Type-I-interferon-responsive microglia shape cortical development and behavior.

Microglia are brain-resident macrophages that shape neural circuit development and are implicated in neurodevelopmental diseases. Multiple microglial transcriptional states have been defined, but their functional significance is unclear. Here, we identify a type I interferon (IFN-I)-responsive microglial state in the developing somatosensory cortex (postnatal day 5) that is actively engulfing whole neurons. This population expands during cortical remodeling induced by partial whisker deprivation. Global or microglial-specific loss of the IFN-I receptor resulted in microglia with phagolysosomal dysfunction and an accumulation of neurons with nuclear DNA damage. IFN-I gain of function increased neuronal engulfment by microglia in both mouse and zebrafish and restricted the accumulation of DNA-damaged neurons. Finally, IFN-I deficiency resulted in excess cortical excitatory neurons and tactile hypersensitivity. These data define a role for neuron-engulfing microglia during a critical window of brain development and reveal homeostatic functions of a canonical antiviral signaling pathway in the brain.

APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFß-mediated checkpoints.

The APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD). Here, we show in mice and humans a negative role of microglial APOE4 in the induction of the MGnD response to neurodegeneration. Deletion of microglial APOE4 restores the MGnD phenotype associated with neuroprotection in P301S tau transgenic mice and decreases pathology in APP/PS1 mice. MGnD-astrocyte cross-talk associated with ß-amyloid (Aß) plaque encapsulation and clearance are mediated via LGALS3 signaling following microglial APOE4 deletion. In the brains of AD donors carrying the APOE4 allele, we found a sex-dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, compared to individuals homozygous for the APOE3 allele. Mechanistically, APOE4-mediated induction of ITGB8-transforming growth factor-ß (TGFß) signaling impairs the MGnD response via upregulation of microglial homeostatic checkpoints, including Inpp5d, in mice. Deletion of Inpp5d in microglia restores MGnD-astrocyte cross-talk and facilitates plaque clearance in APP/PS1 mice. We identify the microglial APOE4-ITGB8-TGFß pathway as a negative regulator of microglial response to AD pathology, and restoring the MGnD phenotype via blocking ITGB8-TGFß signaling provides a promising therapeutic intervention for AD.

Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.

PTP1B mediates the inhibitory effect of MFGE8 on insulin signaling through the ß5 integrin.

Integrins are cell adhesion receptors that dimerize to mediate cell-cell interactions and regulate processes, including proliferation, inflammation, and tissue repair. The role of integrins in regulating insulin signaling is incompletely understood. We have previously shown that binding of the integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) to the αvß5 integrin promotes termination of insulin receptor signaling in mice. Upon ligation of MFGE8, integrin ß5 complexes with the insulin receptor beta (IRß) in skeletal muscle, resulting in dephosphorylation of IRß and reduction of insulin-stimulated glucose uptake. Here, we investigate the mechanism by which the interaction between ß5 and IRß impacts IRß phosphorylation status. We show in in vitro and in vivo in skeletal muscle in mice that antibody-mediated blockade of the ß5 integrin inhibits and recombinant MFGE8 promotes PTP1B binding to and dephosphorylation of IRß resulting in increased or reduced insulin-stimulated glucose uptake, respectively. The ß5-PTP1B complex is recruited by MFGE8 to IRß leading to termination of canonical insulin signaling. ß5 blockade enhances insulin-stimulated glucose uptake in wildtype but not Ptp1b KO mice indicating that PTP1B functions downstream of MFGE8 in modulating insulin receptor signaling. Furthermore, in a human cohort, we report serum MFGE8 levels correlate with indices of insulin resistance. These data provide mechanistic insights into the role of MFGE8 and ß5 in regulating insulin signaling.

An Arginine-Rich Motif in the ORF2 capsid protein regulates the hepatitis E virus lifecycle and interactions with the host cell.

Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.

Associations of social and cognitive-behavioral variables with disinhibited eating and anxiety: An ecological momentary assessment study.

