Endogenous Protein-Protein Interaction Network of the NPC Cholesterol Transporter 1 in the Cerebral Cortex.

NPC intracellular cholesterol transporter 1 (NPC1) is a multipass, transmembrane glycoprotein mostly recognized for its key role in facilitating cholesterol efflux. Mutations in the NPC1 gene result in Niemann-Pick disease, type C (NPC), a fatal, lysosomal storage disease. Due to the progressively expanding implications of NPC1-related disorders, we investigated endogenous NPC1 protein-protein interactions in the mouse cortex and human-derived iPSCs neuronal models of the disease through coimmunoprecipitation-coupled with LC-MS based proteomics. The current study investigated protein-protein interactions specific to the wild-type and the most prevalent NPC1 mutation (NPC1I1061T) while filtering out any protein interactor identified in the Npc1-/- mouse model. Additionally, the results were matched across the two species to map the parallel interactome of wild-type and mutant NPC1I1061T. Most of the identified wild-type NPC1 interactors were related to cytoskeleton organization, synaptic vesicle activity, and translation. We found many putative NPC1 interactors not previously reported, including two SCAR/WAVE complex proteins that regulate ARP 2/3 complex actin nucleation and multiple membrane proteins important for neuronal activity at synapse. Moreover, we identified proteins important in trafficking specific to wild-type and mutant NPC1I1061T. Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.

Predicting suicidal ideation in psychiatrically hospitalized veterans using the death/suicide Implicit Association Test: A prospective cohort study.

INTRODUCTION: We investigated whether the Death/Suicide Implicit Association Test (D/S-IAT) predicted suicidal ideation (SI) in psychiatric inpatients. METHODS: One hundred eighty veterans admitted for either SI or suicidal behavior (SB) (the primary sample) (N = 90) or alcohol detoxification (N = 90) completed the D/S-IAT and scales measuring SI. Correlation and regression coefficients were measured between the D/S-IAT (as a full-scale or dichotomized score [D > 0]) and self-reported current or imminent SI (over the next 1-3 days). RESULTS: In the primary sample, the full-scale D/S-IAT was significantly correlated with the intensity of current SI (r = 0.22, p = 0.04) and especially with wishes to be dead (r = 0.35, p < 0.001). The intensity of imminent SI was significantly predicted by the full-scale (p = 0.02) and dichotomized D/S-IAT score (p = 0.05) in a multiple regression model. However, no significant associations were observed when both the D/S-IAT score and current (present/absent) or imminent SI (occurred/did not occur) were dichotomous measures. In participants receiving alcohol detoxification, the D/S-IAT significantly predicted only wishes to be dead (r = 0.33, p < 0.001). CONCLUSION: The full-scale D/S-IAT score predicted the current intensity of wishes to be dead in both inpatient samples, and current and imminent SI in participants admitted for SI/SB. The dichotomized D/S-IAT score did not predict the simple occurrence of SI.

Apolipoprotein-mimetic nanodiscs reduce lipid accumulation and improve liver function in acid sphingomyelinase deficiency.

Acid sphingomyelinase deficiency (ASMD) is a severe lipid storage disorder caused by the diminished activity of the acid sphingomyelinase enzyme. ASMD is characterized by the accumulation of sphingomyelin in late endosomes and lysosomes leading to progressive neurological dysfunction and hepatosplenomegaly. Our objective was to investigate the utility of synthetic apolipoprotein A-I (ApoA-I) mimetics designed to act as lipid scavengers for the treatment of ASMD. We determined the lead peptide, 22A, could reduce sphingomyelin accumulation in ASMD patient skin fibroblasts in a dose dependent manner. Intraperitoneal administration of 22A formulated as a synthetic high-density lipoprotein (sHDL) nanodisc mobilized sphingomyelin from peripheral tissues into circulation and improved liver function in a mouse model of ASMD. Together, our data demonstrates that apolipoprotein mimetics could serve as a novel therapeutic strategy for modulating the pathology observed in ASMD.

An ISO-certified genomics workflow for identification and surveillance of antimicrobial resistance.

