Factors influencing the successful implementation of a novel digital health application to streamline multidisciplinary communication across multiple organisations for emergency care.

RATIONALE: Delivering optimal patient health care requires interdisciplinary clinician communication. A single communication tool across multiple pre-hospital and hospital settings, and between hospital departments is a novel solution to current systems. Fit-for-purpose, secure smartphone applications allow clinical information to be shared quickly between health providers. Little is known as to what underpins their successful implementation in an emergency care context. AIMS: To identify (a) whether implementing a single, digital health communication application across multiple health care organisations and hospital departments is feasible; (b) the barriers and facilitators to implementation; and (c) which factors are associated with clinicians' intentions to use the technology. METHODS: We used a multimethod design, evaluating the implementation of a secure, digital communication application (Pulsara™). The technology was trialled in two Australian regional hospitals and 25 Ambulance Victoria branches (AV). Post-training, clinicians involved in treating patients with suspected stroke or cardiac events were administered surveys measuring perceived organisational readiness (Organisational Readiness for Implementing Change), clinicians' intentions (Unified Theory of Acceptance and Use of Technology) and internal motivations (Self-Determination Theory) to use Pulsara™, and the perceived benefits and barriers of use. Quantitative data were descriptively summarised with multivariable associations between factors and intentions to use Pulsara™ examined with linear regression. Qualitative data responses were subjected to directed content analysis (two coders). RESULTS: Participants were paramedics (n = 82, median 44 years) or hospital-based clinicians (n = 90, median 37 years), with organisations perceived to be similarly ready. Regression results (F(11, 136) = 21.28, p = <0.001, Adj R2 = 0.60) indicated Habit, Effort Expectancy, Perceived Organisational Readiness, Performance Expectancy and Organisation membership (AV) as predictors of intending to use Pulsara™. Themes relating to benefits (95% coder agreement) included improved communication, procedural efficiencies and faster patient care. Barriers (92% coder agreement) included network accessibility and remembering passwords. PulsaraTM was initiated 562 times. CONCLUSION: Implementing multiorganisational, digital health communication applications is feasible, and facilitated when organisations are change-ready for an easy-to-use, effective solution. Developing habitual use is key, supported through implementation strategies (e.g., hands-on training). Benefits should be emphasised (e.g., during education sessions), including streamlining communication and patient flow, and barriers addressed (e.g., identify champions and local technical support) at project commencement.

Transgenic overexpression of heat shock protein (HSP83) enhances protein kinase A activity, disrupts GP63 surface protease expression and alters promastigote morphology in Leishmania amazonensis.

Leishmania parasites undergo morphological changes during their infectious life cycle, including developmental transitions within the sandfly vector, culminating in metacyclic stages that are pre-adapted for infection. Upon entering vertebrate host phagocytes, Leishmania differentiate into intracellular amastigotes, the form that is ultimately transmitted back to the vector to complete the life cycle. Although environmental conditions that induce these cellular transitions are well-established, molecular mechanisms governing Leishmania morphologic differentiation in response to these cues remain largely uncharacterized. Previous studies indicate a key role for HSP83 in both promastigote metacyclogenesis and amastigote differentiation. To further elucidate HSP83 functions in the Leishmania lifecycle, we examined the biological impact of experimentally elevating HSP83 gene expression in Leishmania. Significantly, HSP83 overexpression was associated with altered metacyclic morphology, increased protein kinase A (PKA) activity and decreased expression of the Leishmania major surface protease, GP63. Corroborating these findings, overexpression of the L. amazonensis PKA catalytic subunit resulted in a largely similar phenotype. Our findings demonstrate for the first time in Leishmania, a functional link between HSP83 and PKA in the control of Leishmania gene expression, replication and morphogenesis.

Infantile metastatic ependymoma with a novel molecular profile and favorable outcome to intensive chemotherapy without irradiation: Case-based review.

