GRT-X Stimulates Dorsal Root Ganglia Axonal Growth in Culture via TSPO and Kv7.2/3 Potassium Channel Activation.

GRT-X, which targets both the mitochondrial translocator protein (TSPO) and the Kv7.2/3 (KCNQ2/3) potassium channels, has been shown to efficiently promote recovery from cervical spine injury. In the present work, we investigate the role of GRT-X and its two targets in the axonal growth of dorsal root ganglion (DRG) neurons. Neurite outgrowth was quantified in DRG explant cultures prepared from wild-type C57BL6/J and TSPO-KO mice. TSPO was pharmacologically targeted with the agonist XBD173 and the Kv7 channels with the activator ICA-27243 and the inhibitor XE991. GRT-X efficiently stimulated DRG axonal growth at 4 and 8 days after its single administration. XBD173 also promoted axonal elongation, but only after 8 days and its repeated administration. In contrast, both ICA27243 and XE991 tended to decrease axonal elongation. In dissociated DRG neuron/Schwann cell co-cultures, GRT-X upregulated the expression of genes associated with axonal growth and myelination. In the TSPO-KO DRG cultures, the stimulatory effect of GRT-X on axonal growth was completely lost. However, GRT-X and XBD173 activated neuronal and Schwann cell gene expression after TSPO knockout, indicating the presence of additional targets warranting further investigation. These findings uncover a key role of the dual mode of action of GRT-X in the axonal elongation of DRG neurons.

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.

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.

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.

Calcium-axonemal microtubuli interactions underlie mechanism(s) of primary cilia morphological changes.

We have used cell culture of astrocytes aligned within microchannels to investigate calcium effects on primary cilia morphology. In the absence of calcium and in the presence of flow of media (10 µL.s-1) the majority (90%) of primary cilia showed reversible bending with an average curvature of 2.1 ± 0.9 × 10-4 nm-1. When 1.0 mM calcium was present, 90% of cilia underwent bending. Forty percent of these cilia demonstrated strong irreversible bending, resulting in a final average curvature of 3.9 ± 1 × 10-4 nm-1, while 50% of cilia underwent bending similar to that observed during calcium-free flow. The average length of cilia was shifted toward shorter values (3.67 ± 0.34 µm) when exposed to excess calcium (1.0 mM), compared to media devoid of calcium (3.96 ± 0.26 µm). The number of primary cilia that became curved after calcium application was reduced when the cell culture was pre-incubated with 15 µM of the microtubule stabilizer, taxol, for 60 min prior to calcium application. Calcium caused single microtubules to curve at a concentration ≈1.0 mM in vitro, but at higher concentration (≈1.5 mM) multiple microtubule curving occurred. Additionally, calcium causes microtubule-associated protein-2 conformational changes and its dislocation from the microtubule wall at the location of microtubule curvature. A very small amount of calcium, that is 1.45 × 1011 times lower than the maximal capacity of TRPPs calcium channels, may cause gross morphological changes (curving) of primary cilia, while global cytosol calcium levels are expected to remain unchanged. These findings reflect the non-linear manner in which primary cilia may respond to calcium signaling, which in turn may influence the course of development of ciliopathies and cancer.

Investigating the interactions of the 18kDa translocator protein and its ligand PK11195 in planar lipid bilayers.

The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug-membrane interactions may contribute to the apparent affinity of TSPO ligands.

Guwiyang Wurra--'Fire Mouse': a global gene knockout model for TSPO/PBR drug development, loss-of-function and mechanisms of compensation studies.

The highly conserved 18-kDa translocator protein (TSPO) or peripheral benzodiazepine receptor (PBR), is being investigated as a diagnostic and therapeutic target for disease conditions ranging from inflammation to neurodegeneration and behavioural illnesses. Many functions have been attributed to TSPO/PBR including a role in the mitochondrial permeability transition pore (MPTP), steroidogenesis and energy metabolism. In this review, we detail the recent developments in determining the physiological role of TSPO/PBR, specifically based on data obtained from the recently generated Tspo knockout mouse models. In addition to defining the role of TSPO/PBR, we also describe the value of Tspo knockout mice in determining the selectivity, specificity and presence of any off-target effects of TSPO/PBR ligands.

Protein-ligand and membrane-ligand interactions in pharmacology: the case of the translocator protein (TSPO).

The targets of many small molecule drugs are membrane proteins, and traditionally the focus of pharmacology is on the interaction between such receptors and their small molecule drug ligands. However, the lipid membranes of cells and organelles are increasingly appreciated as diverse and dynamic structures that also specifically interact with small molecule drugs and peptides, causing profound changes in the properties of these membranes, and modulating the function of the membrane and the proteins within it. Drug-membrane interactions are likely to have a role in both the therapeutic and toxic activity of a variety of compounds, and their role in the overall pharmacological effect of a drug needs to be understood more clearly. This is the case for the 18 kDa translocator protein (TSPO) and its ligands, where functions that were established based on pharmacological studies are being called into question. Re-examining the putative functions of the TSPO and the effects of its ligands reveals a need to consider in more detail the interplay between protein-ligand and membrane-ligand interactions, and the modulatory relationship between TSPO and the lipid membrane.

Green fluorescent protein alters the transcriptional regulation of human mitochondrial genes after gamma irradiation.

Green fluorescent proteins (GFP), extensively used as reporters in biological and imaging studies, are assumed to be mostly biologically inert. Here, we test the assumption in regard to the transcriptional regulation of 18 mitochondrially encoded genes in GFP expressing human T-cell line (JURKAT cells) exposed to gamma radiation. Using quantitative polymerase chain reaction, we demonstrate that wild type and GFP expressing JURKAT cells have different baseline mitochondrial transcript expression (10 out of the 18 tested genes) and after a single dose of radiation (100 Gy) show a significantly different transcriptional regulation of their mitochondrial genes. While in wild type cells, ten of the tested genes are up-regulated in response to radiation exposure, GFP expressing cells show less transcriptional regulation with a small down-regulation in five genes. Our results indicate that the presence of GFP in the cytoplasm can alter the cellular response to ionizing radiation.

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.

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.

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.

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.

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.

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.

Using water quality and isotope studies to inform research in chronic kidney disease of unknown aetiology endemic areas in Sri Lanka.

Chronic Kidney Disease of unknown aetiology (CKDu) is a major public health concern in dry climatic, agricultural regions of Sri Lanka. The chemistry of groundwater (the main source of drinking water) in the area has been studied extensively, in relation to the occurrence of CKDu. This paper investigates water quality studies published in CKDu affected areas of Sri Lanka and also presents a new data set of 27 hydrochemical and isotopic samples collected from groundwater wells in selected CKDu endemic areas in Sri Lanka. The study outcomes do not provide evidence of pollutants such as heavy metals in groundwater. However, the study identifies elevated concentrations of silica which requires further investigation. Two groups of groundwater have been identified based on the isotopic results suggesting different sources or origins. The available water quality data, including the data from this study, is not sufficient to answer questions on whether the chemistry of groundwater is related to the CKDu occurrence. However, this study identifies the importance of detailed investigation into degradation products of agrochemicals, the organic matter content and the influence of elevate silica concentration in groundwater. The study also provides research directions in the form of isotopic tracers and the frequency of sampling that is needed to capture potential pollutants in future groundwater quality studies in CKDu endemic areas in Sri Lanka.

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.