Mutation of negative regulatory gene CEHC1 encoding an FBXO3 protein results in normoxic expression of HYDA genes in Chlamydomonas reinhardtii.

Oxygen is known to prevent hydrogen production in Chlamydomonas, both by inhibiting the hydrogenase enzyme and by preventing the accumulation of HYDA-encoding transcripts. We developed a screen for mutants showing constitutive accumulation of HYDA1 transcripts in the presence of oxygen. A reporter gene required for ciliary motility, placed under the control of the HYDA1 promoter, conferred motility only in hypoxic conditions. By selecting for mutants able to swim even in the presence of oxygen we obtained strains that express the reporter gene constitutively. One mutant identified a gene encoding an F-box only protein 3 (FBXO3), known to participate in ubiquitylation and proteasomal degradation pathways in other eukaryotes. Transcriptome profiles revealed that the mutation, termed cehc1-1 , leads to constitutive expression of HYDA1 and other genes regulated by hypoxia, and of many genes known to be targets of CRR1, a transcription factor in the nutritional copper signaling pathway. CRR1 was required for the constitutive expression of the HYDA1 reporter gene in cehc1-1 mutants. The CRR1 protein, which is normally degraded in Cu-supplemented cells, was stabilized in cehc1-1 cells, supporting the conclusion that CEHC1 acts to facilitate the degradation of CRR1. Our results reveal a novel negative regulator in the CRR1 pathway and possibly other pathways leading to complex metabolic changes associated with response to hypoxia.

The LRRK2 kinase substrates RAB8a and RAB10 contribute complementary but distinct disease-relevant phenotypes in human neurons.

Mutations in the LRRK2 gene cause familial Parkinson's disease presenting with pleomorphic neuropathology that can involve α-synuclein or tau accumulation. LRRK2 mutations are thought to converge upon a pathogenic increase in LRRK2 kinase activity. A subset of small RAB GTPases has been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in RAB inactivation. We used CRISPR-Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well-validated LRRK2 substrates, RAB8a and RAB10, from deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed opposing effects of RAB8a and RAB10 deficiency on lysosomal pH and Golgi organization, with isolated effects of RAB8a and RAB10 ablation on α-synuclein and tau, respectively. Our data demonstrate largely antagonistic effects of genetic RAB8a or RAB10 inactivation, which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation in human disease.

Cellular and subcellular localization of Rab10 and phospho-T73 Rab10 in the mouse and human brain.

Autosomal dominant pathogenic mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease (PD). The most common mutation, G2019S-LRRK2, increases the kinase activity of LRRK2 causing hyper-phosphorylation of its substrates. One of these substrates, Rab10, is phosphorylated at a conserved Thr73 residue (pRab10), and is one of the most abundant LRRK2 Rab GTPases expressed in various tissues. The involvement of Rab10 in neurodegenerative disease, including both PD and Alzheimer's disease makes pinpointing the cellular and subcellular localization of Rab10 and pRab10 in the brain an important step in understanding its functional role, and how post-translational modifications could impact function. To establish the specificity of antibodies to the phosphorylated form of Rab10 (pRab10), Rab10 specific antisense oligonucleotides were intraventricularly injected into the brains of mice. Further, Rab10 knock out induced neurons, differentiated from human induced pluripotent stem cells were used to test the pRab10 antibody specificity. To amplify the weak immunofluorescence signal of pRab10, tyramide signal amplification was utilized. Rab10 and pRab10 were expressed in the cortex, striatum and the substantia nigra pars compacta. Immunofluorescence for pRab10 was increased in G2019S-LRRK2 knockin mice. Neurons, astrocytes, microglia and oligodendrocytes all showed Rab10 and pRab10 expression. While Rab10 colocalized with endoplasmic reticulum, lysosome and trans-Golgi network markers, pRab10 did not localize to these organelles. However, pRab10, did overlap with markers of the presynaptic terminal in both mouse and human cortex, including α-synuclein. Results from this study suggest Rab10 and pRab10 are expressed in all brain areas and cell types tested in this study, but pRab10 is enriched at the presynaptic terminal. As Rab10 is a LRRK2 kinase substrate, increased kinase activity of G2019S-LRRK2 in PD may affect Rab10 mediated membrane trafficking at the presynaptic terminal in neurons in disease.

The LRRK2 kinase substrates Rab8a and Rab10 contribute complementary but distinct disease-relevant phenotypes in human neurons.

Mutations in the LRRK2 gene cause familial Parkinson's disease presenting with pleomorphic neuropathology that can involve α-synuclein or tau accumulation. LRRK2 mutations are thought to converge toward a pathogenic increase in LRRK2 kinase activity. A subset of small Rab GTPases have been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in Rab inactivation. We used CRISPR/Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well validated LRRK2 substrates, Rab8a and Rab10, from two independent, deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed divergent effects of Rab8a and Rab10 deficiency on lysosomal pH, LAMP1 association with Golgi, α-synuclein insolubility and tau phosphorylation, while parallel effects on lysosomal numbers and Golgi clustering were observed. Our data demonstrate largely antagonistic effects of genetic Rab8a or Rab10 inactivation which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation.

Parkin coregulates glutathione metabolism in adult mammalian brain.

We recently discovered that the expression of PRKN, a young-onset Parkinson disease-linked gene, confers redox homeostasis. To further examine the protective effects of parkin in an oxidative stress model, we first combined the loss of prkn with Sod2 haploinsufficiency in mice. Although adult prkn-/-//Sod2± animals did not develop dopamine cell loss in the S. nigra, they had more reactive oxidative species and a higher concentration of carbonylated proteins in the brain; bi-genic mice also showed a trend for more nitrotyrosinated proteins. Because these redox changes were seen in the cytosol rather than mitochondria, we next explored the thiol network in the context of PRKN expression. We detected a parkin deficiency-associated increase in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in murine brain, PRKN-linked human cortex and several cell models. This shift resulted from enhanced recycling of GSSG back to GSH via upregulated glutathione reductase activity; it also correlated with altered activities of redox-sensitive enzymes in mitochondria isolated from mouse brain (e.g., aconitase-2; creatine kinase). Intriguingly, human parkin itself showed glutathione-recycling activity in vitro and in cells: For each GSSG dipeptide encountered, parkin regenerated one GSH molecule and was S-glutathionylated by the other (GSSG + P-SH [Formula: see text] GSH + P-S-SG), including at cysteines 59, 95 and 377. Moreover, parkin's S-glutathionylation was reversible by glutaredoxin activity. In summary, we found that PRKN gene expression contributes to the network of available thiols in the cell, including by parkin's participation in glutathione recycling, which involves a reversible, posttranslational modification at select cysteines. Further, parkin's impact on redox homeostasis in the cytosol can affect enzyme activities elsewhere, such as in mitochondria. We posit that antioxidant functions of parkin may explain many of its previously described, protective effects in vertebrates and invertebrates that are unrelated to E3 ligase activity.

Polymerization of recombinant tau core fragments in vitro and seeding studies in cultured cells.

The relative polymerization of specific tau protein cores that define Alzheimer's disease, Pick's disease and corticobasal degeneration were investigated using amyloid fluorometry and electron microscopy. In addition, the relative prion-like activities of polymers comprised of these respective tau protein segments were investigated in a cell-based assay. It is demonstrated that the seeding activities of specific tau core fibrils are affected by the presence of pathogenic tau missense mutations and the microtubule binding domain composition of tau. The unique impact of tau phosphorylation on seeding propensity was also investigated by altering stretches of phospho-mimetic and phospho-null residues in the presence of Alzheimer's disease tau core fibrils. These results have important mechanistic implications for mutation and isoform-specific driven pathogenesis.

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

[This corrects the article DOI: 10.1371/journal.pbio.3001480.].

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

Is there a special relationship between complex I activity and nigral neuronal loss in Parkinson's disease? A critical reappraisal.

Parkinson's disease (PD) is a progressive neurodegenerative disease manifesting both motor and non-motor symptoms. The motor features are generally ascribed to the selective loss of dopamine neurons within the substantia nigra pars compacta. While the precise etiology of PD remains elusive, multiple genetic and environmental elements have emerged as contributing factors. The discovery of MPTP-induced parkinsonism directed intense inquiry towards mitochondrial pathways, with a specific focus on mitochondrial complex I. Consisting of more than 40 subunits, complex I is the first enzyme of the electron transport chain that is required for mitochondrial ATP production. In this review, we present a critical analysis of studies assessing the prevalence and specificity of mitochondrial complex I deficiency in PD. In addition, we take the novel view of incorporating the features of genetically-defined bona fide complex I disorders and the prevalence of nigral involvement in such cases. Through this innovative bi-directional view, we consider both complex I changes in a disease of the substantia nigra and nigral changes in diseases of complex I. We assess the strength of association between nigral cell loss and complex I deficits, as well as the oft under-appreciated heterogeneity of complex I deficiency disorders and the variability of the PD data.

A Unique Case of COVID-19-related Acute Coronary Thrombosis Complicated by Severe Hypokalemia.

We report the case of a 52-year-old white male who was recently diagnosed with symptomatic coronavirus disease-2019 (COVID-19) and presented to the hospital with ventricular tachycardia/ventricular fibrillation cardiac arrest, ST elevation myocardial infarction, and profound hypokalemia. The patient was successfully treated with primary percutaneous coronary intervention and concurrent aggressive potassium repletion. To the authors' knowledge, this is the first case of COVID-19 presenting not only with an acute coronary thrombosis but also severe hypokalemia, both of which contributed to his cardiac arrest. The association of COVID-19 with acute coronary thrombosis, including the challenges surrounding the diagnosis and management in this patient population, is discussed. Additionally, the effect of COVID-19 on the renin-angiotensin-aldosterone system is reviewed with a focus on hypokalemic presentations.

Cell Type-Specific Transcriptomics Reveals that Mutant Huntingtin Leads to Mitochondrial RNA Release and Neuronal Innate Immune Activation.

The mechanisms by which mutant huntingtin (mHTT) leads to neuronal cell death in Huntington's disease (HD) are not fully understood. To gain new molecular insights, we used single nuclear RNA sequencing (snRNA-seq) and translating ribosome affinity purification (TRAP) to conduct transcriptomic analyses of caudate/putamen (striatal) cell type-specific gene expression changes in human HD and mouse models of HD. In striatal spiny projection neurons, the most vulnerable cell type in HD, we observe a release of mitochondrial RNA (mtRNA) (a potent mitochondrial-derived innate immunogen) and a concomitant upregulation of innate immune signaling in spiny projection neurons. Further, we observe that the released mtRNAs can directly bind to the innate immune sensor protein kinase R (PKR). We highlight the importance of studying cell type-specific gene expression dysregulation in HD pathogenesis and reveal that the activation of innate immune signaling in the most vulnerable HD neurons provides a novel framework to understand the basis of mHTT toxicity and raises new therapeutic opportunities.

LRRK2 Kinase Inhibition Rescues Deficits in Lysosome Function Due to Heterozygous GBA1 Expression in Human iPSC-Derived Neurons.

A growing number of genes associated with Parkinson's disease are implicated in the regulation of lysosome function, including LRRK2, whose missense mutations are perhaps the most common monogenic cause of this neurodegenerative disease. These mutations are collectively thought to introduce a pathologic increase in LRRK2 kinase activity, which is currently a major target for therapeutic intervention. Heterozygous carriers of many missense mutations in the GBA1 gene have dramatically increased risk of Parkinson's disease. A critical question has recently emerged regarding the potential interplay between the proteins encoded by these two disease-linked genes. Our group has recently demonstrated that knockin mutation of a Parkinson's-linked GBA1 variant induces severe lysosomal and cytokine abnormalities in murine astrocytes and that these deficits were normalized via inhibition of wild-type LRRK2 kinase activity in these cells. Another group independently found that LRRK2 inhibition increases glucocerebrosidase activity in wild-type human iPSC-derived neurons, as well as those whose activity is disrupted by GBA1 or LRRK2 mutation. Fundamental questions remain in terms of the lysosomal abnormalities and the effects of LRRK2 kinase inhibition in human neurons deficient in glucocerebrosidase activity. Here, we further elucidate the physiological crosstalk between LRRK2 signaling and glucocerebrosidase activity in human iPSC-derived neurons. Our studies show that the allelic loss of GBA1 manifests broad defects in lysosomal morphology and function. Furthermore, our data show an increase in both the accumulation and secretion of oligomeric α-synuclein protein in these GBA1-heterozygous-null neurons, compared to isogenic controls. Consistent with recent findings in murine astrocytes, we observed that multiple indices of lysosomal dysfunction in GBA1-deficient human neurons were normalized by LRRK2 kinase inhibition, while some defects were preserved. Our findings demonstrate a selective but functional intersection between glucocerebrosidase dysfunction and LRRK2 signaling in the cell and may have implications in the pathogenesis and treatment of Parkinson's disease.

Subacute combined degeneration: a case of pernicious anaemia without haematological manifestations.

Vitamin B12 deficiency can be caused by a diverse group of aetiologies. One of the less common of these is an autoimmune condition pernicious anaemia, so named after the most common physiological manifestation of B12 deficiency: anaemia. However, B12 is also necessary for nervous system function and its depletion can lead to dysfunction of the posterior columns of the spinal cord resulting in subacute combined degeneration (SCD). This disease, while debilitating in its acute phase, can usually be mostly if not fully reversed if caught early and treated appropriately. Early detection can prove challenging if there are no haematological manifestations of B12 deficiency and the only guidance is the high index of suspicion. We present a case of pernicious anaemia leading to SCD without any clinical or laboratory findings of anaemia in this report.

Lysosome and Inflammatory Defects in GBA1-Mutant Astrocytes Are Normalized by LRRK2 Inhibition.

BACKGROUND: Autosomal recessive mutations in the glucocerebrosidase gene, Beta-glucocerebrosidase 1 (GBA1), cause the lysosomal storage disorder Gaucher's disease. Heterozygous carriers of most GBA1 mutations have dramatically increased Parkinson's disease (PD) risk, but the mechanisms and cells affected remain unknown. Glucocerebrosidase expression is relatively enriched in astrocytes, yet the impact of its mutation in these cells has not yet been addressed. OBJECTIVES: Emerging data supporting non-cell-autonomous mechanisms driving PD pathogenesis inspired the first characterization of GBA1-mutant astrocytes. In addition, we asked whether LRRK2, likewise linked to PD and enriched in astrocytes, intersected with GBA1 phenotypes. METHODS: Using heterozygous and homozygous GBA1 D409V knockin mouse astrocytes, we conducted rigorous biochemical and image-based analyses of lysosomal function and morphology. We also examined basal and evoked cytokine response at the transcriptional and secretory levels. RESULTS: The D409V knockin astrocytes manifested broad deficits in lysosomal morphology and function, as expected. This, however, is the first study to show dramatic defects in basal and TLR4-dependent cytokine production. Albeit to different extents, both the lysosomal dysfunction and inflammatory responses were normalized by inhibition of LRRK2 kinase activity, suggesting functional intracellular crosstalk between glucocerebrosidase and LRRK2 activities in astrocytes. CONCLUSIONS: These data demonstrate novel pathologic effects of a GBA1 mutation on inflammatory responses in astrocytes, indicating the likelihood of broader immunologic changes in GBA-PD patients. Our findings support the involvement of non-cell-autonomous mechanisms contributing to the pathogenesis of GBA1-linked PD and identify new opportunities to correct these changes with pharmacological intervention. © 2020 International Parkinson and Movement Disorder Society.

Use of Simulation Based Training to Enhance Cardiac Auscultation Proficiency.

This article was migrated. The article was marked as recommended. INTRODUCTION: Cardiopulmonary auscultation is an important skill for medical professionals with deficiencies being well documented with broad implications on healthcare. Providing ideal bedside auscultatory teaching presents many difficulties. Simulation has been used as a means to improve cardiac auscultatory training and circumvent some of these limitations. METHODS: We studied the use of Harvey © simulation in teaching cardiac murmurs and whether there was improvement in short term knowledge, as well as testing long term knowledge retention, as measured through a standardized test. From 2014-2019, 124 medical students in their 2 nd and 3 rd year of school (during the clinical portion of medical school curriculum) rotating through an Internal Medicine rotation completed a 2 hour training course on cardiac auscultation using Harvey © to identify six common cardiac murmurs. The session contained a pretest, didactic session, and posttest. RESULTS: 124 students participated in the auscultatory training session. Of them, 42 (34%) underwent the session a second time at an average of 1.29 months from their first session. There was statistically significant improvement between tests. Notably, the most often missed murmurs were mitral stenosis and benign (innocent) flow murmur. DISCUSSION/CONCLUSIONS: As shown in our study, simulation based cardiac auscultatory education is feasible and likely beneficial in medical education as it can be delivered to a large group of trainees and overcomes the challenges of bedside teaching.

Asymptomatic bilateral common iliac artery dissections in previously undiagnosed vascular Ehlers-Danlos syndrome.

We report a case of a 35-year-old woman found to have vascular Ehlers-Danlos syndrome (vEDS) after family history of sudden death due to aortic dissection in her otherwise healthy brother prompted further imaging workup and consideration of an underlying heritable genetic condition. CT angiogram of the aorta with intravenous contrast revealed an abdominal aortic artery dissection below the level of the renal arteries extending from the bifurcation into the left common iliac artery with an additional focal dissection of the right common iliac artery. To the author's knowledge, this is the first report of asymptomatic bilateral common iliac artery dissections as a part of the initial presentation of a patient with underlying vEDS. Additionally, this case highlights the importance of familial diagnostic screening in inherited vasculopathies. Clinical history, genetic testing and management are discussed.

Nocturnal dialysis improves sleep apnea more than daytime dialysis: a meta-analysis of crossover studies.

PURPOSE: To systematically review the literature for articles evaluating differences in polysomnography (PSG) data when patients are on primarily daytime hemodialysis (conventional hemodialysis or continuous ambulatory peritoneal dialysis) versus nocturnal hemodialysis (nocturnal hemodialysis or nocturnal peritoneal dialysis). Then to perform a meta-analysis on the available PSG data, specifically evaluating differences in apnea hypopnea index (AHI) and mean saturation of oxygen (SpO2) between these two groups. METHODS: Two authors systematically searched MEDLINE/Pubmed, Scopus, EMBASE, CINAHL, and Cochrane. Searches were performed through December 6, 2018. RESULTS: A total of four adult crossover studies (91 patients, age 50.4 ± 12.4, BMI 25.1 ± 5.3) reported PSG data. The daytime hemodialysis (DHD) and nocturnal hemodialysis (NHD) AHI decreased from 24.6 ± 18.2 to 12.6 ± 11.8 (events/hour) with a mean difference of -11.9 [95% CI -13.47, -10.37], Z score of 15.07 (P < 0.00001). The standardized mean difference was -1.35 [95% CI -2.70, 0.01]. Two studies reported mean SpO2 changes during PSG. The DHD and NHD SpO2 increased from 92.7 ± 2.4 to 94.7 ± 2.2 with a mean difference of 2.26 [95% CI -0.18, 4.71], Z score 1.82 (P = 0.07). CONCLUSION: In the current literature, nocturnal hemodialysis improves AHI more than daytime hemodialysis. A trend towards improvement in mean SpO2 with nocturnal dialysis was noted, but did not reach statistical significance. Consideration can be given for transitioning patients who have end stage renal disease and sleep apnea from daytime to nocturnal hemodialysis as an adjunct to other treatment modalities.

Can health promotion videos 'go viral'? A non-randomised, controlled, before-and-after pilot study to measure the spread and impact of local language mobile videos in Burkina Faso.

BACKGROUND: Mobile phones present a new health communications opportunity but use of mobile videos warrants more exploration. Our study tested a new idea: to produce health promotion videos in languages for which films have never previously been produced to see if they were widely shared. OBJECTIVE: To investigate whether the novelty of films in local languages focusing on health messages would be shared 'virally' among the target population. METHODS: A non-randomised, controlled, before-and-after study was used to evaluate the reach and impact of the intervention. We gave short health promotion videos on memory cards to distributors in eight intervention villages. Ten control villages, where no video distribution took place were randomly selected. We conducted cluster-level difference-in-difference logistic regression to assess self-reported knowledge indicators. We calculated odds ratios for intervention relative to control at baseline and endline and p-values for the change in odds ratios. RESULTS: Seven hundred and eight mothers were interviewed across all villages at baseline and 728 different mothers and 726 men were interviewed in the same villages a year later in October 2015. At endline, 32% of women and 44% of men in the intervention arm had ever seen a film on a mobile phone in Lobiri, compared to 1% of women and 2% of men in the control arm. There was a significant increase in the odds of knowing about giving Orasel to a child with diarrhoea in the intervention area relative to the control area. Awareness of the need to take a child with fever or symptoms of pneumonia to a health centre increased in the intervention area, but not significantly. CONCLUSIONS: Viral sharing of films on mobile phones has the potential to be an effective health promotion tool for communities whose languages are not served by existing mass media channels.

Modelling the effect of a mass radio campaign on child mortality using facility utilisation data and the Lives Saved Tool (LiST): findings from a cluster randomised trial in Burkina Faso.

BACKGROUND: A cluster randomised trial (CRT) in Burkina Faso was the first to demonstrate that a radio campaign increased health-seeking behaviours, specifically antenatal care attendance, health facility deliveries and primary care consultations for children under 5 years. METHODS: Under-five consultation data by diagnosis was obtained from primary health facilities in trial clusters, from January 2011 to December 2014. Interrupted time-series analyses were conducted to assess the intervention effect by time period on under-five consultations for separate diagnosis categories that were targeted by the media campaign. The Lives Saved Tool was used to estimate the number of under-five lives saved and the per cent reduction in child mortality that might have resulted from increased health service utilisation. Scenarios were generated to estimate the effect of the intervention in the CRT study areas, as well as a national scale-up in Burkina Faso and future scale-up scenarios for national media campaigns in five African countries from 2018 to 2020. RESULTS: Consultations for malaria symptoms increased by 56% in the first year (95% CI 30% to 88%; p<0.001) of the campaign, 37% in the second year (95% CI 12% to 69%; p=0.003) and 35% in the third year (95% CI 9% to 67%; p=0.006) relative to the increase in the control arm. Consultations for lower respiratory infections increased by 39% in the first year of the campaign (95% CI 22% to 58%; p<0.001), 25% in the second (95% CI 5% to 49%; p=0.010) and 11% in the third year (95% CI -20% to 54%; p=0.525). Diarrhoea consultations increased by 73% in the first year (95% CI 42% to 110%; p<0.001), 60% in the second (95% CI 12% to 129%; p=0.010) and 107% in the third year (95% CI 43% to 200%; p<0.001). Consultations for other diagnoses that were not targeted by the radio campaign did not differ between intervention and control arms. The estimated reduction in under-five mortality attributable to the radio intervention was 9.7% in the first year (uncertainty range: 5.1%-15.1%), 5.7% in the second year and 5.5% in the third year. The estimated number of under-five lives saved in the intervention zones during the trial was 2967 (range: 1110-5741). If scaled up nationally, the estimated reduction in under-five mortality would have been similar (9.2% in year 1, 5.6% in year 2 and 5.5% in year 3), equating to 14 888 under-five lives saved (range: 4832-30 432). The estimated number of lives that could be saved by implementing national media campaigns in other low-income settings ranged from 7205 in Burundi to 21 443 in Mozambique. CONCLUSION: Evidence from a CRT shows that a child health radio campaign increased under-five consultations at primary health centres for malaria, pneumonia and diarrhoea (the leading causes of postneonatal child mortality in Burkina Faso) and resulted in an estimated 7.1% average reduction in under-five mortality per year. These findings suggest important reductions in under-five mortality can be achieved by mass media alone, particularly when conducted at national scale.