Dissection of Besnoitia besnoiti intermediate host life cycle stages: From morphology to gene expression.

Cyst-forming Apicomplexa (CFA) of the Sarcocystidae have a ubiquitous presence as pathogens of humans and farm animals transmitted through the food chain between hosts with few notable exceptions. The defining hallmark of this family of obligate intracellular protists consists of their ability to remain for very long periods as infectious tissue cysts in chronically infected intermediate hosts. Nevertheless, each closely related species has evolved unique strategies to maintain distinct reservoirs on global scales and ensuring efficient transmission to definitive hosts as well as between intermediate hosts. Here, we present an in-depth comparative mRNA expression analysis of the tachyzoite and bradyzoite stages of Besnoitia besnoiti strain Lisbon14 isolated from an infected farm animal based on its annotated genome sequence. The B. besnoiti genome is highly syntenic with that of other CFA and also retains the capacity to encode a large majority of known and inferred factors essential for completing a sexual cycle in a yet unknown definitive host. This work introduces Besnoitia besnoiti as a new model for comparative biology of coccidian tissue cysts which can be readily obtained in high purity. This model provides a framework for addressing fundamental questions about the evolution of tissue cysts and the biology of this pharmacologically intractable infectious parasite stage.

Microbiomes, Their Function, and Cancer: How Metatranscriptomics Can Close the Knowledge Gap.

The interaction between the microbial communities in the human body and the onset and progression of cancer has not been investigated until recently. The vast majority of the metagenomics research in this area has concentrated on the composition of microbiomes, attempting to link the overabundance or depletion of certain microorganisms to cancer proliferation, metastatic behaviour, and its resistance to therapies. However, studies elucidating the functional implications of the microbiome activity in cancer patients are still scarce; in particular, there is an overwhelming lack of studies assessing such implications directly, through analysis of the transcriptome of the bacterial community. This review summarises the contributions of metagenomics and metatranscriptomics to the knowledge of the microbial environment associated with several cancers; most importantly, it highlights all the advantages that metatranscriptomics has over metagenomics and suggests how such an approach can be leveraged to advance the knowledge of the cancer bacterial environment.

Genome-wide transcriptomics identifies an early preclinical signature of prion infection.

The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance and splicing alterations during the course of disease in prion-inoculated mice. Prion infection induced PrP-dependent transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical signs, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, could be the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed signs in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets.

Alkali therapy protects renal function, suppresses inflammation, and improves cellular metabolism in kidney disease.

Chronic kidney disease (CKD) affects approximately 10-13% of the population worldwide and halting its progression is a major clinical challenge. Metabolic acidosis is both a consequence and a possible driver of CKD progression. Alkali therapy counteracts these effects in CKD patients, but underlying mechanisms remain incompletely understood. Here we show that bicarbonate supplementation protected renal function in a murine CKD model induced by an oxalate-rich diet. Alkali therapy had no effect on the aldosterone-endothelin axis but promoted levels of the anti-aging protein klotho; moreover, it suppressed adhesion molecules required for immune cell invasion along with reducing T-helper cell and inflammatory monocyte invasion. Comparing transcriptomes from the murine crystallopathy model and from human biopsies of kidney transplant recipients (KTRs) suffering from acidosis with or without alkali therapy unveils parallel transcriptome responses mainly associated with lipid metabolism and oxidoreductase activity. Our data reveal novel pathways associated with acidosis in kidney disease and sensitive to alkali therapy and identifies potential targets through which alkali therapy may act on CKD and that may be amenable for more targeted therapies.

Evidence for Effects of Extracellular Vesicles on Physical, Inflammatory, Transcriptome and Reward Behaviour Status in Mice.

Immune-inflammatory activation impacts extracellular vesicles (EVs), including their miRNA cargo. There is evidence for changes in the EV miRNome in inflammation-associated neuropsychiatric disorders. This mouse study investigated: (1) effects of systemic lipopolysaccharide (LPS) and chronic social stress (CSS) on plasma EV miRNome; and (2) physiological, transcriptional, and behavioural effects of peripheral or central delivered LPS-activated EVs in recipient mice. LPS or CSS effects on the plasma EV miRNome were assessed by using microRNA sequencing. Recipient mice received plasma EVs isolated from LPS-treated or SAL-treated donor mice or vehicle only, either intravenously or into the nucleus accumbens (NAc), on three consecutive days. Bodyweight, spleen or NAc transcriptome and reward (sucrose) motivation were assessed. LPS and CSS increased the expression of 122 and decreased expression of 20 plasma EV miRNAs, respectively. Peripheral LPS-EVs reduced bodyweight, and both LPS-EVs and SAL-EVs increased spleen expression of immune-relevant genes. NAc-infused LPS-EVs increased the expression of 10 immune-inflammatory genes. Whereas motivation increased similarly across test days in all groups, the effect of test days was more pronounced in mice that received peripheral or central LPS-EVs compared with other groups. This study provides causal evidence that increased EV levels impact physiological and behavioural processes and are of potential relevance to neuropsychiatric disorders.

