Prenatal AAV9-GFP administration in fetal lambs results in transduction of female germ cells and maternal exposure to virus.

Prenatal somatic cell gene therapy (PSCGT) could potentially treat severe, early-onset genetic disorders such as spinal muscular atrophy (SMA) or muscular dystrophy. Given the approval of adeno-associated virus serotype 9 (AAV9) vectors in infants with SMA by the U.S. Food and Drug Administration, we tested the safety and biodistribution of AAV9-GFP (clinical-grade and dose) in fetal lambs to understand safety and efficacy after umbilical vein or intracranial injection on embryonic day 75 (E75) . Umbilical vein injection led to widespread biodistribution of vector genomes in all examined lamb tissues and in maternal uteruses at harvest (E96 or E140; term = E150). There was robust GFP expression in brain, spinal cord, dorsal root ganglia (DRGs), without DRG toxicity and excellent transduction of diaphragm and quadriceps muscles. However, we found evidence of systemic toxicity (fetal growth restriction) and maternal exposure to the viral vector (transient elevation of total bilirubin and a trend toward elevation in anti-AAV9 antibodies). There were no antibodies against GFP in ewes or lambs. Analysis of fetal gonads demonstrated GFP expression in female (but not male) germ cells, with low levels of integration-specific reads, without integration in select proto-oncogenes. These results suggest potential therapeutic benefit of AAV9 PSCGT for neuromuscular disorders, but warrant caution for exposure of female germ cells.

Prenatal delivery of a therapeutic antisense oligonucleotide achieves broad biodistribution in the brain and ameliorates Angelman syndrome phenotype in mice.

Angelman syndrome (AS), an early-onset neurodevelopmental disorder characterized by abnormal gait, intellectual disabilities, and seizures, occurs when the maternal allele of the UBE3A gene is disrupted, since the paternal allele is silenced in neurons by the UBE3A antisense (UBE3A-AS) transcript. Given the importance of early treatment, we hypothesized that prenatal delivery of an antisense oligonucleotide (ASO) would downregulate the murine Ube3a-AS, resulting in increased UBE3A protein and functional rescue. Using a mouse model with a Ube3a-YFP allele that reports on-target ASO activity, we found that in utero, intracranial (IC) injection of the ASO resulted in dose-dependent activation of paternal Ube3a, with broad biodistribution. Accordingly, in utero injection of the ASO in a mouse model of AS also resulted in successful restoration of UBE3A and phenotypic improvements in treated mice on the accelerating rotarod and fear conditioning. Strikingly, even intra-amniotic (IA) injection resulted in systemic biodistribution and high levels of UBE3A reactivation throughout the brain. These findings offer a novel strategy for early treatment of AS using an ASO, with two potential routes of administration in the prenatal window. Beyond AS, successful delivery of a therapeutic ASO into neurons has implications for a clinically feasible prenatal treatment for numerous neurodevelopmental disorders.

A Cdh3-ß-catenin-laminin signaling axis in a subset of breast tumor leader cells control leader cell polarization and directional collective migration.

Carcinoma dissemination can occur when heterogeneous tumor and tumor-stromal cell clusters migrate together via collective migration. Cells at the front lead and direct collective migration, yet how these leader cells form and direct migration are not fully appreciated. From live videos of primary mouse and human breast tumor organoids in a 3D microfluidic system mimicking native breast tumor microenvironment, we developed 3D computational models, which hypothesize that leader cells need to generate high protrusive forces and overcome extracellular matrix (ECM) resistance at the leading edge. From single-cell sequencing analyses, we find that leader cells are heterogeneous and identify and isolate a keratin 14- and cadherin-3-positive subpopulation sufficient to lead collective migration. Cdh3 controls leader cell protrusion dynamics through local production of laminin, which is required for integrin/focal adhesion function. Our findings highlight how a subset of leader cells interact with the microenvironment to direct collective migration.

