna (Thunnus spp.), swordfish (Xiphias gladius), marlin (Kajijia audax), and Atlantic Pomfret (Brama brama). Further research is required to validate how eDNA and other molecular monitoring tools can be scaled and applied to provide solutions for monitoring challenges in the fisheries sector.

Detect Cytochrome C Oxidase- and Glutathione-S-Transferase-Mediated Detoxification in a Permethrin-Resistant Population of Lygus lineolaris.

Frequent sprays on cotton prompted resistance development in the tarnished plant bug (TPB). Knowledge of global gene regulation is highly desirable to better understand resistance mechanisms and develop molecular tools for monitoring and managing resistance. Novel microarray expressions of 6688 genes showed 3080 significantly up- or down-regulated genes in permethrin-treated TPBs. Among the 1543 up-regulated genes, 255 code for 39 different enzymes, and 15 of these participate in important pathways and metabolic detoxification. Oxidase is the most abundant and over-expressed enzyme. Others included dehydrogenases, synthases, reductases, and transferases. Pathway analysis revealed several oxidative phosphorylations associated with 37 oxidases and 23 reductases. One glutathione-S-transferase (GST LL_2285) participated in three pathways, including drug and xenobiotics metabolisms and pesticide detoxification. Therefore, a novel resistance mechanism of over-expressions of oxidases, along with a GST gene, was revealed in permethrin-treated TPB. Reductases, dehydrogenases, and others may also indirectly contribute to permethrin detoxification, while two common detoxification enzymes, P450 and esterase, played less role in the degradation of permethrin since none was associated with the detoxification pathway. Another potential novel finding from this study and our previous studies confirmed multiple/cross resistances in the same TPB population with a particular set of genes for different insecticide classes.

Environmental nucleic acids: A field-based comparison for monitoring freshwater habitats using eDNA and eRNA.

Nucleic acids released by organisms and isolated from environmental substrates are increasingly being used for molecular biomonitoring. While environmental DNA (eDNA) has received much attention, the potential of environmental RNA as a biomonitoring tool remains under-explored. Several recent studies using paired DNA and RNA metabarcoding of bulk samples suggest that RNA might better reflect "metabolically active" parts of the community. However, such studies mainly capture organismal eDNA and eRNA. For larger eukaryotes, isolation of extra-organismal RNA will be important, but viability needs to be examined in a field-based setting. In this study we evaluate (a) whether extra-organismal eRNA release from macroeukaryotes can be detected given its supposedly rapid degradation, and (b) if the same field collection methods for eDNA can be applied to eRNA. We collected eDNA and eRNA from water in lakes where fish community composition is well documented, enabling a comparison between the two nucleic acids in two different seasons with monitoring using conventional methods. We found that eRNA is released from macroeukaryotes and can be filtered from water and metabarcoded in a similar manner as eDNA to reliably provide species composition information. eRNA had a small but significantly greater true positive rate than eDNA, indicating that it correctly detects more species known to exist in the lakes. Given relatively small differences between the two molecules in describing fish community composition, we conclude that if eRNA provides significant advantages in terms of lability, it is a strong candidate to add to the suite of molecular monitoring tools.

Installing Reduction Responsiveness into Biomolecules by Introducing Nitroaryl Groups.

Artificially synthesized stimuli-responsive biomolecules are attractive as molecular tools for monitoring and modulating biological systems. In biological systems, redox stimuli are common, and their dysregulation is typically linked to various abnormal or disease states. In this Concept article, the molecular design of reduction-responsive biomolecules, such as peptides, nucleic acids, and saccharides, which are produced by introducing nitroaryl groups into them, is reviewed with a special emphasis on simple 4-nitrobenzene-based motifs.

Cross-Sectional Variations in Structure and Function of Coral Reef Microbiome With Local Anthropogenic Impacts on the Kenyan Coast of the Indian Ocean.

