Blood identified and quantified in formalin fixed paraffin embedded lung sections using eosin fluorescence.

Eosin Y is a common stain in histology. Although usually used for colourimetric imaging where the dye is used to stain pink/red a range of structures in the tissue, Eosin Y is also a fluorochrome, and has been used in this manner for decades. In this study our aim was to investigate the fluorescence properties of the dye to enable quantification of structures within formalin-fixed paraffin-embedded (FFPE) tissue sections. To do this, FFPE sections of hamster tissue were prepared with haematoxylin and eosin Y dyes. Spectral detection on a confocal laser scanning microscope was used to obtain the fluorescence emission spectra of the eosin Y under blue light. This showed clear spectral differences between the red blood cells and congealed blood, compared to the rest of the section. The spectra were so distinct that it was possible to discern these in fluorescence and multi-photon microscopy. An image analysis algorithm was used to quantify the red blood cells. These analyses could have broad applications in histopathology where differentiation is required, such as the analysis of clotting disorders to haemorrhage or damage from infectious disease.

Civilian science: the potential of participatory environmental monitoring in areas affected by armed conflicts.

Legal and policy initiatives to address the environmental dimensions of armed conflicts and their impact on people, ecosystems and sustainable development are highly dependent on the availability of environmental data from conflict-affected areas. Socio-political and security conditions in these areas often impede data collection, while traditional models of post-conflict environmental assessments are limited in scope. In response, an increasing range of actors is utilising remote sensing and open source data collection to identify and estimate health and ecological risks during and after conflicts. This paper considers the role of participatory citizen science methodologies in complementing both remote monitoring and post-conflict assessments. It examines existing models and mechanisms for environmental data collection and utilisation in conflict contexts, and the extent to which the core values and principles of citizen science are transferable. We find that 'civilian science' is feasible and could be well-suited to conflict conditions. In addition to addressing gaps in data collection, it may also empower communities affected by environmental degradation, enhance their environmental human rights, supplement the often limited monitoring capacity of governmental agencies and facilitate cooperation and peacebuilding. The paper concludes by proposing methodological approaches for three common forms of environmental degradation associated with armed conflicts.

The frequency of mucosal-associated invariant T cells is selectively increased in dermatitis herpetiformis.

BACKGROUND/OBJECTIVES: Mucosal-associated invariant T (MAIT) cells are a novel subset of innate-like T-cells that are enriched in mucosal tissues. Their presence in human skin has only recently been recognised. We describe the expression of skin-tropic molecules on human skin MAIT cells at steady state and investigate their contribution to various dermatoses with known T-cell involvement. METHODS: To examine the expression of skin-tropic molecules by MAIT cells at steady state, we performed a flow cytometric analysis of blood and skin samples from healthy donors. To investigate any potential wider contribution of MAIT cells to skin disease, we examined psoriasis, alopecia areata and dermatitis herpetiformis biopsies using immunofluorescent staining to identify the proportion of T-cells expressing MAIT cell surface markers. RESULTS: We found that MAIT cells constituted a small population of T-cells in normal human skin, similar to the percentage found in peripheral blood. Like other skin T-cells, skin MAIT cells expressed high levels of the skin-associated markers, cutaneous lymphocyte antigen and CD103. In psoriasis and alopecia areata the proportion of MAIT cells was similar to that found in normal skin, but in dermatitis herpetiformis it was significantly elevated. CONCLUSIONS: The expression of skin-tropic molecules by skin MAIT cells is consistent with their resident status in normal human skin. Our results suggest that MAIT cells may play a role in the pathogenesis of dermatitis herpetiformis.

Ca²âº and calpain control membrane expansion during the rapid cell spreading of neutrophils.

Following adherence of neutrophils to the endothelium, neutrophils undergo a major morphological change that is a necessary prelude to their extravasation. We show here that this shape change is triggered by an elevation of cytosolic inositol (1,4,5)-trisphosphate (IP3), to provoke physiological Ca(2+) influx through a store-operated mechanism. This transition from a spherical to 'flattened' neutrophil morphology is rapid (∼100 seconds) and is accompanied by an apparent rapid expansion of the area of the plasma membrane. However, no new membrane is added into the plasma membrane. Pharmacological inhibition of calpain-activation, which is triggered by Ca(2+) influx during neutrophil spreading, prevents normal cell flattening. In calpain-suppressed cells, an aberrant form of cell spreading can occur where an uncoordinated and localised expansion of the plasma membrane is evident. These data show that rapid neutrophil spreading is triggered by Ca(2+) influx, which causes activation of calpain and release of furled plasma membrane to allow its apparent 'expansion'.

