[Show abstract][Hide abstract] ABSTRACT: Problem:
In 2008, the prevalence of paediatric asthma in Zambia was unknown and the national treatment guideline was outdated.
We created an international partnership between Zambian clinicians, the Zambian Government and a pharmaceutical company to address shortcomings in asthma treatment. We did two studies, one to estimate prevalence in the capital of Lusaka and one to assess attitudes and practices of patients. Based on the information obtained, we educated health workers and the public. The information from the studies was also used to modernize government policy for paediatric asthma management.
The health-care system in Zambia is primarily focused on acute care delivery with a focus on infectious diseases. Comprehensive services for noncommunicable diseases are lacking. Asthma management relies on treatment of acute exacerbations instead of disease control.
Seven percent of children surveyed had asthma (255/3911). Of the 120 patients interviewed, most (82/120, 68%) used oral short-acting β2-agonists for symptom control; almost half (59/120, 49%) did not think the symptoms were preventable and 43% (52/120) thought inhalers were addictive. These misconceptions informed broad-based educational programmes. We used a train-the-trainer model to educate health-care workers and ran public awareness campaigns. Access to inhalers was increased and the Zambian standard treatment guideline for paediatric asthma was revised to include steroid inhalers as a control treatment.
Joint activities were required to change paediatric asthma care in Zambia. Success will depend on local sustainability, and it may be necessary to shift resources to mirror the disease burden.
Preview · Article · Oct 2015 · Bulletin of the World Health Organisation
[Show abstract][Hide abstract] ABSTRACT: Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.
No preview · Article · Jun 2015 · Nature Chemical Biology
[Show abstract][Hide abstract] ABSTRACT: Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology.
Full-text · Article · Dec 2013 · Microbiological Research
[Show abstract][Hide abstract] ABSTRACT: Aside from established genetic evidence, the best proof of a model for disease pathogenesis rests on predicted perturbation via targeted medicines in clinical trials. Here, I discuss the strategy of performing exploratory first-in-human clinical studies on mechanistically homogeneous populations (often small groups of patients with rare diseases) as a routine entrance to full-registration clinical trials. Over the past decade, this approach has proved some pathogenic theories, disproved others, and guided investigators in new scientific directions. The immediate advantages have been smaller trials and provision of new treatments for rare diseases. Later, indications often can be expanded to subsets of more common diseases.
No preview · Article · Sep 2013 · Science translational medicine
[Show abstract][Hide abstract] ABSTRACT: With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.
[Show abstract][Hide abstract] ABSTRACT: With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver-and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood-and liver-stage prolif-eration. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collec-tion, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, clado-sporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.
[Show abstract][Hide abstract] ABSTRACT: The zebrafish is proving to be highly amenable to in vivo small molecule screening. With a growing number of screens successfully completed, a rich interface is being created between disciplines that have historically used zebrafish (e.g., embryology and genetics) and disciplines focused on small molecules (e.g., chemistry and pharmacology). Navigating this interface requires consideration of the unique demands of conducting high-throughput screening in vivo. In this chapter, we discuss design elements of successful zebrafish screens, established screening methods, and approaches for mechanism of action studies following discovery of novel small molecules. These methods are enabling the zebrafish to have an increasingly positive impact on biomedical research and drug development.
Preview · Article · Dec 2011 · Methods in cell biology
[Show abstract][Hide abstract] ABSTRACT: Assembly, maintenance and renewal of sarcomeres require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, the molecules that mediate these processes are largely unknown. Here, we isolated the zebrafish mutant flatline (fla), which shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. By positional cloning we identified a nonsense mutation within the SET- and MYND-domain-containing protein 1 gene (smyd1) to be responsible for the fla phenotype. We found SMYD1 expression to be restricted to the heart and fast-twitch skeletal muscle cells. Within these cell types, SMYD1 localizes to both the sarcomeric M-line, where it physically associates with myosin, and the nucleus, where it supposedly represses transcription through its SET and MYND domains. However, although we found transcript levels of thick filament chaperones, such as Hsp90a1 and UNC-45b, to be severely upregulated in fla, its histone methyltransferase activity - mainly responsible for the nuclear function of SMYD1 - is dispensable for sarcomerogenesis. Accordingly, sarcomere assembly in fla mutant embryos can be reconstituted by ectopically expressing histone methyltransferase-deficient SMYD1. By contrast, ectopic expression of myosin-binding-deficient SMYD1 does not rescue fla mutants, implicating an essential role for the SMYD1-myosin interaction in cardiac and fast-twitch skeletal muscle thick filament assembly.
