A curvature-enhanced random walker segmentation method for detailed capture of 3D cell surface Membranes

High-resolution 3D microscopy is a fast advancing field and requires new techniques in image analysis to handle these new datasets. In this work, we focus on detailed 3D segmentation of Dictyostelium cells undergoing macropinocytosis captured on an iSPIM microscope. We propose a novel random walker-based method with a curvature-based enhancement term, with the aim of capturing fine protrusions, such as filopodia and deep invaginations, such as macropinocytotic cups, on the cell surface. We tested our method on both real and synthetic 3D image volumes, demonstrating that the inclusion of the curvature enhancement term can improve the segmentation of the aforementioned features. We show that our method performs better than other state of the art segmentation methods in 3D images of Dictyostelium cells, and performs competitively against CNN-based methods in two Cell Tracking Challenge datasets, demonstrating the ability to obtain accurate segmentations without the requirement of large training datasets. We also present an automated seeding method for microscopy data, which, combined with the curvature-enhanced random walker method, enables the segmentation of large time series with minimal input from the experimenter

Image based modelling of bleb site selection

Cellular blebs are fast, pressure-driven protrusions of the cell membrane that are initially devoid of F-actin. Although blebs have often been overlooked as a functional part of cell motility, blebbing has been shown to play an important role in migration in 3D mechanically resistive environments, such as movement through densely packed tissues. The location of bleb nucleation sites is often assumed to be entirely stochastic, however, cells migrating using blebbing motility have been repeatedly observed to perform persistent, directional movement. Given the compelling evidence on the role of blebbing in directional cell migration, relatively little is known on the mechanisms of bleb site selection; how bleb sites are determined and directed to the cell front remains an open question. Previously, Tyson et al. found that chemotaxing Dictyostelium cells preferentially bleb from concave regions, where membrane tension could facilitate membrane-cortex detachment. Based on this, a biophysical model for curvature dependent bleb nucleation was proposed, hinting at the possibility that polarised blebbing was due to physical forces alone. We develop a novel image based modeling approach, using real cell contours from image data to initialise the model. This enables us to link quantitative experimental data and predictive modeling on the spatial distribution of blebs for the first time. We proceed to show that the extent to which cell geometry is a good predictor of bleb site selection is highly dependent on the degree of mechanical resistance the cells experience. For cells in highly resistive environments, where we observe the front and rear of the cell to be geometrically distinct, our novel modeling approach demonstrates that physical forces are sufficient to polarise blebbing activity in chemotaxing cells. For cells in low resistive environments however, where the front and rear of the cell are not geometrically distinct, we show that an additional mechanism is required to restrict blebbing to the cell front. We propose this additional mechanism to be a front-to-rear gradient in the linker protein TalinA. Creation of a TalinA-mNeon construct allows us to experimentally confirm the existence of an asymmetric linker distribution. We incorporate the observed exponential linker gradient within the model. Inclusion of this mechanism increases the predictive power of the model, with regard to the spatial distribution of experimentally observed bleb sites, through efficiently directing blebbing activity to the cell front. We have created a method which links quantitative experimental data and predictive modeling, this tool allows us to disentangle the role of physical forces and biological mechanisms in the prediction of bleb nucleation sites

Generative adversarial networks for augmenting training data of microscopic cell images

Generative adversarial networks (GANs) have recently been successfully used to create realistic synthetic microscopy cell images in 2D and predict intermediate cell stages. In the current paper we highlight that GANs can not only be used for creating synthetic cell images optimized for different fluorescent molecular labels, but that by using GANs for augmentation of training data involving scaling or other transformations the inherent length scale of biological structures is retained. In addition, GANs make it possible to create synthetic cells with specific shape features, which can be used, for example, to validate different methods for feature extraction. Here, we apply GANs to create 2D distributions of fluorescent markers for F-actin in the cell cortex of Dictyostelium cells (ABD), a membrane receptor (cAR1), and a cortex-membrane linker protein (TalA). The recent more widespread use of 3D lightsheet microscopy, where obtaining sufficient training data is considerably more difficult than in 2D, creates significant demand for novel approaches to data augmentation. We show that it is possible to directly generate synthetic 3D cell images using GANs, but limitations are excessive training times, dependence on high-quality segmentations of 3D images, and that the number of z-slices cannot be freely adjusted without retraining the network. We demonstrate that in the case of molecular labels that are highly correlated with cell shape, like F-actin in our example, 2D GANs can be used efficiently to create pseudo-3D synthetic cell data from individually generated 2D slices. Because high quality segmented 2D cell data are more readily available, this is an attractive alternative to using less efficient 3D networks