Realising the promise of genomics to revolutionise identification and surveillance of antimicrobial resistance (AMR) has been a long-standing challenge in clinical and public health microbiology. Here, we report the creation and validation of abritAMR, an ISO-certified bioinformatics platform for genomics-based bacterial AMR gene detection. The abritAMR platform utilises NCBI's AMRFinderPlus, as well as additional features that classify AMR determinants into antibiotic classes and provide customised reports. We validate abritAMR by comparing with PCR or reference genomes, representing 1500 different bacteria and 415 resistance alleles. In these analyses, abritAMR displays 99.9% accuracy, 97.9% sensitivity and 100% specificity. We also compared genomic predictions of phenotype for 864 Salmonella spp. against agar dilution results, showing 98.9% accuracy. The implementation of abritAMR in our institution has resulted in streamlined bioinformatics and reporting pathways, and has been readily updated and re-verified. The abritAMR tool and validation datasets are publicly available to assist laboratories everywhere harness the power of AMR genomics in professional practice.

Species-specific differences in NPC1 protein trafficking govern therapeutic response in Niemann-Pick type C disease.

The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and are compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, drug development efforts have been unsuccessful in mouse models. Here, we demonstrated unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We established that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the endoplasmic reticulum-folding environment. Moreover, we demonstrated direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum-folding environment by affecting Ca++ concentration. Finally, we developed a panel of isogenic human NPC1 iNeurons expressing WT, I1061T-, and R934L-NPC1 and demonstrated their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1's response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.

Epidemiology of carbapenem resistant Acinetobacter baumannii in New Zealand.

AIM: Carbapenem resistant Acinetobacter baumannii have limited treatment options and a propensity to cause hospital outbreaks. In recent years an increase in their detection has been observed in New Zealand. This study aimed to describe the molecular epidemiology of these isolates. METHOD: This study utilised carbapenem resistant A. baumannii complex isolates identified across New Zealand between January 2010 to April 2018. Whole genome sequence analysis and associated demographic information was used to contextualise local isolates within the global epidemiology and establish the relationship between isolates. RESULTS: Thirty-three carbapenem resistant A. baumannii complex isolates (31 A. baumannii sensu stricto) were identified. Twenty-four (73%) were from January 2015 onwards. Twenty-four (73%) had an identifiable epidemiological link to overseas hospitalisation. Twenty-three (74%) of 31 A. baumannii sensu stricto were sequence type (ST) 2 (Pasteur scheme). Phylogenetic analysis identified three ST2 clusters. The largest cluster, of 12 isolates, was from 2015 onwards; with nine (75%) associated with recent hospitalisation in Fiji or Samoa. CONCLUSION: Increasing numbers of carbapenem resistant A. baumannii are being identified in New Zealand. Our data show that this is in large part associated with transnational spread of a single A. baumannii sensu stricto ST 2 strain between Fiji, Samoa and New Zealand.

Loss of POGZ alters neural differentiation of human embryonic stem cells.

POGZ is a pogo transposable element derived protein with multiple zinc finger domains. Many de novo loss-of-function (LoF) variants of the POGZ gene are associated with autism and other neurodevelopmental disorders. However, the role of POGZ in human cortical development remains poorly understood. Here we generated multiple POGZ LoF lines in H9 human embryonic stem cells (hESCs) using CRISPR/CAS9 genome editing. These lines were then differentiated into neural structures, similar to those found in early to mid-fetal human brain, a critical developmental stage for studying disease mechanisms of neurodevelopmental disorders. We found that the loss of POGZ reduced neural stem cell proliferation in excitatory cortex-patterned neural rosettes, structures analogous to the cortical ventricular zone in human fetal brain. As a result, fewer intermediate progenitor cells and early born neurons were generated. In addition, neuronal migration from the apical center to the basal surface of neural rosettes was perturbed due to the loss of POGZ. Furthermore, cortical-like excitatory neurons derived from multiple POGZ homozygous knockout lines exhibited a more simplified dendritic architecture compared to wild type lines. Our findings demonstrate how POGZ regulates early neurodevelopment in the context of human cells, and provide further understanding of the cellular pathogenesis of neurodevelopmental disorders associated with POGZ variants.

Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy, improves lysosomal function, and reduces cholesterol storage.

