Impairment in Functioning and Quality of Life in Patients with Idiopathic Hypersomnia: The Real World Idiopathic Hypersomnia Outcomes Study (ARISE).

Purpose: Idiopathic hypersomnia is a debilitating neurologic sleep disorder characterized by excessive daytime sleepiness, sleep inertia, and prolonged sleep. Its impact on patients' quality of life and daily functioning has not been fully elucidated. The Real World Idiopathic Hypersomnia Outcomes Study (ARISE) evaluated the daily functioning, relationships, cognition, emotional well-being, and productivity/employment of participants with idiopathic hypersomnia. Patients and Methods: ARISE was a US-based virtual cross-sectional survey comprising multiple patient-reported outcome measures (Functional Outcomes of Sleep Questionnaire, short version [FOSQ-10], Quality of Life in Neurological Disorders [Neuro-QoL] Social Roles and Stigma domains, British Columbia Cognitive Complaints Inventory [BC-CCI], Patient Health Questionnaire [PHQ-9], and the Work Productivity and Activity Impairment Questionnaire: Specific Health Problem [WPAI:SHP]). Participants were adults 21-65 years of age with idiopathic hypersomnia. Data were analyzed for all participants and for subgroups with/without long sleep time (LST; self-reported sleep ≥11 hours in 24 hours). Results: Of 75 participants enrolled, most were female (81.3%) and the mean (SD) age was 34.1 (10.7) years. Participants' scores on the FOSQ-10 (mean [SD] score: 10.7 [2.8]) and the Neuro-QoL Social Roles (43.4 [4.2]) and Stigma (57.3 [5.9]) domains reflected impairments in daily functioning and quality of life. More than half of participants reported moderate to severe cognitive complaints (BC-CCI; 62.7%) and moderate to severe depressive symptoms (PHQ-9; 66.7%). Scores on the WPAI:SHP showed substantial impairments in absenteeism, presenteeism, overall work productivity, and overall regular daily activity (mean percent [SD]: 12.3 [23.6], 47.6 [22.7], 51.4 [24.7], and 64.0 [21.9], respectively). These considerable impairments were found in participants with and without LST. Conclusion: ARISE participants with idiopathic hypersomnia demonstrated poor quality of life and impaired functioning across multiple symptom domains.

Symptom Severity and Treatment Satisfaction in Patients with Idiopathic Hypersomnia: The Real World Idiopathic Hypersomnia Outcomes Study (ARISE).

Objective: Idiopathic hypersomnia is a debilitating sleep disorder characterized by excessive daytime sleepiness, sleep inertia, and prolonged sleep duration. The patient burden of idiopathic hypersomnia is poorly understood. The Real World Idiopathic Hypersomnia Outcomes Study (ARISE) evaluated symptoms and treatment effectiveness/satisfaction in participants with idiopathic hypersomnia. Methods: ARISE was a United States-based virtual cross-sectional survey. Participants were adults 21-65 years of age with idiopathic hypersomnia recruited from social media, the Hypersomnia Foundation website, and a patient panel. Self-assessments included the Epworth Sleepiness Scale (ESS), Idiopathic Hypersomnia Severity Scale (IHSS), Treatment Satisfaction Questionnaire for Medication, version II (TSQM-vII), and additional treatment questions. Data were analyzed for all participants and for subgroups with/without long sleep time (LST; ≥11 hours in 24 hours). Results: Of 75 participants enrolled, most were female (81.3%). The mean (SD) age was 34.1 (10.7) years and 49% had LST. Most participants took off-label prescription medications (89.3%) and/or used other measures (93.3%) to manage their symptoms. The mean (SD) ESS score was 14.5 (3.5) and the mean IHSS score was 35.2 (7.6). Treatment satisfaction was low (mean [SD] TSQM-vII score: overall, 61.9 [21.2]; with LST, 57.9 [21.4]; without LST, 66.7 [20.3]), primarily driven by dissatisfaction with treatment effectiveness. The most common classes of prescription medications used were stimulants (61.3%), wake-promoting agents (28.0%), and antidepressants (18.7%); non-prescription measures used to manage symptoms included caffeine (73.3%), planned naps (34.7%), and individual accommodations (32.0%). Conclusion: Overall, participants with idiopathic hypersomnia, with or without LST, had substantial symptom burden despite most of the study population taking off-label medications and using nonprescription measures to manage symptoms.

Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderia pseudomallei.

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se.

Lymphostatin, a virulence factor of attaching and effacing Escherichia coli, inhibits proliferation and cytokine responses of human T cells in a manner associated with cell cycle arrest but not apoptosis or necrosis.

Lymphostatin is a virulence factor of enteropathogenic E. coli (EPEC) and non-O157 serogroup enterohaemorrhagic E. coli. Previous studies using whole-cell lysates of EPEC showed that lymphostatin inhibits the mitogen-activated proliferation of bulk human peripheral blood mononuclear cells (PBMCs) and the production of cytokines IL-2, IL-4, IL-5, and IFN-γ. Here, we used highly purified lymphostatin and PBMC-derived T cells to show that lymphostatin inhibits anti-CD3/anti-CD28-activated proliferation of human CD4+ and CD8+ T cells and blocks the synthesis of IL-2, IL-4, IL-10 and IFN-γ without affecting cell viability and in a manner dependent on an N-terminal DTD glycosyltransferase motif. Such inhibition was not observed with T cells activated by phorbol 12-myristate 13-acetate and ionomycin, implying that lymphostatin targets T cell receptor signaling. Analysis of the expression of CD69 indicated that lymphostatin suppresses T cell activation at an early stage and no impacts on apoptosis or necrosis were observed. Flow cytometric analysis of the DNA content of lymphostatin-treated CD4+ and CD8+ T cells showed a concentration- and DTD-dependent accumulation of the cells in the G0/G1 phase of the cell cycle, and corresponding reduction of the percentage of cells in S phase. Consistent with this, we found a marked reduction in the abundance of cyclins D3, E and A and loss of phosphorylated Rb over time in activated T cells from 8 donors treated with lymphostatin. Moreover, the cyclin-dependent kinase (cdk) inhibitor p27kip1, which inhibits progression of the cell cycle at G1 by acting on cyclin E-cdk2 or cyclin D-cdk4 complexes, was found to be accumulated in lymphostatin-treated T cells. Analysis of the abundance of phosphorylated kinases involved in signal transduction found that 30 of 39 were reduced in abundance following lymphostatin treatment of T cells from 5 donors, albeit not significantly so. Our data provide novel insights into the mode of action of lymphostatin on human T lymphocytes.

The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis.

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's Disease, a chronic enteritis of ruminants prevalent across the world. It is estimated that approximately 50% of UK dairy herds are infected with MAP, but this is likely an underestimate of the true prevalence. Infection can result in reduced milk yield, infertility and premature culling of the animal, leading to significant losses to the farming economy and negatively affecting animal welfare. Understanding the initial interaction between MAP and the host is critical to develop improved diagnostic tools and novel vaccines. Here we describe the characterisation of three different multicellular in vitro models derived from bovine intestinal tissue, and their use for the study of cellular interactions with MAP. In addition to the previously described basal-out 3D bovine enteroids, we have established viable 2D monolayers and 3D apical-out organoids. The apical-out enteroids differ from previously described bovine enteroids as the apical surface is exposed on the exterior surface of the 3D structure, enabling study of host-pathogen interactions at the epithelial surface without the need for microinjection. We have characterised the cell types present in each model system using RT-qPCR to detect predicted cell type-specific gene expression, and confocal microscopy for cell type-specific protein expression. Each model contained the cells present in the original bovine intestinal tissue, confirming they were representative of the bovine gut. Exposure of the three model systems to the K10 reference strain of MAP K10, and a recent Scottish isolate referred to as C49, led to the observation of intracellular bacteria by confocal microscopy. Enumeration of the bacteria by quantification of genome copy number, indicated that K10 was less invasive than C49 at early time points in infection in all model systems. This study shows that bovine enteroid-based models are permissive to infection with MAP and that these models may be useful in investigating early stages of MAP pathogenesis in a physiologically relevant in vitro system, whilst reducing the use of animals in scientific research. Bos taurus: urn:lsid:zoobank.org:act:4C90C4FA-6296-4972-BE6A-5EF578677D64.

Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes.

Species A rotavirus (RVA) vaccines based on live attenuated viruses are used worldwide in humans. The recent establishment of a reverse genetics system for rotoviruses (RVs) has opened the possibility of engineering chimeric viruses expressing heterologous peptides from other viral or microbial species in order to develop polyvalent vaccines. We tested the feasibility of this concept by two approaches. First, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian RV SA11 strain viral protein (VP) 4. Second, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C terminus of nonstructural protein (NSP) 3 of the bovine RV RF strain, with or without an intervening Thosea asigna virus 2A (T2A) peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants, no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RV RF strain. Except for the RBD mutant that demonstrated a rescue defect, all NSP3 mutants delivered endpoint infectivity titers and exhibited replication kinetics comparable to that of the wild-type virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross-reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants, but cross-reactivity with SARS-CoV-2 RBD antibody was only detected for the RBM mutant. The tolerance of large SARS-CoV-2 peptide insertions at the C terminus of NSP3 in the presence of T2A element highlights the potential of this approach for the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCE We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an example. Small SARS-CoV-2 peptide insertions (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titer and replication, demonstrating a limited tolerance for peptide insertions at this site. To test the RV RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids in length. With a T2A-separated 193 amino acid tag on NSP3, there was no significant effect on the viral rescue efficiency, endpoint titer, and replication kinetics. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.

Pilot evaluation of a consumer wearable device to assess sleep in a clinical polysomnography trial of suvorexant for treating insomnia in patients with Alzheimer's disease.

The orexin receptor antagonist suvorexant was previously reported to significantly improve total sleep time (TST), by 28 min per night versus placebo after 4 weeks, in a sleep laboratory polysomnography (PSG) study of patients with Alzheimer's disease and insomnia. The study included an exploratory evaluation of a consumer-grade wearable "watch" device for assessing sleep that we report on here. Participants who met diagnostic criteria for both probable Alzheimer's disease dementia and insomnia were randomized to suvorexant 10-20 mg (N = 142) or placebo (N = 143) in a double-blind, 4-week trial. Patients were provided with a consumer-grade wearable watch device (Garmin vívosmart® HR) to be worn continuously. Overnight sleep laboratory PSG was performed on three nights: screening, baseline and Night 29 (last dose). Watch treatment effects were assessed by change-from-baseline in watch TST at Week 4 (average TST per night). We also analysed Night 29 data only, with watch data restricted to the PSG recording time. In the 193 participants included in the Week 4 watch analysis (suvorexant = 97, placebo = 96), the suvorexant-placebo difference in watch TST was 4 min (p = .622). In patients with usable data for both assessments at the baseline and Night 29 PSG (suvorexant = 57, placebo = 50), the watch overestimated TST compared to PSG (e.g., placebo baseline = 412 min for watch and 265 min for PSG) and underestimated change-from-baseline treatment effects: the suvorexant-placebo difference was 20 min for watch TST (p = .405) and 35 min for PSG TST (p = .057). These findings show that the watch was less sensitive than PSG for evaluating treatment effects on TST.

Detection and differentiation of Burkholderia species with pathogenic potential in environmental soil samples.

