The Opto-inflammasome in zebrafish as a tool to study cell and tissue responses to speck formation and cell death.

The inflammasome is a conserved structure for the intracellular detection of danger or pathogen signals. As a large intracellular multiprotein signaling platform, it activates downstream effectors that initiate a rapid necrotic programmed cell death (PCD) termed pyroptosis and activation and secretion of pro-inflammatory cytokines to warn and activate surrounding cells. However, inflammasome activation is difficult to control experimentally on a single-cell level using canonical triggers. We constructed Opto-ASC, a light-responsive form of the inflammasome adaptor protein ASC (Apoptosis-Associated Speck-Like Protein Containing a CARD) which allows tight control of inflammasome formation in vivo. We introduced a cassette of this construct under the control of a heat shock element into zebrafish in which we can now induce ASC inflammasome (speck) formation in individual cells of the skin. We find that cell death resulting from ASC speck formation is morphologically distinct from apoptosis in periderm cells but not in basal cells. ASC-induced PCD can lead to apical or basal extrusion from the periderm. The apical extrusion in periderm cells depends on Caspb and triggers a strong Ca2+ signaling response in nearby cells.

Targeted and whole-genome sequencing reveal a north-south divide in P. falciparum drug resistance markers and genetic structure in Mozambique.

Mozambique is one of the four African countries which account for over half of all malaria deaths worldwide, yet little is known about the parasite genetic structure in that country. We performed P. falciparum amplicon and whole genome sequencing on 2251 malaria-infected blood samples collected in 2015 and 2018 in seven provinces of Mozambique to genotype antimalarial resistance markers and interrogate parasite population structure using genome-wide microhaplotyes. Here we show that the only resistance-associated markers observed at frequencies above 5% were pfmdr1-184F (59%), pfdhfr-51I/59 R/108 N (99%) and pfdhps-437G/540E (89%). The frequency of pfdhfr/pfdhps quintuple mutants associated with sulfadoxine-pyrimethamine resistance increased from 80% in 2015 to 89% in 2018 (p < 0.001), with a lower expected heterozygosity and higher relatedness of microhaplotypes surrounding pfdhps mutants than wild-type parasites suggestive of recent selection. pfdhfr/pfdhps quintuple mutants also increased from 72% in the north to 95% in the south (2018; p < 0.001). This resistance gradient was accompanied by a concentration of mutations at pfdhps-436 (17%) in the north, a south-to-north increase in the genetic complexity of P. falciparum infections (p = 0.001) and a microhaplotype signature of regional differentiation. The parasite population structure identified here offers insights to guide antimalarial interventions and epidemiological surveys.

It takes two to remyelinate: A bioengineered platform to study astrocyte-oligodendrocyte crosstalk and potential therapeutic targets in remyelination.

The loss of the myelin sheath insulating axons is the hallmark of demyelinating diseases. These pathologies often lead to irreversible neurological impairment and patient disability. No effective therapies are currently available to promote remyelination. Several elements contribute to the inadequacy of remyelination, thus understanding the intricacies of the cellular and signaling microenvironment of the remyelination niche might help us to devise better strategies to enhance remyelination. Here, using a new in vitro rapid myelinating artificial axon system based on engineered microfibres, we investigated how reactive astrocytes influence oligodendrocyte (OL) differentiation and myelination ability. This artificial axon culture system enables the effective uncoupling of molecular cues from the biophysical properties of the axons, allowing the detailed study of the astrocyte-OL crosstalk. Oligodendrocyte precursor cells (OPCs) were cultured on poly(trimethylene carbonate-co-ε-caprolactone) copolymer electrospun microfibres that served as surrogate axons. This platform was then combined with a previously established tissue engineered glial scar model of astrocytes embedded in 1 % (w/v) alginate matrices, in which astrocyte reactive phenotype was acquired using meningeal fibroblast conditioned medium. OPCs were shown to adhere to uncoated engineered microfibres and differentiate into myelinating OL. Reactive astrocytes were found to significantly impair OL differentiation ability, after six and eight days in a co-culture system. Differentiation impairment was seen to be correlated with astrocytic miRNA release through exosomes. We found significantly reduction on the expression of pro-myelinating miRNAs (miR-219 and miR-338) and an increase in anti-myelinating miRNA (miR-125a-3p) content between reactive and quiescent astrocytes. Additionally, we show that OPC differentiation inhibition could be reverted by rescuing the activated astrocytic phenotype with ibuprofen, a chemical inhibitor of the small rhoGTPase RhoA. Overall, these findings show that modulating astrocytic function might be an interesting therapeutic avenue for demyelinating diseases. The use of these engineered microfibres as an artificial axon culture system will enable the screening for potential therapeutic agents that promote OL differentiation and myelination while providing valuable insight on the myelination/remyelination processes.

