Single metabarcoding multiplex captures community‐level freshwater biodiversity and beyond

Cost‐effective and accurate quantification of biodiversity is important for biodiversity conservation, resource management, and forecasting. Traditional monitoring approaches have relied on direct observations, remote sensing, and mark‐recapture techniques, providing insights into species ecology and the impact of pollution and climate change on indicator species. However, these techniques are typically low throughput, expensive, and can be invasive. In addition, they cannot detect cryptic diversity and are biased toward species that leave identifiable remains. DNA‐based methods, such as metabarcoding or single marker gene assays, have enabled high throughput screening of a wide range of taxonomic groups, including ones without well‐preserved remains. When compared with traditional techniques, these approaches have high throughput, can resolve cryptic diversity, do not require taxonomic specialist skills, and are non‐invasive. However, although they are comparatively cheaper than traditional approaches, they are expensive when applied at the community‐level as single marker assays are amplified and sequenced independently. Multilocus approaches in which multiple gene markers are amplified in a single reaction are desirable to deliver community‐level assessments in a cost‐effective manner. Yet, they are uncommon because of technical challenges that may lead to biases in downstream analyses, such as index hopping and unbalanced representation of taxonomic groups. Here, we developed a highly multiplexed protocol that combines the early pooling of marker genes that target broad taxonomic groups and taxon‐specific markers in a single tube reaction. This step is followed by the pooling of up to 384 samples per locus (N = 15,636 samples) with unique dual‐indexed sequencing adapters in a single sequencing run. This approach dramatically reduces the costs of community‐level biodiversity quantification and lowers the need for input DNA without compromising output quality. We optimized the multiplex assay on lake freshwater sediment samples and benchmarked the assay on samples from other environmental matrices, demonstrating its direct application to the river and marine communities

Multi-species occupancy modeling reveals methodological and environmental effects on eDNA detection of amphibians in temporary ponds

Aquatic environmental DNA is increasingly used for biodiversity monitoring, such as surveying threatened and invasive species. Mainstreaming these methods in practi- cal applications, however, still requires significant standardization and optimisation, namely regarding DNA capture methods. Here we evaluated how filter type (standard disc filters vs high-capacity capsules), number of sampling sites, volume of water fil- tered and environmental factors affected amphibian detection in Mediterranean tem - porary ponds. The study involved water filtering until clogging at one (capsules) and five (discs) sites from 16 small and shallow ponds, where three urodele and seven anu- ran species were recorded through sweep- netting and adult observations. Detection probabilities were estimated from site occupancy models based on replicate sampling and from an adaptation of time-to-detection models relating detection probability to volume of water filtered. Discs filtered relatively small volumes (15–1250 mL), with detection probabilities of the two abundant species (Pelobates cultripes, Hyla meridi- onalis) increasing rapidly with sample size and water volume, reaching almost per- fect detection (0.95) at four and seven discs, and 420 mL and 1860 mL, respectively. However, reaching high detection probabilities for rare species (Pelodytes atlanticus, Pleurodeles waltl, Triturus pygmaeus) would require larger sampling effort than that used in our study. Despite filtering much larger volumes (600–5300 mL), filtering with capsules at a single site per pond provided lower detection probabilities for abun- dant species than filtering with discs at five sites. Rarer species showed no difference between methods, which may be due to small sample sizes and reduced statistical power for species with few detections. The effect of conductivity on species detect- ability was largely negative, while the influence of water clarity varied across species, and pH had no effects. Overall, our results suggest that eDNA amphibian surveys in Mediterranean temporary ponds need to consider filter clogging, heterogeneous DNA distribution, and highly conductive watersinfo:eu-repo/semantics/publishedVersio

Using molecular diet analysis to inform invasive species management: A case study of introduced rats consuming endemic New Zealand frogs

