Random mutagenesis of Phaeodactylum tricornutum using ultraviolet, chemical, and X-radiation demonstrates the need for temporal analysis of phenotype stability.

We investigated two non-ionising mutagens in the form of ultraviolet radiation (UV) and ethyl methanosulfonate (EMS) and an ionising mutagen (X-ray) as methods to increase fucoxanthin content in the model diatom Phaeodactylum tricornutum. We implemented an ultra-high throughput method using fluorescence-activated cell sorting (FACS) and live culture spectral deconvolution for isolation and screening of potential pigment mutants, and assessed phenotype stability by measuring pigment content over 6 months using high-performance liquid chromatography (HPLC) to investigate the viability of long-term mutants. Both UV and EMS resulted in significantly higher fucoxanthin within the 6 month period after treatment, likely as a result of phenotype instability. A maximum fucoxanthin content of 135 ± 10% wild-type found in the EMS strain, a 35% increase. We found mutants generated using all methods underwent reversion to the wild-type phenotype within a 6 month time period. X-ray treatments produced a consistently unstable phenotype even at the maximum treatment of 1000 Grays, while a UV mutant and an EMS mutant reverted to wild-type after 4 months and 6 months, respectively, despite showing previously higher fucoxanthin than wild-type. This work provides new insights into key areas of microalgal biotechnology, by (i) demonstrating the use of an ionising mutagen (X-ray) on a biotechnologically relevant microalga, and by (ii) introducing temporal analysis of mutants which has substantial implications for strain creation and utility for industrial applications.

Concentration and Distribution of Toxic and Essential Elements in Traditional Rice Varieties of Sri Lanka Grown on an Anuradhapura District Farm.

Toxic heavy metals have been the focus of many investigations into chronic kidney disease of unknown aetiology (CKDu) within Sri Lanka. It has been hypothesised that exposure to nephrotoxic arsenic, cadmium and lead could play a role in the development of CKDu, and these metals have previously been found in unsafe concentrations in Sri Lankan rice. Traditional varieties of Sri Lankan rice remain popular due to their perceived health benefits, but their uptake of trace and toxic heavy metals remained unexplored. Here, we report a one-time, cross-sectional dataset on the concentrations of essential and toxic elements present in eleven samples of polished and unpolished traditional rice varieties, all regularly grown and sold in the Anuradhapura district, a CKDu hotspot. All rice was sourced from the same farm, with the exception of one store bought sample grown on another, unidentified farm. Cadmium concentrations varied significantly between varieties, and potentially unsafe concentrations of cadmium were detected in the store-bought sample (Suwadel, 113±13 µg kg-1). Elemental imaging of the grains revealed lead to be stored mainly in the rice bran, which is removed during polishing, while cadmium was distributed in the edible portion of the grain. Essential elements were generally higher in the traditional rice varieties than those reported for non-traditional varieties and are a potential source of trace elements for nutrient-deficient communities. The concentration of selenium, an element that plays a protective role in the kidneys, was too low to provide the minimum recommended intake. The methods developed in this study could be applied to a more comprehensive study of elemental uptake of rice under controlled growing conditions.

Developing Culturally Appropriate Content for a Child-Rearing App to Support Young Children's Socioemotional and Cognitive Development in Afghanistan: Co-Design Study.