Functionalized retinoic acid lipid nanocapsules promotes a two-front attack on inflammation and lack of demyelination on neurodegenerative disorders.

Demyelinating disorders, with a particular focus on multiple sclerosis (MS), have a multitude of detrimental cognitive and physical effects on the patients. Current treatment options that involve substances promoting remyelination fail in the clinics due to difficulties in reaching the central nervous system (CNS). Here, the dual encapsulation of retinoic acid (RA) into lipid nanocapsules with a nominal size of 70 nm, and a low PdI of 0.1, coupled with super paramagnetic iron oxide nanoparticles (SPIONs) was accomplished, and joined by an external functionalization process with a transferrin-receptor binding peptide. This nanosystem showed a 3-fold improved internalization by endothelial cells compared to the free drug, ability to interact with oligodendrocyte progenitor cells and microglia, and improvements in the permeability through the blood-brain barrier by 5-fold. The lipid nanocapsules also induced the differentiation of oligodendrocyte progenitor cells into more mature, myelin producing oligodendrocytes, as evaluated by high-throughput image screening, by 3-5-fold. Furthermore, the ability to tame the inflammatory response was verified in lipopolysaccharide-stimulated microglia, suppressing the production of pro-inflammatory cytokines by 50-70%. Overall, the results show that this nanosystem can act in both the inflammatory microenvironment present at the CNS of affected patients, but also stimulate the differentiation of new oligodendrocytes, paving the way for a promising platform in the therapy of MS.

Eumelanin decorated poly(lactic acid) electrospun substrates as a new strategy for spinal cord injury treatment.

Spinal cord injury (SCI) is characterized by neuroinflammatory processes that are marked by an uncontrolled activation of microglia, which directly damages neurons. Natural and synthetic melanins represent an effective tool to treat neuroinflammation because they possess immunomodulatory properties. Here, the main objective was to evaluate the effect of eumelanin-coated poly(lactic acid) (EU@PLA) aligned microfibers on in vitro model of neuroinflammation related to spinal cord injury in terms of inflammatory mediators' modulation. Aligned fibers were chosen to provide physical cues to guide axonal growth in a specific direction thus restoring the synaptic connection. Eumelanin decorated PLA electrospun substrates were produced combining electrospinning, spin coating and solid-state polymerization processes (oxidative coupling under oxygen atmosphere). Biological response in terms of antioxidant and anti-inflammatory activity was analyzed on an in vitro model of neuroinflammation [microglial cells stimulated with lipopolysaccharide (LPS)]. Cell morphology and EU@PLA mechanism of action, in terms of toll-like receptor-4 (TLR-4) involvement were assessed. The results show that EU@PLA fibers were able to decrease reactive oxygen species, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) expression >50 % compared to PLA + LPS and interleukin 6 (IL-6) secretion about 20 %. Finally, the mechanism of action of EU@PLA in microglia was found to be dependent on the TLR-4 signaling. Protein expression analysis revealed a decreased in TLR-4 production induced by LPS stimulation in presence of EU@PLA. Overall, our results show that EU@PLA represents an innovative and effective strategy for the control of inflammatory response in central nervous system.

Feasibility, Acceptability, and Protective Efficacy of Seasonal Malaria Chemoprevention Implementation in Nampula Province, Mozambique: Protocol for a Hybrid Effectiveness-Implementation Study.