The Burkholderia pseudomallei phylogenetic cluster includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, B. humptydooensis and B. singularis. Regarded as the only pathogenic members of this group, B. pseudomallei and B. mallei cause the diseases melioidosis and glanders, respectively. Additionally, variant strains of B. pseudomallei and B. thailandensis exist that include the geographically restricted B. pseudomallei that express a B. mallei-like BimA protein (BPBM), and B. thailandensis that express a B. pseudomallei-like capsular polysaccharide (BTCV). To establish a PCR-based assay for the detection of pathogenic Burkholderia species or their variants, five PCR primers were designed to amplify species-specific sequences within the bimA (Burkholderia intracellular motility A) gene. Our multiplex PCR assay could distinguish pathogenic B. pseudomallei and BPBM from the non-pathogenic B. thailandensis and the BTCV strains. A second singleplex PCR successfully discriminated the BTCV from B. thailandensis. Apart from B. humptydooensis, specificity testing against other Burkholderia spp., as well as other Gram-negative and Gram-positive bacteria produced a negative result. The detection limit of the multiplex PCR in soil samples artificially spiked with known quantities of B. pseudomallei and B. thailandensis were 5 and 6 CFU/g soil, respectively. Furthermore, comparison between standard bacterial culture and the multiplex PCR to detect B. pseudomallei from 34 soil samples, collected from an endemic area of melioidosis, showed high sensitivity and specificity. This robust, sensitive, and specific PCR assay will be a useful tool for epidemiological study of B. pseudomallei and closely related members with pathogenic potential in soil.

Functional redundancy of Burkholderia pseudomallei phospholipase C enzymes and their role in virulence.

Phospholipase C (PLC) enzymes are key virulence factors in several pathogenic bacteria. Burkholderia pseudomallei, the causative agent of melioidosis, possesses at least three plc genes (plc1, plc2 and plc3). We found that in culture medium plc1 gene expression increased with increasing pH, whilst expression of the plc3 gene was pH (4.5 to 9.0) independent. Expression of the plc2 gene was not detected in culture medium. All three plc genes were expressed during macrophage infection by B. pseudomallei K96243. Comparing B. pseudomallei wild-type with plc mutants revealed that plc2, plc12 or plc123 mutants showed reduced intracellular survival in macrophages and reduced plaque formation in HeLa cells. However, plc1 or plc3 mutants showed no significant differences in plaque formation compared to wild-type bacteria. These findings suggest that Plc2, but not Plc1 or Plc3 are required for infection of host cells. In Galleria mellonella, plc1, plc2 or plc3 mutants were not attenuated compared to the wild-type strain, but multiple plc mutants showed reduced virulence. These findings indicate functional redundancy of the B. pseudomallei phospholipases in virulence.

Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction.

Burkholderia pseudomallei, the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5' end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis.

Polysomnographic assessment of suvorexant in patients with probable Alzheimer's disease dementia and insomnia: a randomized trial.

INTRODUCTION: We evaluated the clinical profile of the orexin receptor antagonist suvorexant for treating insomnia in patients with mild-to-moderate probable Alzheimer's disease (AD) dementia. METHODS: Randomized, double-blind, 4-week trial of suvorexant 10 mg (could be increased to 20 mg based on clinical response) or placebo in patients who met clinical diagnostic criteria for both probable AD dementia and insomnia. Sleep was assessed by overnight polysomnography in a sleep laboratory. The primary endpoint was change-from-baseline in polysomnography-derived total sleep time (TST) at week 4. RESULTS: Of 285 participants randomized (suvorexant, N = 142; placebo, N = 143), 277 (97%) completed the trial (suvorexant, N = 136; placebo, N = 141). At week 4, the model-based least squares mean improvement-from-baseline in TST was 73 minutes for suvorexant and 45 minutes for placebo; (difference = 28 minutes [95% confidence interval 11-45], p < 0.01). Somnolence was reported in 4.2% of suvorexant-treated patients and 1.4% of placebo-treated patients. DISCUSSION: Suvorexant improved TST in patients with probable AD dementia and insomnia.

Quantifying Mycobacterium avium subspecies paratuberculosis infection of bovine monocyte derived macrophages by confocal microscopy.