Ependymal tumors are the third most common brain tumor under 14 years old. Even though metastatic disease is a rare event, it affects mostly young children and carries an adverse prognosis. The factors associated with dissemination and the best treatment approach have not yet been established and there is limited published data on how to manage metastatic disease, especially in patients under 3 years of age. We provide a review of the literature on clinical characteristics and radiation-sparing treatments for metastatic ependymoma in children under 3 years of age treated. The majority (73%) of the identified cases were above 12 months old and had the PF as the primary site at diagnosis. Chemotherapy-based approaches, in different regimens, were used with radiation reserved for progression or relapse. The prognosis varied among the studies, with an average of 50%-58% overall survival. This study also describes the case of a 7-month-old boy with metastatic posterior fossa (PF) ependymoma, for whom we identified a novel SPECC1L-RAF1 gene fusion using a patient-centric comprehensive molecular profiling protocol. The patient was successfully treated with intensive induction chemotherapy followed by high-dose chemotherapy and autologous hematopoietic progenitor cell rescue (AuHSCR). Currently, the patient is in continuous remission 5 years after his diagnosis, without radiation therapy. The understanding of the available therapeutic approaches may assist physicians in their management of such patients. This report also opens the perspective of newly identified molecular alterations in metastatic ependymomas that might drive more chemo-sensitive tumors.

Real-world, feasibility study to investigate the use of a multidisciplinary app (Pulsara) to improve prehospital communication and timelines for acute stroke/STEMI care.

OBJECTIVES: To determine if a digital communication app improves care timelines for patients with suspected acute stroke/ST-elevation myocardial infarction (STEMI). DESIGN: Real-world feasibility study, quasi-experimental design. SETTING: Prehospital (25 Ambulance Victoria branches) and within-hospital (2 hospitals) in regional Victoria, Australia. PARTICIPANTS: Paramedics or emergency department (ED) clinicians identified patients with suspected acute stroke (onset <4.5 hours; n=604) or STEMI (n=247). INTERVENTION: The Pulsara communication app provides secure, two-way, real-time communication. Assessment and treatment times were recorded for 12 months (May 2017-April 2018), with timelines compared between 'Pulsara initiated' (Pulsara) and 'not initiated' (no Pulsara). PRIMARY OUTCOME MEASURE: Door-to-treatment (needle for stroke, balloon for STEMI) Secondary outcome measures: ambulance and hospital processes. RESULTS: Stroke (no Pulsara n=215, Pulsara n=389) and STEMI (no Pulsara n=76, Pulsara n=171) groups were of similar age and sex (stroke: 76 vs 75 years; both groups 50% male; STEMI: 66 vs 63 years; 68% and 72% male). When Pulsara was used, patients were off ambulance stretcher faster for stroke (11(7, 17) vs 19(11, 29); p=0.0001) and STEMI (14(7, 23) vs 19(10, 32); p=0.0014). ED door-to-first medical review was faster (6(2, 14) vs 23(8, 67); p=0.0001) for stroke but only by 1 min for STEMI (3 (0, 7) vs 4 (0, 14); p=0.25). Door-to-CT times were 44 min faster (27(18, 44) vs 71(43, 147); p=0.0001) for stroke, and percutaneous intervention door-to-balloon times improved by 17 min, but non-significant (56 (34, 88) vs 73 (49, 110); p=0.41) for STEMI. There were improvements in the proportions of patients treated within 60 min for stroke (12%-26%, p=0.15) and 90 min for STEMI (50%-78%, p=0.20). CONCLUSIONS: In this Australian-first study, uptake of the digital communication app was strong, patient-centred care timelines improved, although door-to-treatment times remained similar.

Somatic variation as an incidental finding in the pediatric next-generation sequencing era.

The methodologic approach used in next-generation sequencing (NGS) affords a high depth of coverage in genomic analysis. Inherent in the nature of genomic testing, there exists potential for identifying genomic findings that are incidental or secondary to the indication for clinical testing, with the frequency dependent on the breadth of analysis and the tissue sample under study. The interpretation and management of clinically meaningful incidental genomic findings is a pressing issue particularly in the pediatric population. Our study describes a 16-mo-old male who presented with profound global delays, brain abnormality, progressive microcephaly, and growth deficiency, as well as metopic craniosynostosis. Clinical exome sequencing (ES) trio analysis revealed the presence of two variants in the proband. The first was a de novo variant in the PPP2R1A gene (c.773G > A, p.Arg258His), which is associated with autosomal dominant (AD) intellectual disability, accounting for the proband's clinical phenotype. The second was a recurrent hotspot variant in the CBL gene (c.1111T > C, p.Tyr371His), which was present at a variant allele fraction of 11%, consistent with somatic variation in the peripheral blood sample. Germline pathogenic variants in CBL are associated with AD Noonan syndrome-like disorder with or without juvenile myelomonocytic leukemia. Molecular analyses using a different tissue source, buccal epithelial cells, suggest that the CBL alteration may represent a clonal population of cells restricted to leukocytes. This report highlights the laboratory methodologic and interpretative processes and clinical considerations in the setting of acquired variation detected during clinical ES in a pediatric patient.