BACKGROUND: Seasonal malaria chemoprevention (SMC) is a highly effective community-based intervention to prevent malaria infections in areas where the malaria burden is high and transmission occurs mainly during the rainy season. In Africa, so far, SMC has been implemented in the Sahel region. Mozambique contributes 4% of the global malaria cases, and malaria is responsible for one-quarter of all deaths in the country. Based on recommendations in the Malaria Strategic Plan, the Malaria Consortium, in partnership with the National Malaria Control Programme in Mozambique, initiated a phased SMC implementation study in the northern province of Nampula. The first phase of this 2-year implementation study was conducted in 2020-2021 and focused on the feasibility and acceptability of SMC. The second phase will focus on demonstrating impact. This paper describes phase 2 of the implementation study. OBJECTIVE: Specific objectives include the following: (1) to determine the effectiveness of SMC in terms of its reduction in incidence of malaria infection among children aged 3 to 59 months; (2) to determine the chemoprevention efficacy of sulfadoxine-pyrimethamine plus amodiaquine (SP+AQ) when used for SMC in Nampula Province, Mozambique, and the extent to which efficacy is impacted by drug resistance and drug concentrations; (3) to investigate the presence and change in SP+AQ- and piperaquine-resistance markers over time as a result of SMC implementation; and (4) to understand the impact of the SMC implementation model, determining the process and acceptability outcomes for the intervention. METHODS: This type 2, hybrid, effectiveness-implementation study uses a convergent mixed methods approach. SMC will be implemented in four monthly cycles between December 2021 and March 2022 in four districts of Nampula Province. Phase 2 will include four components: (1) a cluster randomized controlled trial to establish confirmed malaria cases, (2) a prospective cohort to determine the chemoprevention efficacy of the antimalarials used for SMC and whether drug concentrations or resistance influence the duration of protection, (3) a resistance marker study in children aged 3 to 59 months to describe changes in resistance marker prevalence over time, and (4) a process evaluation to determine feasibility and acceptability of SMC. RESULTS: Data collection began in mid-January 2022, and data analysis is expected to be completed by October 2022. CONCLUSIONS: This is the first effectiveness trial of SMC implemented in Mozambique. The findings from this trial will be crucial to policy change and program expansion to other suitable geographies outside of the Sahel. The chemoprevention efficacy cohort study is a unique opportunity to better understand SMC drug efficacy in this new SMC environment. TRIAL REGISTRATION: ClinicalTrials.gov NCT05186363; https://clinicaltrials.gov/ct2/show/NCT05186363. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/36403.

Revisiting the origin of interleukin 1 in anamniotes and sub-functionalization of interleukin 1 in amniotes.

The cytokine interleukin 1 (IL-1) is an evolutionary innovation of vertebrates. Fish and amphibian have one IL1 gene, while mammals have two copies of IL1, IL1A and IL1B, with distinct expression patterns and differences in their proteolytic activation. Our current understanding of the evolutionary history of IL-1 is mainly based on phylogenetic analysis, but this approach provides no information on potentially different functions of IL-1 homologues, and it remains unclear which biological activities identified for IL-1α and IL-1ß in mammals are present in lower vertebrates. Here, we use in vitro and in vivo experimental models to examine the expression patterns and cleavage of IL-1 proteins from various species. We found that IL-1 in the teleost medaka shares the transcriptional patterns of mammalian IL-1α, and its processing also resembles that of mammalian IL-1α, which is sensitive to cysteine protease inhibitors specific for the calpain and cathepsin families. By contrast, IL-1 proteins in reptiles also include biological properties of IL-1ß. Therefore, we propose that the duplication of the ancestral IL1 gene led to the segregation of expression patterns and protein processing that characterizes the two extant forms of IL-1 in mammals.

Mechanotransduction: Exploring New Therapeutic Avenues in Central Nervous System Pathology.

Cells are continuously exposed to physical forces and the central nervous system (CNS) is no exception. Cells dynamically adapt their behavior and remodel the surrounding environment in response to forces. The importance of mechanotransduction in the CNS is illustrated by exploring its role in CNS pathology development and progression. The crosstalk between the biochemical and biophysical components of the extracellular matrix (ECM) are here described, considering the recent explosion of literature demonstrating the powerful influence of biophysical stimuli like density, rigidity and geometry of the ECM on cell behavior. This review aims at integrating mechanical properties into our understanding of the molecular basis of CNS disease. The mechanisms that mediate mechanotransduction events, like integrin, Rho/ROCK and matrix metalloproteinases signaling pathways are revised. Analysis of CNS pathologies in this context has revealed that a wide range of neurological diseases share as hallmarks alterations of the tissue mechanical properties. Therefore, it is our belief that the understanding of CNS mechanotransduction pathways may lead to the development of improved medical devices and diagnostic methods as well as new therapeutic targets and strategies for CNS repair.