BACKGROUND: Optimal child-rearing practices can help mitigate the consequences of detrimental social determinants of health in early childhood. Given the ubiquity of personal digital technologies worldwide, the direct delivery of evidence-based information about early childhood development holds great promise. However, to make the content of these novel systems effective, it is crucial to incorporate place-based cultural beliefs, traditions, circumstances, and value systems of end users. OBJECTIVE: This paper describes the iterative approach used to develop the Thrive by Five child-rearing app in collaboration with Afghan parents, caregivers (eg, grandparents, aunts, and nannies), and subject matter experts (SMEs). We outline how co-design methodologies informed the development and cultural contextualization of content to meet the specific needs of Afghan parents and the content was tested and refined in collaboration with key Afghan stakeholders. METHODS: The preliminary content was developed based on a comprehensive literature review of the historical and sociocultural contexts in Afghanistan, including factors that influence child-rearing practices and early childhood development. After an initial review and refinement based on feedback from SMEs, this content was populated into a beta app for testing. Overall, 8 co-design workshops were conducted in July and August 2021 and February 2022 with 39 Afghan parents and caregivers and 6 SMEs to collect their feedback on the app and its content. The workshops were audio recorded and transcribed; detailed field notes were taken by 2 scribes. A theoretical thematic analysis using semantic codes was conducted to inform the refinement of existing content and development of new content to fulfill the needs identified by participants. RESULTS: The following 4 primary themes were identified: child-rearing in the Afghan sociocultural context, safety concerns, emotion and behavior management, and physical health and nutrition. Overall, participants agreed that the app had the potential to deliver valuable information to Afghan parents; however, owing to the volatility in the country, participants recommended including more activities that could be safely done indoors, as mothers and children are required to spend most of their time at home. Additionally, restrictions on public engagement in music required the removal of activities referencing singing that might be performed outside the home. Further, activities to help parents reduce their children's screen time, promote empathy, manage emotions, regulate behavior, and improve physical health and nutrition were requested. CONCLUSIONS: Direct engagement with Afghan parents, caregivers, and SMEs through co-design workshops enabled the development and refinement of evidence-based, localized, and contextually relevant child-rearing activities promoting healthy social, emotional, and cognitive development during the first 5 years of children's lives. Importantly, the content was adapted for the ongoing conflict in Afghanistan with the aim of empowering Afghan parents and caregivers to support their children's developmental potential despite the security concerns and situational stressors.

Preparation of matrix-matched standards for the analysis of teeth via laser ablation-inductively coupled plasma-mass spectrometry.

Mineralised tissue such as teeth can serve as a retrospective, chronological bioindicator of past exposure to toxic metals. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) can be used to determine the presence and spatial distribution of toxic metals in teeth, giving a record of when an exposure occurred. Concentrations of these metals are often determined by a one-point calibration against NIST glass using an equation that requires an internal standard factor that accounts for differences in ablation behaviour between the glass and the tooth. However, an ideal external calibration would contain multiple matrix-matched standards to obtain a calibration curve. Here, we investigated optimal procedures for preparing synthetic hydroxyapatite (HA) doped with elements of interest as a calibration material. The materials were examined for homogeneity of metal incorporation, matrix-matched ablation characteristics, linearity, and limits of detection. A homogenised and pelleted HA was the most suitable material, providing improved ablation characteristics over previous HA materials and NIST glass for the analysis of teeth. An ablation yield of 1.1 showed its suitability to analyse teeth, the metals were homogeneously incorporated, and it produced excellent linearity with limits of detection ranging from 0.1-2 µg kg-1 for magnesium, aluminium, nickel, copper, zinc, cadmium, barium and lead. A juvenile incisor from a remote indigenous community in Australia and an adult molar from Sri Lanka were assessed for toxic metal exposure. The molar showed evidence of exposure to cadmium and lead. The synthetic HA material was straightforward to prepare, and will improve confidence in the analysis of teeth and other biomineralised material when assessing toxic metal exposure.

Designing an App for Parents and Caregivers to Promote Cognitive and Socioemotional Development and Well-being Among Children Aged 0 to 5 Years in Diverse Cultural Settings: Scientific Framework.

Recent years have seen remarkable progress in our scientific understanding of early childhood social, emotional, and cognitive development, as well as our capacity to widely disseminate health information by using digital technologies. Together, these scientific and technological advances offer exciting opportunities to deliver high-quality information about early childhood development (ECD) to parents and families globally, which may ultimately lead to greater knowledge and confidence among parents and better outcomes among children (particularly in lower- and middle-income countries). With these potential benefits in mind, we set out to design, develop, implement, and evaluate a new parenting app-Thrive by Five-that will be available in 30 countries. The app will provide caregivers and families with evidence-based and culturally appropriate information about ECD, accompanied by sets of collective actions that go beyond mere tips for parenting practices. Herein, we describe this ongoing global project and discuss the components of our scientific framework for developing and prototyping the app's content. Specifically, we describe (1) 5 domains that are used to organize the content and goals of the app's information and associated practices; (2) 5 neurobiological systems that are relevant to ECD and can be behaviorally targeted to potentially influence social, emotional, and cognitive development; (3) our anthropological and cultural framework for learning about local contexts and appreciating decolonization perspectives; and (4) our approach to tailoring the app's content to local contexts, which involves collaboration with in-country partner organizations and local and international subject matter experts in ECD, education, medicine, psychology, and anthropology, among others. Finally, we provide examples of the content that was incorporated in Thrive by Five when it launched globally.