Dynamic modulation of Leishmania cytochrome c oxidase subunit IV (LmCOX4) expression in response to mammalian temperature.

The Leishmania LACK antigen is a ribosome-associated protein that facilitates expression of mitochondrial cytochrome c oxidase subunit IV (LmCOX4) to support parasite mitochondrial fitness and virulence within the vertebrate host. To further examine the relationship between LACK, its putative ribosome binding motif and LmCOX4, we compared the kinetics of LmCOX4 expression following temperature elevation in wildtype LACK (LACK WT) and LACK-putative ribosome-binding mutant (LACKDDE) L. major. We found that, after initial exposure to mammalian temperature, LmCOX4 levels became undetectable in LACKDDE L. major and also, surprisingly, in wild type (WT) control strains. Upon sustained exposure to mammalian temperature, LmCOX4 expression returned in WT control strains only. The initial loss of LmCOX4 in WT L. major was substantially reversed by treatment with the proteasome inhibitor MG132. Our findings indicate that initial loss of LmCOX4 under mammalian conditions is dependent upon proteasome degradation and LmCOX4 re-expression is dependent upon LACK possessing a WT putative ribosome binding motif.

Transcriptomic Analysis of Extracellular RNA Governed by the Endocytic Adaptor Protein Cin1 of Cryptococcus deneoformans.

Membrane vesicles are considered virulence cargoes as they carry capsular and melanin components whose secretory transport is critical for the virulence of the human fungal pathogen Cryptococcus species. However, other components of the vesicles and their function in the growth and virulence of the fungus remain unclear. We have previously found that the cryptococcal intersectin protein Cin1 governs a unique Cin1-Wsp1-Cdc42 endocytic pathway required for intracellular transport and virulence. Using RNA sequencing, we compared the profiles of extracellular RNA (exRNA), including microRNA (miRNA), small interference RNA (siRNA), long noncoding RNA (lncRNA), and messenger RNA (mRNA) between the wild-type (WT), and derived Δcin1 mutant strains of Cryptococcus deneoformans. Seven hundred twelve miRNAs and 88 siRNAs were identified from WT, whereas 799 miRNAs and 66 siRNAs were found in Δcin1. Also, 572 lncRNAs and 7,721 mRNAs were identified from WT and 584 lncRNAs and 7,703 mRNAs from Δcin1. Differential expression analysis revealed that the disruption of CIN1 results in many important cellular changes, including those in exRNA expression, transport, and function. First, for miRNA target genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that cellular processes, components, and macromolecular functions are the most affected pathways. A higher number of genes were involved in the intracellular transport of endocytosis. Second, the results of GO term and KEGG analysis of differentially expressed lncRNA target genes and mRNA genes were consistent with those of miRNA targets. In particular, protein export is the topmost affected pathway among lncRNA target genes and one of the affected pathways among mRNA genes. The result of quantitative real-time reverse transcription PCR (qRT-PCR) from 12 mRNAs tested is largely agreeable with that of RNA-Seq. Taken together, our studies provide a comprehensive reference that Cryptococcus secretes abundant RNAs and that Cin1 plays a critical role in regulating their secretion. Given the growing clinical importance of exRNAs, our studies illuminate the significance of exploring this cutting-edge technology in studies of cryptococcal pathogenesis for the discovery of novel therapeutic strategies.

Whole Exome Sequencing Identifies Candidate Genes Associated with Hereditary Predisposition to Uveal Melanoma.