Coral reefs face an increased number of environmental threats from anthropomorphic climate change and pollution from agriculture, industries and sewage. Because environmental changes lead to their compositional and functional shifts, coral reef microbial communities can serve as indicators of ecosystem impacts through development of rapid and inexpensive molecular monitoring tools. Little is known about coral reef microbial communities of the Western Indian Ocean (WIO). We compared taxonomic and functional diversity of microbial communities inhabiting near-coral seawater and sediments from Kenyan reefs exposed to varying impacts of human activities. Over 19,000 species (bacterial, viral and archaeal combined) and 4,500 clusters of orthologous groups of proteins (COGs) were annotated. The coral reefs showed variations in the relative abundances of ecologically significant taxa, especially copiotrophic bacteria and coliphages, corresponding to the magnitude of the neighboring human impacts in the respective sites. Furthermore, the near-coral seawater and sediment metagenomes had an overrepresentation of COGs for functions related to adaptation to diverse environments. Malindi and Mombasa marine parks, the coral reef sites closest to densely populated settlements were significantly enriched with genes for functions suggestive of mitigation of environment perturbations including the capacity to reduce intracellular levels of environmental contaminants and repair of DNA damage. Our study is the first metagenomic assessment of WIO coral reef microbial diversity which provides a much-needed baseline for the region, and points to a potential area for future research toward establishing indicators of environmental perturbations.

Longitudinal analysis of cell-free mutated KRAS and CA 19\u20139 predicts survival following curative resection of pancreatic cancer

BackgroundNovel biomarkers and molecular monitoring tools hold potential to improve outcome for patients following resection of pancreatic ductal adenocarcinoma (PDAC). We hypothesized that the combined longitudinal analysis of mutated cell-free plasma KRAS (cfKRASmut) and CA 19–9 during adjuvant treatment and follow-up might more accurately predict disease course than hitherto available parameters.MethodsBetween 07/2015 and 10/2018, we collected 134 plasma samples from 25 patients after R0/R1-resection of PDAC during adjuvant chemotherapy and post-treatment surveillance at our institution. Highly sensitive discriminatory multi-target ddPCR assays were employed to screen plasma samples for cfKRASmut. cfKRASmut and CA 19–9 dynamics were correlated with recurrence-free survival (RFS) and overall survival (OS). Patients were followed-up until 01/2020.ResultsOut of 25 enrolled patients, 76% had undergone R0 resection and 48% of resected PDACs were pN0. 17/25 (68%) of patients underwent adjuvant chemotherapy. Median follow-up was 22.0 months, with 19 out of 25 (76%) patients relapsing during study period. Median RFS was 10.0 months, median OS was 22.0 months. Out of clinicopathologic variables, only postoperative CA 19–9 levels and administration of adjuvant chemotherapy correlated with survival endpoints. cfKRASmut. was detected in 12/25 (48%) of patients, and detection of high levels inversely correlated with survival endpoint. Integration of cfKRASmut and CA 19–9 levels outperformed either individual marker. cfKRASmut outperformed CA 19–9 as dynamic marker since increase during adjuvant chemotherapy and follow-up was highly predictive of early relapse and poor OS.ConclusionsIntegrated analysis of cfKRASmut and CA 19–9 levels is a promising approach for molecular monitoring of patients following resection of PDAC. Larger prospective studies are needed to further develop this approach and dissect each marker’s specific potential.

Towards real-time kinetic monitoring of wastewater treatment: A case study of sunlight and ozone treatment of unconcentrated wastewater using flow NMR

Wastewater contains complex organic signatures whose components can be harmful to both environmental and human health. Nuclear Magnetic Resonance (NMR) spectroscopy is an excellent tool for molecular monitoring, with the ability to follow total organics, subcategories (aromatics, aliphatic etc.) and, resolution permitting, even individual compounds. However, due to the relatively low sensitivity of NMR, studies of wastewater to date have involved pre-concentration (often many liters) before analysis. This proof of concept study asks, “Is NMR sensitive enough for on-line wastewater monitoring without pre-concentration?” Here, a continuous flow 500 MHz NMR system, coupled with a sunlight simulator and ozone generator, was used on unaltered and unconcentrated wastewater to assess the potential of on-line NMR for understanding wastewater treatment processes. Wastewater from 3 different treatment stages were analyzed. In general, combined ozone and sunlight was more effective at removing organics, while differences in behavior of the same molecules within different wastewater fractions suggest that the chemistry is more complex than just the action of “reactants” (light, ozone) on the target chemicals, and to some extent, involves other components in the wastewater. As such, the nature of the effluent itself could also have important impacts on the rate and completion of its degradation. In summary, on-line NMR of wastewater treatment could be carried out without pre-concentration of the wastewater, in line with important future applications for understanding wastewater treatment processes at the molecular level. Over the last decade, there has been considerable development in low field, low footprint, low cost, NMR spectrometers. Portable versions of those systems could potentially, in the future, be applied for on-site, on-line monitoring of waste streams at water treatment plants, as described here.

Relationship Between Inactivation and Genome Damage of Human Enteroviruses Upon Treatment by UV254, Free Chlorine, and Ozone.

