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PurposeAccording to standard procedure recommended by the Water Framework Directive (WFD), dissolved concentrations of potentially toxic elements (PTEs) in river water are determined by inductively coupled plasma mass spectrometry (ICP-MS) in filtered (0.45 µm) and acidified (pH 2) samples. Properly prepared and stored composite samples can enhance the temporal representativity of monitoring without increasing analytical costs. For this purpose, the WFD recommends freezing, which can preserve the species integrity and prevent adsorption processes of PTEs.MethodsLow storage temperature in hard water samples can trigger precipitation of calcium carbonate (CaCO3) and subsequent co-precipitation of PTEs. To test and determine to what extent co-precipitation with CaCO3 can influence the determination of PTE concentrations, composite river water samples from two case study catchments in Hungary (Zagyva and Koppány) were prepared following two different sample preservation procedures. To study the behavior of PTEs in river water during storage, in the first procedure, samples were frozen, and they were thawed, filtered, and acidified directly prior to the analysis. In the second procedure, samples were filtered on-site and acidified prior to freezing to prevent precipitation of CaCO3 and then only thawed to carry out the chemical analyses. Concentrations of PTEs were determined by ICP-MS.ResultsA statistical evaluation of the results using Student’s t-test revealed significant differences between the two sample preservation procedures, suggesting that PTEs were largely co-precipitated with CaCO3 if the samples were not acidified prior to freezing.ConclusionWhen establishing protocols for sample preservation procedures, the phenomenon of co-precipitation of PTEs with CaCO3 should be considered if the samples were not acidified before freezing. Therefore, to prevent co-precipitation of PTEs with CaCO3, samples should be filtered and acidified before freezing.

Abstract Background Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests. Methods We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT. Results Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation. Conclusion Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.

With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron tomography provides an accurate representation of molecular architecture of cells. However, detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membrane-bound complexes and an unsupervised classification (PySeg). Applications to intact cells and isolated endoplasmic reticulum (ER) allowed us to detect and classify small protein complexes. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Spatial distribution analysis showed that ER complexes have different localization patterns forming nanodomains. Therefore, this procedure allows a comprehensive detection and structural analysis of complexes in situ.

Abstract First insights on the human gut microbiome have been gained from medium-sized, cross-sectional studies. However, given the modest portion of explained variance of currently identified covariates and the small effect size of gut microbiota modulation strategies, upscaling seems essential for further discovery and characterisation of the multiple influencing factors and their relative contribution. In order to guide future research projects and standardisation efforts, we here review currently applied collection and preservation methods for gut microbiome research. We discuss aspects such as sample quality, applicable omics techniques, user experience and time and cost efficiency. In addition, we evaluate the protocols of a large-scale microbiome cohort initiative, the Flemish Gut Flora Project, to give an idea of perspectives, and pitfalls of large-scale faecal sampling studies. Although cryopreservation can be regarded as the gold standard, freezing protocols generally require more resources due to cold chain management. However, here we show that much can be gained from an optimised transport chain and sample aliquoting before freezing. Other protocols can be useful as long as they preserve the microbial signature of a sample such that relevant conclusions can be drawn regarding the research question, and the obtained data are stable and reproducible over time. The authors review currently applied collection and preservation methods for gut microbiome research, discussing aspects such as sample quality, applicable omics techniques, user experience and time and cost-efficiency, and evaluate the protocols of the Flemish Gut Flora Project, a large-scale gut microbiome sampling effort in Belgium, to give an idea of perspectives, and pitfalls of population-wide studies implementing faecal sampling for gut microbiome research.

Point-scanning imaging systems are among the most widely used tools for high-resolution cellular and tissue imaging, benefiting from arbitrarily defined pixel sizes. The resolution, speed, sample preservation and signal-to-noise ratio (SNR) of point-scanning systems are difficult to optimize simultaneously. We show these limitations can be mitigated via the use of deep learning-based supersampling of undersampled images acquired on a point-scanning system, which we term point-scanning super-resolution (PSSR) imaging. We designed a ‘crappifier’ that computationally degrades high SNR, high-pixel resolution ground truth images to simulate low SNR, low-resolution counterparts for training PSSR models that can restore real-world undersampled images. For high spatiotemporal resolution fluorescence time-lapse data, we developed a ‘multi-frame’ PSSR approach that uses information in adjacent frames to improve model predictions. PSSR facilitates point-scanning image acquisition with otherwise unattainable resolution, speed and sensitivity. All the training data, models and code for PSSR are publicly available at 3DEM.org.Point-scanning super-resolution imaging uses deep learning to supersample undersampled images and enable time-lapse imaging of subcellular events. An accompanying ‘crappifier’ rapidly generates quality training data for robust performance.