PURPOSE: To identify susceptibility genes associated with hereditary predisposition to uveal melanoma (UM) in patients with no detectable germline BAP1 alterations. DESIGN: Retrospective case series from academic referral centers. PARTICIPANTS: Cohort of 154 UM patients with high risk of hereditary cancer defined as patients with 1 or more of the following: (1) familial UM, (2) young age (<35 years) at diagnosis, (3) personal history of other primary cancers, and (4) family history of 2 or more primary cancers with no detectable mutation or deletion in BAP1 gene. METHODS: Whole exome sequencing, a cancer gene panel, or both were carried out. Probands included 27 patients with familial UM, 1 patient with bilateral UM, 1 patient with congenital UM, and 125 UM patients with strong personal or family histories, or both, of cancer. Functional validation of variants was carried out by immunohistochemistry, reverse-transcriptase polymerase chain reaction, and genotyping. MAIN OUTCOME MEASURES: Clinical characterization of UM patients with germline alterations in known cancer genes. RESULTS: We identified actionable pathogenic variants in 8 known hereditary cancer predisposition genes (PALB2, MLH1, MSH6, CHEK2, SMARCE1, ATM, BRCA1, and CTNNA1) in 9 patients, including 3 of 27 patients (11%) with familial UM and 6 of 127 patients (4.7%) with a high risk for cancer. Two patients showed pathogenic variants in CHEK2 and PALB2, whereas variants in the other genes each occurred in 1 patient. Biallelic inactivation of PALB2 and MLH1 was observed in tumors from the respective patients. The frequencies of pathogenic variants in PALB2, MLH1, and SMARCE1 in UM patients were significantly higher than the observed frequencies in noncancer controls (PALB2: P = 0.02; odds ratio, 8.9; 95% confidence interval, 1.5-30.6; MLH1: P = 0.04; odds ratio, 25.4; 95% confidence interval, 1.2-143; SMARCE1: P = 0.001; odds ratio, 2047; 95% confidence interval, 52-4.5e15, respectively). CONCLUSIONS: The study provided moderate evidence of gene and disease association of germline mutations in PALB2 and MLH1 with hereditary predisposition to UM. It also identified several other candidate susceptibility genes. The results suggest locus heterogeneity in predisposition to UM. Genetic testing for hereditary predisposition to cancer is warranted in UM patients with strong personal or family history of cancers, or both.

Prediction of short-term neonatal complications in preterm infants using exome-wide genetic variation and gestational age: a pilot study.

BACKGROUND: Preterm birth is the leading cause of mortality and morbidity in young children, with over a million deaths per year worldwide arising from neonatal complications (NCs). NCs are moderately heritable although the genetic causes are largely unknown. Therefore, we investigated the impact of accumulated genetic variation (burden) on NCs in non-Hispanic White (NHW) and non-Hispanic Black (NHB) preterm infants. METHODS: We sequenced 182 exomes from infants with gestational ages from 26 to 31 weeks. These infants were cared for in the same time period and hospital environment. Eighty-one preterm infants did not develop NCs, whereas 101 developed at least one severe complication. We measured the effect of burden at the single-gene and exome-wide levels and derived a polygenic risk score (PRS) from the top 10 genes to predict NCs. RESULTS: Burden across the exome was associated with NCs in NHW (p = 0.05) preterm infants suggesting that multiple genes influence susceptibility. In a post hoc analysis, we find that PRS alone predicts NCs (AUC = 0.67) and that PRS is uncorrelated with GA ([Formula: see text] = 0.05; p = 0.53). When PRS and GA at birth are combined, the AUC is 0.87. CONCLUSIONS: Our results support the hypothesis that genetic burden influences NCs in NHW preterm infants.

Disruption of the Putative Ribosome-Binding Motif of a Scaffold Protein Impairs Cytochrome c Oxidase Subunit Expression in Leishmania major.

During their parasitic life cycle, through sandflies and vertebrate hosts, Leishmania parasites confront strikingly different environments, including abrupt changes in pH and temperature, to which they must rapidly adapt. These adaptations include alterations in Leishmania gene expression, metabolism, and morphology, allowing them to thrive as promastigotes in the sandfly and as intracellular amastigotes in the vertebrate host. A critical aspect of Leishmania metabolic adaptation to these changes is maintenance of efficient mitochondrial function in the hostile vertebrate environment. Such functions, including generation of ATP, depend upon the expression of many mitochondrial proteins, including subunits of cytochrome c oxidase (COX). Significantly, under mammalian temperature conditions, expression of Leishmania major COX subunit IV (LmCOX4) and virulence are dependent upon two copies of LACK, a gene that encodes the ribosome-associated scaffold protein, LACK (Leishmania ortholog of RACK1 [receptor for activated C kinase]). Targeted replacement of an endogenous LACK copy with a putative ribosome-binding motif-disrupted variant (LACKR34D35G36→LACKD34D35E36) resulted in thermosensitive parasites that showed diminished LmCOX4 expression, mitochondrial fitness, and replication in macrophages. Surprisingly, despite these phenotypes, LACKD34D35E36 associated with monosomes and polysomes and showed no major impairment of global protein synthesis. Collectively, these data suggest that wild-type (WT) LACK orchestrates robust LmCOX4 expression and mitochondrial fitness to ensure parasite virulence, via optimized functional interactions with the ribosome.IMPORTANCELeishmania parasites are trypanosomatid protozoans that persist in infected human hosts to cause a spectrum of pathologies, from cutaneous and mucocutaneous manifestations to visceral leishmaniasis caused by Leishmania donovani The latter is usually fatal if not treated. Persistence of L. major in the mammalian host depends upon maintaining gene-regulatory programs to support essential parasite metabolic functions. These include expression and assembly of mitochondrial L. major cytochrome c oxidase (LmCOX) subunits, important for Leishmania ATP production. Significantly, under mammalian conditions, WT levels of LmCOX subunits require threshold levels of the Leishmania ribosome-associated scaffold protein, LACK. Unexpectedly, we find that although disruption of LACK's putative ribosome-binding motif does not grossly perturb ribosome association or global protein synthesis, it nonetheless impairs COX subunit expression, mitochondrial function, and virulence. Our data indicate that the quality of LACK's interaction with Leishmania ribosomes is critical for LmCOX subunit expression and parasite mitochondrial function in the mammalian host. Collectively, these findings validate LACK's ribosomal interactions as a potential therapeutic target.