Quality control of oblique incidence optical coatings based on normal incidence measurement data.

We demonstrate selection of reliable approaches for post-production characterization of oblique incidence multilayer optical coatings. The approaches include choice of input information, selection of adequate coating model, corresponding numerical characterization algorithm, and verification of the results. Applications of the approaches are illustrated with post-production characterization of oblique incidence edge filter, oblique incidence beam splitter and oblique incidence 43-layer quarter-wave mirror.

Optical characterization and reverse engineering based on multiangle spectroscopy.

We perform characterization of thin films and reverse engineering of multilayer coatings on the basis of multiangle spectral photometric data provided by a new advanced spectrophotometer accessory. Experimental samples of single thin films and multilayer coatings are produced by magnetron sputtering and electron-beam evaporation. Reflectance and transmittance data at two polarization states are measured at incidence angles from 7 to 40 deg. We demonstrate that multiangle reflectance and transmittance data provide reliable characterization and reverse-engineering results.

Systematic Mapping and Review of Landscape Fire Smoke (LFS) Exposure Impacts on Insects.

Landscape fire activity is changing in many regions because of climate change. Smoke emissions from landscape fires contain many harmful air pollutants, and beyond the potential hazard posed to human health, these also have ecological impacts. Insects play essential roles in most ecosystems worldwide, and some work suggests they may also be sensitive to smoke exposure. There is therefore a need for a comprehensive review of smoke impacts on insects. We systematically reviewed the scientific literature from 1930 to 2022 to synthesize the current state of knowledge of the impacts of smoke exposure from landscape fires on the development, behavior, and mortality of insects. We found: (1) 42 relevant studies that met our criteria, with 29% focused on the United States of America and 19% on Canada; (2) of these, 40 insect species were discussed, all of which were sensitive to smoke pollution; (3) most of the existing research focuses on how insect behavior responds to landscape fire smoke (LFS); (4) species react differently to smoke exposure, with for example some species being attracted to the smoke (e.g., some beetles) while others are repelled (e.g., some bees). This review consolidates the current state of knowledge on how smoke impacts insects and highlights areas that may need further investigation. This is particularly relevant since smoke impacts on insect communities will likely worsen in some areas due to increasing levels of biomass burning resulting from the joint pressures of climate change, land use change, and more intense land management involving fire.

Developing whole cell standards for the microbiome field.

BACKGROUND: Effective standardisation of the microbiome field is essential to facilitate global translational research and increase the reproducibility of microbiome studies. In this study, we describe the development and validation of a whole cell reference reagent specific to the gut microbiome by the UK National Institute for Biological Standards and Control. We also provide and test a two-step reporting framework to allow microbiome researchers to quickly and accurately validate choices of DNA extraction, sequencing, and bioinformatic pipelines. RESULTS: Using 20 strains that are commonly found in the gut, we developed a whole cell reference reagent (WC-Gut RR) for the evaluation of the DNA extraction protocols commonly used in microbiome pipelines. DNA was first analysed using the physicochemical measures of yield, integrity, and purity, which demonstrated kits widely differed in the quality of the DNA they produced. Importantly, the combination of the WC-Gut RR and the three physicochemical measures allowed us to differentiate clearly between kit performance. We next assessed the ability of WC-Gut RR to evaluate kit performance in the reconstitution of accurate taxonomic profiles. We applied a four-measure framework consisting of Sensitivity, false-positive relative abundance (FPRA), Diversity, and Similarity as previously described for DNA reagents. Using the WC-Gut RR and these four measures, we could reliably identify the DNA extraction kits' biases when using with both 16S rRNA sequencing and shotgun sequencing. Moreover, when combining this with complementary DNA standards, we could estimate the relative bias contributions of DNA extraction kits vs bioinformatic analysis. Finally, we assessed WC-Gut RR alongside other commercially available reagents. The analysis here clearly demonstrates that reagents of lower complexity, not composed of anaerobic and hard-to-lyse strains from the gut, can artificially inflate the performance of microbiome DNA extraction kits and bioinformatic pipelines. CONCLUSIONS: We produced a complex whole cell reagent that is specific for the gut microbiome and can be used to evaluate and benchmark DNA extractions in microbiome studies. Used alongside a DNA standard, the NIBSC DNA-Gut-Mix RR helps estimating where biases occur in microbiome pipelines. In the future, we aim to establish minimum thresholds for data quality through an interlaboratory collaborative study. Video Abstract.