Differential Expression of Serum Extracellular Vesicle miRNAs in Multiple Sclerosis: Disease-Stage Specificity and Relevance to Pathophysiology.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Its first clinical presentation (clinically isolated syndrome, CIS) is often followed by the development of relapsing-remitting MS (RRMS). The periphery-to-CNS transmission of inflammatory molecules is a major pathophysiological pathway in MS. This could include signalling via extracellular vesicle (EV) microRNAs (miRNAs). In this study, we investigated the serum EV miRNome in CIS and RRMS patients and matched controls, with the aims to identify MS stage-specific differentially expressed miRNAs and investigate their biomarker potential and pathophysiological relevance. miRNA sequencing was conducted on serum EVs from CIS-remission, RRMS-relapse, and viral inflammatory CNS disorder patients, as well as from healthy and hospitalized controls. Differential expression analysis was conducted, followed by predictive power and target-pathway analysis. A moderate number of dysregulated serum EV miRNAs were identified in CIS-remission and RRMS-relapse patients, especially relative to healthy controls. Some of these miRNAs were also differentially expressed between the two MS stages and had biomarker potential for patient-control and CIS-RRMS separations. For the mRNA targets of the RRMS-relapse-specific EV miRNAs, biological processes inherent to MS pathophysiology were identified using in silico analysis. Study findings demonstrate that specific serum EV miRNAs have MS stage-specific biomarker potential and contribute to the identification of potential targets for novel, efficacious therapies.

Germ-free and microbiota-associated mice yield small intestinal epithelial organoids with equivalent and robust transcriptome/proteome expression phenotypes.

Intestinal epithelial organoids established from gut tissue have become a widely used research tool. However, it remains unclear how environmental cues, divergent microbiota composition and other sources of variation before, during and after establishment confound organoid properties, and how these properties relate to the original tissue. While environmental influences cannot be easily addressed in human organoids, mice offer a controlled assay-system. Here, we probed the effect of donor microbiota differences, previously identified as a confounding factor in murine in vivo studies, on organoids. We analysed the proteomes and transcriptomes of primary organoid cultures established from two colonised and one germ-free mouse colony of C57BL/6J genetic background, and compared them to their tissue of origin and commonly used cell lines. While an imprint of microbiota-exposure was observed on the proteome of epithelial samples, the long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead, stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts and closely mimicked expression patterns in the gut epithelium. This included the inflammasome components ASC, Naip1-6, Nlrc4 and Caspase-1, which were highly expressed in all organoids compared to the reference cell line m-ICc12 or mouse embryonic fibroblasts. Taken together, these results reveal that the donor microbiota has little effect on the organoid phenotype and suggest that organoids represent a more suitable culture model than immortalised cell lines, in particular for studies of intestinal epithelial inflammasomes.

Acidosis and alkali therapy in patients with kidney transplant is associated with transcriptional changes and altered abundance of genes involved in cell metabolism and acid-base balance.

BACKGROUND: Metabolic acidosis occurs frequently in patients with kidney transplant and is associated with a higher risk for and accelerated loss of graft function. To date, it is not known whether alkali therapy in these patients improves kidney function and whether acidosis and its therapy are associated with altered expression of proteins involved in renal acid-base metabolism. METHODS: We retrospectively collected kidney biopsies from 22 patients. Of these patients, nine had no acidosis, nine had metabolic acidosis [plasma bicarbonate (HCO3- <22 mmol/L) and four had acidosis and received alkali therapy. We performed transcriptome analysis and immunohistochemistry for proteins involved in renal acid-base handling. RESULTS: We found that the expression of 40 transcripts significantly changed between kidneys from non-acidotic and acidotic patients. These genes are mostly involved in proximal tubule (PT) amino acid and lipid metabolism and energy homoeostasis. Three transcripts were fully recovered by alkali therapy: the Kir4.2 potassium channel, an important regulator of PT HCO3- metabolism and transport, acyl-CoA dehydrogenase short/branched chain and serine hydroxymethyltransferase 1, genes involved in beta oxidation and methionine metabolism. Immunohistochemistry showed reduced staining for the PT NBCe1 HCO3- transporter in kidneys from acidotic patients who recovered with alkali therapy. In addition, the HCO3- exchanger pendrin was affected by acidosis and alkali therapy. CONCLUSIONS: Metabolic acidosis in kidney transplant recipients is associated with alterations in the renal transcriptome that are partly restored by alkali therapy. Acid-base transport proteins mostly from PT were also affected by acidosis and alkali therapy, suggesting that the downregulation of critical players contributes to metabolic acidosis in these patients.

