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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.

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.

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.

Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive. Immediate freezing at −20°C or below has been considered the gold standard for microbiome preservation, yet this approach is not feasible for many field studies, ranging from anthropology to wildlife conservation. Here we tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including such types of variation as freeze-thaw cycles and the high temperature fluctuations often encountered under field conditions. We found that three of the methods—95% ethanol, FTA cards, and the OMNIgene Gut kit—can preserve samples sufficiently well at ambient temperatures such that differences at 8 weeks are comparable to differences among technical replicates. However, even the worst methods, including those with no fixative, were able to reveal microbiome differences between species at 8 weeks and between individuals after a week, allowing meta-analyses of samples collected using various methods when the effect of interest is expected to be larger than interindividual variation (although use of a single method within a study is strongly recommended to reduce batch effects). Encouragingly for FTA cards, the differences caused by this method are systematic and can be detrended. As in other studies, we strongly caution against the use of 70% ethanol. The results, spanning 15 individuals and over 1,200 samples, provide our most comprehensive view to date of storage effects on stool and provide a paradigm for the future studies of other sample types that will be required to provide a global view of microbial diversity and its interaction among humans, animals, and the environment. IMPORTANCE Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive.

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.

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.

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. A template-free image processing approach automatically detects and classifies membrane-bound protein complexes in cryo-electron tomograms of isolated endoplasmic reticulum and in intact cells.

The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.

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.

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.