Prevalence of disordered eating, eating disorders and risk of low energy availability in professional, competitive and recreational female athletes based in the United Kingdom.

Eating disorders (ED), disordered eating (DE) and low energy availability (LEA) can be detrimental to health and performance. Previous studies have independently investigated the prevalence of ED, DE or LEA; however, few combined methods have identified risk within female athletes. The aim of this study was to identify the prevalence of ED, DE and LEA in UK-based female athletes and investigate whether associations exist between age, competition level and primary sport. The Female Athlete Screening Tool (FAST) and Low Energy Availability in Females Questionnaire (LEAF-Q) were used in a cross-sectional study design. A total of 112 responses eligible for analysis were received. A total of 16%, 44% and 53% of female athletes were at risk of ED (FAST: >94), DE and LEA, respectively. Competition level (recreational, competitive or professional athletes; fishers, p ≤ 0.05) influenced and was a predictor of FAST (R2 = 0.076, F(1,110) = 10.067, p ≤ 0.05, variance inflation value; VIF = 1.0) whereas age influenced (age: H(2) = 13.128, p ≤ 0.05), and was a predictor (R2 = 0.144, F(2,109) = 9.170, p ≤ 0.05, VIF = 1.0) of LEAF-Q. A positive correlation was observed between FAST and LEAF-Q scores (R = 0.496, p ≤ 0.05). Age and competition level may be predicting risk factors of ED/DE and LEA within female athletes; however, further research is required to support the findings of this present study.

Intrinsic host susceptibility among multiple species to intranasal SARS-CoV-2 identifies diverse virological, biodistribution and pathological outcomes.

SARS-CoV-2 exhibits a diverse host species range with variable outcomes, enabling differential host susceptibility studies to assess suitability for pre-clinical countermeasure and pathogenesis studies. Baseline virological, molecular and pathological outcomes were determined among multiple species-one Old World non-human primate (NHP) species (cynomolgus macaques), two New World NHP species (red-bellied tamarins; common marmosets) and Syrian hamsters-following single-dose, atraumatic intranasal administration of SARS-CoV-2/Victoria-01. After serial sacrifice 2, 10 and 28-days post-infection (dpi), hamsters and cynomolgus macaques displayed differential virus biodistribution across respiratory, gastrointestinal and cardiovascular systems. Uniquely, New World tamarins, unlike marmosets, exhibited high levels of acute upper airway infection, infectious virus recovery associated with mild lung pathology representing a host previously unrecognized as susceptible to SARS-CoV-2. Across all species, lung pathology was identified post-clearance of virus shedding (antigen/RNA), with an association of virus particles within replication organelles in lung sections analysed by electron microscopy. Disrupted cell ultrastructure and lung architecture, including abnormal morphology of mitochondria 10-28 dpi, represented on-going pathophysiological consequences of SARS-CoV-2 in predominantly asymptomatic hosts. Infection kinetics and host pathology comparators using standardized methodologies enables model selection to bridge differential outcomes within upper and lower respiratory tracts and elucidate longer-term consequences of asymptomatic SARS-CoV-2 infection.

Active DLP hyperspectral illumination: a noninvasive, in vivo, system characterization visualizing tissue oxygenation at near video rates.

We report use of a novel hyperspectral imaging system utilizing digital light processing (DLP) technology to noninvasively visualize in vivo tissue oxygenation during surgical procedures. The system's novelty resides in its method of illuminating tissue with precisely predetermined continuous complex spectra. The Texas Instruments digital micromirror device, DMD, chip consisting of 768 by 1024 mirrors, each 16 µm square, can be switched between two positions at 12.5 kHz. Switching the appropriate mirrors controls the intensity of light illuminating the tissue as a function of wavelength, active spectral illumination. Meaning, the tissue can be illuminated with a different spectrum of light within 80 µs. Precisely, predetermined spectral illumination penetrates into patient tissue, its chemical composition augments the spectral properties of the light, and its reflected spectra are detected and digitized at each pixel detector of a silicon charge-coupled device, CCD. Using complex spectral illumination, digital signal processing and chemometric methods produce chemically relevant images at near video rates. Specific to this work, tissue is illuminated spectrally with light spanning the visible electromagnetic spectrum (380 to 780 nm). Spectrophotometric images are detected and processed visualizing the percentage of oxyhemoglobin at each pixel detector and presented continuously, in real time, at 3 images per second. As a proof of principle application, kidneys of four live anesthetized pigs were imaged before, during, and after renal vascular occlusion. DLP Hyperspectral Imaging with active spectral illumination detected a 64.73 ± 1.5% drop in the oxygenation of hemoglobin within 30 s of renal arterial occlusion. Producing chemically encoded images at near video rate, time-resolved hyperspectral imaging facilitates monitoring renal blood flow during animal surgery and holds considerable promise for doing the same during human surgical interventions.