Exposure to adversity and inflammatory outcomes in mid and late childhood

Background We aimed to estimate the association between exposure to adversity and inflammatory markers in mid (4 years) and late (11-12 years) childhood, and whether effects differ by type and timing of exposure. Methods Data sources: Barwon Infant Study (BIS; N = 510 analyzed) and Longitudinal Study of Australian Children (LSAC; N = 1156 analyzed). Exposures: Adversity indicators assessed from 0 to 4 (BIS) and 0-11 years (LSAC): parent legal problems, mental illness and substance abuse, anger in parenting responses, separation/divorce, unsafe neighborhood, and family member death; a count of adversities; and, in LSAC only, early (0-3), middle (4-7), or later (10-11) initial exposure. Outcomes: Inflammation quantified by high sensitivity C-reactive protein (hsCRP, Log (ug/ml)) and glycoprotein acetyls (GlycA, Log (umol/L)). Analyses: Linear regression was used to estimate relative change in inflammatory markers, adjusted for sociodemographic characteristics, with exposure to adversity. Outcomes were log-transformed. Results Evidence of an association between adversity and hsCRP was weak and inconsistent (e.g., 3+ versus no adversity: BIS: 12% higher, 95%CI -49.4, 147.8; LSAC 4.6% lower, 95%CI: −36.6, 48.3). A small positive association between adversity and GlycA levels was observed at both 4 years (e.g., 3+ versus no adversity: 3.3% higher, 95%CI -3.0, 9.9) and 11-12 years (3.2% higher, 95%CI 0.8, 5.8). In LSAC, we did not find evidence that inflammatory outcomes differed by initial timing of adversity exposure. Conclusions Small positive associations between adversity and inflammation were consistently observed for GlycA, across two cohorts with differing ages. Further work is needed to understand mechanisms, clinical relevance, and to identify opportunities for early intervention.Meredith O'Connor and Sarah Arnup were supported by the Melbourne Children's LifeCourse initiative, funded by a Royal Children's Hospital Foundation Grant (2018-984). Anne-Louise Ponsonby is supported by a National Health and Medical Research Council (NHMRC) Fellowship (1110200). Peter Sly is sup- ported by an NHMRC Fellowship (APP1102590). Naomi Priest is sup- ported by an NHMRC Career Development Fellowship (APP1123677). Kate Lycett is supported by an NHMRC Early Career Fellowship (APP1091124) and Honorary National Heart Foundation Postdoctoral Fellowship (101239). Sharon Goldfeld is supported by an NHMRC Career Development Fellowship (1082922). David Burgner is supported by an NHMRC Investigator Grant (1175744)

Light Stress Responses by the Eelgrass, Zostera marina (L)

Zostera marina is the dominant seagrass species in the Northern Hemisphere where it grows in sheltered bays and estuaries. As a consequence of its distribution its conservation is commonly threatened by poor coastal water quality. The high minimum light requirements of seagrasses results in water quality degradation (high turbidity and eutrophication) being a significant risk. Bioindicators of light stress can be used to interpret seagrass responses to light limitation and therefore act as sentinels for conservation management. However, there exists limited experimental inter-comparison of the effectiveness of multiple individual bioindicator responses. Meta-analysis suggests that rhizome sugars, shoot C:N, shoot growth, and number of leaves per shoot provide the most consistent response variables to increasing light limitation in seagrass, but this premise remains largely untested at the plant level as a direct comparison of multiple bioindicators. The present study aimed to test the morphological, physiological, and photo-physiological bioindicator responses of Z. marina to light stress applied within controlled laboratory conditions. These bioindicators were used to assign minimum light thresholds. Growth rate and photophysiological parameters (alpha, Ek, and ETRmax) were rapidly (1st week) and drastically affected by low light shade treatments (20.12 μmol photons m−2s−1 and lower). After 3 weeks at low light, significant reductions in maximum leaf length and leaf width were observed. Principal Component Analysis identified leaf length, shoot growth, shoot surface area, ETRmax, Ek, and alpha as having the strongest responses to reduced light. Shoot growth, ETRmax, Ek, and alpha were found to provide the best early warning of light limitation after 5–8 days. These results provide evidence for bioindicators of light stress in Z. marina and highlights the importance of understanding these responses for the successful management and conservation of this species

