Phage-induced efflux down-regulation boosts antibiotic efficacy.

The interactions between a virus and its host vary in space and time and are affected by the presence of molecules that alter the physiology of either the host or the virus. Determining the molecular mechanisms at the basis of these interactions is paramount for predicting the fate of bacterial and phage populations and for designing rational phage-antibiotic therapies. We study the interactions between stationary phase Burkholderia thailandensis and the phage ΦBp-AMP1. Although heterogeneous genetic resistance to phage rapidly emerges in B. thailandensis, the presence of phage enhances the efficacy of three major antibiotic classes, the quinolones, the beta-lactams and the tetracyclines, but antagonizes tetrahydrofolate synthesis inhibitors. We discovered that enhanced antibiotic efficacy is facilitated by reduced antibiotic efflux in the presence of phage. This new phage-antibiotic therapy allows for eradication of stationary phase bacteria, whilst requiring reduced antibiotic concentrations, which is crucial for treating infections in sites where it is difficult to achieve high antibiotic concentrations.

Molecular mechanisms of embryonic tail development in the self-fertilizing mangrove killifish Kryptolebias marmoratus.

Using the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.

The Arabinose 5-Phosphate Isomerase KdsD Is Required for Virulence in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, which is endemic primarily in Southeast Asia and northern Australia but is increasingly being seen in other tropical and subtropical regions of the world. Melioidosis is associated with high morbidity and mortality rates, which is mediated by the wide range of virulence factors encoded by B. pseudomallei. These virulence determinants include surface polysaccharides such as lipopolysaccharide (LPS) and capsular polysaccharides (CPS). Here, we investigated a predicted arabinose-5-phosphate isomerase (API) similar to KdsD in B. pseudomallei strain K96243. KdsD is required for the production of the highly conserved 3-deoxy-d-manno-octulosonic acid (Kdo), a key sugar in the core region of LPS. Recombinant KdsD was expressed and purified, and API activity was determined. Although a putative API paralogue (KpsF) is also predicted to be encoded, the deletion of kdsD resulted in growth defects, loss of motility, reduced survival in RAW 264.7 murine macrophages, and attenuation in a BALB/c mouse model of melioidosis. Suppressor mutations were observed during a phenotypic screen for motility, revealing single nucleotide polymorphisms or indels located in the poorly understood CPS type IV cluster. Crucially, suppressor mutations did not result in reversion of attenuation in vivo. This study demonstrates the importance of KdsD for B. pseudomallei virulence and highlights further the complex nature of the polysaccharides it produces. IMPORTANCE The intrinsic resistance of B. pseudomallei to many antibiotics complicates treatment. This opportunistic pathogen possesses a wide range of virulence factors, resulting in severe and potentially fatal disease. Virulence factors as targets for drug development offer an alternative approach to combat pathogenic bacteria. Prior to initiating early drug discovery approaches, it is important to demonstrate that disruption of the target gene will prevent the development of disease. This study highlights the fact that KdsD is crucial for virulence of B. pseudomallei in an animal model of infection and provides supportive phenotypic characterization that builds a foundation for future therapeutic development.

Cutaneous Malignant Melanoma With Rhabdomyosarcomatous Dedifferentiation: an Immunohistological and Molecular Case Study With Literature Review.

ABSTRACT: Cutaneous malignant melanoma can show a wide range of cytomorphological variability, in particular exhibiting a rhabdoid appearance is not uncommon in melanoma cells; however, the phenomenon of "dedifferentiation" with loss of melanocytic immunohistochemical properties and expression of skeletal muscle immunomarkers is exceedingly rare. Owing to the rarity of such melanomas, their clinicopathological features and molecular profile remain largely unknown. In this report, we describe the clinical, immunomorphological, and molecular features of melanomas with rhabdomyosarcomatous dedifferentiation by presenting a new case and exploring the literature for the previously reported cases.

AWZ1066S, a highly specific anti-Wolbachia drug candidate for a short-course treatment of filariasis.

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

Artemisinin inspired synthetic endoperoxide drug candidates: Design, synthesis, and mechanism of action studies.