Urban reconciliation ecology: the potential of living roofs and walls.

Reconciling human and non-human use of urban regions to support biological conservation represents a major challenge for the 21st century. The concept of reconciliation ecology, by which the anthropogenic environment may be modified to encourage non-human use and biodiversity preservation without compromising societal utilization, potentially represents an appropriate paradigm for urban conservation given the generally poor opportunities that exist for reserve establishment and ecological restoration in urban areas. Two habitat improvement techniques with great potential for reconciliation ecology in urban areas are the installation of living roofs and walls, which have been shown to support a range of taxa at local scales. This paper evaluates the reconciliation potential of living roofs and walls, in particular highlighting both ecological and societal limitations that need to be overcome for application at the landscape scale. We further consider that successful utilization of living roofs and walls for urban reconciliation ecology will rely heavily on the participation of urban citizens, and that a 'citizen science' model is needed to facilitate public participation and support and to create an evidence base to determine their effectiveness. Living roofs and walls are just one aspect of urban reconciliation ecology, but are particularly important 'bottom-up' techniques for improving urban biodiversity that can be performed directly by the citizenry.

The impacts of modern warfare on freshwater ecosystems.

There is increasing recognition and concern regarding the impacts of modern industrial warfare on the environment. Freshwater ecosystems are perhaps the most vulnerable to warfare-related impacts, which is of concern given that they provide so many essential environmental resources and services to society. Despite this, there has been little work to establish and quantify the types of impacts (both negative and positive) that warfare may have on such systems. This paper firstly highlights why rivers and lakes may be susceptible to warfare-related impacts, before synthesizing the available literature to explore the following main themes: intensification of wartime resource acquisition, use of water as an offensive or defensive weapon, direct and indirect effects of explosive ordnance, increased pollution, introduction of invasive alien species, and positive ecological impacts. This is then followed by a discussion of the implications of such impacts in relation to future warfare, including a consideration of the efficacy of existing legal instruments to protect the environment during conflict, and the trend for war to become more localized and 'informal', and therefore less regulated. Finally, the paper identifies key research foci for understanding and mitigating the effects of warfare on freshwater ecosystems.

Fibrinogen protects neutrophils from the cytotoxic effects of histones and delays neutrophil extracellular trap formation induced by ionomycin.

Neutrophils are pivotal players in immune defence which includes a process of release of histones and DNA as neutrophil extracellular traps (NETs). Histones, while toxic to invading pathogens, also kill host cells, including neutrophils. Bacteria have evolved mechanisms to escape neutrophils, including the secretion of leucocidins (e.g. ionomycin). Live cell video microscopy showed how fibrinogen and fibrin influence NETosis and neutrophil responses to extracellular histones. Histones were rapidly lethal to neutrophils after binding to cells, but formation of fibrinogen/fibrin-histone aggregates prevented cell death. Histone cytotoxicity was also reduced by citrullination by peptidyl arginine deiminase 4, or digestion by serine proteases. Ionomycin and phorbol 12-myristate 13 acetate (PMA) are used to trigger NETosis. Fibrinogen was responsible for a second distinct mechanism of neutrophil protection after treatment with ionomycin. Fibrinogen clustered on the surface of ionomycin-stimulated neutrophils to delay NETosis; and blocking the ß integrin receptor, αMß2, abolished fibrinogen protection. Fibrinogen did not bind to or protect neutrophils stimulated with PMA. Fibrinogen is an acute phase protein that will protect exposed cells from damaging circulating histones or leucocidins; but fibrinogen depletion/consumption, as in trauma or sepsis will reduce protection. It is necessary to consider the role of fibrinogen in NETosis.

Human iPSC-Derived Neural Crest Stem Cells Exhibit Low Immunogenicity.

