Probing the mechanisms of two exonuclease domain mutators of DNA polymerase ϵ.

We report the properties of two mutations in the exonuclease domain of the Saccharomyces cerevisiae DNA polymerase ϵ. One, pol2-Y473F, increases the mutation rate by about 20-fold, similar to the catalytically dead pol2-D290A/E290A mutant. The other, pol2-N378K, is a stronger mutator. Both retain the ability to excise a nucleotide from double-stranded DNA, but with impaired activity. pol2-Y473F degrades DNA poorly, while pol2-N378K degrades single-stranded DNA at an elevated rate relative to double-stranded DNA. These data suggest that pol2-Y473F reduces the capacity of the enzyme to perform catalysis in the exonuclease active site, while pol2-N378K impairs partitioning to the exonuclease active site. Relative to wild-type Pol ϵ, both variants decrease the dNTP concentration required to elicit a switch between proofreading and polymerization by more than an order of magnitude. While neither mutation appears to alter the sequence specificity of polymerization, the N378K mutation stimulates polymerase activity, increasing the probability of incorporation and extension of a mismatch. Considered together, these data indicate that impairing the primer strand transfer pathway required for proofreading increases the probability of common mutations by Pol ϵ, elucidating the association of homologous mutations in human DNA polymerase ϵ with cancer.

The underlying sex differences in neuroendocrine adaptations relevant to Myalgic Encephalomyelitis Chronic Fatigue Syndrome.

INTRODUCTION: Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) is a complex multisystem disease characterised by severe and disabling new-onset symptoms of post-exertional malaise (PEM), fatigue, brain fog, and sleep dysfunction that lasts for at least six months. Accumulating evidence suggests that sex and endocrine events have a significant influence on symptom onset and moderation of ME/CFS, with female sex being one of the most consistent and credible predictive risk factors associated with diagnosis. Such sex differences suggest sex chromosomes and sex steroids may play a part in the development of the condition or moderation of symptoms, although this has yet to be explored in detail. METHODS/AIMS: This narrative review outlines sex differences in ME/CFS in terms of vulnerability factors and clinical phenotype and explores the known sex differences in neuroendocrine systems affected in ME/CFS and how this may relate to disease risk, onset, pathophysiology, and potential treatment avenues. CONCLUSIONS: There is clear evidence of a sex dimorphism with regards to prevalence (3:1 female preponderance), clinical phenotypes, and aetiological triggers prior to symptom onset of ME/CFS. Endocrinological events, particularly those throughout the female lifespan, are associated with ME/CFS and include reproductive menstrual cycle fluctuations, pregnancy, post-partum and perimenopause. Further, there is evidence for gonadal sex, adrenal stress and renal neuroendocrine systems as implicated in ME/CFS, including changes in estrogen, progesterone compounds, aldosterone, and cortisol levels, of which there are established sex differences. The broad effects of steroid hormones on the physiological systems may also speak to the diversity of ME/CFS symptomatology observed in patients. Further attention must be paid to sex, age, and steroid biology in ME/CFS.

The relationship between cognitive clusters and telomere length in bipolar-schizophrenia spectrum disorders.

