Deductive automated pollen classification in environmental samples via exploratory deep learning and imaging flow cytometry.

Pollen and tracheophyte spores are ubiquitous environmental indicators at local and global scales. Palynology is typically performed manually by microscopic analysis; a specialised and time-consuming task limited in taxonomical precision and sampling frequency, therefore restricting data quality used to inform climate change and pollen forecasting models. We build on the growing work using AI (artificial intelligence) for automated pollen classification to design a flexible network that can deal with the uncertainty of broad-scale environmental applications. We combined imaging flow cytometry with Guided Deep Learning to identify and accurately categorise pollen in environmental samples; here, pollen grains captured within c. 5500 Cal yr BP old lake sediments. Our network discriminates not only pollen included in training libraries to the species level but, depending on the sample, can classify previously unseen pollen to the likely phylogenetic order, family and even genus. Our approach offers valuable insights into the development of a widely transferable, rapid and accurate exploratory tool for pollen classification in 'real-world' environmental samples with improved accuracy over pure deep learning techniques. This work has the potential to revolutionise many aspects of palynology, allowing a more detailed spatial and temporal understanding of pollen in the environment with improved taxonomical resolution.

The evolution of atmospheric particulate matter in an urban landscape since the Industrial Revolution.

Atmospheric particulate matter (PM) causes 3.7 million annual deaths worldwide and potentially damages every organ in the body. The cancer-causing potential of fine particulates (PM2.5) highlights the inextricable link between air quality and human health. With over half of the world's population living in cities, PM2.5 emissions are a major concern, however, our understanding of exposure to urban PM is restricted to relatively recent (post-1990) air quality monitoring programmes. To investigate how the composition and toxicity of PM has varied within an urban region, over timescales encompassing changing patterns of industrialisation and urbanisation, we reconstructed air pollution records spanning 200 years from the sediments of urban ponds in Merseyside (NW England), a heartland of urbanisation since the Industrial Revolution. These archives of urban environmental change across the region demonstrate a key shift in PM emissions from coarse carbonaceous 'soot' that peaked during the mid-twentieth century, to finer combustion-derived PM2.5 post-1980, mirroring changes in urban infrastructure. The evolution of urban pollution to a recent enhanced PM2.5 signal has important implications for understanding lifetime pollution exposures for urban populations over generational timescales.

Uptake of microplastics by marine worms depends on feeding mode and particle shape but not exposure time.

The uptake of microplastics into marine species has been widely documented across trophic levels. Feeding mode is suggested as playing an important role in determining different contamination loads across species, but this theory is poorly supported with empirical evidence. Here we use the two distinct feeding modes of the benthic polychaete, Hediste diversicolor (The Harbour Ragworm) (O.F. Müller, 1776), to test the hypothesis that filter feeding will lead to a greater uptake of microplastic particles than deposit feeding. Worms were exposed to both polyamide microfragments and microfibres in either water (as filter feeders) or sediment (as deposit feeders) for 1 week. No effect of exposure time was found between 1 day and 1 week (p > 0.19) but feeding mode was found to significantly affect the number of microfibres recovered from each worm (p < 0.001). When exposed to microfibers, filter feeding worms took up ≈15,000 % more fibres than deposit feeding worms (p < 0.001), whereas when feeding on microfragments there was no difference between feeding modes. Our data demonstrate that both feeding mode and particle characteristics significantly influence the uptake of microplastics by H. diversicolor. Using imaging flow cytometry, filter feeders were found to take up a broader size range of particles, with significantly more smaller and larger particles than deposit feeders (p < 0.05), commensurate with the range of plastics isolated from the guts of ragworms recovered from the environment. These results demonstrate that biological traits are useful in understanding the uptake of plastics into marine worms and warrant further exploration as a tool for understanding the bioaccessibility of plastics to marine organisms.

In-situ sequencing reveals the effect of storage on lacustrine sediment microbiome demographics and functionality.