The Comprehensive Steroidome in Complete TSPO/PBR Knockout Mice under Basal Conditions.

The 18 kDa translocator protein (TSPO/PBR) is a multifunctional evolutionary highly conserved outer mitochondrial membrane protein. Decades of research has reported an obligatory role of TSPO/PBR in both mitochondrial cholesterol transport and, thus, steroid production. However, the strict dependency of steroidogenesis on TSPO/PBR has remained controversial. The aim of this study was to provide insight into the steroid profile in complete C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout male mice (TSPO-KO) under basal conditions. The steroidome in the brain, adrenal glands, testes and plasma was measured by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). We found that steroids present in wild-type (WT) mice were also detected in TSPO-KO mice, including pregnenolone (PREG), progestogens, mineralo-glucocorticosteroids and androgens. The concentrations of PREG and most metabolites were similar between genotypes, except a significant decrease in the levels of the 5α-reduced metabolites of progesterone (PROG) in adrenal glands and plasma and of the 5α-reduced metabolites of corticosterone (B) in plasma in TSPO-KO compared to WT animals, suggesting other regulatory functions for the TSPO/PBR. The expression levels of the voltage-dependent anion-selective channel (VDAC-1), CYP11A1 and 5α-reductase were not significantly different between both groups. Thus, the complete deletion of the tspo gene in male mice does not impair de novo steroidogenesis in vivo.

Long-term diazepam treatment enhances microglial spine engulfment and impairs cognitive performance via the mitochondrial 18 kDa translocator protein (TSPO).

Benzodiazepines are widely administered drugs to treat anxiety and insomnia. In addition to tolerance development and abuse liability, their chronic use may cause cognitive impairment and increase the risk for dementia. However, the mechanism by which benzodiazepines might contribute to persistent cognitive decline remains unknown. Here we report that diazepam, a widely prescribed benzodiazepine, impairs the structural plasticity of dendritic spines, causing cognitive impairment in mice. Diazepam induces these deficits via the mitochondrial 18 kDa translocator protein (TSPO), rather than classical γ-aminobutyric acid type A receptors, which alters microglial morphology, and phagocytosis of synaptic material. Collectively, our findings demonstrate a mechanism by which TSPO ligands alter synaptic plasticity and, as a consequence, cause cognitive impairment.

Long term food stability for extended space missions: a review.

At present, human spaceflight is confined to low Earth orbit but, in future, will again go to the Moon and, beyond, to Mars. The provision of food during these extended missions will need to meet the special nutritional and psychosocial needs of the crew. Terrestrially grown and processed food products, currently provided for consumption by astronauts/cosmonauts, have not yet been systematically optimised to maintain their nutritional integrity and reach the shelf-life necessary for extended space voyages. Notably, space food provisions for Mars exploration will be subject to extended exposure to galactic cosmic radiation and solar particle events, the impact of which is not fully understood. In this review, we provide a summary of the existing knowledge about current space food products, the impact of radiation and storage on food composition, the identification of radiolytic biomarkers and identify gaps in our knowledge that are specific in relation to the effect of the cosmic radiation on food in space.

The effect of thermal pasteurization, freeze-drying, and gamma irradiation on donor human milk.

The availability of donor human milk (DHM) is currently limited by the volumes that can be thermally pasteurized and kept in long-term cold storage. This study assesses the application of freeze-drying followed by low-dose gamma irradiation of DHM for simplified, safe long-term storage. Solid-phase microextraction (SPME) GC-MS, SDS and native PAGE gel electrophoresis demonstrated that the overall changes in volatile and protein profiles in Holder pasteurized and freeze-dried DHM was negligible compared to the natural variations in DHM. Freeze-dried DHM samples (moisture < 2.2 %) processed with 2 kGy gamma irradiation did not show any significant lipid oxidation end-products and variation in protein profile. Therefore, freeze-drying followed by in-packaging gamma irradiation could be a safe method for pasteurization, convenient storage and delivery of DHM at ambient temperature. These methods may generate a means to create a reserve stock of DHM for emergencies and humanitarian aid.