OBJECTIVE: Among adolescents, disinhibited eating and anxiety commonly co-occur. Precision intervention approaches targeting unique mechanistic vulnerabilities that contribute to disinhibited eating and anxiety may therefore be helpful. However, the effectiveness of such interventions hinges on knowledge of between- and within-person associations related to disinhibited eating, anxiety, and related processes. METHOD: A sample of 39 adolescent females (12-17 years) with elevated anxiety and above-average weight (BMI %ile ≥ 75th) completed measures of theoretically driven social and cognitive-behavioral variables, disinhibited eating, and anxiety via ecological momentary assessment over 7 days. Data were analyzed using mixed-effects models. RESULTS: Between-person differences in social stressors were linked to emotional eating, eating in the absence of hunger, and anxiety, whereas between-person differences in negative thoughts were associated with all disinhibited eating variables and anxiety. Between-person differences in avoidance were not related to any outcome. Additionally, between-person differences in social stressors and negative thoughts-as well as within-person deviations (from person-average levels) of social stressors, negative thoughts, and avoidance-were associated with anxiety. In turn, between-person differences in anxiety predicted eating in the absence of hunger and emotional eating, and within-person deviations in anxiety were associated with emotional eating at any given time point. DISCUSSION: Findings support elements of both the interpersonal and cognitive-behavioral models of disinhibited eating. Differential trigger effects on anxiety, both at the between- and within-person levels, and significant associations between anxiety and all eating-related outcomes, highlight the potential utility of interventions targeting individual differences in sensitivity to anxiety triggers. PUBLIC SIGNIFICANCE: Findings provide support for the interpersonal and cognitive-behavioral models of disinhibited eating, highlighting anxiety as a salient vulnerability and potential mechanistic factor underlying disinhibited eating. Social, cognitive, and behavioral variables were differentially related to anxiety across participants, suggesting potential for future intervention tailoring and intervention selection based on adolescents' sensitivity to anxiety as a trigger for disinhibited eating behavior.

Low-field MRI: Clinical promise and challenges.

Modern MRI scanners have trended toward higher field strengths to maximize signal and resolution while minimizing scan time. However, high-field devices remain expensive to install and operate, making them scarce outside of high-income countries and major population centers. Low-field strength scanners have drawn renewed academic, industry, and philanthropic interest due to advantages that could dramatically increase imaging access, including lower cost and portability. Nevertheless, low-field MRI still faces inherent limitations in image quality that come with decreased signal. In this article, we review advantages and disadvantages of low-field MRI scanners, describe hardware and software innovations that accentuate advantages and mitigate disadvantages, and consider clinical applications for a new generation of low-field devices. In our review, we explore how these devices are being or could be used for high acuity brain imaging, outpatient neuroimaging, MRI-guided procedures, pediatric imaging, and musculoskeletal imaging. Challenges for their successful clinical translation include selecting and validating appropriate use cases, integrating with standards of care in high resource settings, expanding options with actionable information in low resource settings, and facilitating health care providers and clinical practice in new ways. By embracing both the promise and challenges of low-field MRI, clinicians and researchers have an opportunity to transform medical care for patients around the world. LEVEL OF EVIDENCE: 5 TECHNICAL EFFICACY: Stage 6.

SARS-CoV-2 viral protein Nsp2 stimulates translation under normal and hypoxic conditions.

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

Effect of Anionic Lipids on Mammalian Plasma Cell Membrane Properties.

The effect of lipid composition on models of the inner leaflet of mammalian cell membranes has been investigated. Grazing incidence X-ray diffraction and X-ray and neutron reflectivity have been used to characterize lipid packing and solvation, while electrochemical and infrared spectroscopic methods have been employed to probe phase behavior in an applied electric field. Introducing a small quantity of the anionic lipid dimyristoylphosphatidylserine (DMPS) into bilayers of zwitterionic dimyristoylphosphatidylethanolamine (DMPE) results in a significant change in the bilayer response to an applied field: the tilt of the hydrocarbon chains increases before returning to the original tilt angle on detachment of the bilayer. Equimolar mixtures, with slightly closer chain packing, exhibit a similar but weaker response. The latter also tend to incorporate more solvent during this electrochemical phase transition, at levels similar to those of pure DMPS. Reflectivity measurements reveal greater solvation of lipid layers for DMPS > 30 mol %, matching the greater propensity for DMPS-rich bilayers to incorporate water. Taken together, the data indicate that the range of 10-35 mol % DMPS provides optimum bilayer properties (in flexibility and function as a barrier), which may explain why the DMPS content of cell membranes tends to be found within this range.

Influence of the Lipid Backbone on Electrochemical Phase Behavior.