Optogenetic activators of apoptosis, necroptosis, and pyroptosis.

Targeted and specific induction of cell death in an individual or groups of cells hold the potential for new insights into the response of tissues or organisms to different forms of death. Here, we report the development of optogenetically controlled cell death effectors (optoCDEs), a novel class of optogenetic tools that enables light-mediated induction of three types of programmed cell death (PCD)-apoptosis, pyroptosis, and necroptosis-using Arabidopsis thaliana photosensitive protein Cryptochrome-2. OptoCDEs enable a rapid and highly specific induction of PCD in human, mouse, and zebrafish cells and are suitable for a wide range of applications, such as sub-lethal cell death induction or precise elimination of single cells or cell populations in vitro and in vivo. As the proof-of-concept, we utilize optoCDEs to assess the differences in neighboring cell responses to apoptotic or necrotic PCD, revealing a new role for shingosine-1-phosphate signaling in regulating the efferocytosis of the apoptotic cell by epithelia.

A paradigm shift: Bioengineering meets mechanobiology towards overcoming remyelination failure.

Therapeutic strategies aimed at overcoming the loss of myelin sheath in central nervous system demyelinating diseases are often unsuccessful due to nescience underlying the mechanisms of remyelination failure. The environment surrounding a demyelination lesion is seen as a hostile terrain, characterized by factors that can inhibit myelin production by oligodendrocytes (OLs). The formation of a glial scar containing reactive astrocytes producing high amounts of altered matrix proteins can compromise OL remyelination. Allied to glial scar, mechanical properties of the tissue are altered. The paradigms in the remyelination failure are changing. We point mechanobiology as a missing key towards unravelling the nature of (de)myelination. Mechanical cues as stiffness, axonal tension or physical constraints are emerging as dictators of tissue homeostasis and pathology. Here we delve into an in-depth characterization of the preeminent models to study mechanobiology events of (de)myelination and remyelination. Alternatives to in vivo systems are provided, either through the exploration of simpler animal models, creation of in vitro models using tissue engineered approaches or through in silico tools. We discuss how bioengineering is being explored to generate relevant models to dissect new mechanobiology mechanisms and identify novel therapeutic targets, being expected to profoundly impact the treatment of demyelinating diseases.

Notch1 deficiency alters the migratory behavior of developing T cells and calcium signaling in the thymus of medaka.

The differentiation of T cells from lymphoid progenitors in the thymus follows sequential developmental stages that constantly require interaction with thymic epithelial cells. Several distinct aspects of early T cell development depend on the activation of Notch receptors on thymocytes, while the selection of thymocytes at later stages are believed to be Notch independent. Using reverse genetic approaches and whole-thymus live imaging in an in vivo teleost model, the medaka, we report that Notch1 signals is required for proliferation and specification of developing T cells as well as involved in their selection in the thymus. We reveal that Notch1 controls the migratory behavior of thymocytes through controlling the chemokine receptor Ccr9b and thereby influence the T cell receptor (TCR) activation. Hence, we propose that, in lower vertebrates, the function of Notch signaling extends to all stages of T cell development, except when thymocytes undergo TCRß rearrangement.

In vivo efficacy and safety of artemether-lumefantrine and amodiaquine-artesunate for uncomplicated Plasmodium falciparum malaria in Mozambique, 2018.