Mitochondrial Translocator Protein (TSPO) Expression in the Brain After Whole Body Gamma Irradiation.

The brain's early response to low dose ionizing radiation, as may be encountered during diagnostic procedures and space exploration, is not yet fully characterized. In the brain parenchyma, the mitochondrial translocator protein (TSPO) is constitutively expressed at low levels by endothelial cells, and can therefore be used to assess the integrity of the brain's vasculature. At the same time, the inducible expression of TSPO in activated microglia, the brain's intrinsic immune cells, is a regularly observed early indicator of subtle or incipient brain pathology. Here, we explored the use of TSPO as a biomarker of brain tissue injury following whole body irradiation. Post-radiation responses were measured in C57BL/6 wild type (Tspo +/+) and TSPO knockout (Tspo -/-) mice 48 h after single whole body gamma irradiations with low doses 0, 0.01, and 0.1 Gy and a high dose of 2 Gy. Additionally, post-radiation responses of primary microglial cell cultures were measured at 1, 4, 24, and 48 h at an irradiation dose range of 0 Gy-2 Gy. TSPO mRNA and protein expression in the brain showed a decreased trend after 0.01 Gy relative to sham-irradiated controls, but remained unchanged after higher doses. Immunohistochemistry confirmed subtle decreases in TSPO expression after 0.01 Gy in vascular endothelial cells of the hippocampal region and in ependymal cells, with no detectable changes following higher doses. Cytokine concentrations in plasma after whole body irradiation showed differential changes in IL-6 and IL-10 with some variations between Tspo-/- and Tspo +/+ animals. The in vitro measurements of TSPO in primary microglial cell cultures showed a significant reduction 1 h after low dose irradiation (0.01 Gy). In summary, acute low and high doses of gamma irradiation up to 2 Gy reduced TSPO expression in the brain's vascular compartment without de novo induction of TSPO expression in parenchymal microglia, while TSPO expression in directly irradiated, isolated, and thus highly activated microglia, too, was reduced after low dose irradiation. The potential link between TSPO, its role in mitochondrial energy metabolism and the selective radiation sensitivity, notably of cells with constitutive TSPO expression such as vascular endothelial cells, merits further exploration.

Control of Neuroinflammation through Radiation-Induced Microglial Changes.

Microglia, the innate immune cells of the central nervous system, play a pivotal role in the modulation of neuroinflammation. Neuroinflammation has been implicated in many diseases of the CNS, including Alzheimer's disease and Parkinson's disease. It is well documented that microglial activation, initiated by a variety of stressors, can trigger a potentially destructive neuroinflammatory response via the release of pro-inflammatory molecules, and reactive oxygen and nitrogen species. However, the potential anti-inflammatory and neuroprotective effects that microglia are also thought to exhibit have been under-investigated. The application of ionising radiation at different doses and dose schedules may reveal novel methods for the control of microglial response to stressors, potentially highlighting avenues for treatment of neuroinflammation associated CNS disorders, such as Alzheimer's disease and Parkinson's disease. There remains a need to characterise the response of microglia to radiation, particularly low dose ionising radiation.

The Effects of Thermal Pasteurisation, Freeze-Drying, and Gamma-Irradiation on the Antibacterial Properties of Donor Human Milk.