The sediment microbiome is a demographically diverse and functionally active biosphere. Ensuring that data acquired from sediment is truly representative of the microbiome is critical to achieving robust analyses. Sample storage and the processing and timing of nucleic acid purification after environmental sample extraction may fundamentally affect the detectable microbial community and thereby significantly alter resultant data. Direct sequencing of environmental samples is increasingly commonplace due to the advent of the portable Oxford Nanopore MinION sequencing device. Here we demonstrate that storing sediment subsamples at - 20 °C or storing the cores at 4 °C for 10 weeks prior to analysis, has a significant effect on the sediment microbiome analysed using sedimentary DNA (sedDNA), especially for Alpha-, Beta- and Deltaproteobacteria species. Furthermore, these significant differences are observed regardless of sediment type. We show that the taxa which are predominantly affected by storage are Proteobacteria, and therefore recommend on-site purifications are performed to ensure an accurate representation of these taxa are observed in the microbiome. Comparisons of sedimentary RNA (sedRNA) analyses, revealed substantial differences between samples purified and sequenced immediately on-site, samples that were frozen before transportation, and cores that were stored at 4 °C prior to analysis. Our data therefore suggest that a more accurate representation of the sediment microbiome demography and functionality may be achieved by environmental sequencing as rapidly as possible to minimise confounding effects of storage.

The Application of Imaging Flow Cytometry for Characterisation and Quantification of Bacterial Phenotypes.

Bacteria modify their morphology in response to various factors including growth stage, nutrient availability, predation, motility and long-term survival strategies. Morphological changes may also be associated with specific physiological phenotypes such as the formation of dormant or persister cells in a "viable but non-culturable" (VBNC) state which frequently display different shapes and size compared to their active counterparts. Such dormancy phenotypes can display various degrees of tolerance to antibiotics and therefore a detailed understanding of these phenotypes is crucial for combatting chronic infections and associated diseases. Cell shape and size are therefore more than simple phenotypic characteristics; they are important physiological properties for understanding bacterial life-strategies and pathologies. However, quantitative studies on the changes to cell morphologies during bacterial growth, persister cell formation and the VBNC state are few and severely constrained by current limitations in the most used investigative techniques of flow cytometry (FC) and light or electron microscopy. In this study, we applied high-throughput Imaging Flow Cytometry (IFC) to characterise and quantify, at single-cell level and over time, the phenotypic heterogeneity and morphological changes in cultured populations of four bacterial species, Bacillus subtilis, Lactiplantibacillus plantarum, Pediococcus acidilactici and Escherichia coli. Morphologies in relation to growth stage and stress responses, cell integrity and metabolic activity were analysed. Additionally, we were able to identify and morphologically classify dormant cell phenotypes such as VBNC cells and monitor the resuscitation of persister cells in Escherichia coli following antibiotic treatment. We therefore demonstrate that IFC, with its high-throughput data collection and image capture capabilities, provides a platform by which a detailed understanding of changes in bacterial phenotypes and their physiological implications may be accurately monitored and quantified, leading to a better understanding of the role of phenotypic heterogeneity in the dynamic microbiome.

In Vivo Characterization of Endogenous Cardiovascular Extracellular Vesicles in Larval and Adult Zebrafish.

Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.

Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival.

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.

A new method for isolating and analysing coccospheres within sediment.

Size is a fundamental cellular trait that is important in determining phytoplankton physiological and ecological processes. Fossil coccospheres, the external calcite structure produced by the excretion of interlocking plates by the phytoplankton coccolithophores, can provide a rare window into cell size in the past. Coccospheres are delicate however and are therefore poorly preserved in sediment. We demonstrate a novel technique combining imaging flow cytometry and cross-polarised light (ISX+PL) to rapidly and reliably visually isolate and quantify the morphological characteristics of coccospheres from marine sediment by exploiting their unique optical and morphological properties. Imaging flow cytometry combines the morphological information provided by microscopy with high sample numbers associated with flow cytometry. High throughput imaging overcomes the constraints of labour-intensive manual microscopy and allows statistically robust analysis of morphological features and coccosphere concentration despite low coccosphere concentrations in sediments. Applying this technique to the fine-fraction of sediments, hundreds of coccospheres can be visually isolated quickly with minimal sample preparation. This approach has the potential to enable rapid processing of down-core sediment records and/or high spatial coverage from surface sediments and may prove valuable in investigating the interplay between climate change and coccolithophore physiological/ecological response.