Sphingolipids are an important class of lipids found in mammalian cell membranes with important structural and signaling roles. They differ from another major group of lipids, the glycerophospholipids, in the connection of their hydrocarbon chains to their headgroups. In this study, a combination of electrochemical and structural methods has been used to elucidate the effect of this difference on sphingolipid behavior in an applied electric field. N-Palmitoyl sphingomyelin forms bilayers of similar coverage and thickness to its close analogue di-palmitoyl phosphatidylcholine. Grazing incidence diffraction data show slightly closer packing and a smaller chain tilt angle from the surface normal. Electrochemical IR results at low charge density show that the difference in tilt angle is retained on deposition to form bilayers. The bilayers respond differently to increasing electric field strength: chain tilt angles increase for both molecules, but sphingomyelin chains remain tilted as field strength is further increased. This behavior is correlated with disruption of the hydrogen-bonding network of small groups of sphingomyelin molecules, which may have significance for the behavior of molecules in lipid rafts in the presence of strong fields induced by ion gradients or asymmetric distribution of charged lipids.

Structural investigation of sulfobetaines and phospholipid monolayers at the air-water interface.

Mixtures of sulfobetaine based lipids with phosphocholine phospholipids are of interest in order to study the interactions between zwitterionic surfactants and the phospholipids present in cell membranes. In this study we have investigated the structure of mixed monolayers of sulfobetaines and phosphocholine phospholipids. The sulfobetaine used has a single 18-carbon tail, and is referred to as SB3-18, and the phospholipid used is DMPC. Surface pressure-area isotherms of the samples were used to determine whether any phase transitions were present during the compression of the monolayers. Neutron and X-ray reflectometry were then used to investigate the structure of these monolayers perpendicular to the interface. We found that the average headgroup and tail layer thickness was reasonably consistent across all mixtures, with a variation of less than 3 Å reported in the total thickness of the monolayers at each surface pressure. However, by selective deuteration of the two components of the monolayers, it was found that the two components have different tail layer thicknesses. For the mixture with equal compositions of DMPC and SB3-18 or with a higher composition of DMPC the tail tilts were found to be constant, resulting in a greater tail layer thickness for SB3-18 due to its longer tail. For the mixture higher in SB3-18 this was not the case, the tail tilt angle for the two components was found to be different and DMPC was found to have a greater tail layer thickness than SB3-18 as a result.

Comment on Paolo Euron's "Uncanny Beauty: Aesthetics of Companionship, Love, and Sex Robots".

Paolo Euron's "Uncanny Beauty: Aesthetics of Companionship, Love, and Sex Robots" lays out a vision for appreciating sex robots in aesthetic terms, centering the concept of "beauty" as a measure of what they can inspire culturally and existentially. In these comments I turn toward the field of human-robot interaction and the ethical challenges that inhabit the core of such an aesthetic turn.

Scleral Lens-Induced Corneal Edema After Radial Keratotomy.

ABSTRACT: This case series examines the magnitude and regional variation in scleral lens-induced corneal edema in postradial keratotomy (RK) eyes. Scheimpflug imaging was used to measure corneal thickness across the central 6 mm before and after scleral lens wear in nine post-RK eyes (median age 64 years). Variations in corneal edema were examined as a function of distance from the corneal center (six 0.5-mm annuli) and from 0° to 360°. The median central corneal edema was 2.19% (interquartile range 1.03%-3.18%; P=0.02) and increased in magnitude and variability further from the central cornea (from 1.30% in the central 0-0.5 mm to 3.12% in the 2.5-3.0 mm midperiphery) (P=0.02). Scleral lens-induced corneal edema in post-RK eyes was greater in magnitude further from the corneal center and at the approximate location of corneal incisions.

Carbonyl-protein content increases in brain and blood of female rats after chronic oxycodone treatment.