BACKGROUND: Artemisinin-based combination therapy (ACT) has been the recommended first-line treatment for uncomplicated malaria in Mozambique since 2006, with artemether-lumefantrine (AL) and amodiaquine-artesunate (AS-AQ) as the first choice. To assess efficacy of currently used ACT, an in vivo therapeutic efficacy study was conducted. METHODS: The study was conducted in four sentinel sites: Montepuez, Moatize, Mopeia and Massinga. Patients between 6 and 59 months old with uncomplicated Plasmodium falciparum malaria (2000-200,000 parasites/µl) were enrolled between February and September of 2018, assigned to either an AL or AS-AQ treatment arm, and monitored for 28 days. A Bayesian algorithm was applied to differentiate recrudescence from new infection using genotyping data of seven neutral microsatellites. Uncorrected and PCR-corrected efficacy results at day 28 were calculated. RESULTS: Totals of 368 and 273 patients were enrolled in the AL and AS-AQ arms, respectively. Of these, 9.5% (35/368) and 5.1% (14/273) were lost to follow-up in the AL and AS-AQ arms, respectively. There were 48 and 3 recurrent malaria infections (late clinical and late parasitological failures) in the AL and AS-AQ arms, respectively. The day 28 uncorrected efficacy was 85.6% (95% confidence interval (CI) 81.3-89.2%) for AL and 98.8% (95% CI 96.7-99.8%) for AS-AQ, whereas day 28 PCR-corrected efficacy was 97.9% (95% CI 95.6-99.2%) for AL and 99.6% (95% CI 97.9-100%) for AS-AQ. Molecular testing confirmed that 87.4% (42/48) and 33.3% (1/3) of participants with a recurrent malaria infection in the AL and AS-AQ arms were new infections; an expected finding in a high malaria transmission area. Adverse events were documented in less than 2% of participants for both drugs. CONCLUSION: Both AL and AS-AQ have therapeutic efficacies well above the 90% WHO recommended threshold and remain well-tolerated in Mozambique. Routine monitoring of therapeutic efficacy should continue to ensure the treatments remain efficacious. Trial registration Clinicaltrials.gov: NCT04370977.

Molecular surveillance for polymorphisms associated with artemisinin-based combination therapy resistance in Plasmodium falciparum isolates collected in Mozambique, 2018.

BACKGROUND: Due to the threat of emerging anti-malarial resistance, the World Health Organization recommends incorporating surveillance for molecular markers of anti-malarial resistance into routine therapeutic efficacy studies (TESs). In 2018, a TES of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) was conducted in Mozambique, and the prevalence of polymorphisms in the pfk13, pfcrt, and pfmdr1 genes associated with drug resistance was investigated. METHODS: Children aged 6-59 months were enrolled in four study sites. Blood was collected and dried on filter paper from participants who developed fever within 28 days of initial malaria treatment. All samples were first screened for Plasmodium falciparum using a multiplex real-time PCR assay, and polymorphisms in the pfk13, pfcrt, and pfmdr1 genes were investigated by Sanger sequencing. RESULTS: No pfk13 mutations, associated with artemisinin partial resistance, were observed. The only pfcrt haplotype observed was the wild type CVMNK (codons 72-76), associated with chloroquine sensitivity. Polymorphisms in pfmdr1 were only observed at codon 184, with the mutant 184F in 43/109 (39.4%) of the samples, wild type Y184 in 42/109 (38.5%), and mixed 184F/Y in 24/109 (22.0%). All samples possessed N86 and D1246 at these two codons. CONCLUSION: In 2018, no markers of artemisinin resistance were documented. Molecular surveillance should continue to monitor the prevalence of these markers to inform decisions on malaria treatment in Mozambique.

In vivo efficacy and safety of artemether-lumefantrine and amodiaquine-artesunate for uncomplicated Plasmodium falciparum malaria in Mozambique, 2018

Artemisinin-based combination therapy (ACT) has been the recommended first-line treatment for uncomplicated malaria in Mozambique since 2006, with artemether-lumefantrine (AL) and amodiaquine-artesunate (AS-AQ) as the first choice. To assess efficacy of currently used ACT, an in vivo therapeutic efficacy study was conducted. Methods: The study was conducted in four sentinel sites: Montepuez, Moatize, Mopeia and Massinga. Patients between 6 and 59 months old with uncomplicated Plasmodium falciparum malaria (2000-200,000 parasites/µl) were enrolled between February and September of 2018, assigned to either an AL or AS-AQ treatment arm, and monitored for 28 days. A Bayesian algorithm was applied to differentiate recrudescence from new infection using genotyping data of seven neutral microsatellites. Uncorrected and PCR-corrected efficacy results at day 28 were calculated. Results: Totals of 368 and 273 patients were enrolled in the AL and AS-AQ arms, respectively. Of these, 9.5% (35/368) and 5.1% (14/273) were lost to follow-up in the AL and AS-AQ arms, respectively. There were 48 and 3 recurrent malaria infections (late clinical and late parasitological failures) in the AL and AS-AQ arms, respectively. The day 28 uncorrected efficacy was 85.6% (95% confidence interval (CI) 81.3-89.2%) for AL and 98.8% (95% CI 96.7-99.8%) for AS-AQ, whereas day 28 PCR-corrected efficacy was 97.9% (95% CI 95.6-99.2%) for AL and 99.6% (95% CI 97.9-100%) for AS-AQ. Molecular testing confirmed that 87.4% (42/48) and 33.3% (1/3) of participants with a recurrent malaria infection in the AL and AS-AQ arms were new infections; an expected finding in a high malaria transmission area. Adverse events were documented in less than 2% of participants for both drugs. Conclusion: Both AL and AS-AQ have therapeutic efficacies well above the 90% WHO recommended threshold and remain well-tolerated in Mozambique. Routine monitoring of therapeutic efficacy should continue to ensure the treatments remain efficacious. Trial registration Clinicaltrials.gov