Niemann-Pick C (NPC) is an autosomal recessive disorder characterized by mutations in the NPC1 or NPC2 genes encoding endolysosomal lipid transport proteins, leading to cholesterol accumulation and autophagy dysfunction. We have previously shown that enrichment of NPC1-deficient cells with the anionic lipid lysobisphosphatidic acid (LBPA; also called bis(monoacylglycerol)phosphate) via treatment with its precursor phosphatidylglycerol (PG) results in a dramatic decrease in cholesterol storage. However, the mechanisms underlying this reduction are unknown. In the present study, we showed using biochemical and imaging approaches in both NPC1-deficient cellular models and an NPC1 mouse model that PG incubation/LBPA enrichment significantly improved the compromised autophagic flux associated with NPC1 disease, providing a route for NPC1-independent endolysosomal cholesterol mobilization. PG/LBPA enrichment specifically enhanced the late stages of autophagy, and effects were mediated by activation of the lysosomal enzyme acid sphingomyelinase. PG incubation also led to robust and specific increases in LBPA species with polyunsaturated acyl chains, potentially increasing the propensity for membrane fusion events, which are critical for late-stage autophagy progression. Finally, we demonstrated that PG/LBPA treatment efficiently cleared cholesterol and toxic protein aggregates in Purkinje neurons of the NPC1I1061T mouse model. Collectively, these findings provide a mechanistic basis supporting cellular LBPA as a potential new target for therapeutic intervention in NPC disease.

Explaining Service Use and Residential Stability in Supported Housing: Problems, Preferences, Peers.

BACKGROUND: The behavioral model of health service use identified health needs, service preferences (predispositions), and service availability (enabling factors) as important predictors, but research has not conceptualized consistently each type of influence nor identified their separate effects on use of substance abuse and mental health services or their value in predicting service outcomes. OBJECTIVES: To test hypotheses predicting use of substance abuse and mental health services and residential stability and evaluate peer specialists' impact. RESEARCH DESIGN: Randomized trial of peer support added to standard case management in VA-supported housing program (Housing and Urban Development-VA Supportive Housing program). SUBJECTS: One hundred sixty-six dually diagnosed Veterans in Housing and Urban Development-VA Supportive Housing program in 2 cities. MEASURES: Average VA service episodes for substance abuse and mental illness; residential instability; preferences for alcohol, drug, and psychological services; extent of alcohol, drug, and psychological problems; availability of a peer specialist. RESULTS: Self-assessed health needs, mediated by service preferences, and assignment to a peer specialist predicted use of VA behavioral health services and residential stability, as did chronic medical problems, sex, and race. CONCLUSIONS: The behavioral model identifies major predictors of health service use and residential stability, but must recognize the mediating role of service preferences, the differing effects of alcohol and drug use, the unique influences of social background, and the importance of clinical judgment in needs assessment. Service availability and residential stability can be increased by proactive efforts involving peer specialists even in a health care system that provides services without a financial barrier.

Treatment of acetylcholinesterase inhibitor-induced seizures with polytherapy targeting GABA and glutamate receptors.

The initiation and maintenance of cholinergic-induced status epilepticus (SE) are associated with decreased synaptic gamma-aminobutyric acid A receptors (GABAAR) and increased N-methyl-d-aspartate receptors (NMDAR) and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). We hypothesized that trafficking of synaptic GABAAR and glutamate receptors is maladaptive and contributes to the pharmacoresistance to antiseizure drugs; targeting these components should ameliorate the pathophysiological consequences of refractory SE (RSE). We review studies of rodent models of cholinergic-induced SE, in which we used a benzodiazepine allosteric GABAAR modulator to correct loss of inhibition, concurrent with the NMDA antagonist ketamine to reduce excitation caused by increased synaptic localization of NMDAR and AMPAR, which are NMDAR-dependent. Models included lithium/pilocarpine-induced SE in rats and soman-induced SE in rats and in Es1-/- mice, which similar to humans lack plasma carboxylesterase, and may better model soman toxicity. These model human soman toxicity and are refractory to benzodiazepines administered at 40 min after seizure onset, when enough synaptic GABAAR may not be available to restore inhibition. Ketamine-midazolam combination reduces seizure severity, epileptogenesis, performance deficits and neuropathology following cholinergic-induced SE. Supplementing that treatment with valproate, which targets a non-benzodiazepine site, effectively terminates RSE, providing further benefit against cholinergic-induced SE. The therapeutic index of drug combinations is also reviewed and we show the improved efficacy of simultaneous administration of midazolam, ketamine and valproate compared to sequential drug administration. These data suggest that future clinical trials should treat both the lack of sufficient inhibition and the excess excitation that characterize RSE, and include early combination drug therapies. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

Health service preferences among veterans in supported housing in relation to needs expressed and services used.