The decline of amphibians has been of international concern for more than two decades, and the global spread of introduced fauna is a major factor in this decline. Conservation management decisions to implement control of introduced fauna are often based on diet studies. One of the most common metrics to report in diet studies is Frequency of Occurrence (FO), but this can be difficult to interpret, as it does not include a temporal perspective. Here, we examine the potential for FO data derived from molecular diet analysis to inform invasive species management, using invasive ship rats (Rattus rattus) and endemic frogs (Leiopelma spp.) in New Zealand as a case study. Only two endemic frog species persist on the mainland. One of these, Leiopelma archeyi, is Critically Endangered (IUCN 2017) and ranked as the world\u27s most evolutionarily distinct and globally endangered amphibian (EDGE, 2018). Ship rat stomach contents were collected by kill-trapping and subjected to three methods of diet analysis (one morphological and two DNA-based). A new primer pair was developed targeting all anuran species that exhibits good coverage, high taxonomic resolution, and reasonable specificity. Incorporating a temporal parameter allowed us to calculate the minimum number of ingestion events per rat per night, providing a more intuitive metric than the more commonly reported FO. We are not aware of other DNA-based diet studies that have incorporated a temporal parameter into FO data. The usefulness of such a metric will depend on the study system, in particular the feeding ecology of the predator. Ship rats are consuming both species of native frogs present on mainland New Zealand, and this study provides the first detections of remains of these species in mammalian stomach contents

Patient-reported outcome measures for emotional functioning in cancer patients : content comparison of the EORTC CAT Core, FACT-G, HADS, SF-36, PRO-CTCAE, and PROMIS instruments

Background: Cancer and its treatment can have substantial impact on patients' emotional functioning. Several patient-reported outcome measures (PROMs) assessing emotional functioning are available, but differences in content limit the comparability of results. To better understand conceptual (dis)similarities, we conducted a content comparison of commonly used PROMs. Methods: We included emotional functioning items, scales, and item banks from the EORTC CAT Core, EORTC QLQ-C30, FACT-G, Hospital anxiety and depression scale (HADS), SF-36, PRO-CTCAE, and PROMIS (item banks for anxiety, depression, and anger). Item content was linked to the International Classification of Functioning, Disability, and Health (ICF) and a hierarchical framework established for PROMIS. Single items could be coded with more than one ICF category but were solely assigned to one facet within the PROMIS framework. Results: The measures comprise 132 unique items covering the ICF components ‘Body functions’ (136/153 codings, 88.9%) and ‘Activities and participation’ (15/153, 9.8%). Most ICF codings (112/153, 73.2%) referred to the third-level category ‘b1528 Emotional functions, other specified’. According to the PROMIS framework 48.5% of the items assessed depression (64/132 items), followed by anxiety (41/132, 31.1%) and anger (26/132, 19.7%). The EORTC measures covered depression, anxiety, and anger in a single measure, while the PROMIS inventory provides separate item banks for these concepts. The FACT-G, SF-36, PRO-CTCAE and HADS covered depression and anxiety, but not anger. Conclusion: Our results provide an in-depth conceptual understanding of selected PROMs and important qualitative information going beyond psychometric evidence. Such information supports the identification of PROMs for which scores can be meaningfully linked with quantitative methods

Comparing the contents of patient-reported outcome measures for fatigue : EORTC CAT Core, EORTC QLQ-C30, EORTC QLQ-FA12, FACIT, PRO-CTCAE, PROMIS, brief fatigue inventory, multidimensional fatigue inventory, and piper fatigue scale