A Hybrid Sequencing Approach Completes the Genome Sequence of Thermoanaerobacter ethanolicus JW 200.

Thermoanaerobacter ethanolicus JW 200 has been identified as a potential sustainable biofuel producer due to its ability to readily ferment carbohydrates to ethanol. A hybrid sequencing approach, combining Oxford Nanopore and Illumina DNA sequence reads, was applied to produce a single contiguous genome sequence of 2,911,280 bp.

Using urban man-made ponds to reconstruct a 150-year history of air pollution in northwest England.

A regional pollution history has been reconstructed for the borough of Halton (northwest England) from four urban ponds in north Cheshire and south Merseyside, using environmental analyses of lake sediment stratigraphies. Mineral magnetism, geochemistry and radiometric dating have produced profiles of pollution characteristics dating from the mid-nineteenth century to present day. These pollution profiles reflect the atmospheric deposition of a range of pollutants over 150 years of intensified industry. Distinct phases of pollution deposition and characteristics are identified reflecting: (1) intensification of industry in the nineteenth century; (2) expansion of industry during the twentieth century; (3) post 1956 Clean Air Acts. This work promotes the potential use of these pollution archives for use in epidemiology to better understand links between human health and environmental pollution, especially for diseases with long latency times, where retrospective pollution exposure assessments are important.

Magneto-biomonitoring of intra-urban spatial variations of particulate matter using tree leaves.

Preliminary mineral magnetic results from a pilot project investigating the suitability of roadside tree leaves as depositories of vehicular pollution are presented. Tree leaf surfaces (Lime: Tilia europaea; Sycamore: Acer pseudoplatanus) at four roadside and one woodland location in Wolverhampton, UK, have been monitored (July 2003 to November 2003). Mineral magnetic technologies have revealed spatial variations of particulate pollution concentration throughout the conurbation and data analysis indicates that magnetic concentration parameters are suitable proxies for fine particulate pollution, which are particularly hazardous to health. Site-specific traffic management and associated vehicle behaviour appear to be chiefly responsible for the magnetic concentration differences between sites. Magneto-biomonitoring in this way allows the high-resolution spatial mapping of particulate matter (PM) pollution, which may also benefit epidemiology in better assessing exposure to vehicular-derived particulates. Given the speed, measurement sensitivity and non-destructive nature of the technique, it is proposed that this low-cost approach offers some advantages over centralised monitoring stations to monitor urban roadside particulate pollution.

M1 muscarinic acetylcholine receptor agonism alters sleep without affecting memory consolidation.

Preclinical studies have implicated cholinergic neurotransmission, specifically M1 muscarinic acetylcholine receptor (mAChR) activation, in sleep-associated memory consolidation. In the present study, we investigated the effects of administering the direct M1 mAChR agonist RS-86 on pre-post sleep memory consolidation. Twenty healthy human participants were tested in a declarative word-list task and a procedural mirror-tracing task. RS-86 significantly reduced rapid eye movement (REM) sleep latency and slow wave sleep (SWS) duration in comparison with placebo. Presleep acquisition and postsleep recall rates were within the expected ranges. However, recall rates in both tasks were almost identical for the RS-86 and placebo conditions. These results indicate that selective M1 mAChR activation in healthy humans has no clinically relevant effect on pre-post sleep consolidation of declarative or procedural memories at a dose that reduces REM sleep latency and SWS duration.

Margins and outcome of screen-detected breast cancer with extensive in situ component.