The most common pasteurisation method used by human milk banks is Holder pasteurisation. This involves thermal processing, which can denature important proteins and can potentially reduce the natural antimicrobial properties found in human milk. This study assesses the application of a hybrid method comprised of freeze-drying followed by low-dose gamma-irradiation for nonthermal donor human milk pasteurisation. Freeze-drying donor human milk followed by gamma-irradiation at 2 kGy was as efficient as Holder pasteurisation in the reduction of bacterial inoculants of Staphylococcus aureus (106 cfu/mL) and Salmonella typhimurium (106 cfu/mL) in growth inhibition assays. These assays also demonstrated that human milk naturally inhibits the growth of bacterial inoculants S. aureus, S. typhimurium, and Escherichia coli. Freeze drying (without gamma-irradiation) did not significantly reduce this natural growth inhibition. By contrast, Holder pasteurisation significantly reduced the milk's natural antimicrobial effect on S. aureus growth after 6 h (-19.8% p = 0.01). Freeze-dried and then gamma-irradiated donor human milk showed a strong antimicrobial effect across a dose range of 2-50 kGy, with only a minimal growth of S. aureus observed after 6 h incubation. Thus, a hybrid method of freeze-drying followed by 2 kGy of gamma-irradiation preserves antimicrobial properties and enables bulk pasteurisation within sealed packaging of powderised donor human milk. This work forwards a goal of increasing shelf life and simplifying storage and transportation, while also preserving functionality and antimicrobial properties.

Ground state depletion microscopy as a tool for studying microglia-synapse interactions.

Ground state depletion followed by individual molecule return microscopy (GSDIM) has been used in the past to study the nanoscale distribution of protein co-localization in living cells. We now demonstrate the successful application of GSDIM to archival human brain tissue sections including from Alzheimer's disease cases as well as experimental tissue samples from mouse and zebrafish larvae. Presynaptic terminals and microglia and their cell processes were visualized at a resolution beyond diffraction-limited light microscopy, allowing clearer insights into their interactions in situ. The procedure described here offers time and cost savings compared to electron microscopy and opens the spectrum of molecular imaging using antibodies and super-resolution microscopy to the analysis of routine formalin-fixed paraffin sections of archival human brain. The investigation of microglia-synapse interactions in dementia will be of special interest in this context.

PathoFusion: An Open-Source AI Framework for Recognition of Pathomorphological Features and Mapping of Immunohistochemical Data.

We have developed a platform, termed PathoFusion, which is an integrated system for marking, training, and recognition of pathological features in whole-slide tissue sections. The platform uses a bifocal convolutional neural network (BCNN) which is designed to simultaneously capture both index and contextual feature information from shorter and longer image tiles, respectively. This is analogous to how a microscopist in pathology works, identifying a cancerous morphological feature in the tissue context using first a narrow and then a wider focus, hence bifocal. Adjacent tissue sections obtained from glioblastoma cases were processed for hematoxylin and eosin (H&E) and immunohistochemical (CD276) staining. Image tiles cropped from the digitized images based on markings made by a consultant neuropathologist were used to train the BCNN. PathoFusion demonstrated its ability to recognize malignant neuropathological features autonomously and map immunohistochemical data simultaneously. Our experiments show that PathoFusion achieved areas under the curve (AUCs) of 0.985 ± 0.011 and 0.988 ± 0.001 in patch-level recognition of six typical pathomorphological features and detection of associated immunoreactivity, respectively. On this basis, the system further correlated CD276 immunoreactivity to abnormal tumor vasculature. Corresponding feature distributions and overlaps were visualized by heatmaps, permitting high-resolution qualitative as well as quantitative morphological analyses for entire histological slides. Recognition of more user-defined pathomorphological features can be added to the system and included in future tissue analyses. Integration of PathoFusion with the day-to-day service workflow of a (neuro)pathology department is a goal. The software code for PathoFusion is made publicly available.

The risk of infectious pathogens in breast-feeding, donated human milk and breast milk substitutes.

OBJECTIVE: This review collates the published reports that focus on microbial and viral illnesses that can be transmitted by breast milk, donor milk and powdered infant formula (PIF). In this context, we attempt to define a risk framework encompassing those hazards, exposure scenarios, vulnerability and protective factors. DESIGN: A literature search was performed for reported cases of morbidity and mortality associated with different infant feeding modes. SETTING: Exclusive breast-feeding is the recommended for infant feeding under 6 months, or failing that, provision of donated human milk. However, the use of PIF remains high despite its intrinsic and extrinsic risk of microbial contamination, as well as the potential for adverse physiological effects, including infant gut dysbiosis. RESULTS: Viable pathogen transmission via breast-feeding or donor milk (pasteurised and unpasteurised) is rare. However, transmission of HIV and human T-cell lymphotropic virus-1 is a concern for breast-feeding mothers, particularly for mothers undertaking a mixed feeding mode (PIF and breast-feeding). In PIF, intrinsic and extrinsic microbial contamination, such as Cronobacter and Salmonella, remain significant identifiable causes of infant morbidity and mortality. CONCLUSIONS: Disease transmission through breast-feeding or donor human milk is rare, most likely owing to its complex intrinsically protective composition of human milk and protection of the infant gut lining. Contamination of PIF and the morbidity associated with this is likely underappreciated in terms of community risk. A better system of safe donor milk sharing that also establishes security of supply for non-hospitalised healthy infants in need of breast milk would reduce the reliance on PIF.