BACKGROUND: Schizophrenia and bipolar disorder are complex mental illnesses that are associated with cognitive deficits. There is considerable cognitive heterogeneity that exists within both disorders. Studies that cluster schizophrenia and bipolar patients into subgroups based on their cognitive profile increasingly demonstrate that, relative to healthy controls, there is a severely compromised subgroup and a relatively intact subgroup. There is emerging evidence that telomere shortening, a marker of cellular senescence, may be associated with cognitive impairments. The aim of this study was to explore the relationship between cognitive subgroups in bipolar-schizophrenia spectrum disorders and telomere length against a healthy control sample. METHODS: Participants included a transdiagnostic group diagnosed with bipolar, schizophrenia or schizoaffective disorder (n = 73) and healthy controls (n = 113). Cognitive clusters within the transdiagnostic patient group, were determined using K-means cluster analysis based on current cognitive functioning (MATRICS Consensus Cognitive Battery scores). Telomere length was determined using quantitative PCRs genomic DNA extracted from whole blood. Emergent clusters were then compared to the healthy control group on telomere length. RESULTS: Two clusters emerged within the patient group that were deemed to reflect a relatively intact cognitive group and a cognitively impaired subgroup. Telomere length was significantly shorter in the severely impaired cognitive subgroup compared to the healthy control group. CONCLUSIONS: This study replicates previous findings of transdiagnostic cognitive subgroups and associates shorter telomere length with the severely impaired cognitive subgroup. These findings support emerging literature associating cognitive impairments in psychiatric disorders to accelerated cellular aging as indexed by telomere length.

A network meta-analysis of stress mediators in suicide behaviour.

Stress homeostatic mediators are the most consistently anomalous biomarkers observed in suicide and may therefore point to a common 'core biology' of stress susceptibility, and suicidal behaviour. Previously reported meta-analyses have demonstrated aberrant levels of stress cortisol and inflammatory cytokines in suicide patients compared to controls, and significant associations between the stress regulator FK506-binding protein 51 (FKBP5) gene and suicidal behaviour. Although these independent studies were investigated as separate entities in suicide, stress mediators interact in a dynamic system, collectively giving rise to system changes physiologically, and ultimately psychologically and behaviourally. It is therefore important to study the dynamic network these stress mediators. Network meta-analysis allows for the simultaneous comparison of more than two biological mediators, and for comparisons to be made between mediators that have not been directly compared before, using previously reported, pooled meta data. Such network approaches may help study the complex biological phenomena of suicide and may provide better prediction of biological risk of suicidal states. METHODS: This study aimed to establish the comparative relationships between key stress mediators in suicidal patients compared to non-suicidal controls using a random-effects network meta-analysis approach.. The key stress mediators included cortisol, six inflammatory markers (interleukin-6 (IL-6), interleukin-4 (IL-4), interleukin-2 (IL-2), tumour necrosis factor-a (TNF-α), interferon (IFN-y) and transforming growth factor ß (TGF-ß), and the FKBP5 single nucleotide polymorphism (SNP) allele. Data was derived from three previously published meta-analysis. The study population comprised of 1348 suicidal patients, defined as suicide attempters, completers, or patients with severe suicidal ideation, and 1750 non-suicidal controls, defined as healthy controls and psychiatric patients without suicidal ideation or previous attempts. RESULTS: Pair-wise indirect effects of stress mediators in suicide compared to controls demonstrated that relative to the effect of the FKBP5 risk SNP allele on suicide risk, the magnitude of differences (suicide vs control) for the levels of IL-2 (SMD -0.72; 95% CI, -0.135 to -0.09 and IL-4 (SMD -0.71; 95% CI, -1.34 to -0.08) were significantly smaller (with 95% confidence intervals not crossing the null). The comparative relationships between stress mediators in suicidal behaviour demonstrates that the dynamic stress network relationship is dysregulated in suicide patients when compared to controls. CONCLUSIONS: This model suggests that a genetic stress susceptibility with downstream abnormal cortisol stress axis functioning, together with anomalous interactions between the inflammatory system, may be one of the neurobiological correlates of suicide behaviour. This biological state may leave the individual physiologically susceptible and unable to cope with environmental stressors, which is consistent with the stress-diathesis hypothesis of suicide behaviour.

Influence of cortisol awakening response on telomere length: Trends for males and females.