BACKGROUND: In situ disease surrounding invasive tumours is an important consideration in the management of patients with early breast cancer. This study of screen-detected breast cancers assessed the influence of in situ disease including an extensive in situ component (defined as ductal carcinoma in situ involving more than 25% of the area within the invasive tumour) on surgical management, local recurrence and survival of a group of patients. METHODS: A total of 595 cases of invasive breast cancer detected at St Vincent's BreastScreen were retrospectively reviewed to determine presence and extent of in situ disease, the surgical procedure and adequacy of excision. Outcome was examined in a cohort of 126 cases. RESULTS: A total of 438 (74%) patients had in situ foci in or around the invasive tumour and 107 (18%) were defined as extensive in situ component (EIC)-positive. The initial procedure was mastectomy in 20% of the cases and breast-conserving surgery in 80% including 18% who underwent further surgery. Re-excision (P = 0.02) or mastectomy (P = 0.01) was more often required in patients with EIC. After definitive local excision, margins were close or involved with invasive disease in 3% but the patients with EIC were more likely to have margins close or involved with in situ disease (16 vs 2%; P = 0.001). There were seven deaths and one local invasive recurrence in the follow-up group and none of the deaths were in patients who were EIC-positive. CONCLUSIONS: EIC predicts for a higher rate of re-excision and/or mastectomy. For patients with EIC, there is an acceptably low risk of local recurrence if margins are clear.

Anisomycin infused into the hippocampus fails to block "reconsolidation" but impairs extinction: the role of re-exposure duration.

Recent studies have reported new evidence consistent with the hypothesis that reactivating a memory by re-exposure to a training context destabilizes the memory and induces "reconsolidation." In the present experiments, rats' memory for inhibitory avoidance (IA) training was tested 6 h (Test 1), 2 d (Test 2), and 6 d (Test 3) after training. On Test 1 the rats were either removed from the shock compartment immediately after entry or retained in the shock context for 200 sec, and intrahippocampal infusions of the protein synthesis inhibitor anisomycin (75 microg/side) were administered immediately after the test. Anisomycin infusions administered after Test 1 impaired IA performance on Test 2 in animals given the brief re-exposure, but impaired extinction in animals exposed to the context for 200 sec. Rats with anisomycin-induced retention impairment on Test 2 demonstrated spontaneous recovery of retention performance on Test 3, whereas rats showing extinction on Test 2 showed further extinction on Test 3. The findings indicate that post-retrieval administration of anisomycin impairs subsequent retention performance only in the absence of extinction and that this impairment is temporary.

Muscarinic cholinergic influences in memory consolidation.

The central cholinergic system and muscarinic cholinergic receptor (mR) activation have long been associated with cognitive function. Although mR activation is no doubt involved in many aspects of cognitive functioning, the extensive evidence that memory is influenced by cholinergic treatments given after training either systemically or intra-cranially clearly indicates that cholinergic activation via mRs is a critical component in modulation of memory consolidation. Furthermore, the evidence indicates that activation of mRs in the basolateral amygdala (BLA) plays an essential role in enabling other neuromodulatory influences on memory consolidation. Memory can also be affected by posttraining activation of mRs in the hippocampus, striatum and cortex. Evidence of increases in hippocampal and cortical acetylcholine (ACh) levels following learning experiences support the view that endogenous ACh release is involved in long-term memory consolidation. Furthermore, the findings indicating that mR drug treatments influence plasticity in the hippocampus and in sensory cortices strongly suggest that mR activation is involved in the storage of information in these brain regions.

Role of the basolateral amygdala in memory consolidation.

Memories of emotionally arousing events tend to be more vivid and to persist longer than do memories of neutral or trivial events. Moreover, memories of emotionally influenced information may endure after a single experience. Recent findings strongly suggest that the influence of emotional arousal on memory consolidation is mediated by the release of adrenal stress hormones (epinephrine and glucocorticoids) and neurotransmitters that converge in modulating the noradrenergic system within the amygdala. Considerable evidence also indicates that amygdala activation influences memory by regulating consolidation in other brain regions. The findings suggest further that this memory-modulatory system may be involved in the formation of traumatic memories and posttraumatic stress disorder in human subjects.

Cholinergic activation of the basolateral amygdala regulates unlearned freezing behavior in rats.