Artemisinin combination therapies (ACTs) have been used as the first-line treatments against Plasmodium falciparum malaria for decades. Recent advances in chemical proteomics have shed light on the complex mechanism of action of semi-synthetic artemisinin (ARTs), particularly their promiscuous alkylation of parasite proteins via previous heme-mediated bioactivation of the endoperoxide bond. Alarmingly, the rise of resistance to ART in South East Asia and the synthetic limitations of the ART scaffold have pushed the course for the necessity of fully synthetic endoperoxide-based antimalarials. Several classes of synthetic endoperoxide antimalarials have been described in literature utilizing various endoperoxide warheads including 1,2-dioxanes, 1,2,4-trioxanes, 1,2,4-trioxolanes, and 1,2,4,5-tetraoxanes. Two of these classes, the 1,2,4-trioxolanes (arterolane and artefenomel) and the 1,2,4,5-tetraoxanes (N205 and E209) based antimalarials, have been explored extensively and are still in active development. In contrast, the most recent publication pertaining to the development of the 1,2-dioxane, Arteflene, and 1,2,4-trioxanes fenozan-50F, DU1301, and PA1103/SAR116242 was published in 2008. This review summarizes the synthesis, biological and clinical evaluation, and mechanistic studies of the most developed synthetic endoperoxide antimalarials, providing an update on those classes still in active development.

Dose prediction for repurposing nitazoxanide in SARS-CoV-2 treatment or chemoprophylaxis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared a global pandemic and urgent treatment and prevention strategies are needed. Nitazoxanide, an anthelmintic drug, has been shown to exhibit in vitro activity against SARS-CoV-2. The present study used physiologically based pharmacokinetic (PBPK) modelling to inform optimal doses of nitazoxanide capable of maintaining plasma and lung tizoxanide exposures above the reported SARS-CoV-2 EC90 . METHODS: A whole-body PBPK model was validated against available pharmacokinetic data for healthy individuals receiving single and multiple doses between 500 and 4000 mg with and without food. The validated model was used to predict doses expected to maintain tizoxanide plasma and lung concentrations above the EC90 in >90% of the simulated population. PopDes was used to estimate an optimal sparse sampling strategy for future clinical trials. RESULTS: The PBPK model was successfully validated against the reported human pharmacokinetics. The model predicted optimal doses of 1200 mg QID, 1600 mg TID and 2900 mg BID in the fasted state and 700 mg QID, 900 mg TID and 1400 mg BID when given with food. For BID regimens an optimal sparse sampling strategy of 0.25, 1, 3 and 12 hours post dose was estimated. CONCLUSION: The PBPK model predicted tizoxanide concentrations within doses of nitazoxanide already given to humans previously. The reported dosing strategies provide a rational basis for design of clinical trials with nitazoxanide for the treatment or prevention of SARS-CoV-2 infection. A concordant higher dose of nitazoxanide is now planned for investigation in the seamless phase I/IIa AGILE trial.

Synthesis of Non-symmetrical Dispiro-1,2,4,5-Tetraoxanes and Dispiro-1,2,4-Trioxanes Catalyzed by Silica Sulfuric Acid.

A novel protocol for the preparation of non-symmetrical 1,2,4,5-tetraoxanes and 1,2,4-trioxanes, promoted by the heterogeneous silica sulfuric acid (SSA) catalyst, is reported. Different ketones react under mild conditions with gem-dihydroperoxides or peroxysilyl alcohols/ß-hydroperoxy alcohols to generate the corresponding endoperoxides in good yields. Our mechanistic proposal, assisted by molecular orbital calculations, at the ωB97XD/def2-TZVPP/PCM(DCM)//B3LYP/6-31G(d) level of theory, enhances the role of SSA in the cyclocondensation step. This novel procedure differs from previously reported methods by using readily available and inexpensive reagents, with recyclable properties, thereby establishing a valid alternative approach for the synthesis of new biologically active endoperoxides.

Modification of the cyclopropyl moiety of abacavir provides insight into the structure activity relationship between HLA-B*57:01 binding and T-cell activation.

BACKGROUND: Abacavir is associated with hypersensitivity reactions in individuals positive for the HLA-B*57:01 allele. The drug binds within the peptide binding groove of HLA-B*57:01 altering peptides displayed on the cell surface. Presentation of these HLA-abacavir-peptide complexes to T-cells is hypothesized to trigger a CD8+ T-cell response underpinning the hypersensitivity. Thus, the aim of this study was to explore the relationship between the structure of abacavir with HLA-B*57:01 binding and the CD8+ T-cell activation. METHODS: Seventeen abacavir analogues were synthesized and cytokine secretion from abacavir/abacavir analogue-responsive CD8+ T-cell clones was measured using IFN-γ ELIspot. In silico docking studies were undertaken to assess the predicted binding poses of the abacavir analogues within the HLA-B*57:01 peptide binding groove. In parallel, the effect of selected abacavir analogues on the repertoire of self-peptides presented by cellular HLA-B*57:01 was characterized using mass spectrometry. RESULTS: Abacavir and ten analogues stimulated CD8+ T-cell IFN-γ release. Molecular docking of analogues that retained antiviral activity demonstrated a relationship between predicted HLA-B*57:01 binding orientations and the ability to induce a T-cell response. Analogues that stimulated T-cells displayed a perturbation of the natural peptides displayed by HLA-B*57:01. The antigen-specific CD8+ T-cell response was dependent on the enantiomeric form of abacavir at both cyclopropyl and cyclopentyl regions. CONCLUSION: Alteration of the chemical constitution of abacavir generates analogues that retain a degree of pharmacological activity, but have variable ability to activate T-cells. Modelling and immunopeptidome analysis delineate how drug HLA-B*57:01 binding and peptide display by antigen presenting cells relate to the activation of CD8+ T-cells.