Bolaamphiphile Analogues of 12-bis-THA Cl2 Are Potent Antimicrobial Therapeutics with Distinct Mechanisms of Action against Bacterial, Mycobacterial, and Fungal Pathogens.

12-Bis-THA Cl2 [12,12'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regarding their toxicity and which could be more useful if such risks are reduced or eliminated. We modified a bolalipid antiseptic used in throat lozenges to see if it could be made more effective against some of the highest-priority bacteria and less toxic. We found that structural modifications that rendered the lipid more toxic against human cells made it less toxic in infection models and we could effectively treat caterpillars infected with either Mycobacterium tuberculosis, methicillin resistant Staphylococcus aureus, or Acinetobacter baumannii. The study provides a rationale for further adaptation toward diversifying the range of indications in which this class of antimicrobial may be used.

Synergy between Winter Flounder antimicrobial peptides.

Some antimicrobial peptides (AMPs) have potent bactericidal activity and are being considered as potential alternatives to classical antibiotics. In response to an infection, such AMPs are often produced in animals alongside other peptides with low or no perceivable antimicrobial activity, whose role is unclear. Here we show that six AMPs from the Winter Flounder (WF) act in synergy against a range of bacterial pathogens and provide mechanistic insights into how this increases the cooperativity of the dose-dependent bactericidal activity and potency that enable therapy. Only two WF AMPs have potent antimicrobial activity when used alone but we find a series of two-way combinations, involving peptides which otherwise have low or no activity, yield potent antimicrobial activity. Weakly active WF AMPs modulate the membrane interactions of the more potent WF AMPs and enable therapy in a model of Acinetobacter baumannii burn wound infection. The observed synergy and emergent behaviour may explain the evolutionary benefits of producing a family of related peptides and are attractive properties to consider when developing AMPs towards clinical applications.

Temporin B Forms Hetero-Oligomers with Temporin L, Modifies Its Membrane Activity, and Increases the Cooperativity of Its Antibacterial Pharmacodynamic Profile.

The pharmacodynamic profile of antimicrobial peptides (AMPs) and their in vivo synergy are two factors that are thought to restrict resistance evolution and ensure their conservation. The frog Rana temporaria secretes a family of closely related AMPs, temporins A-L, as an effective chemical dermal defense. The antibacterial potency of temporin L has been shown to increase synergistically in combination with both temporins B and A, but this is modest. Here we show that the less potent temporin B enhances the cooperativity of the in vitro antibacterial activity of the more potent temporin L against EMRSA-15 and that this may be associated with an altered interaction with the bacterial plasma membrane, a feature critical for the antibacterial activity of most AMPs. Addition of buforin II, a histone H2A fragment, can further increase the cooperativity. Molecular dynamics simulations indicate temporins B and L readily form hetero-oligomers in models of Gram-positive bacterial plasma membranes. Patch-clamp studies show transmembrane ion conductance is triggered with lower amounts of both peptides and more quickly when used in combination, but conductance is of a lower amplitude and pores are smaller. Temporin B may therefore act by forming temporin L/B hetero-oligomers that are more effective than temporin L homo-oligomers at bacterial killing and/or by reducing the probability of the latter forming until a threshold concentration is reached. Exploration of the mechanism of synergy between AMPs isolated from the same organism may therefore yield antibiotic combinations with advantageous pharmacodynamic properties.

Study protocol for CELLAR (COVID-related Eating Limitations and Latent dietary effects in the Atlantic Region): population-based observational study to monitor dietary intakes and purchasing during COVID-19 in four Atlantic Canadian provinces.