Recent evidence suggests that the basolateral amygdala (BLA) is involved in the expression of freezing behavior in rats. This study investigated the effects of unilateral phthalic acid (PA) lesions of the nucleus basalis magnocellularis (NBM) on fear-motivated behavior in response to a natural predator-stimulus. Such lesions preferentially disrupt the cholinergic projection to the BLA. Rats were placed in a chamber containing either real or fake cat hair, and the amount of time spent freezing and the number of contacts made with the stimulus were measured. Compared with Sham control rats, the PA NBM-lesioned rats displayed significantly less freezing in the presence of the cat hair. Both the Sham and lesioned rats made fewer contacts with the real than the fake cat hair. Pre-testing intra-BLA infusion of the direct muscarinic cholinergic agonist oxotremorine ipsilateral to the PA NBM-lesion attenuated the freezing deficit. The indirect non-specific cholinergic agonist physostigmine increased the time spent freezing in Sham rats, but did not attenuate the freezing deficit in the NBM-lesioned rats. Sham and NBM-lesioned rats given oxotremorine infusions made fewer contacts with either the real or the fake cat hair. The PA NBM-lesion did not affect open field activity. These findings indicate that muscarinic cholinergic activation in the BLA from the NBM influences fear-motivated freezing behavior.

Phthalic acid amygdalopetal lesion of the nucleus basalis magnocellularis induces reversible memory deficits in rats.

The basolateral amygdala (BLA) is extensively implicated in emotional learning and memory. The current study investigated the contribution of cholinergic afferents to the BLA from the nucleus basalis magnocellularis in influencing aversive learning and memory. Sprague-Dawley rats were given permanent unilateral phthalic acid (300 ng) lesions of the nucleus basalis magnocellularis and were chronically implanted with cannulas aimed at the ipsilateral BLA. Lesioned rats showed a pronounced inhibitory avoidance task retention deficit that was attenuated by acute posttraining infusions of the muscarinic cholinergic agonist oxotremorine (4 ng) or the indirect agonist physostigmine (1 microg) into the BLA. Continuous multiple-trial inhibitory avoidance training and testing revealed that lesioned rats have a mild acquisition deficit, requiring approximately 1 additional shock to reach the criterion, and a pronounced consolidation deficit as indicated by a shorter latency to enter the shock compartment on the retention test. Because lesioned rats did not differ from sham-operated controls in performance on a spatial water maze task or in shock sensitivity, it is not likely that the memory impairments produced by the phthalic acid lesions are due to any general sensory or motor deficits. These findings suggest that the dense cholinergic projection from the nucleus basalis magnocellularis to the BLA is involved in both the acquisition and the consolidation of the aversive inhibitory avoidance task.

Lesions of the nucleus basalis magnocellularis induced by 192 IgG-saporin block memory enhancement with posttraining norepinephrine in the basolateral amygdala.

Extensive evidence indicates that drugs and stress hormones act in the basolateral amygdala (BLA) to modulate memory consolidation. The BLA projects to the nucleus basalis magnocellularis (NBM), which sends broad cholinergic projections to the neocortex. NBM-cortex projections have been implicated in learning, memory storage, and plasticity. The current study investigated whether the cholinergic NBM-cortex projections are involved in BLA-mediated modulation of memory consolidation. Bilateral cholinergic cell lesions of the NBM were induced in rats with infusions of 192 IgG-saporin (0.1 microg/0.5 microl per side). Additionally, cannulae were implanted bilaterally in the BLA. One week after surgery, the rats were trained in an inhibitory avoidance task and, immediately after training, norepinephrine (0.3 microg, 1.0 microg, or 3.0 microg in 0.2 microl) or vehicle (PBS) was infused bilaterally into the BLA. Norepinephrine infusions produced a dose-dependent enhancement of 48-h retention (0.3 microg and 1.0 microg doses enhanced) in nonlesioned rats but did not affect retention in NBM-lesioned rats. Choline acetyltransferase assays of frontal and occipital cortices confirmed the NBM lesions. These findings indicate that cholinergic NBM-cortex projections are required for BLA-mediated modulation of memory consolidation.