A multi-centre investigation of delivering national guidelines on exercise training for men with advanced prostate cancer undergoing androgen deprivation therapy in the UK NHS.

BACKGROUND: National guidelines (NICE-CG175) recommended 12 weeks of supervised exercise training for men treated with androgen deprivation therapy (ADT) for prostate cancer to counter debilitating adverse effects of castration. As with other chronic conditions where exercise is indicated, it is uncertain if these services are being delivered in the health services. The aim of this multi-centre investigation was to examine what exercise referral is currently available for men on ADT as provided by the NHS and if a supervised, individually-tailored exercise training package (as per the national NICE guidelines CG175) is embedded within prostate cancer care. METHODS: A multi-centre investigation of current National Health Service (NHS) care involving a web-based survey of NHS prostate cancer care, five focus groups involving 26 men on ADT and 37 semi-structured interviews with healthcare professionals (HCPs) involved in the management of prostate cancer. Descriptive statistics and thematic analysis evaluated quantitative and qualitative data, respectively. Qualitative methods followed COREQ standards. RESULTS: HCPs and men on ADT asserted that medical castration has a serious and debilitating impact on many features of men's quality of life. There is support for exercise training programmes as part of cancer care and patients would support their initiation soon after diagnosis. Involving the Multidisciplinary Team (MDT) is proposed as key to this. Critically, traditional values in oncology would need to be overcome for widespread acceptance. Specialist further training for HCPs around behaviour change support could encourage this. Given that these schemes are seen as a fundamental part of cancer care, it is felt the NHS should commission and support provision. 79 representatives of 154 NHS trusts (51%) provided survey data on current delivery: only 17% could provide supervised exercise as per CG175. CONCLUSIONS: Evidence-based national exercise guidelines are not being delivered to men on ADT as intended. Traditional values in oncology and the need for NHS financial support are seen as major barriers to provision of current best practice guidelines. Despite this both HCPs and men on ADT are in favour of such programmes being a fundamental part of their cancer care.

Comparative proteomic analysis of Gib2 validating its adaptor function in Cryptococcus neoformans.

Cryptococcus neoformans causes often-fatal fungal meningoencephalitis in immunocompromised individuals. While the exact disease mechanisms remain elusive, signal transduction pathways mediated by key elements such as G-protein α subunit Gpa1, small GTPase Ras1, and atypical Gß-like/RACK1 protein Gib2 are known to play important roles in C. neoformans virulence. Gib2 is important for normal growth, differentiation, and pathogenicity, and it also positively regulates cAMP levels in conjunction with Gpa1. Interestingly, Gib2 displays a scaffold protein property by interacting with a wide variety of cellular proteins. To explore Gib2 global regulatory functions, we performed two-dimensional differential gel electrophoresis (DIGE) analysis and found that GIB2 disruption results in an increased expression of 304 protein spots (43.4%) and a decreased expression of 396 protein spots (56.6%). Analysis of 96 proteins whose expression changes were deemed significant (≥ +/- 1.5- fold) revealed that 75 proteins belong to at least 12 functional protein groups. Among them, eight groups have the statistical stringency of p ≤ 0.05, and four groups, including Hsp70/71 heat shock protein homologs and ribosomal proteins, survived the Bonferroni correction. This finding is consistent with earlier established roles for the human Gß-like/RACK1 and the budding yeast Saccharomyces cerevisiae Asc1. It suggests that Gib2 could also be part of the complex affecting ribosomal biogenesis and protein translation in C. neoformans. Since eukaryotic Hsp70/71 proteins are involved in the facilitation of nascent protein folding, processing, and protection of cells against stress, we also propose that Gib2-regulated stress responses are linked to fungal virulence. Collectively, our study supports a conserved role of Gß-like/RACK/Gib2 proteins in the essential cellular process of ribosomal biogenesis and protein translation. Our study also highlights a multifaceted regulatory role of Gib2 in the growth and pathogenicity of C. neoformans.

A novel model for IFN-γ-mediated autoinflammatory syndromes.

Autoinflammatory disease and hyperinflammatory syndromes represent a growing number of diseases associated with inappropriately controlled inflammation in multiple organs. Systemic inflammation commonly results from dysregulated activation of innate immune cells, and therapeutic targeting of the IL-1ß pathway has been used to ameliorate some of these diseases. Some hyperinflammatory syndromes, however, such as hemophagocytic lymphohistiocytosis and the newly classified proteasome disability syndromes, are refractory to such treatments, suggesting that other factors or environmental stressors may be contributing. In comparing two cytokine reporter mouse strains, we identify IFN-γ as a mediator of systemic autoinflammatory disease. Chronically elevated levels of IFN-γ resulted in progressive multiorgan inflammation and two copies of the mutant allele resulted in increased mortality accompanied by myeloproliferative disease. Disease was alleviated by genetic deletion of T-bet. These studies raise the possibility that therapeutics targeting the IFN-γ pathway might be effective in hyperinflammatory conditions refractory to IL-1ß-targeted therapies.

LACK, a RACK1 ortholog, facilitates cytochrome c oxidase subunit expression to promote Leishmania major fitness.

Leishmania are kinetoplastid parasites that cause the sandfly-transmitted disease leishmaniasis. To maintain fitness throughout their infectious life cycle, Leishmania must undergo rapid metabolic adaptations to the dramatically distinct environments encountered during transition between sandfly and vertebrate hosts. We performed proteomic and immunoblot analyses of attenuated L. major strains deficient for LACK, the Leishmania ortholog of the mammalian receptor for activated c kinase (RACK1), that is important for parasite thermotolerance and virulence. This approach identified cytochrome c oxidase (LmCOX) subunit IV as a LACK-dependent fitness protein. Consistent with decreased levels of LmCOX subunit IV at mammalian temperature, and in amastigotes, LmCOX activity and mitochondrial function were also impaired in LACK-deficient L. major under these conditions. Importantly, overexpression of LmCOX subunit IV in LACK-deficient L. major restored thermotolerance and macrophage infectivity. Interestingly, overexpression of LmCOX subunit IV enhanced LmCOX subunit VI expression at mammalian temperature. Collectively, our data suggest LACK promotes Leishmania adaptation to the mammalian host environment by sustaining LmCOX subunit IV expression and hence energy metabolism in response to stress stimuli such as heat. These findings extend the repertoire of RACK1 protein utility to include a role in mitochondrial function.

Molecular and cellular characterization of an AT-hook protein from Leishmania.

AT-rich DNA, and the proteins that bind it (AT-hook proteins), modulate chromosome structure and function in most eukaryotes. Unlike other trypanosomatids, the genome of Leishmania species is unusually GC-rich, and the regulation of Leishmania chromosome structure, replication, partitioning is not fully understood. Because AT-hook proteins modulate these functions in other eukaryotes, we examined whether AT-hook proteins are encoded in the Leishmania genome, to test their potential functions. Several Leishmania ORFs predicted to be AT-hook proteins were identified using in silico approaches based on sequences shared between eukaryotic AT-hook proteins. We have used biochemical, molecular and cellular techniques to characterize the L. amazonensis ortholog of the L. major protein LmjF06.0720, a potential AT-hook protein that is highly conserved in Leishmania species. Using a novel fusion between the AT-hook domain encoded by LmjF06.0720 and a herpesviral protein, we have demonstrated that LmjF06.0720 functions as an AT-hook protein in mammalian cells. Further, as observed for mammalian and viral AT-hook proteins, the AT-hook domains of LmjF06.0720 bind specific regions of condensed mammalian metaphase chromosomes, and support the licensed replication of DNA in mammalian cells. LmjF06.0720 is nuclear in Leishmania, and this localization is disrupted upon exposure to drugs that displace AT-hook proteins from AT-rich DNA. Coincidentally, these drugs dramatically alter the cellular physiology of Leishmania promastigotes. Finally, we have devised a novel peptido-mimetic agent derived from the sequence of LmjF06.0720 that blocks the proliferation of Leishmania promastigotes, and lowers amastigote parasitic burden in infected macrophages. Our results indicate that AT-hook proteins are critical for the normal biology of Leishmania. In addition, we have described a simple technique to examine the function of Leishmania chromatin-binding proteins in a eukaryotic context amenable to studying chromosome structure and function. Lastly, we demonstrate the therapeutic potential of compounds directed against AT-hook proteins in Leishmania.