INTRODUCTION: Poor diet is a leading preventable risk for the global burden of non-communicable disease. Robust measurement is needed to determine the effect of COVID-19 on dietary intakes and consumer purchasing, given the widespread changes to consumer food environments and economic precarity. The research objectives are as follows: (1) describe dietary intakes of foods, beverages and nutrients of concern during the COVID-19 pandemic; (2) quantify change in diet during COVID-19 as compared with prepandemic, previously captured in the provincial samples of the population-representative 2015 Canadian Community Health Survey-Nutrition and (3) examine how household purchasing practices predict dietary intakes during COVID-19. METHODS AND ANALYSES: Observational study of diet, using a population-based stratified probability sampling strategy allocated via dual-frame (landline and cellphone) calls to random-digit dialled numbers, followed by age-sex group quotas. The base population comprises the four provinces of the Atlantic region of Canada, jurisdictions with an excess burden of pre-existing dietary risk, compared with the rest of Canada. Our aim is n=1000 to obtain reliable estimates at a regional level to describe intakes and compare with prepandemic baseline. Data collection entails 12 weeks participation: (1) enrolment with sociodemographics (key dietary risk predictors such as age, sex, gender, pre-COVID-19 income, employment, household composition, receipt of economic relief, rural residence); (2) two 24hour diet recalls using the online ASA-24 Canada 2018 tool; and (3) online uploads of household food purchase receipts over the 12 weeks enrolled. Participation incentives will be offered. ETHICS AND DISSEMINATION: This research protocol received funding from the Canadian Institutes of Health Research (FRN VR5 172691) and ethics review approval from the Dalhousie University Research Ethics Board. Study protocol and instruments and a de-identified dataset will be made publicly available. We will submit the findings to peer-reviewed journals, as well as conferences geared towards scientific and decision-maker audiences.

Pseudomonas aeruginosa adapts to octenidine via a combination of efflux and membrane remodelling.

Pseudomonas aeruginosa is an opportunistic pathogen capable of stably adapting to the antiseptic octenidine by an unknown mechanism. Here we characterise this adaptation, both in the laboratory and a simulated clinical setting, and identify a novel antiseptic resistance mechanism. In both settings, 2 to 4-fold increase in octenidine tolerance was associated with stable mutations and a specific 12 base pair deletion in a putative Tet-repressor family gene (smvR), associated with a constitutive increase in expression of the Major Facilitator Superfamily (MFS) efflux pump SmvA. Adaptation to higher octenidine concentrations led to additional stable mutations, most frequently in phosphatidylserine synthase pssA and occasionally in phosphatidylglycerophosphate synthase pgsA genes, resulting in octenidine tolerance 16- to 256-fold higher than parental strains. Metabolic changes were consistent with mitigation of oxidative stress and altered plasma membrane composition and order. Mutations in SmvAR and phospholipid synthases enable higher level, synergistic tolerance of octenidine.

Tyrosine kinase-independent actions of DDR2 in tumor cells and cancer-associated fibroblasts influence tumor invasion, migration and metastasis.

Both tumor cell-intrinsic signals and tumor cell-extrinsic signals from cells within the tumor microenvironment influence tumor cell dissemination and metastasis. The fibrillar collagen receptor tyrosine kinase (RTK) discoidin domain receptor 2 (DDR2) is essential for breast cancer metastasis in mouse models, and high expression of DDR2 in tumor and tumor stromal cells is strongly associated with poorer clinical outcomes. DDR2 tyrosine kinase activity has been hypothesized to be required for the metastatic activity of DDR2; however, inhibition of DDR2 tyrosine kinase activity, along with that of other RTKs, has failed to provide clinically relevant responses in metastatic patients. Here, we show that tyrosine kinase activity-independent action of DDR2 in tumor cells can support Matrigel invasion and in vivo metastasis. Paracrine actions of DDR2 in tumor cells and cancer-associated fibroblasts (CAFs) also support tumor invasion, migration and lung colonization in vivo. These data suggest that tyrosine kinase-independent functions of DDR2 could explain failures of tyrosine kinase inhibitor treatment in metastatic breast cancer patients and highlight the need for alternative therapeutic strategies that inhibit both tyrosine kinase-dependent and -independent actions of RTKs in the treatment of breast cancer. This article has an associated First Person interview with the first author of the paper.