BACKGROUND: Understanding consumer service preferences is important for recovery-oriented care. AIMS: To test the influence of perceived service needs on importance attached to treatment for alcohol, drug, mental health, and physical health problems and identify the influence of service needs and preferences on service use. METHODS: Formerly homeless dually diagnosed Veterans in supported housing were surveyed in three waves for 1 year, with measures of treatment interests, health problems, social support, clinician-assessed risk of housing loss, and sociodemographics. Multiple regression analysis was used to identify independent influences on preferences in each wave. Different health services at the VA were distinguished in administrative records and baseline predictors for services used throughout the project were identified with multiple regression analysis. RESULTS: Self-assessed problem severity was associated with the importance of treatment for alcohol, drug, mental health, and physical health problems. Social support also had some association with treatment interest for alcohol abuse, as did baseline clinician risk rating at the project's end. Preferences, but not perceived problem severity, predicted the use of the corresponding health services. CONCLUSIONS: The health beliefs model of service interests was supported, but more integrated service delivery models may be needed to strengthen the association of health needs with service use.

Search and Contain: Impact of an Integrated Genomic and Epidemiological Surveillance and Response Program for Control of Carbapenemase-producing Enterobacterales.

BACKGROUND: Multiresistant organisms (MROs) pose a critical threat to public health. Population-based programs for control of MROs such as carbapenemase-producing Enterobacterales (CPE) have emerged and evaluation is needed. We assessed the feasibility and impact of a statewide CPE surveillance and response program deployed across Victoria, Australia (population 6.5 million). METHODS: A prospective multimodal intervention including active screening, carrier isolation, centralized case investigation, and comparative pathogen genomics was implemented. We analyzed trends in CPE incidence and clinical presentation, risk factors, and local transmission over the program's first 3 years (2016-2018). RESULTS: CPE case ascertainment increased over the study period to 1.42 cases/100 000 population, linked to increased screening without a concomitant rise in active clinical infections (0.45-0.60 infections/100 000 population, P = .640). KPC-2 infection decreased from 0.29 infections/100 000 population prior to intervention to 0.03 infections/100 000 population in 2018 (P = .003). Comprehensive case investigation identified instances of overseas community acquisition. Median time between isolate referral and genomic and epidemiological assessment for local transmission was 11 days (IQR, 9-14). Prospective surveillance identified numerous small transmission networks (median, 2; range, 1-19 cases), predominantly IMP and KPC, with median pairwise distance of 8 (IQR, 4-13) single nucleotide polymorphisms; low diversity between clusters of the same sequence type suggested genomic cluster definitions alone are insufficient for targeted response. CONCLUSIONS: We demonstrate the value of centralized CPE control programs to increase case ascertainment, resolve risk factors, and identify local transmission through prospective genomic and epidemiological surveillance; methodologies are transferable to low-prevalence settings and MROs globally.

Tracking the COVID-19 pandemic in Australia using genomics.

Genomic sequencing has significant potential to inform public health management for SARS-CoV-2. Here we report high-throughput genomics for SARS-CoV-2, sequencing 80% of cases in Victoria, Australia (population 6.24 million) between 6 January and 14 April 2020 (total 1,333 COVID-19 cases). We integrate epidemiological, genomic and phylodynamic data to identify clusters and impact of interventions. The global diversity of SARS-CoV-2 is represented, consistent with multiple importations. Seventy-six distinct genomic clusters were identified, including large clusters associated with social venues, healthcare and cruise ships. Sequencing sequential samples from 98 patients reveals minimal intra-patient SARS-CoV-2 genomic diversity. Phylodynamic modelling indicates a significant reduction in the effective viral reproductive number (Re) from 1.63 to 0.48 after implementing travel restrictions and physical distancing. Our data provide a concrete framework for the use of SARS-CoV-2 genomics in public health responses, including its use to rapidly identify SARS-CoV-2 transmission chains, increasingly important as social restrictions ease globally.