To accurately define the role of the gut microbiota in health and disease pathogenesis, the preservation of stool sample integrity, in terms of microbial community composition and metabolic function, is critical. This presents a challenge for any studies which rely on participants self-collecting and returning stool samples as this introduces variability and uncertainty of sample storage/handling. Here, we tested the performance of three stool sample collection/preservation buffers when storing human stool samples at different temperatures (room temperature [20 °C], 4 °C and – 80 °C) for up to three days. We compared and quantified differences in 16S rRNA sequencing composition and short-chain fatty acid profiles compared against immediately snap-frozen stool. We found that the choice of preservation buffer had the largest effect on the resulting microbial community and metabolomic profiles. Collectively analysis confirmed that PSP and RNAlater buffered samples most closely recapitulated the microbial diversity profile of the original (immediately – 80 °C frozen) sample and should be prioritised for human stool microbiome studies.

In an effort to better understand the ancestral state of the human distal gut microbiome, we examine feces retrieved from archaeological contexts (coprolites). To accomplish this, we pyrosequenced the 16S rDNA V3 region from duplicate coprolite samples recovered from three archaeological sites, each representing a different depositional environment: Hinds Cave (~8000 years B.P.) in the southern United States, Caserones (1600 years B.P.) in northern Chile, and Rio Zape in northern Mexico (1400 years B.P.). Clustering algorithms grouped samples from the same site. Phyletic representation was more similar within sites than between them. A Bayesian approach to source-tracking was used to compare the coprolite data to published data from known sources that include, soil, compost, human gut from rural African children, human gut, oral and skin from US cosmopolitan adults and non-human primate gut. The data from the Hinds Cave samples largely represented unknown sources. The Caserones samples, retrieved directly from natural mummies, matched compost in high proportion. A substantial and robust proportion of Rio Zape data was predicted to match the gut microbiome found in traditional rural communities, with more minor matches to other sources. One of the Rio Zape samples had taxonomic representation consistent with a child. To provide an idealized scenario for sample preservation, we also applied source tracking to previously published data for Ötzi the Iceman and a soldier frozen for 93 years on a glacier. Overall these studies reveal that human microbiome data has been preserved in some coprolites, and these preserved human microbiomes match more closely to those from the rural communities than to those from cosmopolitan communities. These results suggest that the modern cosmopolitan lifestyle resulted in a dramatic change to the human gut microbiome.

The gut microbiome has the potential to be an effective indicator of individual and population health in fish given its sensitivity to internal and external stressors. However, without consistent and tested validated sampling protocols, the gut microbiome’s potential as a reliable indicator may be limited. Routine sampling of wild free-living fish caught by commercial fisheries rarely occurs at the time of capture and more commonly occurs hours, days, or weeks after fish capture when the catch is unloaded in port. This delay in sampling provides time for the degradation and decomposition of the microbiome community potentially compromising the reliability of gut microbiome analyses. Unfortunately, comprehensive and systematic analyses on post-capture changes in the gut microbiome communities of wild marine fish are lacking, limiting the reliability of microbiome studies. Here, we investigated the post-mortem changes in the gut microbiome of one wild-caught marine fish, skipjack tuna ( Katsuwonus pelamis ), at five different time points (immediately, 2 h, 24 h, 12 days, and 24 days after fish capture). The decomposition of the gut microbiome community occurred within the first 24 h if the samples were not preserved immediately. Over time, the relative abundance of Vibrionaceae decreased while Bradyrhizobiaceae increased, indicating the potential of these two families to serve as gut microbiome degradation indicators. Our findings highlight the importance of timely preservation in gut microbiome studies of wild-caught fish. Without appropriate preservation, both the diversity of the gut microbiome and the relative abundance of key microbial families change significantly within 24 h. To obtain reliable and representative results, we recommend preserving gut samples as soon as possible after capture, ideally within two hours, and no later than 24 h.s.

Collectively, we have been reviewers for microbial ecology, genetics and genomics studies that include environmental DNA (eDNA), microbiome studies, and whole bacterial genome biology for Microbial Ecology and other journals for about three decades. Here, we wish to point out trends and point to areas of study that readers, especially those moving into the next generation of microbial ecology research, might learn and consider. In this communication, we are not saying the work currently being accomplished in microbial ecology and restoration biology is inadequate. What we are saying is that a significant milestone in microbial ecology has been reached, and approaches that may have been overlooked or were unable to be completed before should be reconsidered in moving forward into a new more ecological era where restoration of the ecological trajectory of systems has become critical. It is our hope that this introduction, along with the papers that make up this special issue, will address the sense of immediacy and focus needed to move into the next generation of microbial ecology study.

Advanced personalized medical diagnostics depend on the availability of high-quality biological samples. These are typically biofluids, such as blood, saliva, or urine; and their collection and storage is critical to obtain reliable results. Without proper temperature regulation, protein biomarkers in particular can degrade rapidly in blood samples, an effect that ultimately compromises the quality and reliability of laboratory tests. Here, we present the use of silk fibroin as a solid matrix to encapsulate blood analytes, protecting them from thermally induced damage that could be encountered during nonrefrigerated transportation or freeze–thaw cycles. Blood samples are recovered by simple dissolution of the silk matrix in water. This process is demonstrated to be compatible with a number of immunoassays and provides enhanced sample preservation in comparison with traditional air-drying paper approaches. Additional processing can remediate interactions with conformational structures of the silk protein to further enhance blood stabilization and recovery. This approach can provide expanded utility for remote collection of blood and other biospecimens empowering new modalities of temperature- independent remote diagnostics.