Re-evaluation of single nucleotide variants and identification of structural variants in a cohort of 45 sudden unexplained death cases.

Sudden unexplained death (SUD) takes up a considerable part in overall sudden death cases, especially in adolescents and young adults. During the past decade, many channelopathy- and cardiomyopathy-associated single nucleotide variants (SNVs) have been identified in SUD studies by means of postmortem molecular autopsy, yet the number of cases that remain inconclusive is still high. Recent studies had suggested that structural variants (SVs) might play an important role in SUD, but there is no consensus on the impact of SVs on inherited cardiac diseases. In this study, we searched for potentially pathogenic SVs in 244 genes associated with cardiac diseases. Whole-exome sequencing and appropriate data analysis were performed in 45 SUD cases. Re-analysis of the exome data according to the current ACMG guidelines identified 14 pathogenic or likely pathogenic variants in 10 (22.2%) out of the 45 SUD cases, whereof 2 (4.4%) individuals had variants with likely functional effects in the channelopathy-associated genes SCN5A and TRDN and 1 (2.2%) individual in the cardiomyopathy-associated gene DTNA. In addition, 18 structural variants (SVs) were identified in 15 out of the 45 individuals. Two SVs with likely functional impairment were found in the coding regions of PDSS2 and TRPM4 in 2 SUD cases (4.4%). Both were identified as heterozygous deletions, which were confirmed by multiplex ligation-dependent probe amplification. In conclusion, our findings support that SVs could contribute to the pathology of the sudden death event in some of the cases and therefore should be investigated on a routine basis in suspected SUD cases.

Cell Cycle Constraints and Environmental Control of Local DNA Hypomethylation in α-Proteobacteria.

Heritable DNA methylation imprints are ubiquitous and underlie genetic variability from bacteria to humans. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. Despite the importance of local (hypo)methylation at specific loci, how and when these patterns are established during the cell cycle remains poorly characterized. Taking advantage of the small genomes and the synchronizability of α-proteobacteria, we discovered that conserved determinants of the cell cycle transcriptional circuitry establish specific hypomethylation patterns in the cell cycle model system Caulobacter crescentus. We used genome-wide methyl-N6-adenine (m6A-) analyses by restriction-enzyme-cleavage sequencing (REC-Seq) and single-molecule real-time (SMRT) sequencing to show that MucR, a transcriptional regulator that represses virulence and cell cycle genes in S-phase but no longer in G1-phase, occludes 5'-GANTC-3' sequence motifs that are methylated by the DNA adenine methyltransferase CcrM. Constitutive expression of CcrM or heterologous methylases in at least two different α-proteobacteria homogenizes m6A patterns even when MucR is present and affects promoter activity. Environmental stress (phosphate limitation) can override and reconfigure local hypomethylation patterns imposed by the cell cycle circuitry that dictate when and where local hypomethylation is instated.

Exome analysis in 34 sudden unexplained death (SUD) victims mainly identified variants in channelopathy-associated genes.

Sudden cardiac death (SCD) is one of the major causes of mortality worldwide, mostly involving coronary artery disease in the elderly. In contrary, sudden death events in young victims often represent the first manifestation of undetected genetic cardiac diseases, which remained without any symptoms during lifetime. Approximately 30% of these sudden death cases have no definite cardiac etiology after a comprehensive medicolegal investigation and are therefore termed as sudden unexplained death (SUD) cases. Advances in high-throughput sequencing approaches have provided an efficient diagnostic tool to identify likely pathogenic variants in cardiovascular disease-associated genes in otherwise autopsy-negative SUD cases. The aim of this study was to genetically investigate a cohort of 34 unexplained death cases by focusing on candidate genes associated with cardiomyopathies and channelopathies. Exome analysis identified potentially disease-causing sequence alterations in 29.4% of the 34 SUD cases. Six (17.6%) individuals had variants with likely functional effects in the channelopathy-associated genes AKAP9, KCNE5, RYR2, and SEMA3A. Interestingly, four of these six SUD individuals were younger than 18 years of age. Since the total SUD cohort of this study included five children and adolescents, post-mortem molecular autopsy screening indicates a high diagnostic yield within this age group. Molecular genetic testing represents a valuable approach to uncover the cause of death in some of the SUD victims; however, 70-80% of the cases still remain elusive, emphasizing the importance of additional research to better understand the pathological mechanisms leading to a sudden death event.