BACKGROUND: Opioids are the most effective drugs commonly prescribed to treat pain. Due to their addictive nature, opioid pain relievers are now second to marijuana, ahead of cocaine with respect to dependence. Ours and other studies suggest potential toxic effects of chronic opioid administration leading to neuronal degeneration. It has been suggested that protein carbonylation may represent a sensitive biomarker of cellular degeneration. To evaluate whether prolonged oxycodone administration is associated with accumulation of protein aggregates that may contribute to neuronal degeneration we measured protein carbonylation levels in brain and also in blood plasma of rats after 30-days of 15 mg/kg daily oxycodone administration. RESULTS: We observed a significant increase in the level of carbonylated proteins in rat brain cortex after 30-days of oxycodone treatment compare to that in water treated animals. Also, oxycodone treated rats demonstrated accumulation of insoluble carbonyl-protein aggregates in blood plasma. CONCLUSIONS: Our data suggests that tests detecting insoluble carbonyl-protein aggregates in blood may serve as an inexpensive and minimally invasive method to monitor neuronal degeneration in patients with a history of chronic opioid use. Such methods could be used to detect toxic side effects of other medications and monitor progression of aging and neurodegenerative diseases.

Effect of HIV-1 TAT Peptide Fusion on 5' mRNA Cap Analogs Cell Membrane Permeability and Translation Inhibition.

The development of targeted anticancer drugs has been one of the most challenging goals of current research. Eukaryotic translation initiation factor 4E (eIF4E) is an oncogene that stimulates mRNA translation via binding to the 5' endcap structure. It is well documented that eIF4E is overexpressed in many cancers including breast, prostate, head and neck, and stomach malignancies and leads to oncogenic transformation and metastasis. One approach to block eIF4E function in cancer cells is based on the disruption of the interaction between eIF4E and the 5' mRNA cap structure using cap analog inhibitors. Since analogs are cell-impermeable due to their anionic nature, we used a cell penetrating peptide (CPP) for delivery of model cap analogs into cancer cells. The human immunodeficiency virus I (HIV-1) transactivator of transcription derived peptide (TAT) was conjugated with the analogs m7GMP and m7GpppG using click chemistry methodology. We observed that both conjugates (m7GMP-TAT and m7GpppG-TAT), contrary to TAT alone, did not translocate through the artificial phospholipid membrane of giant unilamellar vesicles. This suggests that passive transport is not the mechanism by which translocation of cap analogs occurs. In contrast, synthesized fluorescently labeled m7GpppG-TAT translocated into the human breast adenocarcinoma cancer cell line MCF-7. Furthermore, we demonstrated that m7GMP-TAT and m7GpppG-TAT inhibited cap-dependent translation up to 30% both in vivo and in vitro while simultaneously not affecting cell growth and viability. These results demonstrate the usefulness of cell penetration peptides as carriers for the internalization of cap analogs.

Disruption of the Extracellular Matrix Progressively Impairs Central Nervous System Vascular Maturation Downstream of ß-Catenin Signaling.

Objective- The Wnt/ß-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/ß-catenin signaling by induced overexpression of Axin1, an inhibitor of ß-catenin signaling, specifically in endothelial cells ( Axin1 iEC- OE). AOE (Axin1 overexpression) in Axin1 iEC- OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/ß-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC- OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, ß-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific ß-catenin-responsive ECM signature was also repressed in Axin1 iEC- OE and endothelial cell-specific ß-catenin-knockout mice ( Ctnnb1-KOiEC) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/ß-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-ß-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.

Trauma-Induced Nanohydroxyapatite Deposition in Skeletal Muscle is Sufficient to Drive Heterotopic Ossification.

Heterotopic ossification (HO), or the pathologic formation of bone within soft tissues, is a significant complication following severe injuries as it impairs joint motion and function leading to loss of the ability to perform activities of daily living and pain. While soft tissue injury is a prerequisite of developing HO, the exact molecular pathology leading to trauma-induced HO remains unknown. Through prior investigations aimed at identifying the causative factors of HO, it has been suggested that additional predisposing factors that favor ossification within the injured soft tissues environment are required. Considering that chondrocytes and osteoblasts initiate physiologic bone formation by depositing nanohydroxyapatite crystal into their extracellular environment, we investigated the hypothesis that deposition of nanohydroxyapatite within damaged skeletal muscle is likewise sufficient to predispose skeletal muscle to HO. Using a murine model genetically predisposed to nanohydroxyapatite deposition (ABCC6-deficient mice), we observed that following a focal muscle injury, nanohydroxyapatite was robustly deposited in a gene-dependent manner, yet resolved via macrophage-mediated regression over 28 days post injury. However, if macrophage-mediated regression was inhibited, we observed persistent nanohydroxyapatite that was sufficient to drive the formation of HO in 4/5 mice examined. Together, these results revealed a new paradigm by suggesting the persistent nanohydroxyapatite, referred to clinically as dystrophic calcification, and HO may be stages of a pathologic continuum, and not discrete events. As such, if confirmed clinically, these findings support the use of early therapeutic interventions aimed at preventing nanohydroxyapatite as a strategy to evade HO formation.