Impacts of Metabolism and Organic Acids on Cell Wall Composition and Pseudomonas aeruginosa Susceptibility to Membrane Active Antimicrobials.

Reliable antimicrobial susceptibility testing is essential in informing both clinical antibiotic therapy decisions and the development of new antibiotics. Mammalian cell culture media have been proposed as an alternative to bacteriological media, potentially representing some critical aspects of the infection environment more accurately. Here, we use a combination of NMR metabolomics and electron microscopy to investigate the response of Escherichia coli and Pseudomonas aeruginosa to growth in differing rich media to determine whether and how this determines metabolic strategies, the composition of the cell wall, and consequently susceptibility to membrane active antimicrobials including colistin and tobramycin. The NMR metabolomic approach is first validated by characterizing the expected E. coli acid stress response to fermentation and the accompanying changes in the cell wall composition, when cultured in glucose rich mammalian cell culture media. Glucose is not a major carbon source for P. aeruginosa but is associated with a response to osmotic stress and a modest increase in colistin tolerance. Growth of P. aeruginosa in a range of bacteriological media is supported by consumption of formate, an important electron donor in anaerobic respiration. In mammalian cell culture media, however, the overall metabolic strategy of P. aeruginosa is instead dependent on consumption of glutamine and lactate. Formate doping of mammalian cell culture media does not alter the overall metabolic strategy but is associated with polyamine catabolism, remodelling of both inner and outer membranes, and a modest sensitization of P. aeruginosa PAO1 to colistin. Further, in a panel of P. aeruginosa isolates an increase between 2- and 3-fold in sensitivity to tobramycin is achieved through doping with other organic acids, notably propionate which also similarly enhances the activity of colistin. Organic acids are therefore capable of nonspecifically influencing the potency of membrane active antimicrobials.

Optimization of PEGylated KL4 Peptide for siRNA Delivery with Improved Pulmonary Tolerance.

Pulmonary delivery of small interfering RNA (siRNA) is a promising therapeutic strategy for treating various respiratory diseases but an effective carrier for the delivery of siRNA into the cells of the lungs and a robust gene-silencing effect is still lacking. Previously, we reported that the KL4 peptide, a synthetic cationic peptide with a repeating KLLLL sequence, can mediate effective siRNA transfection in lung epithelial cells but its high hydrophobic leucine content, and hence poor water solubility, limits its application as a delivery vector. Here, we show that the covalent attachment of monodisperse poly(ethylene glycol) (PEG) improves the solubility of KL4 and the uptake of its complex with siRNA into lung epithelial cells, such that very robust silencing is produced. All PEGylated KL4 peptides, with PEG length varying between 6 and 24 monomers, could bind and form nanosized complexes with siRNA, but the interaction between siRNA and peptides became weaker as the PEG chain length increased. All PEGylated KL4 peptides exhibited satisfactory siRNA transfection efficiency on three human lung epithelial cell lines, including A549 cells, Calu-3 cells, and BEAS-2B cells. The PEG12KL4 peptide, which contains 12 monomers of PEG, was optimal for siRNA delivery and also demonstrated a low risk of inflammatory response and toxicity in vivo following pulmonary administration.

A pleurocidin analogue with greater conformational flexibility, enhanced antimicrobial potency and in vivo therapeutic efficacy.

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.

Cytoglobin deficiency potentiates Crb1-mediated retinal degeneration in rd8 mice.