Fbxo2 mediates clearance of damaged lysosomes and modifies neurodegeneration in the Niemann-Pick C brain.

A critical response to lysosomal membrane permeabilization (LMP) is the clearance of damaged lysosomes through a selective form of macroautophagy known as lysophagy. Although regulators of this process are emerging, whether organ- and cell-specific components contribute to the control of lysophagy remains incompletely understood. Here, we examined LMP and lysophagy in Niemann-Pick type C (NPC) disease, an autosomal recessive disorder characterized by the accumulation of unesterified cholesterol within late endosomes and lysosomes, leading to neurodegeneration and early death. We demonstrated that NPC human fibroblasts show enhanced sensitivity to lysosomal damage as a consequence of lipid storage. Moreover, we described a role for the glycan-binding F-box protein 2 (Fbxo2) in CNS lysophagy. Fbxo2 functions as a component of the S phase kinase-associated protein 1-cullin 1-F-box protein (SKP1-CUL1-SCF) ubiquitin ligase complex. Loss of Fbxo2 in mouse primary cortical cultures delayed clearance of damaged lysosomes and decreased viability after lysosomal damage. Moreover, Fbxo2 deficiency in a mouse model of NPC exacerbated deficits in motor function, enhanced neurodegeneration, and reduced survival. Collectively, our data identified a role for Fbxo2 in CNS lysophagy and establish its functional importance in NPC.

Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA.

Introduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals.Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (sd±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 ml-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.

Isolation and rapid sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia.

OBJECTIVES: To describe the first isolation and sequencing of SARS-CoV-2 in Australia and rapid sharing of the isolate. SETTING: SARS-CoV-2 was isolated from a 58-year-old man from Wuhan, China who arrived in Melbourne on 19 January 2020 and was admitted to the Monash Medical Centre, Melbourne from the emergency department on 24 January 2020 with fever, cough, and progressive dyspnoea. MAJOR OUTCOMES: Clinical course and laboratory features of the first reported case of COVID-19 (the illness caused by SARS-CoV-2) in Australia; isolation, whole genome sequencing, imaging, and rapid sharing of virus from the patient. RESULTS: A nasopharyngeal swab and sputum collected when the patient presented to hospital were each positive for SARS-CoV-2 (reverse transcription polymerase chain reaction). Inoculation of Vero/hSLAM cells with material from the nasopharyngeal swab led to the isolation of SARS-CoV-2 virus in culture. Electron microscopy of the supernatant confirmed the presence of virus particles with morphology characteristic of viruses of the family Coronaviridae. Whole genome sequencing of the viral isolate and phylogenetic analysis indicated the isolate exhibited greater than 99.99% sequence identity with other publicly available SARS-CoV-2 genomes. Within 24 hours of isolation, the first Australian SARS-CoV-2 isolate was shared with local and overseas reference laboratories and major North American and European culture collections. CONCLUSIONS: The ability to rapidly identify, propagate, and internationally share our SARS-CoV-2 isolate is an important step in collaborative scientific efforts to deal effectively with this international public health emergency by developing better diagnostic procedures, vaccine candidates, and antiviral agents.

Impact of Peer Specialist Services on Residential Stability and Behavioral Health Status Among Formerly Homeless Veterans With Cooccurring Mental Health and Substance Use Conditions.

OBJECTIVES: This study tested the impacts of peer specialists on housing stability, substance abuse, and mental health status for previously homeless Veterans with cooccurring mental health issues and substance abuse. METHODS: Veterans living in the US Housing and Urban Development-Veterans Administration Supported Housing (HUD-VASH) program were randomized to peer specialist services that worked independently from HUD-VASH case managers (ie, not part of a case manager/peer specialist dyad) and to treatment as usual that included case management services. Peer specialist services were community-based, using a structured curriculum for recovery with up to 40 weekly sessions. Standardized self-report measures were collected at 3 timepoints. The intent-to-treat analysis tested treatment effects using a generalized additive mixed-effects model that allows for different nonlinear relationships between outcomes and time for treatment and control groups. A secondary analysis was conducted for Veterans who received services from peer specialists that were adherent to the intervention protocol. RESULTS: Treated Veterans did not spend more days in housing compared with control Veterans during any part of the study at the 95% level of confidence. Veterans assigned to protocol adherent peer specialists showed greater housing stability between about 400 and 800 days postbaseline. Neither analysis detected significant effects for the behavioral health measures. CONCLUSIONS: Some impact of peer specialist services was found for housing stability but not for behavioral health problems. Future studies may need more sensitive measures for early steps in recovery and may need longer time frames to effectively impact this highly challenged population.