Recent clinical trials are evaluating induced pluripotent stem cells (iPSCs) as a cellular therapy in the field of regenerative medicine. The widespread clinical utility of iPSCs is expected to be realized using allogeneic cells that have undergone thorough safety evaluations, including assessment of their immunogenicity. IPSC-derived neural crest stem cells (NCSCs) have significant potential in regenerative medicine; however, their application in cellular therapy has not been widely studied to date, and no reports on their potential immunogenicity have been published so far. In this study, we have assessed the expression of immune-related antigens in iPSC-NCSCs, including human leukocyte antigen (HLA) class I and II and co-stimulatory molecules. To investigate functional immunogenicity, we used iPSC-NCSCs as stimulator cells in a one-way mixed lymphocyte reaction. In these experiments, iPSC-NCSCs did not stimulate detectable proliferation of CD3+ and CD3+CD8+ T cells or induce cytokine production. We show that this was not a result of any immunosuppressive features of iPSC-NCSCs, but rather more consistent with their non-immunogenic molecular phenotype. These results are encouraging for the potential future use of iPSC-NCSCs as a cellular therapy.

Higher mass meningococcal group C-tetanus toxoid vaccines conjugated with carbodiimide correlate with greater immunogenicity.

To examine the link between meningococcal C (MenC) vaccine size and immunogenic response, a panel of MenC glycoconjugate vaccines were prepared differing in chain length, molar mass and hydrodynamic volume. The preparations consisted of different lengths of MenC polysaccharide (PS) covalently linked to monomeric purified tetanus toxoid (TT) carrier protein using the coupling reagent ethylcarbodiimide hydrochloride (EDC). Size exclusion chromatography with multi-angle light scattering (SEC-MALS) and viscometry analysis confirmed that the panel of MenC-TT conjugates spanned masses of 191,500 to 2,348,000 g/mol, and hydrodynamic radii ranging from 12.1 to 47.9 nm. The two largest conjugates were elliptical in shape, whereas the two smallest conjugates were more spherical. The larger conjugates appeared to fit a model described by multiple TTs with cross-linked PS, typical of lattice-like networks described previously for TT conjugates, while the smaller conjugates were found to fit a monomeric or dimeric TT configuration. The effect of vaccine conjugate size on immune responses was determined using a two-dose murine immunization. The two larger panel vaccine conjugates produced higher anti-MenC IgG1 and IgG2b titres after the second dose. Larger vaccine conjugate size also stimulated greater T-cell proliferative responses in an in vitro recall assay, although cytokines indicative of a T-helper response were not measurable. In conclusion, larger MenC-TT conjugates up to 2,348,000 g/mol produced by EDC chemistry correlate with greater humoral and cellular murine immune responses. These observations suggest that conjugate size can be an important modulator of immune response.

Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining.

Tuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.

Evaluating natural infrastructure for flood management within the watersheds of selected global cities.

Cities are dependent on their upstream watersheds for storage and gradual release of water into river systems. These watersheds act as important flood mitigation infrastructure, providing an essential ecosystem service. In this paper we use metrics from the WaterWorld model to examine the flood management-relevant natural infrastructure of the upstream watersheds of selected global cities. These metrics enable the characterisation of different types, magnitudes and geographical distributions of potential natural flood storage. The storages are categorised as either green (forest canopy, wetland and soil) or blue (water body and floodplain) storages and the proportion of green to blue indicates how different city upstream basin contexts provide different types and levels of storage which may buffer flood risk. We apply the WaterWorld method for examining flood risk as the ratio of accumulated modelled annual runoff volume to accumulated available green and blue water storage capacity. The aim of these metrics is to highlight areas where there is more runoff than storage capacity and thus where the maintenance or restoration of further natural infrastructure (such as canopy cover, wetlands and soil) could aid in storing more water and thus better alleviate flood risks. Such information is needed by urban planners, city authorities and governments to help prepare cities for climate change impacts.

Taking Care of Mine: Can Child Support Become a Family-Building Institution?

U.S. children are more likely to live apart from a biological parent than at any time in history. Although the Child Support Enforcement system has tremendous reach, its policies have not kept pace with significant economic, demographic, and cultural changes. Narrative analysis of in-depth interviews with 429 low-income noncustodial fathers suggests that the system faces a crisis of legitimacy. Visualization of language used to describe all forms child support show that the formal system is considered punitive and to lead to a loss of power and autonomy. Further, it is not associated with coparenting or the father-child bond-themes closely associated with informal and in-kind support. Rather than stoking men's identities as providers, the system becomes "just another bill to pay." Orders must be sustainable, all fathers should have coparenting agreements, and alternative forms of support should count toward fathers' obligations. Recovery of government welfare costs should be eliminated.