Antimalarial Agents as Therapeutic Tools Against Toxoplasmosis-A Short Bridge between Two Distant Illnesses.

Toxoplasmosis is an infectious disease with paramount impact worldwide, affecting many vulnerable populations and representing a significant matter of concern. Current therapies used against toxoplasmosis are based essentially on old chemotypes, which fail in providing a definitive cure for the disease, placing the most sensitive populations at risk for irreversible damage in vital organs, culminating in death in the most serious cases. Antimalarial drugs have been shown to possess key features for drug repurposing, finding application in the treatment of other parasite-borne illnesses, including toxoplasmosis. Antimalarials provide the most effective therapeutic solutions against toxoplasmosis and make up for the majority of currently available antitoxoplasmic drugs. Additionally, other antiplasmodial drugs have been scrutinized and many promising candidates have emanated in recent developments. Available data demonstrate that it is worthwhile to explore the activity of classical and most recent antimalarial chemotypes, such as quinolines, endoperoxides, pyrazolo[1,5-a]pyrimidines, and nature-derived peptide-based parasiticidal agents, in the context of toxoplasmosis chemotherapy, in the quest for encountering more effective and safer tools for toxoplasmosis control or eradication.

Extensive genome analysis of Coxiella burnetii reveals limited evolution within genomic groups.

BACKGROUND: Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. An improved understanding of the genetic diversity of C. burnetii is essential for the development of diagnostics, vaccines and therapeutics, but genotyping data is lacking from many parts of the world. Sporadic outbreaks of Q fever have occurred in the United Kingdom, but the local genetic make-up of C. burnetii has not been studied in detail. RESULTS: Here, we report whole genome data for nine C. burnetii sequences obtained in the UK. All four genomes of C. burnetii from cattle, as well as one sheep sample, belonged to Multi-spacer sequence type (MST) 20, whereas the goat samples were MST33 (three genomes) and MST32 (one genome), two genotypes that have not been described to be present in the UK to date. We established the phylogenetic relationship between the UK genomes and 67 publically available genomes based on single nucleotide polymorphisms (SNPs) in the core genome, which confirmed tight clustering of strains within genomic groups, but also indicated that sub-groups exist within those groups. Variation is mainly achieved through SNPs, many of which are non-synonymous, thereby confirming that evolution of C. burnetii is based on modification of existing genes. Finally, we discovered genomic-group specific genome content, which supports a model of clonal expansion of previously established genotypes, with large scale dissemination of some of these genotypes across continents being observed. CONCLUSIONS: The genetic make-up of C. burnetii in the UK is similar to the one in neighboring European countries. As a species, C. burnetii has been considered a clonal pathogen with low genetic diversity at the nucleotide level. Here, we present evidence for significant variation at the protein level between isolates of different genomic groups, which mainly affects secreted and membrane-associated proteins. Our results thereby increase our understanding of the global genetic diversity of C. burnetii and provide new insights into the evolution of this emerging zoonotic pathogen.

Laryngoscope burn risk in neonatal intubation.

LARYNGOSCOPE BURNS IN NEONATAL INTUBATION: Following burns during neonatal intubation, we mounted an in vitro study of laryngoscopes to determine the temperatures reached during clinical use. The temperature of 10 different bulb laryngoscopes heads and two fibre optic heads were measured with a thermocouple, once opened, and upon closing. Within 60 s, all ten laryngoscopes, with light-bulb sources, had gained significant heat to cause thermal injury to neonatal skin. Laryngoscopes with LED light source and fibre optic heads did not. CONCLUSION: We recommend that the bulb laryngoscope blade, if used, is not left open prior to intubation and that it is closed between intubation attempts. What is Known: • The preterm epidermis is particularly vulnerable to injury. What is New: • Bulb laryngoscope light bulbs consistently reach temperatures sufficient to burn neonatal skin in less than 100 s in an in vitro study. • Bulb light safety advice should be incorporated into intubation guidelines.

Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7.

The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography-MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs.

Revisiting the Structure and Chemistry of 3(5)-Substituted Pyrazoles.

Pyrazoles are known as versatile scaffolds in organic synthesis and medicinal chemistry, often used as starting materials for the preparation of more complex heterocyclic systems with relevance in the pharmaceutical field. Pyrazoles are also interesting compounds from a structural viewpoint, mainly because they exhibit tautomerism. This phenomenon may influence their reactivity, with possible impact on the synthetic strategies where pyrazoles take part, as well as on the biological activities of targets bearing a pyrazole moiety, since a change in structure translates into changes in properties. Investigations of the structure of pyrazoles that unravel the tautomeric and conformational preferences are therefore of upmost relevance. 3(5)-Aminopyrazoles are largely explored as precursors in the synthesis of condensed heterocyclic systems, namely pyrazolo[1,5-a]pyrimidines. However, the information available in the literature concerning the structure and chemistry of 3(5)-aminopyrazoles is scarce and disperse. We provide a revision of data on the present subject, based on investigations using theoretical and experimental methods, together with the applications of the compounds in synthesis. It is expected that the combined information will contribute to a deeper understanding of structure/reactivity relationships in this class of heterocycles, with a positive impact in the design of synthetic methods, where they take part.

α-Methyl-α-phenylsuccinimide ameliorates neurodegeneration in a C. elegans model of TDP-43 proteinopathy.

The antiepileptic drug ethosuximide has recently been shown to be neuroprotective in various Caenorhabditis elegans and rodent neurodegeneration models. It is therefore a promising repurposing candidate for the treatment of multiple neurodegenerative diseases. However, high concentrations of the drug are required for its protective effects in animal models, which may impact on its translational potential and impede the identification of its molecular mechanism of action. Therefore, we set out to develop more potent neuroprotective lead compounds based on ethosuximide as a starting scaffold. Chemoinformatic approaches were used to identify compounds with structural similarity to ethosuximide and to prioritise these based on good predicated blood-brain barrier permeability and C. elegans bioaccumulation properties. Selected compounds were initially screened for anti-convulsant activity in a C. elegans pentylenetetrazol-induced seizure assay, as a rapid primary readout of bioactivity; and then assessed for neuroprotective properties in a C. elegans TDP-43 proteinopathy model based on pan-neuronal expression of human A315T mutant TDP-43. The most potent compound screened, α-methyl-α-phenylsuccinimide (MPS), ameliorated the locomotion defects and extended the shortened lifespan of TDP-43 mutant worms. MPS also directly protected against neurodegeneration by reducing the number of neuronal breaks and cell body losses in GFP-labelled GABAergic motor neurons. Importantly, optimal neuroprotection was exhibited by external application of 50 µM MPS, compared to 8 mM for ethosuximide. This greater potency of MPS was not due to bioaccumulation to higher internal levels within the worm, based on 1H-nuclear magnetic resonance analysis. Like ethosuximide, the activity of MPS was abolished by mutation of the evolutionarily conserved FOXO transcription factor, daf-16, suggesting that both compounds act via the same neuroprotective pathway(s). In conclusion, we have revealed a novel neuroprotective activity of MPS that is >100-fold more potent than ethosuximide. This increased potency will facilitate future biochemical studies to identify the direct molecular target(s) of both compounds, as we have shown here that they share a common downstream DAF-16-dependent mechanism of action. Furthermore, MPS is the active metabolite of another approved antiepileptic drug, methsuximide. Therefore, methsuximide may have repurposing potential for treatment of TDP-43 proteinopathies and possibly other human neurodegenerative diseases.

Synthesis and profiling of benzylmorpholine 1,2,4,5-tetraoxane analogue N205: Towards tetraoxane scaffolds with potential for single dose cure of malaria.

A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed and synthesized in a short synthetic sequence from readily available starting materials. From this series of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84 nM were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205 (10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency (SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles with a representative of this series, N205, an excellent starting point for further lead optimization studies.

Antimalarial 4(1H)-pyridones bind to the Qi site of cytochrome bc1.