Characterization of the Striatal Extracellular Matrix in a Mouse Model of Parkinson's Disease.

Parkinson's disease's etiology is unknown, although evidence suggests the involvement of oxidative modifications of intracellular components in disease pathobiology. Despite the known involvement of the extracellular matrix in physiology and disease, the influence of oxidative stress on the matrix has been neglected. The chemical modifications that might accumulate in matrix components due to their long half-live and the low amount of extracellular antioxidants could also contribute to the disease and explain ineffective cellular therapies. The enriched striatal extracellular matrix from a mouse model of Parkinson's disease was characterized by Raman spectroscopy. We found a matrix fingerprint of increased oxalate content and oxidative modifications. To uncover the effects of these changes on brain cells, we morphologically characterized the primary microglia used to repopulate this matrix and further quantified the effects on cellular mechanical stress by an intracellular fluorescence resonance energy transfer (FRET)-mechanosensor using the U-2 OS cell line. Our data suggest changes in microglia survival and morphology, and a decrease in cytoskeletal tension in response to the modified matrix from both hemispheres of 6-hydroxydopamine (6-OHDA)-lesioned animals. Collectively, these data suggest that the extracellular matrix is modified, and underscore the need for its thorough investigation, which may reveal new ways to improve therapies or may even reveal new therapies.

In vivo efficacy and safety of artemetherlumefantrine and amodiaquine­artesunate for uncomplicated Plasmodium falciparum malaria in Mozambique, 2018

Background: Artemisinin-based combination therapy (ACT) has been the recommended first-line treatment for uncomplicated malaria in Mozambique since 2006, with artemether­lumefantrine (AL) and amodiaquine­artesunate (AS­AQ) as the first choice. To assess efficacy of currently used ACT, an in vivo therapeutic efficacy study was conducted. Methods: The study was conducted in four sentinel sites: Montepuez, Moatize, Mopeia and Massinga. Patients between 6 and 59 months old with uncomplicated Plasmodium falciparum malaria (2000­200,000 parasites/µl) were enrolled between February and September of 2018, assigned to either an AL or AS­AQ treatment arm, and monitored for 28 days. A Bayesian algorithm was applied to differentiate recrudescence from new infection using genotyping data of seven neutral microsatellites. Uncorrected and PCR-corrected efficacy results at day 28 were calculated. Results: Totals of 368 and 273 patients were enrolled in the AL and AS­AQ arms, respectively. Of these, 9.5% (35/368) and 5.1% (14/273) were lost to follow-up in the AL and AS­AQ arms, respectively. There were 48 and 3 recurrent malaria infections (late clinical and late parasitological failures) in the AL and AS­AQ arms, respectively. The day 28 uncorrected efficacy was 85.6% (95% confidence interval (CI) 81.3­89.2%) for AL and 98.8% (95% CI 96.7­99.8%) for AS­AQ, whereas day 28 PCR-corrected efficacy was 97.9% (95% CI 95.6­99.2%) for AL and 99.6% (95% CI 97.9­100%) for AS­AQ. Molecular testing confirmed that 87.4% (42/48) and 33.3% (1/3) of participants with a recurrent malaria infection in the AL and AS­AQ arms were new infections; an expected finding in a high malaria transmission area. Adverse events were documented in less than 2% of participants for both drugs. Conclusion: Both AL and AS­AQ have therapeutic efficacies well above the 90% WHO recommended threshold and remain well-tolerated in Mozambique. Routine monitoring of therapeutic efficacy should continue to ensure the treatments remain efficacious.