An allosteric gating model recapitulates the biophysical properties of IK,L expressed in mouse vestibular type I hair cells.

KEY POINTS: Vestibular type I and type II hair cells and their afferent fibres send information to the brain regarding the position and movement of the head. The characteristic feature of type I hair cells is the expression of a low-voltage-activated outward rectifying K+ current, IK,L , whose biophysical properties and molecular identity are still largely unknown. In vitro, the afferent nerve calyx surrounding type I hair cells causes unstable intercellular K+ concentrations, altering the biophysical properties of IK,L . We found that in the absence of the calyx, IK,L in type I hair cells exhibited unique biophysical activation properties, which were faithfully reproduced by an allosteric channel gating scheme. These results form the basis for a molecular and pharmacological identification of IK,L . ABSTRACT: Type I and type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named the calyx, and by the expression of a low-voltage-activated outward rectifying K+ current, IK,L . The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch-clamp whole-cell technique, we examined the voltage- and time-dependent properties of IK,L in type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential (Veq K+ ). Both the outward and inward K+ currents shifted Veq K+ consistent with K+ accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half-activation at -79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The 'early' open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the 'native' biophysical properties of IK,L in adult mouse vestibular type I hair cells.

SUSHI: an exquisite recipe for fully documented, reproducible and reusable NGS data analysis.

BACKGROUND: Next generation sequencing (NGS) produces massive datasets consisting of billions of reads and up to thousands of samples. Subsequent bioinformatic analysis is typically done with the help of open source tools, where each application performs a single step towards the final result. This situation leaves the bioinformaticians with the tasks to combine the tools, manage the data files and meta-information, document the analysis, and ensure reproducibility. RESULTS: We present SUSHI, an agile data analysis framework that relieves bioinformaticians from the administrative challenges of their data analysis. SUSHI lets users build reproducible data analysis workflows from individual applications and manages the input data, the parameters, meta-information with user-driven semantics, and the job scripts. As distinguishing features, SUSHI provides an expert command line interface as well as a convenient web interface to run bioinformatics tools. SUSHI datasets are self-contained and self-documented on the file system. This makes them fully reproducible and ready to be shared. With the associated meta-information being formatted as plain text tables, the datasets can be readily further analyzed and interpreted outside SUSHI. CONCLUSION: SUSHI provides an exquisite recipe for analysing NGS data. By following the SUSHI recipe, SUSHI makes data analysis straightforward and takes care of documentation and administration tasks. Thus, the user can fully dedicate his time to the analysis itself. SUSHI is suitable for use by bioinformaticians as well as life science researchers. It is targeted for, but by no means constrained to, NGS data analysis. Our SUSHI instance is in productive use and has served as data analysis interface for more than 1000 data analysis projects. SUSHI source code as well as a demo server are freely available.

High-resolution community profiling of arbuscular mycorrhizal fungi.

Community analyses of arbuscular mycorrhizal fungi (AMF) using ribosomal small subunit (SSU) or internal transcribed spacer (ITS) DNA sequences often suffer from low resolution or coverage. We developed a novel sequencing based approach for a highly resolving and specific profiling of AMF communities. We took advantage of previously established AMF-specific PCR primers that amplify a c. 1.5-kb long fragment covering parts of SSU, ITS and parts of the large ribosomal subunit (LSU), and we sequenced the resulting amplicons with single molecule real-time (SMRT) sequencing. The method was applicable to soil and root samples, detected all major AMF families and successfully discriminated closely related AMF species, which would not be discernible using SSU sequences. In inoculation tests we could trace the introduced AMF inoculum at the molecular level. One of the introduced strains almost replaced the local strain(s), revealing that AMF inoculation can have a profound impact on the native community. The methodology presented offers researchers a powerful new tool for AMF community analysis because it unifies improved specificity and enhanced resolution, whereas the drawback of medium sequencing throughput appears of lesser importance for low-diversity groups such as AMF.

Corrigendum: Precision medicine: a new era for inner ear diseases.

[This corrects the article DOI: 10.3389/fphar.2024.1328460.].

Precision medicine: a new era for inner ear diseases.