Quantitative PET in the 2020s: a roadmap.

Positron emission tomography (PET) plays an increasingly important role in research and clinical applications, catalysed by remarkable technical advances and a growing appreciation of the need for reliable, sensitive biomarkers of human function in health and disease. Over the last 30 years, a large amount of the physics and engineering effort in PET has been motivated by the dominant clinical application during that period, oncology. This has led to important developments such as PET/CT, whole-body PET, 3D PET, accelerated statistical image reconstruction, and time-of-flight PET. Despite impressive improvements in image quality as a result of these advances, the emphasis on static, semi-quantitative 'hot spot' imaging for oncologic applications has meant that the capability of PET to quantify biologically relevant parameters based on tracer kinetics has not been fully exploited. More recent advances, such as PET/MR and total-body PET, have opened up the ability to address a vast range of new research questions, from which a future expansion of applications and radiotracers appears highly likely. Many of these new applications and tracers will, at least initially, require quantitative analyses that more fully exploit the exquisite sensitivity of PET and the tracer principle on which it is based. It is also expected that they will require more sophisticated quantitative analysis methods than those that are currently available. At the same time, artificial intelligence is revolutionizing data analysis and impacting the relationship between the statistical quality of the acquired data and the information we can extract from the data. In this roadmap, leaders of the key sub-disciplines of the field identify the challenges and opportunities to be addressed over the next ten years that will enable PET to realise its full quantitative potential, initially in research laboratories and, ultimately, in clinical practice.

The water chemistry and microbiome of household wells in Medawachchiya, Sri Lanka, an area with high prevalence of chronic kidney disease of unknown origin (CKDu).

Chronic kidney disease (CKD) of unknown etiology (CKDu) mostly affects agricultural communities in Central America, South Asia, Africa, but likely also in North America and Australia. One such area with increased CKDu prevalence is the Medawachchiya District Secretariat Division of the Anuradhapura District in the North Central Province of Sri Lanka. Recent research has focused on the presence of various microbial pathogens in drinking water as potential causal or contributing factors to CKDu, yet no study to date has performed a more comprehensive microbial and water chemistry assessment of household wells used for domestic water supply in areas of high CKDu prevalence. In this study, we describe the chemical composition and total microbial content in 30 domestic household wells in the Medawachchiya District Secretariat Division. While the chemical composition in the tested wells mostly lies within standard drinking water limits, except for high levels of fluoride (F), magnesium (Mg), sodium (Na), chloride (Cl) and calcium (Ca) in some samples, we find a frequent presence of cyanotoxin-producing Microcystis, confirming earlier studies in Sri Lanka. Since the total microbial content of drinking water also directly influences the composition of the human gut microbiome, it can be considered an important determinant of health. Several bacterial phyla were previously reported in the gut microbiome of patients with CKD. Using these bacteria phyla to define operational taxonomic units, we found that these bacteria also occur in the microbiome of the sampled well water. Based on available environmental data, our study demonstrates associations between the abundances of these bacteria with geographical distribution, well water temperature and likely fertilizer use in the local surface water catchment area of the individual household wells. Our results reinforce the recommendation that household wells with stagnant or infrequently used water should be purged prior to use for drinking water, bathing and irrigation. The latter is suggested because of the reported potential accumulation of bacterial toxins by agricultural crops. The observation that bacteria previously found in chronic kidney disease patients are also present in household wells requires a more detailed systematic study of both the human gut and drinking water microbiomes in CKDu patients, in relation to disease prevalence and progression.

Author Correction: Selective, high-contrast detection of syngeneic glioblastoma in vivo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.