Bifidobacterium animalis AHC7 protects against pathogen-induced NF-κB activation in vivo

BACKGROUND: Bifidobacteria and lactobacilli are among the early and important colonizers of the gastrointestinal tract and are generally considered to be part of a normal, healthy microbiota. It is believed that specific strains within the microbiota can influence host immune-reactivity and may play a role in protection from infection and aberrant inflammatory activity. One such strain, Bifidobacterium animalis AHC7, has been previously shown to protect against Salmonella typhimurium infection in mice and helps resolve acute idiopathic diarrhea in dogs. The aim of this study was to investigate the potential molecular and cellular mechanisms underpinning the Bifidobacterium animalis AHC7 protective effect. RESULTS: Following 4 hours of infection with Salmonella typhimurium, NF-κB activation was significantly elevated in vivo in placebo and Enterococcus faecium-fed animals while Bifidobacterium animalis AHC7 consumption significantly attenuated the NF-κB response. In vitro anti-CD3/CD28 stimulated Peyer's patch cells secreted significantly less TNF-α and IFN-γ following Bifidobacterium animalis AHC7 consumption. Stimulated cells released more IL-12p70 but this difference did not reach statistical significance. No alteration in mucosal IL-6, IL-10 or MCP-1 levels were observed. No statistically significant change in the cytokine profile of mesenteric lymph node cells was noted. In vitro, Bifidobacterium animalis AHC7 was bound by dendritic cells and induced secretion of both IL-10 and IL-12p70. In addition, co-culture of CD4+ T cells with Bifidobacterium animalis AHC7-stimulated dendritic cells resulted in a significant increase in CD25+Foxp3+ T cell numbers. CONCLUSION: Bifidobacterium animalis AHC7 exerts an anti-inflammatory effect via the attenuation of pro-inflammatory transcription factor activation in response to an infectious insult associated with modulation of pro-inflammatory cytokine production within the mucosa. The cellular mechanism underpinning Bifidobacterium animalis AHC7 mediated attenuation of NF-κB activation may include recognition of the bacterium by dendritic cells and induction of CD25+Foxp3+ T cells

BioTIME: A database of biodiversity time series for the Anthropocene.

MotivationThe BioTIME database contains raw data on species identities and abundances in ecological assemblages through time. These data enable users to calculate temporal trends in biodiversity within and amongst assemblages using a broad range of metrics. BioTIME is being developed as a community-led open-source database of biodiversity time series. Our goal is to accelerate and facilitate quantitative analysis of temporal patterns of biodiversity in the Anthropocene.Main types of variables includedThe database contains 8,777,413 species abundance records, from assemblages consistently sampled for a minimum of 2 years, which need not necessarily be consecutive. In addition, the database contains metadata relating to sampling methodology and contextual information about each record.Spatial location and grainBioTIME is a global database of 547,161 unique sampling locations spanning the marine, freshwater and terrestrial realms. Grain size varies across datasets from 0.0000000158 km2 (158 cm2) to 100 km2 (1,000,000,000,000 cm2).Time period and grainBioTIME records span from 1874 to 2016. The minimal temporal grain across all datasets in BioTIME is a year.Major taxa and level of measurementBioTIME includes data from 44,440 species across the plant and animal kingdoms, ranging from plants, plankton and terrestrial invertebrates to small and large vertebrates.Software format.csv and .SQL

Commensal-Induced Regulatory T Cells Mediate Protection against Pathogen-Stimulated NF-κB Activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-κB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-κB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to naïve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-κB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis–fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-κB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-κB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS