Analysis of the Healthy Platelet Proteome Identifies a New Form of Domain-Specific O-Fucosylation.

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.

Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. METHODS: Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. RESULTS: SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. CONCLUSIONS: This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

What shapes parental feeding decisions over the first 18 months of parenting: Insights into drivers towards commercial and home-prepared foods among different socioeconomic groups in the UK.

Infants born into families experiencing socioeconomic disadvantage follow a high-risk trajectory for obesity and poor health in later life. Differences in early childhood food experiences may be contributing to these inequalities. This study aimed to explore the factors that influence parental decisions on when, how and what food to introduce over the first 18 months of their child's life and identify differences according to families' social position. Particular attention was given to social and environmental determinants within and outside the home. This research utilised a longitudinal qualitative methodology, with interviews and photo-elicitation exercises completed by participants when their children were 4-6; 10-12 and 16-18 months of age. Participants were parents (61 mothers; 1 father), distributed across low, medium and high socioeconomic position (SEP). During analysis, observable differences in factors directing parents to home-prepared or commercial foods were identified. Factors that undermined the provision of home-prepared meals included lack of time after returning to work, insufficient support from partners, uncertainty around infant and young child feeding (defined as the introduction and provision of solids) and an implicit trust in the messaging on branded products. These factors directed parents towards commercial foods and were most persistent among families experiencing socioeconomic disadvantage due to barriers accessing formal childcare, less flexible working conditions and fathers being less involved in infant feeding. To facilitate an enabling environment for healthy infant and young child feeding practices and address dietary inequalities, immediate steps that policy makers and healthcare providers can take include: i) changing the eligibility criteria for shared parental leave, ii) aligning claims on commercial infant food labels with international best practices, and iii) improving access to formal childcare.

Metabolic changes with intermittent fasting.

BACKGROUND: The prevalence of obesity is rising globally and effective strategies to treat obesity are needed. Intermittent fasting, a dietary intervention for weight management, has received growing interest from the general public, as well as healthcare professionals, as a form of lifestyle intervention. METHODS: We executed a rapid review using PUBMED database to identify systematic reviews that examined the impact of intermittent fasting on metabolic indices, published between 2011 and 2022. RESULTS: Intermittent fasting leads to weight loss of a similar magnitude to continuous energy restriction. Most of the evidence shows that intermittent fasting leads to greater fat loss as measured by fat mass (kg) or body fat percentage compared to an ad libitum diet, but fat loss attained during intermittent fasting is not significantly different to continuous energy restriction, although recent evidence shows intermittent fasting to be superior. There is mixed evidence for the impact of intermittent fasting on insulin resistance, fasting glucose and lipid profile. Some studies focused on populations of Muslim people, which showed that Ramadan fasting may lead to weight loss and improvement of metabolic parameters during fasting, although the effects are reversed when fasting is finished. CONCLUSIONS: Intermittent fasting is more effective than an ad libitum dietary intake, and equally or more effective as continuous energy restriction, for weight management. However, there is inconclusive evidence on whether intermittent fasting has a clinically beneficial effect on glucose and lipid metabolism.

Intracellular Amyloid-ß Detection from Human Brain Sections Using a Microfluidic Immunoassay in Tandem with MALDI-MS.

Alzheimer's disease (AD) currently affects more than 30 million people worldwide. The lack of understanding of AD's physiopathology limits the development of therapeutic and diagnostic tools. Soluble amyloid-ß peptide (Aß) oligomers that appear as intermediates along the Aß aggregation into plaques are considered among the main AD neurotoxic species. Although a wealth of data are available about Aß from in vitro and animal models, there is little known about intracellular Aß in human brain cells, mainly due to the lack of technology to assess the intracellular protein content. The elucidation of the Aß species in specific brain cell subpopulations can provide insight into the role of Aß in AD and the neurotoxic mechanism involved. Here, we report a microfluidic immunoassay for in situ mass spectrometry analysis of intracellular Aß species from archived human brain tissue. This approach comprises the selective laser dissection of individual pyramidal cell bodies from tissues, their transfer to the microfluidic platform for sample processing on-chip, and mass spectrometric characterization. As a proof-of-principle, we demonstrate the detection of intracellular Aß species from as few as 20 human brain cells.

Integrated DNA Methylation/RNA Profiling in Middle Temporal Gyrus of Alzheimer's Disease.

Alzheimer's disease is a neurodegenerative disorder clinically defined by gradual cognitive impairment and alteration in executive function. We conducted an epigenome-wide association study (EWAS) of a clinically and neuropathologically characterized cohort of 296 brains, including Alzheimer's disease (AD) and non-demented controls (ND), exploring the relationship with the RNA expression from matched donors. We detected 5246 CpGs and 832 regions differentially methylated, finding overlap with previous EWAS but also new associations. CpGs previously identified in ANK1, MYOC, and RHBDF2 were differentially methylated, and one of our top hits (GPR56) was not previously detected. ANK1 was differentially methylated at the region level, along with APOE and RHBDF2. Only a small number of genes showed a correlation between DNA methylation and RNA expression statistically significant. Multiblock partial least-squares discriminant analysis showed several CpG sites and RNAs discriminating AD and ND (AUC = 0.908) and strongly correlated with each other. Furthermore, the CpG site cg25038311 was negatively correlated with the expression of 22 genes. Finally, with the functional epigenetic module analysis, we identified a protein-protein network characterized by inverse RNA/DNA methylation correlation and enriched for "Regulation of insulin-like growth factor transport", with IGF1 as the hub gene. Our results confirm and extend the previous EWAS, providing new information about a brain region not previously explored in AD DNA methylation studies. The relationship between DNA methylation and gene expression is not significant for most of the genes in our sample, consistently with the complexities in the gene expression regulation.

Targeting miR-27a/VE-cadherin interactions rescues cerebral cavernous malformations in mice.

Cerebral cavernous malformations (CCMs) are vascular lesions predominantly developing in the central nervous system (CNS), with no effective treatments other than surgery. Loss-of-function mutation in CCM1/krev interaction trapped 1 (KRIT1), CCM2, or CCM3/programmed cell death 10 (PDCD10) causes lesions that are characterized by abnormal vascular integrity. Vascular endothelial cadherin (VE-cadherin), a major regulator of endothelial cell (EC) junctional integrity is strongly disorganized in ECs lining the CCM lesions. We report here that microRNA-27a (miR-27a), a negative regulator of VE-cadherin, is elevated in ECs isolated from mouse brains developing early CCM lesions and in cultured ECs with CCM1 or CCM2 depletion. Furthermore, we show miR-27a acts downstream of kruppel-like factor (KLF)2 and KLF4, two known key transcription factors involved in CCM lesion development. Using CD5-2 (a target site blocker [TSB]) to prevent the miR-27a/VE-cadherin mRNA interaction, we present a potential therapy to increase VE-cadherin expression and thus rescue the abnormal vascular integrity. In CCM1- or CCM2-depleted ECs, CD5-2 reduces monolayer permeability, and in Ccm1 heterozygous mice, it restores dermal vessel barrier function. In a neonatal mouse model of CCM disease, CD5-2 normalizes vasculature and reduces vascular leakage in the lesions, inhibits the development of large lesions, and significantly reduces the size of established lesions in the hindbrain. Furthermore, CD5-2 limits the accumulation of inflammatory cells in the lesion area. Our work has established that VE-cadherin is a potential therapeutic target for normalization of the vasculature and highlights that targeting miR-27a/VE-cadherin interaction by CD5-2 is a potential novel therapy for the devastating disease, CCM.

Trends in socioeconomic inequalities in behavioural non-communicable disease risk factors: analysis of repeated cross-sectional health surveys in England between 2003 and 2019.

BACKGROUND: Previous studies have shown that those in lower socioeconomic positions (SEPs) generally have higher levels of behavioural non-communicable disease (NCD) risk factors. However, there are limited studies examining recent trends in inequalities. This study examined trends in socioeconomic inequalities in NCD behavioural risk factors and their co-occurrence in England from 2003-19. METHODS: This time-trend analysis of repeated cross-sectional data from the Health Survey for England examined the relative index of inequalities (RII) and slope index of inequalities (SII) in four NCD behavioural risk factors: smoking; drinking above recommended limits; insufficient fruit and vegetables consumption; and physical inactivity. FINDINGS: Prevalence of risk factors has reduced over time, however, this has not been consistent across SEPs. Absolute and relative inequalities increased for physical inactivity; relative inequalities also increased for smoking; for insufficient fruit and vegetable consumption, the trends in inequalities depended on SEPs measure. Those in lower SEPs experienced persistent socioeconomic inequalities and clustering of behavioural risk factors. In contrast, those in higher SEPs had higher prevalence of excessive alcohol consumption; this inequality widened over the study period. INTERPRETATION: Inequalities in smoking and physical inactivity are persisting or widening. The pattern of higher drinking in higher SEPs obscure the fact that the greatest burden of alcohol-related harm falls on lower SEPs. Policy attention is required to tackle increasing inequalities in smoking prevalence, low fruit and vegetable consumption and physical inactivity, and to reduce alcohol harm.

Blood RNA transcripts reveal similar and differential alterations in fundamental cellular processes in Alzheimer's disease and other neurodegenerative diseases.

BACKGROUND: Dysfunctional processes in Alzheimer's disease and other neurodegenerative diseases lead to neural degeneration in the central and peripheral nervous system. Research demonstrates that neurodegeneration of any kind is a systemic disease that may even begin outside of the region vulnerable to the disease. Neurodegenerative diseases are defined by the vulnerabilities and pathology occurring in the regions affected. METHOD: A random forest machine learning analysis on whole blood transcriptomes from six neurodegenerative diseases generated unbiased disease-classifying RNA transcripts subsequently subjected to pathway analysis. RESULTS: We report that transcripts of the blood transcriptome selected for each of the neurodegenerative diseases represent fundamental biological cell processes including transcription regulation, degranulation, immune response, protein synthesis, apoptosis, cytoskeletal components, ubiquitylation/proteasome, and mitochondrial complexes that are also affected in the brain and reveal common themes across six neurodegenerative diseases. CONCLUSION: Neurodegenerative diseases share common dysfunctions in fundamental cellular processes. Identifying regional vulnerabilities will reveal unique disease mechanisms. HIGHLIGHTS: Transcriptomics offer information about dysfunctional processes. Comparing multiple diseases will expose unique malfunctions within diseases. Blood RNA can be used ante mortem to track expression changes in neurodegenerative diseases. Protocol standardization will make public datasets compatible.

MIMAS: microfluidic platform in tandem with MALDI mass spectrometry for protein quantification from small cell ensembles.

Understanding cell-to-cell variation at the molecular level provides relevant information about biological phenomena and is critical for clinical and biological research. Proteins carry important information not available from single-cell genomics and transcriptomics studies; however, due to the minute amount of proteins in single cells and the complexity of the proteome, quantitative protein analysis at the single-cell level remains challenging. Here, we report an integrated microfluidic platform in tandem with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the detection and quantification of targeted proteins from small cell ensembles (> 10 cells). All necessary steps for the assay are integrated on-chip including cell lysis, protein immunocapture, tryptic digestion, and co-crystallization with the matrix solution for MALDI-MS analysis. We demonstrate that our approach is suitable for protein quantification by assessing the apoptotic protein Bcl-2 released from MCF-7 breast cancer cells, ranging from 26 to 223 cells lysed on-chip (8.75 nL wells). A limit of detection (LOD) of 11.22 nM was determined, equivalent to 5.91 × 107 protein molecules per well. Additionally, the microfluidic platform design was further improved, establishing the successful quantification of Bcl-2 protein from MCF-7 cell ensembles ranging from 8 to 19 cells in 4 nL wells. The LOD in the smaller well designs for Bcl-2 resulted in 14.85 nM, equivalent to 3.57 × 107 protein molecules per well. This work shows the capability of our approach to quantitatively assess proteins from cell lysate on the MIMAS platform for the first time. These results demonstrate our approach constitutes a promising tool for quantitative targeted protein analysis from small cell ensembles down to single cells, with the capability for multiplexing through parallelization and automation.

Food shortages, stockpiling and panic buying ahead of Brexit as reported by the British media: a mixed methods content analysis.

BACKGROUND: On 23 June 2016, the United Kingdom voted to leave the European Union. From that date until the UK left the EU in January 2021, there were frequent warnings from industry and government sources of potential disruption to the food supply chain and possible food shortages. Over this period, the media had an important role in communicating on the potential impacts of Brexit. This study examines how food supply and demand, in the context of Brexit, was portrayed by the British media. METHODS: The study consisted of two components: (1) a quantitative analysis measuring frequency of reporting and information sources for articles on food supply and demand in the context of Brexit, in three daily newspapers, between January 2015 and January 2020; and (2) a content analysis exploring key themes and media framing of relevant issues in a subset of articles. RESULTS: Reports by the media about the impact of Brexit on the UK food system were largely absent in the six months before the UK voted to leave the EU in June 2016, increasing in frequency from mid-2018 onward, peaking in mid-2019 following the appointment of Boris Johnson as prime minister. Five themes were developed from included articles: food shortages/panic buying (appearing in 96% of articles); food chain disruption (86%); economic impacts (80%); preparation and stockpiling by the government/food sector (63%) and preparation and stockpiling by individuals (22%). CONCLUSION: Government messaging sought to reassure the public that even under a worst-case scenario there would be no food shortages. These messages, however, contradicted warnings in the media of disruption to the food supply chain and food shortages. The media further reinforced this narrative of potential food shortages by reporting on the experiences of those preparing for Brexit by stockpiling food. The media must consider the impact of their messaging on public behaviour, as even imagined food shortages can instigate stockpiling and panic buying behaviour, as observed during the COVID-19 pandemic.

Impact of national COVID-19 restrictions on incidence of notifiable communicable diseases in England: an interrupted time series analysis.

BACKGROUND: Non-pharmaceutical interventions (NPIs), such as travel restrictions, social distancing and isolation policies, aimed at controlling the spread of COVID-19 may have reduced transmission of other endemic communicable diseases, such as measles, mumps and meningitis in England. METHODS: An interrupted time series analysis was conducted to examine whether NPIs was associated with trends in endemic communicable diseases, using weekly reported cases of seven notifiable communicable diseases (food poisoning, measles, meningitis, mumps, scarlet fever and pertussis) between 02/01/2017 to 02/01/2021 for England. RESULTS: Following the introduction of COVID-19 restrictions, there was an 81.1% (95% CI; 77.2-84.4) adjusted percentage reduction in the total number of notifiable diseases recorded per week in England. The greatest decrease was observed for measles, with a 90.5% percentage reduction (95% CI; 86.8-93.1) from 42 to 5 cases per week. The smallest decrease was observed for food poisoning, with a 56.4% (95%CI; 42.5-54.2) decrease from 191 to 83 cases per week. CONCLUSIONS: A total reduction in the incidence of endemic notifiable diseases was observed in England following the implementation of public health measures aimed at reducing transmission of SARS-COV-2 on March 23, 2020. The greatest reductions were observed in diseases most frequently observed during childhood that are transmitted via close human-to-human contact, such as measles and pertussis. A less substantive reduction was observed in reported cases of food poisoning, likely due to dining services (i.e., home deliveries and takeaways) remaining open and providing a potential route of transmission. This study provides further evidence of the effectiveness of non-pharmaceutical public health interventions in reducing the transmission of both respiratory and food-borne communicable diseases.

Implementation of novel and conventional outbreak control measures in managing COVID-19 outbreaks in a large UK prison.

BACKGROUND: Outbreak control measures during COVID-19 outbreaks in a large UK prison consisted of standard (e.g., self-isolation) and novel measures, including establishment of: (i) reverse cohorting units for accommodating new prison admissions; (ii) protective isolation unit for isolating symptomatic prisoners, and (iii) a shielding unit to protect medically vulnerable prisoners. METHODS: Single-centre prospective longitudinal study (outbreak control study), implementing novel and traditional outbreak control measures to prevent a SARS-COV-2 outbreak. The prison held 977 prisoners and employed 910 staff at that start of the outbreak. RESULTS: 120 probable and 25 confirmed cases among prisoners and staff were recorded between March and June 2020 during the first outbreak. Over 50% of initial cases among prisoners were on the two wings associated with the index case. During the second outbreak, 182 confirmed cases were recorded after probable reintroduction from a staff member. Widespread testing identified 145 asymptomatic prisoners, 16.9% of the total prisoner cases. The cohorting units prevented re-infection from new prison admissions and the shielding unit had no COVID-19 infections linked to either outbreak. CONCLUSIONS: Identifying and isolating infected prisoners, cohorting new admissions and shielding vulnerable individuals helped prevent uncontrollable spread of SARS-COV-2. These novel and cost-effective approaches can be implemented in correctional facilities globally.

The Discovery of Suvorexant, the First Orexin Receptor Drug for Insomnia.

Historically, pharmacological therapies have used mechanisms such as γ-aminobutyric acid A (GABAA) receptor potentiation to drive sleep through broad suppression of central nervous system activity. With the discovery of orexin signaling loss as the etiology underlying narcolepsy, a disorder associated with hypersomnolence, orexin antagonism emerged as an alternative approach to attenuate orexin-induced wakefulness more selectively. Dual orexin receptor antagonists (DORAs) block the activity of orexin 1 and 2 receptors to both reduce the threshold to transition into sleep and attenuate orexin-mediated arousal. Among DORAs evaluated clinically, suvorexant has pharmacokinetic properties engineered for a plasma half-life appropriate for rapid sleep onset and maintenance at low to moderate doses. Unlike GABAA receptor modulators, DORAs promote both non-rapid eye movement (NREM) and REM sleep, do not disrupt sleep stage-specific quantitative electroencephalogram spectral profiles, and allow somnolence indistinct from normal sleep. The preservation of cognitive performance and the ability to arouse to salient stimuli after DORA administration suggest further advantages over historical therapies.

YAP and the RhoC regulator ARHGAP18, are required to mediate flow-dependent endothelial cell alignment.

BACKGROUND: Vascular endothelial cell alignment in the direction of flow is an adaptive response that protects against aortic diseases such as atherosclerosis. The RhoGTPases are known to regulate this alignment. We have shown previously that ARHGAP18 in endothelial cells is a negative regulator of RhoC and its expression is essential in flow-mediated alignment. Depletion of ARHGAP18 inhibits alignment and results in the induction of a pro-inflammatory phenotype. In embryogenesis, ARHGAP18 was identified as a downstream effector of the Yes-associated protein, YAP, which regulates cell shape and size. METHODS: We have used siRNA technology to deplete either ARHGAP18 or YAP in human endothelial cells. The in vitro studies were performed under athero-protective, laminar flow conditions. The analysis of YAP activity was also investigated, using high performance confocal imaging, in our ARHGAP18 knockout mutant mice. RESULTS: We show here that loss of ARHGAP18, although decreasing the expression of YAP results in its nuclear localisation consistent with activation. We further show that depletion of YAP itself results in its activation as defined by an in increase in its nuclear localisation and an increase in the YAP target gene, CyR61. Depletion of YAP, similar to that observed for ARHGAP18 depletion, results in loss of endothelial cell alignment under high shear stress mediated flow and also in the activation of NFkB, as determined by p65 nuclear localisation. In contrast, ARHGAP18 overexpression results in upregulation of YAP, its phosphorylation, and a decrease in the YAP target gene Cyr61, consistent with YAP inactivation. Finally, in ARHGAP18 deleted mice, in regions where there is a loss of endothelial cell alignment, a situation associated with a priming of the cells to a pro-inflammatory phenotype, YAP shows nuclear localisation. CONCLUSION: Our results show that YAP is downstream of ARHGAP18 in mature endothelial cells and that this pathway is involved in the athero-protective alignment of endothelial cells under laminar shear stress. ARHGAP18 depletion leads to a disruption of the junctions as seen by loss of VE-Cadherin localisation to these regions and a concomitant localisation of YAP to the nucleus.

Identification of potent inhibitors of the sortilin-progranulin interaction.

High-throughput screening methods have been used to identify two novel series of inhibitors that disrupt progranulin binding to sortilin. Exploration of structure-activity relationships (SAR) resulted in compounds with sufficient potency and physicochemical properties to enable co-crystallization with sortilin. These co-crystal structures supported observed SAR trends and provided guidance for additional avenues for designing compounds with additional interactions within the binding site.

Therapeutic regulation of VE-cadherin with a novel oligonucleotide drug for diabetic eye complications using retinopathy mouse models.

AIMS/HYPOTHESIS: A major feature of diabetic retinopathy is breakdown of the blood-retinal barrier, resulting in macular oedema. We have developed a novel oligonucleotide-based drug, CD5-2, that specifically increases expression of the key junctional protein involved in barrier integrity in endothelial cells, vascular-endothelial-specific cadherin (VE-cadherin). CD5-2 prevents the mRNA silencing by the pro-angiogenic microRNA, miR-27a. CD5-2 was evaluated in animal models of ocular neovascularisation and vascular leak to determine its potential efficacy for diabetic retinopathy. METHODS: CD5-2 was tested in three mouse models of retinal dysfunction: conditional Müller cell depletion, streptozotocin-induced diabetes and oxygen-induced retinopathy. Vascular permeability in the Müller cell-knockout model was assessed by fluorescein angiography. The Evans Blue leakage method was used to determine vascular permeability in streptozotocin- and oxygen-induced retinopathy models. The effects of CD5-2 on retinal neovascularisation, inter-endothelial junctions and pericyte coverage in streptozotocin- and oxygen-induced retinopathy models were determined by staining for isolectin-B4, VE-cadherin and neural/glial antigen 2 (NG2). Blockmir CD5-2 localisation in diseased retina was determined using fluorescent in situ hybridisation. The effects of CD5-2 on VE-cadherin expression and in diabetic retinopathy-associated pathways, such as the transforming growth factor beta (TGF-ß) and wingless/integrated (WNT) pathway, were confirmed using western blot of lysates from HUVECs, a mouse brain endothelial cell line and a VE-cadherin null mouse endothelial cell line. RESULTS: CD5-2 penetrated the vasculature of the eye in the oxygen-induced retinopathy model. Treatment of diseased mice with CD5-2 resulted in reduced vascular leak in all three animal models, enhanced expression of VE-cadherin in the microvessels of the eye and improved pericyte coverage of the retinal vasculature in streptozotocin-induced diabetic models and oxygen-induced retinopathy models. Further, CD5-2 reduced the activation of retinal microglial cells in the streptozotocin-induced diabetic model. The positive effects of CD5-2 seen in vivo were further confirmed in vitro by increased protein expression of VE-cadherin, SMAD2/3 activity, and platelet-derived growth factor B (PDGF-B). CONCLUSIONS/INTERPRETATION: CD5-2 has therapeutic potential for individuals with vascular-leak-associated retinal diseases based on its ease of delivery and its ability to reverse vascular dysfunction and inflammatory aspects in three animal models of retinopathy.

Oligomeric amyloid ß preferentially targets neuronal and not glial mitochondrial-encoded mRNAs.

INTRODUCTION: Our laboratories have demonstrated that accumulation of oligomeric amyloid ß (OAß) in neurons is an essential step leading to OAß-mediated mitochondrial dysfunction. METHODS: Alzheimer's disease (AD) and matching control hippocampal neurons, astrocytes, and microglia were isolated by laser-captured microdissection from the same subjects, followed by whole-transcriptome sequencing. Complementary in vitro work was performed in OAß-treated differentiated SH-SY5Y, followed by the use of a novel CoQ10 analogue for protection. This compound is believed to be effective both in suppressing reactive oxygen species and also functioning in mitochondrial electron transport. RESULTS: We report decreases in the same mitochondrial-encoded mRNAs in Alzheimer's disease laser-captured CA1 neurons and in OAß-treated SH-SY5Y cells, but not in laser-captured microglia and astrocytes. Pretreatment with a novel CoQ10 analogue, protects neuronal mitochondria from OAß-induced mitochondrial changes. DISCUSSION: Similarity of expression changes in neurons from Alzheimer's disease brain and neuronal cells treated with OAß, and the effect of a CoQ10 analogue on the latter, suggests a pretreatment option to prevent OAß toxicity, long before the damage is apparent.

The dual orexin receptor antagonist, DORA-22, lowers histamine levels in the lateral hypothalamus and prefrontal cortex without lowering hippocampal acetylcholine.

Chronic insomnia is defined as a persistent difficulty with sleep initiation maintenance or non-restorative sleep. The therapeutic standard of care for this condition is treatment with gamma-aminobutyric acid (GABA)A receptor modulators, which promote sleep but are associated with a panoply of side effects, including cognitive and memory impairment. Dual orexin receptor antagonists (DORAs) have recently emerged as an alternative therapeutic approach that acts via a distinct and more selective wake-attenuating mechanism with the potential to be associated with milder side effects. Given their distinct mechanism of action, the current work tested the hypothesis that DORAs and GABAA receptor modulators differentially regulate neurochemical pathways associated with differences in sleep architecture and cognitive performance induced by these pharmacological mechanisms. Our findings showed that DORA-22 suppresses the release of the wake neurotransmitter histamine in the lateral hypothalamus, prefrontal cortex, and hippocampus with no significant alterations in acetylcholine levels. In contrast, eszopiclone, commonly used as a GABAA modulator, inhibited acetylcholine secretion across brain regions with variable effects on histamine release depending on the extent of wakefulness induction. In normal waking rats, eszopiclone only transiently suppressed histamine secretion, whereas this suppression was more obvious under caffeine-induced wakefulness. Compared with the GABAA modulator eszopiclone, DORA-22 elicits a neurotransmitter profile consistent with wake reduction that does not impinge on neurotransmitter levels associated with cognition and rapid eye movement sleep.