Full-text · Article · Aug 2011 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: The zebrafish mutant silent partner is characterized by a dysmorphic, non-contractile ventricle resulting in an inability to generate normal blood flow. We have identified the genetic lesion in the zebrafish homolog of the slow twitch skeletal/cardiac troponin C gene. Although human troponin C1 (TNNC1) is expressed in both cardiac and skeletal muscle, duplication of this gene in zebrafish has resulted in tissue-specific partitioning of troponin C expression and function. Mutation of the zebrafish paralog tnnc1a, which is expressed predominantly in the heart, results in a loss of contractility and myofibrillar organization within ventricular cardiomyocytes, while skeletal muscle remains functional and intact. We further show that defective contractility in the developing heart results in abnormal atrial and ventricular chamber morphology. Together, our results suggest that tnnc1a is required both for the function and structural integrity of the contractile machinery in cardiomyocytes, helping to clarify potential mechanisms of troponin C-mediated cardiomyopathy.
Preview · Article · Nov 2010 · Developmental Dynamics
[Show abstract][Hide abstract] ABSTRACT: Cilia defects have been implicated in a variety of human diseases and genetic disorders, but how cilia motility contributes to these phenotypes is still unknown. To further our understanding of how cilia function in development, we have cloned and characterized two alleles of seahorse, a zebrafish mutation that results in pronephric cysts. seahorse encodes Lrrc6l, a leucine-rich repeat-containing protein that is highly conserved in organisms that have motile cilia. seahorse is expressed in zebrafish tissues known to contain motile cilia. Although mutants do not affect cilia structure and retain the ability to interact with Disheveled, both alleles of seahorse strongly affect cilia motility in the zebrafish pronephros and neural tube. Intriguingly, although seahorse mutations variably affect fluid flow in Kupffer's vesicle, they can have very weak effects on left-right patterning. Combined with recently published results, our alleles suggest that the function of seahorse in cilia motility is separable from its function in other cilia-related phenotypes.
[Show abstract][Hide abstract] ABSTRACT: Although it is well known that mutations in the cardiac essential myosin light chain-1 (cmlc-1) gene can cause hypertrophic cardiomyopathy, the precise in vivo structural and functional roles of cMLC-1 in the heart are only poorly understood. We have isolated the zebrafish mutant lazy susan (laz), which displays severely reduced contractility of both heart chambers. By positional cloning, we identified a nonsense mutation within the zebrafish cmlc-1 gene to be responsible for the laz phenotype, leading to expression of a carboxyl-terminally truncated cMLC-1. Whereas complete loss of cMLC-1 leads to cardiac acontractility attributable to impaired cardiac sarcomerogenesis, expression of a carboxyl-terminally truncated cMLC-1 in laz mutant hearts is sufficient for normal cardiac sarcomerogenesis but severely impairs cardiac contractility in a cell-autonomous fashion. Whereas overexpression of wild-type cMLC-1 restores contractility of laz mutant cardiomyocytes, overexpression of phosphorylation site serine 195-deficient cMLC-1 (cMLC-1(S195A)) does not reconstitute cardiac contractility in laz mutant cardiomyocytes. By contrast, introduction of a phosphomimetic amino acid on position 195 (cMLC-1(S195D)) rescues cardiomyocyte contractility, demonstrating for the first time an essential role of the carboxyl terminus and especially of serine 195 of cMLC-1 in the regulation of cardiac contractility.
Preview · Article · Feb 2009 · Circulation Research
[Show abstract][Hide abstract] ABSTRACT: Genetic predisposition is believed to be responsible for most clinically significant arrhythmias; however, suitable genetic animal models to study disease mechanisms and evaluate new treatment strategies are largely lacking.
In search of suitable arrhythmia models, we isolated the zebrafish mutation reggae (reg), which displays clinical features of the malignant human short-QT syndrome such as accelerated cardiac repolarization accompanied by cardiac fibrillation. By positional cloning, we identified the reg mutation that resides within the voltage sensor of the zebrafish ether-à-go-go-related gene (zERG) potassium channel. The mutation causes premature zERG channel activation and defective inactivation, which results in shortened action potential duration and accelerated cardiac repolarization. Genetic and pharmacological inhibition of zERG rescues recessive reg mutant embryos, which confirms the gain-of-function effect of the reg mutation on zERG channel function in vivo. Accordingly, QT intervals in ECGs from heterozygous and homozygous reg mutant adult zebrafish are considerably shorter than in wild-type zebrafish.
With its molecular and pathophysiological concordance to the human arrhythmia syndrome, zebrafish reg represents the first animal model for human short-QT syndrome.
[Show abstract][Hide abstract] ABSTRACT: The vertebrate heart possesses autoregulatory mechanisms enabling it first to sense and then to adapt its force of contraction to continually changing demands. The molecular components of the cardiac mechanical stretch sensor are mostly unknown but of immense medical importance, since dysfunction of this sensing machinery is suspected to be responsible for a significant proportion of human heart failure. In the hearts of the ethylnitros-urea (ENU)-induced, recessive embryonic lethal zebrafish heart failure mutant main squeeze (msq), we find stretch-responsive genes such as atrial natriuretic factor (anf) and vascular endothelial growth factor (vegf) severely down-regulated. We demonstrate through positional cloning that heart failure in msq mutants is due to a mutation in the integrin-linked kinase (ilk) gene. ILK specifically localizes to costameres and sarcomeric Z-discs. The msq mutation (L308P) reduces ILK kinase activity and disrupts binding of ILK to the Z-disc adaptor protein beta-parvin (Affixin). Accordingly, in msq mutant embryos, heart failure can be suppressed by expression of ILK, and also of a constitutively active form of Protein Kinase B (PKB), and VEGF. Furthermore, antisense-mediated abrogation of zebrafish beta-parvin phenocopies the msq phenotype. Thus, we provide evidence that the heart uses the Integrin-ILK-beta-parvin network to sense mechanical stretch and respond with increased expression of ANF and VEGF, the latter of which was recently shown to augment cardiac force by increasing the heart's calcium transients.
Full-text · Article · Oct 2006 · Genes & Development
[Show abstract][Hide abstract] ABSTRACT: During embryogenesis, the myocardial layer of the primitive heart tube grows outward from the endocardial-lined lumen, with new cells added to generate concentric thickness to the wall. This is a key evolutionary step, demarcating vertebrates from more primitive chordates, and is essential for normal cardiac function. Zebrafish embryos with the recessive lethal mutations santa (san) and valentine (vtn) do not thicken, but do add the proper number of cells to the myocardium. Consequently, the heart chambers are huge, constituted of a monolayered myocardium lined by endocardium. This phenotype is similar to that of the heart of glass (heg) mutation, which we described previously as a novel endocardial expressed gene. By positional cloning, we here identify san as the zebrafish homolog of human CCM1, and vtn as the homolog of human CCM2. Dominant mutations of either in humans cause vascular anomalies in the brain, known as cerebral cavernous malformations. The synergistic effects of morpholino pairs indicate that san, vtn and heg are in a genetic pathway, and san and vtn contain protein motifs, NPxY and PTB domain, respectively, known to interact. This suggests that concentric growth of the myocardium, crucial for blood pressure generation, is dictated by a heg-san-vtn signaling pathway.
[Show abstract][Hide abstract] ABSTRACT: Although it is well known that mutations in the cardiac regulatory myosin light chain-2 (mlc-2) gene cause hypertrophic cardiomyopathy, the precise in vivo structural and functional roles of MLC-2 in the heart are only poorly understood. We have isolated a mutation in zebrafish, tell tale heart (tel(m225)), which selectively perturbs contractility of the embryonic heart. By positional cloning, we identified tel to encode the zebrafish mlc-2 gene. In contrast to mammals, zebrafish have only 1 cardiac-specific mlc-2 gene, which we find to be expressed in atrial and ventricular cardiomyocytes during early embryonic development, but also in the adult heart. Accordingly, loss of zMLC-2 function cannot be compensated for by upregulation of another mlc-2 gene. Surprisingly, ultrastructural analysis of tel cardiomyocytes reveals complete absence of organized thick myofilaments. Thus, our findings provide the first in vivo evidence that cardiac MLC-2 is required for thick-filament stabilization and contractility in the vertebrate heart.
Full-text · Article · Sep 2006 · Circulation Research
[Show abstract][Hide abstract] ABSTRACT: GAP-43 is a gene expressed only in the nervous system. The protein product is believed to be important to neuronal growth and plasticity. Most, and likely all, neurons express high levels of GAP-43 during periods of neurite elongation. To initiate studies of GAP-43 gene regulation we have cloned the rat gene encoding GAP-43. The GAP-43 gene includes three exons. The first exon encodes only the amino terminal 10 amino acids, which corresponds to the membrane targeting domain of GAP-43. The second exon encodes a putative calmodulin binding domain and a protein kinase C phosphorylation site. The 5′-flanking sequence is unusual in that it lacks CAAT or TATA elements, and directs RNA transcription initiation from several sites. Some of the transcription start sites are used to a different degree in the central and peripheral nervous systems.
No preview · Article · Apr 2006 · European Journal of Neuroscience