Rational polytherapy in the treatment of cholinergic seizures.

The initiation and maintenance phases of cholinergic status epilepticus (SE) are associated with maladaptive trafficking of synaptic GABAA and glutamate receptors. The resulting pharmacoresistance reflects a decrease in synaptic GABAA receptors and increase in NMDA and AMPA receptors, which tilt the balance between inhibition and excitation in favor of the latter. If these changes are important to the pathophysiology of SE, both should be treated, and blocking their consequences should have therapeutic potential. We used a model of benzodiazepine-refractory SE (RSE) (Tetz et al., 2006) and a model of soman-induced SE to test this hypothesis. Treatment of RSE with combinations of the GABAAR agonists midazolam or diazepam and the NMDAR antagonists MK-801 or ketamine terminated RSE unresponsive to high-dose monotherapy with benzodiazepines, ketamine or other antiepileptic drugs (AEDs). It also reduced RSE-associated neuronal injury, spatial memory deficits and the occurrence of spontaneous recurrent seizures (SRS), tested several weeks after SE. Treatment of sc soman-induced SE similarly showed much greater reduction of EEG power by a combination of midazolam with ketamine, compared to midazolam monotherapy. When treating late (40 min after seizure onset), there may not be enough synaptic GABAAR left to be able to restore inhibition with maximal GABAAR stimulation, and further benefit is derived from the addition of an AED which increases inhibition or reduces excitation by a non-GABAergic mechanism. The midazolam-ketamine-valproate combination is effective in terminating RSE. 3-D isobolograms demonstrate positive cooperativity between midazolam, ketamine and valproate, without any interaction between the toxicity of these drugs, so that the therapeutic index is increased by combination therapy between GABAAR agonist, NMDAR antagonist and selective AEDs. We compared this drug combination based on the receptor trafficking hypothesis to treatments based on clinical practice. The midazolam-ketamine-valproate combination is far more effective in stopping RSE than the midazolam-fosphenytoin-valproate combination inspired from clinical guidelines. Furthermore, sequential administration of midazolam, ketamine and valproate is far less effective than simultaneous treatment with the same drugs at the same dose. These data suggest that we should re-evaluate our traditional treatment of RSE, and that treatment should be based on pathophysiology. The search for a better drug has to deal with the fact that most monotherapy leaves half the problem untreated. The search for a better benzodiazepine should acknowledge the main cause of pharmacoresistance, which is loss of synaptic GABAAR. Future clinical trials should consider treating both the failure of inhibition and the runaway excitation which characterize RSE, and should include an early polytherapy arm.

Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann-Pick diseases.

BACKGROUND: Niemann-Pick disease type C is a fatal and progressive neurodegenerative disorder characterized by the accumulation of unesterified cholesterol in late endosomes and lysosomes. We sought to develop new therapeutics for this disorder by harnessing the body's endogenous cholesterol scavenging particle, high-density lipoprotein (HDL). METHODS: Here we design, optimize, and define the mechanism of action of synthetic HDL (sHDL) nanoparticles. RESULTS: We demonstrate a dose-dependent rescue of cholesterol storage that is sensitive to sHDL lipid and peptide composition, enabling the identification of compounds with a range of therapeutic potency. Peripheral administration of sHDL to Npc1 I1061T homozygous mice mobilizes cholesterol, reduces serum bilirubin, reduces liver macrophage size, and corrects body weight deficits. Additionally, a single intraventricular injection into adult Npc1 I1061T brains significantly reduces cholesterol storage in Purkinje neurons. Since endogenous HDL is also a carrier of sphingomyelin, we tested the same sHDL formulation in the sphingomyelin storage disease Niemann-Pick type A. Utilizing stimulated Raman scattering microscopy to detect endogenous unlabeled lipids, we show significant rescue of Niemann-Pick type A lipid storage. CONCLUSIONS: Together, our data establish that sHDL nanoparticles are a potential new therapeutic avenue for Niemann-Pick diseases.