Quantitative PCR (qPCR) is a convenient tool for monitoring virus concentrations in water and wastewater treatment trains, though it only informs about virus presence, but not infectivity. This limitation can be overcome if the relationship between infectivity loss and genome decay induced by a given disinfectant is known. Here, we performed inactivation experiments using two human enteroviruses, Coxsackievirus B5 and Echovirus 11, with three disinfection methods: low-pressure ultraviolet light (UV254), free chlorine (FC), and ozone. We compared the inactivation rates as measured by culturing to the decay rates of the whole genome, to evaluate the extent of qPCR-measurable genome damage as a function of inactivation. To determine genome damage, we used an approach that estimates damage across the full viral genome from the measured decay of multiple amplicons distributed across the viral genome. Correlations between inactivation and genome decay were observed for all viruses and all disinfection treatments, but results showed that even among closely related viruses, disinfection methods can damage the viral genome to different extents and that genome damage does not necessarily translate to inactivation. For both viruses, UV254 treatment had the closest relationship between inactivation and genome decay and with ozone, the rate of genome decay exceeded the inactivation rate. Finally, for FC, the ratios between methods were vastly different between viruses. This work provides the basis to relate qPCR measurements to infectivity loss and enables the establishment of molecular monitoring tools for assessing enterovirus inactivation during disinfection treatments of water and wastewater.

Abstract 4918: Concordance of circulating tumor DNA (ctDNA) and next-generation sequencing (NGS) as molecular monitoring tools in metastatic breast cancer (MBC)

Abstract Background MBC is an incurable disease with complex molecular features including somatic mutations that evolve in relation to genomic instability and selective treatment pressure. Patients with treatment-refractory MBC may benefit from tissue genomic evaluation using next-genomic sequencing (NGS). Furthermore, circulating DNA fragments with tumor-specific sequence alterations (ctDNA) found in the blood of patients with advanced disease offer the possibility of non-invasive molecular monitoring. ctDNA detects actionable mutations with the advantage of serial evaluation and allowing capture of inter- and intra-tumor heterogeneity. Methods This is a retrospective evaluation of 28 patients with MBC who failed standard therapies and had baseline plasma analyzed for ctDNA and tissue analysis (NGS) before starting new therapy. We were interested in the performance of the ctDNA test and the concordance rate of genomic alterations detected in the two tests. Selection criteria: progression of disease after standard therapies, need to detect novel molecular abnormalities for possible therapeutic targeting, or confirmation of persistence of genomic abnormalities already demonstrated in tissue or blood analysis. Guardant360™(Guardant Health) involves comprehensive sequencing of a panel of 54 gene associated with solid tumors using single-molecule digital sequencing technology. FoundationOne®(Foundation Medicine) performed the NGS on tissue evaluates the entire coding sequence of 315 cancer-related genes. Results All patients had biopsy-proved metastatic disease, and had ctDNA and NGS performed. 93% of patients had ctDNA alterations detected (with 0.1%-27.8% circulating tumor fraction), and 9 patients had serial ctDNA. Overall, for the patients we detected mutations in ctDNA and tissue, 89% of patients had a specific alteration on ctDNA that matched the NGS analysis. Among all mutations detected in tumors which are in overlapped genes, 71% of alterations were common (83% excluding gene amplifications). Interestingly, for patients who had both tests done within 8 weeks, 70% of had additional alterations in the ctDNA that were not found on NGS, such as ERBB2 mutations. Conclusions Genomic analysis using ctDNA and NGS detects genomic abnormalities in all patients with MBC with high concordance. However, each method was able to detect alterations that the other did not, suggesting the two methods can be complementary in detecting actionable mutations and expanding therapeutic options. Intriguingly, this occurred more frequently with the ctDNA, demonstrating its utility as an adjunct to tissue sampling which permits capture of the inter- and intra-tumor heterogeneity of the disease, and warrants further investigation prospectively. Citation Format: Laura K. Austin, Tiffany Avery, Rebecca Jaslow, Paolo Fortina, Dragan Sebisanovic, LaiMun Siew, Aubrey Zapanta, AmirAli Talasaz, Massimo Cristofanilli. Concordance of circulating tumor DNA (ctDNA) and next-generation sequencing (NGS) as molecular monitoring tools in metastatic breast cancer (MBC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4918. doi:10.1158/1538-7445.AM2015-4918

MOLTOOLS: a workshop on “Molecular tools for monitoring marine invasive species”