PURPOSE: The purpose of this study is to determine the effect of Cytoglobin (Cygb) deficiency on Crb1-related retinopathy. The Crb1 cell polarity complex is required for photoreceptor function and survival. Crb1-related retinopathies encompass a broad range of phenotypes which are not completely explained by the variability of Crb1 mutations. Genes thought to modify Crb1 function are therefore important targets of research. The biological function of Cygb involves oxygen delivery, scavenging of reactive oxygen species, and nitric oxide metabolism. However, the relationship of Cygb to diseases involving the Crb1 cell polarity complex is unknown. METHODS: Cygb knockout mice homozygous for the rd8 mutation (Cygb-/-rd8/rd8) were screened for ocular abnormalities and imaged using optical coherence tomography and fundus photography. Electroretinography was performed, as was histology and immunohistochemistry. Quantitative PCR was used to determine the effect of Cygb deficiency on transcription of Crb1 related cell polarity genes. RESULTS: Cygb-/-rd8/rd8 mice develop an abnormal retina with severe lamination abnormalities. The retina undergoes progressive degeneration with the ventral retina more severely affected than the dorsal retina. Cygb expression is in neurons of the retinal ganglion cell layer and inner nuclear layer. Immunohistochemical studies suggest that cell death predominates in the photoreceptors. Electroretinography amplitudes show reduced a- and b-waves, consistent with photoreceptor disease. Cygb deficient retinas had only modest transcriptional perturbations of Crb1-related cell polarity genes. Cygb-/- mice without the rd8 mutation did not exhibit obvious retinal abnormalities. CONCLUSIONS: Cygb is necessary for retinal lamination, maintenance of cell polarity, and photoreceptor survival in rd8 mice. These results are consistent with Cygb as a disease modifying gene in Crb1-related retinopathy. Further studies are necessary to investigate the role of Cygb in the human retina.

Biomedical studies on temporal bones of the first multi-channel cochlear implant patient at the University of Melbourne.

OBJECTIVE: To analyse the temporal bones and implant of the first University of Melbourne's (UOM) patient (MC-1) to receive the multi-channel cochlear prosthesis. METHODS: The left cochlea was implanted with the prototype multi-channel cochlear prosthesis on 1 August 1978, and the Cochlear versions CI-22 and CI-24 on 22 June 1983 and 10 November 1998, respectively. MC-1 died in 2007. RESULTS: Plain X-rays of the temporal bones showed that after the CI-22 had been explanted seven electrode bands remained in situ. Micro-CT scans also revealed a partially united fracture transecting the left implanted and right control cochleae. Histology indicated a total loss of the organ of Corti on both sides, and a tear of the left basilar membrane. In addition, there was a dense fibrous capsule with heterotopic bone surrounding one proximal band of the CI-22 array that restricted its removal. This pathology was associated with dark particulate material within macrophages, probably due to the release of platinum from the electrode bands. Scanning electron microscopy (SEM) showed possible corrosion of platinum and surface roughening. Three-dimensional reconstruction of the cochlear histology demonstrated the position of the electrode tracts (C1-22 and CI-24) in relation to the spiral ganglion, which showed 85-90% loss of ganglion cells. DISCUSSION AND CONCLUSIONS: This study confirms our first histopathological findings that our first free-fitting banded electrode array produced moderate trauma to the cochlea when inserted around the scala tympani of the basal turn. The difficulty in extraction was most likely due to one band being surrounded by an unusually large amount of fibrous tissue and bone, with an electrode band caught due to surface irregularities. Some surface corrosion and a small degree of platinum deposition in the tissue may also help explain the outcome for this long-term cochlear implantation.

The p75 neurotrophin receptor protects primary auditory neurons against acoustic trauma in mice.

In the adult rodent inner ear, p75NTR is weakly expressed in primary auditory neurons (PANs) and cochlear Schwann cells. When the organ of Corti is damaged during trauma, its expression dramatically increases. It is unclear what role p75NTR plays under these conditions. Characterisation of p75NTR mutant mice reveals that altering genetic backgrounds can differentially affect the survival of PANs in mutant mice. To conclusively elucidate the physiological role of p75NTR in the cochlea, we challenged wild type (p75NTR +/+) and mutant (p75NTR -/-) mice with an acoustic trauma at 130 dB SPL, 10 kHz for 2 h. This produces a permanent auditory threshold shift >40 dB SPL, damages the organ of Corti and causes secondary degeneration of PANs. After exposure, mice were maintained for 3-9 weeks. Interestingly, survival of PANs in p75NTR -/- mice was significantly compromised in all time-points when compared to wild type mice: 15% reduction after 3 weeks (n = 6), 32% reduction after 6 weeks (n = 6) and 26% reduction after 9 weeks (n = 6-8). Therefore, our data do not support a role of p75NTR as a death inducer in PANs but show its crucial role in protecting PANs.