The inner ear is the organ responsible for hearing and balance. Inner ear dysfunction can be the result of infection, trauma, ototoxic drugs, genetic mutation or predisposition. Often, like for Ménière disease, the cause is unknown. Due to the complex access to the inner ear as a fluid-filled cavity within the temporal bone of the skull, effective diagnosis of inner ear pathologies and targeted drug delivery pose significant challenges. Samples of inner ear fluids can only be collected during surgery because the available procedures damage the tiny and fragile structures of the inner ear. Concerning drug administration, the final dose, kinetics, and targets cannot be controlled. Overcoming these limitations is crucial for successful inner ear precision medicine. Recently, notable advancements in microneedle technologies offer the potential for safe sampling of inner ear fluids and local treatment. Ultrasharp microneedles can reach the inner ear fluids with minimal damage to the organ, collect µl amounts of perilymph, and deliver therapeutic agents in loco. This review highlights the potential of ultrasharp microneedles, combined with nano vectors and gene therapy, to effectively treat inner ear diseases of different etiology on an individual basis. Though further research is necessary to translate these innovative approaches into clinical practice, these technologies may represent a true breakthrough in the clinical approach to inner ear diseases, ushering in a new era of personalized medicine.

Metatranscriptome and Resistome of the Endodontic Microbiome.

INTRODUCTION: In this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance gene expression in endodontic infections. METHODS: Root canal samples were collected from ten teeth, including five primary and five persistent/secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification. RESULTS: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochetes, and Synergistetes were among the nondominant phyla. The top ten species were mainly represented by obligate (or quasiobligate) anaerobes, including Gram-negative (eg, Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia, and Tannerella sp. oral taxon HOT-286) and Gram-positive species (eg, Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (eg, glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/protection were the main resistance mechanisms. CONCLUSIONS: Metatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in AR.

Most genome-wide significant susceptibility loci for schizophrenia and bipolar disorder reported to date cross-traditional diagnostic boundaries.

Recent findings from genetic epidemiology and from genome-wide association studies point strongly to a partial overlap in the genes that contribute susceptibility to schizophrenia and bipolar disorder (BD). Previous data have also directly implicated one of the best supported schizophrenia-associated loci, zinc finger binding protein 804A (ZNF804A), as showing trans-disorder effects, and the same is true for one of the best supported bipolar loci, calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNA1C) which has also been associated with schizophrenia. We have undertaken a cross-phenotype study based upon the remaining variants that show genome-wide evidence for association in large schizophrenia and BD meta-analyses. These comprise in schizophrenia, SNPs in or in the vicinity of transcription factor 4 (TCF4), neurogranin (NRGN) and an extended region covering the MHC locus on chromosome 6. For BD, the strongly supported variants are in the vicinity of ankyrin 3, node of Ranvier (ANK3) and polybromo-1 (PBRM1). Using data sets entirely independent of their original discoveries, we observed strong evidence that the PBRM1 locus is also associated with schizophrenia (P = 0.00015) and nominally significant evidence (P < 0.05) that the NRGN and the extended MHC region are associated with BD. Moreover, considering this highly restricted set of loci as a group, the evidence for trans-disorder effects is compelling (P = 4.7 × 10(-5)). Including earlier reported data for trans-disorder effects for ZNF804A and CACNA1C, six out of eight of the most robustly associated loci for either disorder show trans-disorder effects.

Pimozide Increases a Delayed Rectifier K+ Conductance in Chicken Embryo Vestibular Hair Cells.

Pimozide is a conventional antipsychotic drug largely used in the therapy for schizophrenia and Tourette's syndrome. Pimozide is assumed to inhibit synaptic transmission at the CNS by acting as a dopaminergic D2 receptor antagonist. Moreover, pimozide has been shown to block voltage-gated Ca2+ and K+ channels in different cells. Despite its widespread clinical use, pimozide can cause several adverse effects, including extrapyramidal symptoms and cardiac arrhythmias. Dizziness and loss of balance are among the most common side effects of pimozide. By using the patch-clamp whole-cell technique, we investigated the effect of pimozide [3 µM] on K+ channels expressed by chicken embryo vestibular type-II hair cells. We found that pimozide slightly blocks a transient outward rectifying A-type K+ current but substantially increases a delayed outward rectifying K+ current. The net result was a significant hyperpolarization of type-II hair cells at rest and a strong reduction of their response to depolarizing stimuli. Our findings are consistent with an inhibitory effect of pimozide on the afferent synaptic transmission by type-II hair cells. Moreover, they provide an additional key to understanding the beneficial/collateral pharmacological effects of pimozide. The finding that pimozide can act as a K+ channel opener provides a new perspective for the use of this drug.