Although telomere attrition is associated with the process of normal ageing, shorter telomere length (TL) has been associated with acute and chronic stressors. A neurobiological factor hypothesised to be responsible for this accelerated attrition is the dysregulation of the cortisol stress response, which can induce DNA damage affecting DNA telomeric caps. Marked sex differences are reported in both the cortisol stress response and telomere dynamics, yet no explicit investigation of sex specificity on the relationship between cortisol and TL exists. This study used mathematical equation modelling to describe the relationship between diurnal cortisol levels and telomere length within the context of sex, in a healthy population. Cortisol awakening responses (CAR) were measured via ELISA methodology in fifty-one healthy participants (28 males, 23 females). qPCRs determined TL from genomic DNA extracted from saliva. To assess the effect of free cortisol on relative TL ratio, a semi-log regression plot of the two variables trended for sex were fitted using spline curves. Results demonstrated significant differences between males and females in the relationship defining CAR and TL association (p = 0.03). These results suggest the relationship is not linear and can be represented as a complex arcsin function, and that the patterns are opposite in males and females. Males demonstrate a positive correlation, with higher levels of CAR being associated with longer telomere sequences. Females demonstrated a negative correlation. Future studies must carefully take into consideration moderating factors such as sex, and sex hormones across the lifespan when investigating telomere length.

Screening for phenotypic outliers identifies an unusually low concentration of a ß-lactoglobulin B protein isoform in bovine milk caused by a synonymous SNP.

BACKGROUND: Milk samples from 10,641 dairy cattle were screened by a mass spectrometry method for extreme concentrations of the A or B isoforms of the whey protein, ß-lactoglobulin (BLG), to identify causative genetic variation driving changes in BLG concentration. RESULTS: A cohort of cows, from a single sire family, was identified that produced milk containing a low concentration of the BLG B protein isoform. A genome-wide association study (GWAS) of BLG B protein isoform concentration in milk from AB heterozygous cows, detected a group of highly significant single nucleotide polymorphisms (SNPs) within or close to the BLG gene. Among these was a synonymous G/A variation at position + 78 bp in exon 1 of the BLG gene (chr11:103256256G > A). The effect of the A allele of this SNP (which we named B') on BLG expression was evaluated in a luciferase reporter assay in transfected CHO-K1 and MCF-7 cells. In both cell types, the presence of the B' allele in a plasmid containing the bovine BLG gene from -922 to + 898 bp (relative to the transcription initiation site) resulted in a 60% relative reduction in mRNA expression, compared to the plasmid containing the wild-type B sequence allele. Examination of a mammary RNAseq dataset (n = 391) identified 14 heterozygous carriers of the B' allele which were homozygous for the BLG B protein isoform (BB'). The level of expression of the BLG B' allele was 41.9 ± 1.0% of that of the wild-type BLG B allele. Milk samples from three cows, homozygous for the A allele at chr11:103,256,256 (B'B'), were analysed (HPLC) and showed BLG concentrations of 1.04, 1.26 and 1.83 g/L relative to a mean of 4.84 g/L in milk from 16 herd contemporaries of mixed (A and B) BLG genotypes. The mechanism by which B' downregulates milk BLG concentration remains to be determined. CONCLUSIONS: High-throughput screening and identification of outliers, enabled the discovery of a synonymous G > A mutation in exon 1 of the B allele of the BLG gene (B'), which reduced the milk concentration of ß-lactoglobulin B protein isoform, by more than 50%. Milk from cows carrying the B' allele is expected to have improved processing characteristics, particularly for cheese-making.

Sex Differences and COVID-19.

Biological sex and psychosocial gender both play a role in many disease outcomes, and the novel coronavirus disease (COVID-19) is no different. Clinical observations in COVID-19 patient data delineate clear disparities between males and females, indicating males are at a higher risk for poorer disease outcomes. Although we are yet to understand the sex and gender-based disparities specific to COVID-19, there is evidence for sex-based differences in the endocrine, immune and renin-angiotensin system, all systems implicated in COVID-19 outcomes. Such disparities are largely thought to be driven by sex chromosomes and modulating sex hormones, which are known to vary between sex, and across the reproductive lifespan. Understanding and exploiting these driving factors are critical to understanding the pathobiology of SARS-CoV-2 virus and may lead to the development of novel therapies and increase the efficacy of preventative vaccine strategies currently under development. This chapter focuses on the endocrine, immune and renin-angiotensin system and genetic sex-based differences that could account for the meaningful differences observed in the outcomes of the SARS-CoV-2 infection.