Trypanosomatid RACK1 orthologs show functional differences associated with translation despite similar roles in Leishmania pathogenesis.

RACK1 proteins belong to the eukaryote WD40-repeat protein family and function as spatial regulators of multiple cellular events, including signaling pathways, the cell cycle and translation. For this latter role, structural and genetic studies indicate that RACK1 associates with the ribosome through two conserved positively charged amino acids in its first WD40 domain. Unlike RACK1s, including Trypanosoma brucei RACK1 (TbRACK1), only one of these two positively-charged residues is conserved in the first WD40 domain of the Leishmania major RACK1 ortholog, LACK. We compared virulence-attenuated LACK single copy (LACK/-) L. major, with L. major expressing either two LACK copies (LACK/LACK), or one copy each of LACK and TbRACK1 (LACK/TbRACK1), to evaluate the function of these structurally distinct RACK1 orthologs with respect to translation, viability at host temperatures and pathogenesis. Our results indicate that although the ribosome-binding residues are not fully conserved in LACK, both LACK and TbRACK1 co-sedimented with monosomes and polysomes in LACK/LACK and LACK/TbRACK1 L. major, respectively. LACK/LACK and LACK/TbRACK1 strains differed in their sensitivity to translation inhibitors implying that minor sequence differences between the RACK1 proteins can alter their functional properties. While biochemically distinguishable, both LACK/LACK and LACK/TbRACK1 lines were more tolerant of elevated temperatures, resistant to translation inhibitors, and displayed robust pathogenesis in vivo, contrasting to LACK/- parasites.

The oligopeptidase B of Leishmania regulates parasite enolase and immune evasion.

Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites, in the host-parasite relationship, and in the pathogenesis of parasitic diseases. Furthermore, proteases are targets for the development of new anti-parasitic therapy. Protozoan parasites like Leishmania predominantly express Clan CA cysteine proteases for key life cycle functions. It was therefore unexpected to find a high level of serine protease activity expressed by Leishmania donovani. Purification of this activity followed by mass spectrometry identified oligopeptidase B (OPB; Clan SC, family S9A) as the responsible enzyme. This was confirmed by gene knock-out of OPB, which resulted in the disappearance of the detected serine protease activity of Leishmania extracts. To delineate the specific role of OPB in parasite physiology, proteomic analysis was carried out on OPB(-/-) versus wild type parasites. Four protein species were significantly elevated in OPB(-/-) parasites, and all four were identified by mass spectrometry as enolase. This increased enolase was enzymatically inactive and associated with the parasite membrane. Aside from its classic role in carbohydrate metabolism, enolase was recently found to localize to membranes, where it binds host plasminogen and functions as a virulence factor for several pathogens. As expected, there was a striking alteration in macrophage responses to Leishmania when OPB was deleted. Whereas wild type parasites elicited little, if any, response from infected macrophages, OPB(-/-) parasites induced a massive up-regulation in gene transcription. Additionally, these OPB(-/-) parasites displayed decreased virulence in the murine footpad infection model.

Leishmania subtilisin is a maturase for the trypanothione reductase system and contributes to disease pathology.

Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites, in the host-parasite relationship, and in the pathogenesis of parasitic diseases. Furthermore, proteases are druggable targets for the development of new anti-parasitic therapy. The subtilisin protease (SUB; Clan SB, family S8) of Leishmania donovani was cloned and found to possess a unique catalytic triad. This gene was then deleted by gene knock-out, which resulted in reduced ability by the parasite to undergo promastigote to amastigote differentiation in vitro. Electron microscopy of SUB knock-out amastigotes revealed abnormal membrane structures, retained flagella, and increased binucleation. SUB-deficient Leishmania displayed reduced virulence in both hamster and murine infection models. Histology of spleens from SUB knock-out-infected hamsters revealed the absence of psammoma body calcifications indicative of the granulomatous lesions that occur during Leishmania infection. To delineate the specific role of SUB in parasite physiology, two-dimensional gel electrophoresis was carried out on SUB(-/-) versus wild-type parasites. SUB knock-out parasites showed altered regulation of the terminal peroxidases of the trypanothione reductase system. Leishmania and other trypanosomatids lack glutathione reductase, and therefore rely on the novel trypanothione reductase system to detoxify reactive oxygen intermediates and to maintain redox homeostasis. The predominant tryparedoxin peroxidases were decreased in SUB(-/-) parasites, and higher molecular weight isoforms were present, indicating altered processing. In addition, knock-out parasites showed increased sensitivity to hydroperoxide. These data suggest that subtilisin is the maturase for tryparedoxin peroxidases and is necessary for full virulence.