The 2011–2014 European Community project VECTORS (‘‘Vectors of Change in Oceans and SeasMarine Life, Impact on Economic Sectors’’) was an integrated, multidisciplinary European project which aimed to improve our understanding of how environmental anthropogenic drivers are impacting European marine ecosystems now and in the future. VECTORS investigated how these changes may affect marine biodiversity and ecosystem functioning, the range of goods and services provided by the oceans, the resulting socio-economic impacts and some possible future scenarios for mitigation and adaptation. The VECTORS Work Package WP2.1 (Mechanisms of Change: Outbreaks and Invasives) was committed to identifying mechanisms of species outbreaks and invasions and introduction vectors as both symptoms and drivers of change in European marine ecosystems. VECTORS particularly focused on native outbreak-forming species (OFS) and invasive alien species (IAS) (Box 1) to deliver new information on (1) IAS and OFS distribution in European seas, (2) stochastic and deterministic drivers of invasions and ecophysiological features boosting regime shifts of marine communities, (3) sources and vectors of bioinvasions, and (4) consequences of bioinvasions, in terms of impact on ecosystem functioning and services. These studies provided a substantive contribution to the understanding of the ecological conditions in which outbreaks and alien invasions are more likely to occur, and the determination of critical thresholds/tipping points that mark alternative regime shifts in community structure and organization. The progressive reduction in the inherent costs of molecular data over the past 20 years, along with rapid recent technological and analytical developments, has resulted in molecular methods becoming an inescapable component of investigation on biological invasions, from simple biodiversity assessment to historical reconstruction of invasion histories. Within this context, 60 participants frwom 14 nations gathered in Lecce, Italy on 12–14th September 2012 for MOLTOOLS (Fig. 1), a dedicated workshop to discuss and promote the use of molecular tools for monitoring and understanding marine invasive species. The workshop was designed to address several key questions regarding mechanisms and management J. A. Darling National Exposure Research Laboratory, United States Environmental Protection Agency, Durham, NC 27713, USA

Usefulness of quantitative assessment of Wilms tumor suppressor gene expression in chronic myeloid leukemia patients undergoing imatinib therapy

The Wilms tumor suppressor gene (WT1) is overexpressed in a number of human hematological malignancies, including chronic myeloid leukemia (CML). Although at present, the biological significance of WT1 expression in CML in still unclear, this marker could represent a useful tool for molecular monitoring of CML patients prior to and post imatinib therapy. In fact, the use of real-time polymerase chaine reaction (PCR) to quantitatively measure the WT1 transcript amount may be a predictor of patient response to imatinib therapy. Semin Hematol 40(suppl 2):37-41. © 2003 Elsevier Inc. All rights reserved.

Usefulness of quantitative assessment of Wilms tumor suppressor gene expression in chronic myeloid leukemia patients undergoing imatinib therapy

The Wilms tumor suppressor gene (WT1) is overexpressed in a number of human hematological malignancies, including chronic myeloid leukemia (CML). Although at present, the biological significance of WT1 expression in CML in still unclear, this marker could represent a useful tool for molecular monitoring of CML patients prior to and post imatinib therapy. In fact, the use of real-time polymerase chaine reaction (PCR) to quantitatively measure the WT1 transcript amount may be a predictor of patient response to imatinib therapy. Semin Hematol 40(suppl 2):37-41. © 2003 Elsevier Inc. All rights reserved.

Monitoring minimal residual disease in AML: the right time for real time.

Detection of minimal residual disease (MRD) by polymerase chain reaction (PCR) has become an essential tool for molecular monitoring of acute myeloid leukemia (AML). Currently, specific translocation markers are available for 40-50% of AMLs. Expression markers may widen this spectrum to 70-90%. Quantitative PCR (Q-PCR, real-time PCR) is now as sensitive as conventional two-step PCR and could improve as well as facilitate clinical decision-making. Q-PCR has been applied to a variety of molecular markers, delineating threshold levels early after induction therapy, for postinduction monitoring, as well as for the detection of relapse. For most markers, lack of decline of transcript levels by less than 2 logs after chemotherapy has been established as a poor prognostic sign. Moreover, increases in transcript levels are almost invariably associated with relapse. However, the predictive value of PCR negativity after chemotherapy is not as clear. The major tasks for the future will be standardization of Q-PCR techniques, exact definition of threshold levels, and monitoring schedules in bone marrow (BM) and peripheral blood (PB), as well as investigation of novel markers found by microarray analysis.