The Efficacy of Antivenin Latrodectus (Black Widow) Equine Immune F(ab')2 Versus Placebo in the Treatment of Latrodectism: A Randomized, Double-Blind, Placebo-Controlled, Clinical Trial.

STUDY OBJECTIVE: The antivenom currently available for treatment of systemic black widow envenomation (latrodectism) is composed of equine whole immunoglobin. Although considered effective, it has been associated with anaphylaxis and 2 reported fatalities. We test the efficacy and safety of new equine antivenom composed of purified F(ab')2 antibody fragments. METHODS: A randomized, double-blind, placebo-controlled trial was conducted at 16 sites across the United States. Subjects aged 10 years or older with moderate to severe pain because of black widow spider envenomation received F(ab')2 antivenom or placebo. The primary outcome measure was treatment failure, which was defined as failure to achieve and maintain clinically significant reduction in pain for 48 hours posttreatment. Secondary measures of pain intensity differences and summed pain intensity difference were computed. Adverse events were recorded. RESULTS: Sixty patients were treated (29 antivenom and 31 placebo). The mean age was 39 years and 68% were male. There were 15 treatment failures in the antivenom group and 24 in the placebo group (P=.019). Differences in pain intensity difference between groups were lower at each postbaseline point, and the mean summed pain intensity difference was greater for the antivenom group (difference 2,133; 95% confidence interval 177 to 4,090). No deaths or serious drug-related adverse events were detected. CONCLUSION: The F(ab')2 antivenom met the predefined primary outcome of reduced treatment failures. Secondary outcomes of pain intensity difference and summed pain intensity difference also supported efficacy. The rate of symptom improvement in the placebo group was higher than expected, which may be related to enrollment criteria or placebo effect.

Bayesian determination of the effect of a deep eutectic solvent on the structure of lipid monolayers.

In this work, we present the first example of the self-assembly of phospholipid monolayers at the interface between air and an ionic solvent. Deep eutectic solvents are a novel class of environmentally friendly, non-aqueous, room temperature liquids with tunable properties, that have wide-ranging potential applications and are capable of promoting the self-assembly of surfactant molecules. We use a chemically-consistent Bayesian modelling of X-ray and neutron reflectometry measurements to show that these monolayers broadly behave as they do on water. This method allows for the monolayer structure to be determined, alongside the molecular volumes of the individual monolayer components, without the need for water-specific constraints to be introduced. Furthermore, using this method we are able to better understand the correlations present between parameters in the analytical model. This example of a non-aqueous phospholipid monolayer has important implications for the potential uses of these solvents and for our understanding of how biomolecules behave in the absence of water.

GatewayNet: a form of sequential rule mining.

BACKGROUND: The gateway hypothesis (and particularly the prediction of developmental stages in drug abuse) has been a subject of protracted debate since the 1970s. Extensive research has gone into this subject, but has yielded contradictory findings. We propose an algorithm for detecting both association and causation relationships given a discrete sequence of events, which we believe will be useful in addressing the validity of the gateway hypothesis. To assess the gateway hypothesis, we developed the GatewayNet algorithm, a refinement of sequential rule mining called initiation rule mining. After a brief mathematical definition, we describe how to perform initiation rule mining and how to infer causal relationships from its rules ("gateway rules"). We tested GatewayNet against data for which relationships were known. After constructing a transaction database using a first-order Markov chain, we mined it to produce a gateway network. We then discuss various incarnations of the gateway network. We then evaluated the performance of GatewayNet on urine drug screening data collected from the emergency department at LSU Health Sciences Center in Shreveport. A de-identified database of urine drug screenings ordered by the department between August 1998 and June 2011 was collected and then restricted to patients having at least one screening succeeding their first positive drug screening result. RESULTS: In the synthetic data, a chain of gateway rules was found in the network which demonstrated causation. We did not find any evidence of gateway rules in the empirical data, but we were able to isolate two documented transitions into benzodiazepine use. CONCLUSIONS: We conclude that GatewayNet may show promise not only for substance use data, but other data involving sequences of events. We also express future goals for GatewayNet, including optimizing it for speed.