Quantification of Mycobacterium avium subspecies paratuberculosis (MAP) during in vitro infection experiments is challenging due to limitations of currently utilised methods, such as colony counting. Here we describe quantifying MAP infection of bovine macrophages (Mφ) using confocal microscopy. Bovine monocyte derived macrophages were infected with MAP at a high or low dose and the number of intracellular bacteria calculated at 2 h post infection using confocal microscopy. Bacteria within simultaneously infected Mφ were quantified by colony counting in order to compare confocal microscopy results with results obtained by an established method. Confocal microscopy provided a robust alternative quantification method that allowed for assessment of the infection at the individual Mφ level. This demonstrated that MAP infection was not homogeneous, and that there were higher numbers of both infected Mφ and intracellular bacteria and bacterial aggregates at the high dose compared to the low dose, potentially impacting the Mφ response to infection. Confocal microscopy can therefore provide a level of detail regarding the infection unobtainable by other quantification methods.

Interleukin 10 knock-down in bovine monocyte-derived macrophages has distinct effects during infection with two divergent strains of Mycobacterium bovis.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a cattle disease of global importance. M. bovis infects bovine macrophages (Mø) and subverts the host cell response to generate a suitable niche for survival and replication. We investigated the role of the anti-inflammatory cytokine interleukin (IL) 10 during in vitro infection of bovine monocyte-derived Mø (bMDM) with two divergent UK strains of M. bovis, which differentially modulate expression of IL10. The use of IL10-targeting siRNA revealed that IL10 inhibited the production of IL1B, IL6, tumour necrosis factor (TNF) and interferon gamma (IFNG) during infection of bMDM with the M. bovis strain G18. In contrast, IL10 only regulated a subset of these genes; TNF and IFNG, during infection with the M. bovis reference strain AF2122/97. Furthermore, nitric oxide (NO) production was modulated by IL10 during AF2122/97 infection, but not at the nitric oxide synthase 2 (NOS2) mRNA level, as observed during G18 infection. However, IL10 was found to promote survival of both M. bovis strains during early bMDM infection, but this effect disappeared after 24 h. The role of IL10-induced modulation of TNF, IFNG and NO production in M. bovis survival was investigated using siRNA targeting TNF, IFNG receptor 1 (IFNGR1) and NOS2. Knock-down of these genes individually did not promote survival of either M. bovis strain and therefore modulation of these genes does not account for the effect of IL10 on M. bovis survival. However, TNF knock-down was found to be detrimental to the survival of the M. bovis strain G18 during early infection. The results provide further evidence for the importance of IL10 during M. bovis infection of Mø. Furthermore, they highlight M. bovis strain specific differences in the interaction with the infected bMDM, which may influence the course of infection and progression of bovine TB.

Quantitative Proteomics Reveals Differences in the Response of Neutrophils Isolated from Healthy or Diabetic Subjects to Infection with Capsule-Variant Burkholderia thailandensis.

In Thailand, diabetes mellitus is the most significant risk factor for melioidosis, a severe disease caused by Burkholderia pseudomallei. In this study, neutrophils isolated from healthy or diabetic subjects were infected with B. thailandensis E555, a variant strain with a B. pseudomallei-like capsular polysaccharide used here as a surrogate micro-organism for B. pseudomallei. At 2 h post-infection, neutrophil proteins were subjected to 4-plex iTRAQ-based comparative proteomic analysis. A total of 341 proteins were identified in two or more samples, of which several proteins involved in oxidative stress and inflammation were enriched in infected diabetic neutrophils. We validated this finding by demonstrating that infected diabetic neutrophils generated significantly elevated levels of pro-inflammatory cytokines TNFα, IL-6, IL-1ß, and IL-17 compared to healthy neutrophils. Our data also revealed that infected neutrophils from healthy or diabetic individuals undergo apoptotic cell death at distinctly different rates, with infected diabetic neutrophils showing a diminished ability to delay apoptosis and an increased likelihood of undergoing a lytic form of cell death, compared to infected neutrophils from healthy individuals. Increased expression of inflammatory proteins by infected neutrophils could contribute to the increased susceptibility to infection and inflammation in diabetic patients in melioidosis-endemic areas.