Derivation of familial iPSC lines from three patients with retinitis pigmentosa carrying an autosomal dominant RPE65 mutation (NUIGi027-A, NUIGi028-A, NUIGi029-A).

Retinitis Pigmentosa (RP) is an inherited disorder of retinal degeneration with progressive loss of rod and cone photoreceptors. RPE65 is a gene encoding the trans-cis isomerase which is essential for the classical visual cycle. While most RPE65 mutations associated with RP have been reported as autosome, an Irish c.1430A > G (p.D477G) mutation is the first case reported to cause dominantly inherited RP. In this study, we used the non-integrational Sendai virus to generate induced pluripotent stem cell (iPSC) lines carrying the c.1430A > G (p.D477G) mutation from three familial RP patients.

Integrated community case management: planning for sustainability in five African countries.

BACKGROUND: The World Health Organization (WHO) launched an initiative to plan for the sustainability of integrated community case management (iCCM) programmes supported by the Rapid Access Expansion (RAcE) Programme in five African countries in 2016. WHO contracted experts to facilitate sustainability planning among Ministries of Health, WHO, nongovernmental organisation grantees, and other stakeholders. METHODS: We designed an iterative and unique process for each RAcE project area which involved creating a sustainability framework to guide planning; convening meetings to identify and prioritise elements of the framework; forming technical working groups to build country ownership; and, ultimately, creating roadmaps to guide efforts to fully transfer ownership of the iCCM programmes to host countries. For this analysis, we compared priorities identified in roadmaps across RAcE project sites, examined progress against roadmaps via transition plans, and produced recommendations for short-term actions based on roadmap priorities that were unaddressed or needed further attention. RESULTS: This article describes the sustainability planning process, roadmap priorities, progress against roadmaps, and recommendations made for each project area. We found a few patterns among the prioritised roadmap elements. Overall, every project area identified priorities related to policy and coordination of external stakeholders including funders; supply chain management; service delivery and referral system; and communication and social mobilisation, indicating that these factors have persisted despite iCCM programme maturity, and are also of concern to new programmes. We also found that a facilitated process to identify and document programme priorities in roadmaps, along with deliberately planning for transition from an external implementer to a national system could support the sustainability of iCCM programmes by facilitating teams of stakeholders to accomplish explicit tasks related to transitioning the programme. CONCLUSIONS: Certain common elements are of concern for sustaining iCCM programmes across countries, among them political leadership, supply chain management, data processes, human resources, and community engagement. Adapting and using a sustainability planning approach created an inclusive and comprehensive dialogue about systemic factors that influence the sustainability of iCCM services and facilitated changes to health systems in each country.

Multi-professional simulation and risk perception of health care workers caring for Ebola-infected patients.

INTRODUCTION: Treatment of infections that require high-level isolation can cause anxiety and fear among health care workers. Adequate and complete multi-professional simulation-based training could reduce those feelings and improve patient care. OBJECTIVE: The purpose of this study was to assess the impact of multi-professional simulation-based training on the risk perception and preparedness of health care workers (registered nurses, doctors and ancillary staff) who care for patients assessed to be at risk or confirmed to have Ebola, level 3-4 biohazard. METHODS: A prospective before-after study was designed. Health care workers who participated in a multi-professional simulation training course to improve the care of patients potentially infected with Level 3 and 4 biohazards were evaluated about their risk perception. The training was based on clinical scenarios. The evaluation was conducted using questionnaire based on Likert scale. After the training, a satisfaction survey about the most important aspects of the course was also conducted. RESULTS: Fifty-eight health care workers participated in the training course, 22 of whom were registered nurses. Participants presented positive changes after the training, increasing their sense of security, predisposition and confidence (p < 0.000001 for all). CONCLUSION: Multi-professional simulation-based training significantly improves the perception of safety and preparedness of health care workers regarding the care of patients potentially infected with Ebola virus and other Level 3-4 biohazards. RELEVANCE TO CLINICAL PRACTICE: The implementation of educational training strategies - such as simulations - is beneficial in improving the capacity of response and coping, as well as in reducing feelings of fear and insecurity.