Review of therapeutic agents for burns pruritus and protocols for management in adult and paediatric patients using the GRADE classification.

To review the current evidence on therapeutic agents for burns pruritus and use the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) classification to propose therapeutic protocols for adult and paediatric patients. All published interventions for burns pruritus were analysed by a multidisciplinary panel of burns specialists following the GRADE classification to rate individual agents. Following the collation of results and panel discussion, consensus protocols are presented. Twenty-three studies appraising therapeutic agents in the burns literature were identified. The majority of these studies (16 out of 23) are of an observational nature, making an evidence-based approach to defining optimal therapy not feasible. Our multidisciplinary approach employing the GRADE classification recommends the use of antihistamines (cetirizine and cimetidine) and gabapentin as the first-line pharmacological agents for both adult and paediatric patients. Ondansetron and loratadine are the second-line medications in our protocols. We additionally recommend a variety of non-pharmacological adjuncts for the perusal of clinicians in order to maximise symptomatic relief in patients troubled with postburn itch. Most studies in the subject area lack sufficient statistical power to dictate a 'gold standard' treatment agent for burns itch. We encourage clinicians to employ the GRADE system in order to delineate the most appropriate therapeutic approach for burns pruritus until further research elucidates the most efficacious interventions. This widely adopted classification empowers burns clinicians to tailor therapeutic regimens according to current evidence, patient values, risks and resource considerations in different medical environments.

Mice deficient for the type II transmembrane serine protease, TMPRSS1/hepsin, exhibit profound hearing loss.

Defective proteolysis has been implicated in hearing loss through the discovery of mutations causing autosomal recessive nonsyndromic deafness in a type II transmembrane serine protease gene, TMPRSS3. To investigate their physiological function and the contribution of this family of proteases to the auditory function, we analyzed the hearing status of mice deficient for hepsin, also known as TMPRSS1. These mice exhibited profound hearing loss with elevated hearing thresholds compared with their heterozygous and wild-type littermates. Their cochleae showed abnormal tectorial membrane development, reduction in fiber compaction in the peripheral portion of the auditory nerve, and decreased expression of the myelin proteins myelin basic protein and myelin protein zero. In addition, reduced level of the large conductance voltage- and Ca(2+)-activated K(+) channel was detected in the sensory hair cells of Tmprss1-null mice. We examined thyroid hormone levels in Tmprss1-deficient mice, as similar cochlear defects have been reported in animal models of hypothyroidism, and found significantly reduced free thyroxine levels. These data show that TMPRSS1 is required for normal auditory function. Hearing impairment present in Tmprss1-null mice is characterized by a combination of various structural, cellular, and molecular abnormalities that are likely to affect different cochlear processes.

Cochlear immunochemistry--a new technique based on gelatin embedding.

Histological processing of the cochlea for immunochemistry is often a compromise between good anatomical resolution and preservation of antigenicity. Techniques able to preserve tissue architecture invariably demand elevated temperatures and harsh chemicals or a combination of both. The likely result is reduced antigenicity, enzyme activity and nucleic acid integrity. We have modified an existing embedding medium for use in the cochlea that operates at physiological temperature and avoids denaturing agents and organic solvents. Tissue antigenicity is maximised and anatomical detail preserved, normally two mutually exclusive goals. The method is attractive because of its simplicity, speed and transparency for easy cochlear orientation. It is also likely to be adaptable for the infiltration of other heterogeneous structures prone to distortion during frozen sectioning.