Burkholderia pseudomallei BimC Is Required for Actin-Based Motility, Intracellular Survival, and Virulence.

The intracellular pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans and various animals, is capable of survival and movement within the cytoplasm of host cells by a process known as actin-based motility. The bacterial factor BimA is required for actin-based motility through its direct interaction with actin, and by mediating actin polymerization at a single pole of the bacterium to promote movement both within and between cells. However, little is known about the other bacterial proteins required for this process. Here, we have investigated the role of the bimC gene (bpss1491) which lies immediately upstream of the bimA gene (bpss1492) on the B. pseudomallei chromosome 2. Conserved amongst all B. pseudomallei, B. mallei and B. thailandensis strains sequenced to date, this gene encodes an iron-binding protein with homology to a group of proteins known as the bacterial autotransporter heptosyltransferase (BAHT) family. We have constructed a B. pseudomallei bimC deletion mutant and demonstrate that it is defective in intracellular survival in HeLa cells, but not in J774.1 macrophage-like cells. The bimC mutant is defective in cell to cell spread as demonstrated by ablation of plaque formation in HeLa cells, and by the inability to form multi-nucleated giant cells in J774.1 cells. These phenotypes in intracellular survival and cell to cell spread are not due to the loss of expression and polar localization of the BimA protein on the surface of intracellular bacteria, however they do correlate with an inability of the bacteria to recruit and polymerize actin. Furthermore, we also establish a role for bimC in virulence of B. pseudomallei using a Galleria mellonella larvae model of infection. Taken together, our findings indicate that B. pseudomallei BimC plays an important role in intracellular behavior and virulence of this emerging pathogen.

Determining the effective dose of street-level heroin: A new way to consider fluctuations in heroin purity, mass and potential contribution to overdose.

BACKGROUND & AIMS: Heroin use is associated with a disproportionately high level of morbidity and mortality with most deaths attributable to drug overdose. Aggregate heroin purity data has been used to examine the relationship between overdose and variability in street-level heroin, however heroin purity data alone may not be the most appropriate nor a sensitive enough measurement tool for this assessment. The aim of this study was to measure the variability in effective dose of street-level heroin seizures, accounting for variation in both purity and mass, and determine the proportion of samples with higher than expected effective dose that would not be detected using a purity-only measure. METHODS: Data on Victorian heroin seizures ≤150mg in mass made between 01/01/2012 and 31/12/2013 were obtained from the Victoria Police Forensic Services Department. The effective dose of heroin in each sample was determined by multiplying the mass and purity variables. Effective dose outlier samples were considered as those containing either greater than 1.5-2 times or >2 times the median effective dose of heroin for the sample data. RESULTS: The 983 street-level heroin samples of ≤150mg had a median mass of 92mg (IQR of 43mg), a median purity of 13% (range 3.6%-80.9%) and a median effective dose of 12.0mg of heroin (IQR 6.6mg; range 0.4mg-111mg). Approximately one in 13 samples (8%) and one in 17 samples (6%) contained between 1.5-2 times and >2 times the median effective dose of heroin respectively. CONCLUSION: The effective dose of heroin is a more appropriate measure than purity to identify outlier samples that containing larger than expected doses of heroin compared to typical doses that may be expected by users. Together with other identified risk factors, fluctuation in the effective dose of heroin contained in street-level samples may contribute to the potential for overdose.

BipC, a Predicted Burkholderia pseudomallei Type 3 Secretion System Translocator Protein with Actin Binding Activity.