Cytochrome bc1 is a proven drug target in the prevention and treatment of malaria. The rise in drug-resistant strains of Plasmodium falciparum, the organism responsible for malaria, has generated a global effort in designing new classes of drugs. Much of the design/redesign work on overcoming this resistance has been focused on compounds that are presumed to bind the Q(o) site (one of two potential binding sites within cytochrome bc1 using the known crystal structure of this large membrane-bound macromolecular complex via in silico modeling. Cocrystallization of the cytochrome bc1 complex with the 4(1H)-pyridone class of inhibitors, GSK932121 and GW844520, that have been shown to be potent antimalarial agents in vivo, revealed that these inhibitors do not bind at the Q(o) site but bind at the Q(i )site. The discovery that these compounds bind at the Q(i) site may provide a molecular explanation for the cardiotoxicity and eventual failure of GSK932121 in phase-1 clinical trial and highlight the need for direct experimental observation of a compound bound to a target site before chemical optimization and development for clinical trials. The binding of the 4(1H)-pyridone class of inhibitors to Q(i) also explains the ability of this class to overcome parasite Q(o)-based atovaquone resistance and provides critical structural information for future design of new selective compounds with improved safety profiles.

Sulfatase-mediated manipulation of the astrocyte-Schwann cell interface.

Schwann cell (SC) transplantation following spinal cord injury (SCI) may have therapeutic potential. Functional recovery is limited however, due to poor SC interactions with host astrocytes and the induction of astrogliosis. Olfactory ensheathing cells (OECs) are closely related to SCs, but intermix more readily with astrocytes in culture and induce less astrogliosis. We previously demonstrated that OECs express higher levels of sulfatases, enzymes that remove 6-O-sulfate groups from heparan sulphate proteoglycans, than SCs and that RNAi knockdown of sulfatase prevented OEC-astrocyte mixing in vitro. As human OECs are difficult to culture in large numbers we have genetically engineered SCs using lentiviral vectors to express sulfatase 1 and 2 (SC-S1S2) and assessed their ability to interact with astrocytes. We demonstrate that SC-S1S2s have increased integrin-dependent motility in the presence of astrocytes via modulation of NRG and FGF receptor-linked PI3K/AKT intracellular signaling and do not form boundaries with astrocytes in culture. SC-astrocyte mixing is dependent on local NRG concentration and we propose that sulfatase enzymes influence the bioavailability of NRG ligand and thus influence SC behavior. We further demonstrate that injection of sulfatase expressing SCs into spinal cord white matter results in less glial reactivity than control SC injections comparable to that of OEC injections. Our data indicate that sulfatase-mediated modification of the extracellular matrix can influence glial interactions with astrocytes, and that SCs engineered to express sulfatase may be more OEC-like in character. This approach may be beneficial for cell transplant-mediated spinal cord repair. GLIA 2016 GLIA 2017;65:19-33.

Molecular epidemiology of Pseudomonas aeruginosa in an unsegregated bronchiectasis cohort sharing hospital facilities with a cystic fibrosis cohort.

While Pseudomonas aeruginosa (PA) cross-infection is well documented among patients with cystic fibrosis (CF), the equivalent risk among patients with non-CF bronchiectasis (NCFB) is unclear, particularly those managed alongside patients with CF. We performed analysis of PA within a single centre that manages an unsegregated NCFB cohort alongside a segregated CF cohort. We found no evidence of cross-infection between the two cohorts or within the segregated CF cohort. However, within the unsegregated NCFB cohort, evidence of cross-infection was found between three (of 46) patients. While we do not presently advocate any change in the management of our NCFB cohort, longitudinal surveillance is clearly warranted.

FGFR1-Frs2/3 signalling maintains sensory progenitors during inner ear hair cell formation.

Inner ear mechanosensory hair cells transduce sound and balance information. Auditory hair cells emerge from a Sox2-positive sensory patch in the inner ear epithelium, which is progressively restricted during development. This restriction depends on the action of signaling molecules. Fibroblast growth factor (FGF) signalling is important during sensory specification: attenuation of Fgfr1 disrupts cochlear hair cell formation; however, the underlying mechanisms remain unknown. Here we report that in the absence of FGFR1 signaling, the expression of Sox2 within the sensory patch is not maintained. Despite the down-regulation of the prosensory domain markers, p27(Kip1), Hey2, and Hes5, progenitors can still exit the cell cycle to form the zone of non-proliferating cells (ZNPC), however the number of cells that form sensory cells is reduced. Analysis of a mutant Fgfr1 allele, unable to bind to the adaptor protein, Frs2/3, indicates that Sox2 maintenance can be regulated by MAP kinase. We suggest that FGF signaling, through the activation of MAP kinase, is necessary for the maintenance of sensory progenitors and commits precursors to sensory cell differentiation in the mammalian cochlea.