Background: To assess fatigue in cancer patients, several patient-reported outcome measures (PROMs) are available that differ in content. To support the selection of suitable measures for specific applications and to evaluate possibilities of quantitative linking, the present study provides a content comparison of common fatigue measures, scales, and item banks. We included the EORTC CAT Core, EORTC QLQ-FA12, EORTC QLQ-C30, FACIT-F, PROMIS Fatigue (Cancer item bank v1.0), Brief Fatigue Inventory (BFI), Multidimensional Fatigue Inventory (MFI-20), Piper Fatigue Scale (PFS-12), and PRO-CTCAE. Methods: All items of the included measures were linked to the International Classification of Functioning, Disability and Health (ICF). Additionally, they were categorized as assessing general, physical, emotional, or cognitive fatigue. Descriptive statistics were used to display the contents covered in each measure and to allow for a qualitative comparison. Results: The measures consist of 160 items in total and covered primarily contents of the ICF components ‘Body functions’, ‘Activities and participation’, and ‘Environmental Factors’. Most ICF codings refer to ‘b1300 Energy level’ (9–67% of the codings per instrument; 47% of all coded content). Within the broad categorization of types of fatigue, most items were classified as general fatigue (33–100% of the codings per instrument; 49% of the overall item pool). While the EORTC CAT Core focuses exclusively on physical and general fatigue, FACIT and BFI additionally assess emotional fatigue. The EORTC QLQ-FA12, PROMIS, MFI-20, and PFS-12 cover all fatigue components, including cognitive fatigue. Discussion: The review provides an in-depth content comparison of PROMs assessing cancer-related fatigue. This can inform the selection of suitable measures in different clinical contexts. Furthermore, it will inform quantitative analyses to facilitate comparison of scores obtained with different PROMs

Adhoc Setup of an Online Mental Health Self-Help Program During the COVID-19 Pandemic: Description of the Development and Implementation Processes and Analysis of Its Users’ and Usage Profiles

BackgroundThe COVID-19 pandemic hit Austria in March 2020. This led to a considerable reduction in outpatient psychiatric therapies. People with mental disorders as well as with newly emerging mental health issues found themselves with very limited treatment options. Within only a few days our hospital set up an online mental health self-help program which went online in its first version on the first day of the lockdown in Austria. The process of this development and implementation process alongside with the user’s and usage data for the program are presented here.MethodsA small core team initiated the development of the program on a low-budget basis and using mostly freely available digital resources. The program had to be free of costs for its users and easy to navigate. Each self-help module contains a text description of the topic, a self-rating questionnaire and several psychoeducational 2–5 min videos. These videos explain, e.g., interactions of mental stress and the immune system or the vicious circle of anxiety. Additional videos provide easy to learn techniques like breathing and relaxation exercises.ResultsWe illustrate the implementation of this program following the replicating effective program (REP) model. We provide a detailed description of the implementation process starting from a simple website to a smartphone-based application with registered user area and instantaneous reporting of self-rating questionnaire results to users. The described process could be used as a model for the setup of similar programs in a very short time. As an indicator of acceptance, we report 46,100 unique video views and 3,937 completed questionnaires in the first year of use. The most accessed videos were those on anxiety, relaxation and resilience. Analysis of the sociodemographic user data indicate that they were mostly young (< 45 years; 59.7%), females (77.5%) and previously mentally healthy individuals (74.5%). An example of the collected psychometric questionnaire data over time is given.ConclusionWe show that it is possible to set up an online mental health self-help program ad hoc and without extensive prior planning, which enabled us to dynamically respond to a new situation. We are now planning on keeping the program active for a longer period of time to supplement and expand traditional treatment settings also outside the COVID-19 pandemic

Granulocytes mediates the Fas-L-associated apoptosis during lung metastasis of melanoma that determines the metastatic behaviour

The survival of tumour cells in a new tissue environment is crucial for tumour metastasis. Factors contributing to the death of tumour cells during metastasis are not completely understood. In murine melanoma model, activation of Fas (CD95, APO-1) signal in tumour cells reduces their lung metastasis potential, which may be associated with an induction of apoptosis in tumours. To elucidate the cellular mechanism, we used a Fas-ligand (Fas-L) specific ribozyme (Fas-Lribozyme) to suppress the expression of Fas-L but not Fas or TNF-α in B16F10 melanoma cells. The Fas-Lribozyme-carrying cells grew slightly faster in vitro with better viability than controls. Suppression of Fas-L in B16F10 melanoma cells by Fas-Lribozyme enhanced lung metastasis of the cells in C57BL/6 mice, and that was correlated with reductions in both apoptotic tumour cells and granulocytic infiltration. Mice depleted of granulocytes, but not CD4+ and CD8+ cells, showed a greatly elevated susceptibility to lung metastasis. Moreover, apoptosis in tumour cells was significantly reduced in granulocyte-depleted mice during the course of tumour formation. Taken together, our findings indicate that Fas-L-associated apoptosis in tumour cells determines the metastasis behaviour of melanoma in the lung and this apoptosis is primarily mediated by the cytotoxicity of recruited granulocytes