Burkholderia pseudomallei is an intracellular bacterial pathogen and the causative agent of melioidosis, a severe disease of humans and animals. Like other clinically important Gram-negative bacteria, fundamental to B. pseudomallei pathogenesis is the Bsa Type III Secretion System. The Bsa system injects bacterial effector proteins into the cytoplasm of target host cells subverting cellular pathways for the benefit of the bacteria. It is required for invasion of non-phagocytic host cells, escape from the endocytic compartment into the host cell cytoplasm, and for virulence in murine models of melioidosis. We have recently described the repertoire of effector proteins secreted by the B. pseudomallei Bsa system, however the functions of many of these effector proteins remain an enigma. One such protein is BipC, a homolog of the translocator/effector proteins SipC and IpaC from Salmonella spp. and Shigella flexneri respectively. SipC and IpaC each have separate and distinct roles acting both as translocators, involved in creating a pore in the eukaryotic cell membrane through which effector proteins can transit, and as effectors by interacting with and polymerizing host cell actin. In this study, pull-down assays demonstrate an interaction between BipC and actin. Furthermore, we show that BipC directly interacts with actin, preferentially with actin polymers (F-actin) and has the ability to polymerize actin in a similar manner as that described for SipC. Yet unlike SipC, BipC does not stabilize F-actin filaments, indicating a functionally distinct interaction with actin. Expression of Myc-tagged BipC in HeLa cells induces the formation of pseudopodia similar to that seen for IpaC. This study explores the effector function of BipC and reveals that actin interaction is conserved within the BipC/SipC/IpaC family of translocator/effector proteins.

Type III Secretion in the Melioidosis Pathogen Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative intracellular pathogen and the causative agent of melioidosis, a severe disease of both humans and animals. Melioidosis is an emerging disease which is predicted to be vastly under-reported. Type III Secretion Systems (T3SSs) are critical virulence factors in Gram negative pathogens of plants and animals. The genome of B. pseudomallei encodes three T3SSs. T3SS-1 and -2, of which little is known, are homologous to Hrp2 secretion systems of the plant pathogens Ralstonia and Xanthomonas. T3SS-3 is better characterized and is homologous to the Inv/Mxi-Spa secretion systems of Salmonella spp. and Shigella flexneri, respectively. Upon entry into the host cell, B. pseudomallei requires T3SS-3 for efficient escape from the endosome. T3SS-3 is also required for full virulence in both hamster and murine models of infection. The regulatory cascade which controls T3SS-3 expression and the secretome of T3SS-3 have been described, as well as the effect of mutations of some of the structural proteins. Yet only a few effector proteins have been functionally characterized to date and very little work has been carried out to understand the hierarchy of assembly, secretion and temporal regulation of T3SS-3. This review aims to frame current knowledge of B. pseudomallei T3SSs in the context of other well characterized model T3SSs, particularly those of Salmonella and Shigella.

Investigation of orexin-2 selective receptor antagonists: Structural modifications resulting in dual orexin receptor antagonists.

In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX2R subtype and culminating in the discovery of 23, a highly potent, OX2R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX1R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.

Identification of Candidate Host Cell Factors Required for Actin-Based Motility of Burkholderia pseudomallei.

Intracellular actin-based motility of the melioidosis pathogen Burkholderia pseudomallei requires the bacterial factor BimA. Located at one pole of the bacterium, BimA recruits and polymerizes cellular actin to promote bacterial motility within and between cells. Here, we describe an affinity approach coupled with mass spectrometry to identify cellular proteins recruited to BimA-expressing bacteria under conditions that promote actin polymerization. We identified a group of cellular proteins that are recruited to the B. pseudomallei surface in a BimA-dependent manner, a subset of which were independently validated with specific antisera including the ubiquitous scaffold protein Ras GTPase-activating-like protein (IQGAP1). IQGAP1 integrates several key cellular signaling pathways including those involved in actin dynamics and has been shown to be involved in the adhesion of attaching and effacing Escherichia coli to infected cells and invasion of host cells by Salmonella enterica serovar Typhimurium. Although a direct interaction between BimA and IQGAP1 could not be detected using either conventional pulldown or yeast two hybrid techniques, confocal microscopy revealed that IQGAP1 is recruited to B. pseudomallei actin tails in infected cells, and siRNA-mediated knockdown highlighted a role for this protein in controlling the length and actin density of B. pseudomallei actin tails.