Suppression of LPS-induced inflammatory responses in macrophages infected with Leishmania.

BACKGROUND: Chronic inflammation activated by macrophage innate pathogen recognition receptors such as TLR4 can lead to a range of inflammatory diseases, including atherosclerosis, Crohn's disease, arthritis and cancer. Unlike many microbes, the kinetoplastid protozoan pathogen Leishmania has been shown to avoid and even actively suppress host inflammatory cytokine responses, such as LPS-induced IL-12 production. The nature and scope of Leishmania-mediated inflammatory cytokine suppression, however, is not well characterized. Advancing our knowledge of such microbe-mediated cytokine suppression may provide new avenues for therapeutic intervention in inflammatory disease. METHODS: We explored the kinetics of a range of cytokine and chemokine responses in primary murine macrophages stimulated with LPS in the presence versus absence of two clinically distinct species of Leishmania using sensitive multiplex cytokine analyses. To confirm that these effects were parasite-specific, we compared the effects of Leishmania uptake on LPS-induced cytokine expression with uptake of inert latex beads. RESULTS: Whilst Leishmania uptake alone did not induce significant levels of any cytokine analysed in this study, Leishmania uptake in the presence of LPS caused parasite-specific suppression of certain LPS-induced pro-inflammatory cytokines, including IL-12, IL-17 and IL-6. Interestingly, L. amazonensis was generally more suppressive than L. major. We also found that other LPS-induced proinflammatory cytokines, such as IL-1alpha, TNF-alpha and the chemokines MIP-1alpha and MCP-1 and also the anti-inflammatory cytokine IL-10, were augmented during Leishmania uptake, in a parasite-specific manner. CONCLUSIONS: During uptake by macrophages, Leishmania evades the activation of a broad range of cytokines and chemokines. Further, in the presence of a strong inflammatory stimulus, Leishmania suppresses certain proinflammatory cytokine responses in a parasite-specific manner, however it augments the production of other proinflammatory cytokines. Our findings highlight the complexity of inflammatory cytokine signalling regulation in the context of the macrophage and Leishmania interaction and confirm the utility of the Leishmania/macrophage infection model as an experimental system for further studies of inflammatory regulation. Such studies may advance the development of therapies against inflammatory disease.

Bis-acridines as lead antiparasitic agents: structure-activity analysis of a discrete compound library in vitro.

Parasitic diseases are of enormous public health significance in developing countries-a situation compounded by the toxicity of and resistance to many current chemotherapeutics. We investigated a focused library of 18 structurally diverse bis-acridine compounds for in vitro bioactivity against seven protozoan and one helminth parasite species and compared the bioactivities and the cytotoxicities of these compounds toward various mammalian cell lines. Structure-activity relationships demonstrated the influence of both the bis-acridine linker structure and the terminal acridine heterocycle on potency and cytotoxicity. The bioactivity of polyamine-linked acridines required a minimum linker length of approximately 10 A. Increasing linker length resulted in bioactivity against most parasites but also cytotoxicity toward mammalian cells. N alkylation, but less so N acylation, of the polyamine linker ameliorated cytotoxicity while retaining bioactivity with 50% effective concentration (EC(50)) values similar to or better than those measured for standard drugs. Substitution of the polyamine for either an alkyl or a polyether linker maintained bioactivity and further alleviated cytotoxicity. Polyamine-linked compounds in which the terminal acridine heterocycle had been replaced with an aza-acridine also maintained acceptable therapeutic indices. The most potent compounds recorded low- to mid-nanomolar EC(50) values against Plasmodium falciparum and Trypanosoma brucei; otherwise, low-micromolar potencies were measured. Importantly, the bioactivity of the library was independent of P. falciparum resistance to chloroquine. Compound bioactivity was a function of neither the potential to bis-intercalate DNA nor the inhibition of trypanothione reductase, an important drug target in trypanosomatid parasites. Our approach illustrates the usefulness of screening focused compound libraries against multiple parasite targets. Some of the bis-acridines identified here may represent useful starting points for further lead optimization.