Advancing the use of molecular methods for routine freshwater macroinvertebrate biomonitoring : the need for calibration experiments

Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates

Do neurocognitive impairments explain the differences between brain tumor patients and their proxies when assessing the patient's IADL?

Background Neurocognitive impairments are common among brain tumor patients, and may impact patients' awareness of performance in instrumental activities in daily life (IADL). We examined differences between patient- and proxy-reported assessments of the patient's IADL, and whether the level of (dis)agreement is associated with neurocognitive impairments. Methods Brain tumor patients and their proxies completed the phase 3 version of the EORTC IADL-BN32 questionnaire measuring IADL, and patients completed six neurocognitive measures. Patient-proxy difference scores in IADL were compared between patients who were defined as neurocognitively impaired (>= 2 neurocognitive measures >= 2.0 standard deviations below healthy controls) and non-neurocognitively impaired. With multinomial logistic regression analyses we examined if neurocognitive variables were independently associated with patient-proxy disagreement in IADL ratings. Results Patients (n = 81) did not systematically (P < .01) rate IADL outcomes different than their proxies. Proxies did report more problems on 19/32 individual items and all five scales. This effect was more apparent in dyads with a neurocognitively impaired patient (n = 37), compared to dyads with non-neurocognitively impaired patients (n = 44). Multinomial logistic regression analyses showed that several neurocognitive variables (e.g., cognitive flexibility and verbal fluency) were independently associated with disagreement between patients and proxies on different scales. Conclusion Neurocognitive deficits seem to play a role in the discrepancies between brain tumor patients and their proxies assessment of patient's level of IADL. Although replication of our results is needed, our findings suggests that caution is warranted in interpreting self-reported IADL by patients with neurocognitive impairment, and that such self-reports should be supplemented with proxy ratings.Biological, physical and clinical aspects of cancer treatment with ionising radiatio

Development of an EORTC questionnaire measuring instrumental activities of daily living (IADL) in patients with brain tumours: phase I-III

Purpose Being able to function independently in society is an important aspect of quality of life. This ability goes beyond self-care, requires higher order cognitive functioning, and is typically measured with instrumental activities of daily living (IADL) questionnaires. Cognitive deficits are frequently observed in brain tumour patients, however, IADL is almost never assessed because no valid and reliable IADL measure is available for this patient group. Therefore, this measure is currently being developed. Methods This international multicentre study followed European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Group module development guidelines. Three out of four phases are completed: phases (I) generation of items, (II) construction of the item list, and (III) pre-testing. This paper reports the item selection procedures and preliminary psychometric properties of the questionnaire. Brain tumour patients (gliomas and brain metastases), their informal caregivers, and health care professionals (HCPs) were included. Results Phase I (n = 44 patient-proxy dyads and 26 HCPs) generated 59 relevant and important activities. In phase II, the activities were converted into items. In phase III (n = 85 dyads), the 59 items were pre-tested. Item selection procedures resulted in 32 items. Exploratory factor analysis revealed a preliminary dimensional structure consisting of five scales with acceptable to excellent internal consistency (alpha = 0.73-0.94) and two single items. For three scales, patients with cognitive impairments had significantly more IADL problems than patients without impairments. Conclusion A phase IV validation study is needed to confirm the psychometric properties of the EORTC IADL-BN32 questionnaire in a larger international sample.Biological, physical and clinical aspects of cancer treatment with ionising radiatio