Evaluating the data quality of the Gazepoint GP3 low-cost eye tracker when used independently by study participants.

The portability of low-cost eye trackers makes them attractive for research outside of the laboratory. Such research may require independent eye-tracker use. The present work compared the data quality of the Gazepoint GP3 when used independently by research participants with expert eye-tracking users. Twenty participants completed a training and a testing session 1 week apart. At training visits, participants were taught how to set up and use eye-tracking hardware and software and how to complete two tasks: a calibration task to measure accuracy and precision, as well as a visual search task to assess target fixations. At the testing session, participants set up the Gazepoint eye tracker and completed the two tasks without assistance. Participant accuracy and precision and visual search performance were compared to values obtained from two expert eye-tracking users. Additionally, the eye-tracker sampling rate, which is sensitive to factors such as head motion, was assessed in both participants and the expert users. Participant accuracy and precision closely approximated expert user values. Participant target fixations were detected with a 92.5% sensitivity and 76.8% specificity, closely mirroring expert user sensitivity and specificity. The sampling rate distribution was also similar between the participants and expert user (the means of those distributions were 16.99 ± 3.0 ms and 16.43 ± 2.3 ms, respectively). When used independently, data quality obtained from a low-cost, portable eye-tracking setup closely approximated values obtained from an expert user and was adequate enough to be a feasible option for some studies that require independent use by study participants.

Sex Differences in Aging and Associated Biomarkers.

Aging is a natural process defined by the gradual, time-dependent decline of biological and behavioural functions, for which individuals of the same chronological age show variability. The capacity of biological systems to continuously adjust for optimal functioning despite ever changing environments is essential for healthy aging, and variability in these adaptive homeostatic mechanisms may reflect such heterogeneity in the aging process. With an ever-increasing aging population, interest in biomarkers of aging is growing. Although no universally accepted definition of biomarkers of healthy aging exists, mediators of homeostasis are consistently used as measures of the aging process. As important sex differences are known to underlie many of these systems, it is imperative to consider that this may reflect, to some extent, the sex differences observed in aging and age-related disease states. This chapter aims to outline sex differences in key homeostatic domains thought to be associated with the pathophysiology of aging, often proposed as biomarkers of aging and age-related disease states. This includes considering sex-based differences and hormonal status with regards to the gonadal and adrenal endocrine systems and immune function.

Inner Peace: Evaluating a Complementary Program Promoting Intra-Personal Peace at Adelaide Women's Prison, Australia.

The Peace Education Program, created in 2012, is a complementary program with potential to supplement official rehabilitation interventions offered in correctional centers. The program promotes "inner peace" as an innate and universal human resource, but whilst inner peace is a key concept in positive psychology and the Good Lives Model, there is a paucity of research regarding how to operationalize and evaluate this concept. The program had not previously been the subject of independent theoretically-informed research. Drawing on a mixed methods study conducted in Adelaide Women's Prison, this article explores the impact of the program on participants' learning regarding inner peace. Participants reported a greater understanding about inner peace, which they described as contributing to a stronger sense of their identity, enhanced self-esteem and increased self-regulation skills, resulting in reductions in impulsivity and reactive aggression. The quantitative data indicated there was a significant increase in participants' subjective ratings of inner peace before the program (M = 12.08) and post-program completion (M = 14.00) (p < .001). Growth in affect-regulation and anger-management skills may contribute to reductions in offending.

Evaluation of the Everyday Memory Questionnaire-Revised in a menopausal population: understanding the brain fog during menopause.

OBJECTIVE: Brain fog (ie, memory complaints and concentration difficulties) is frequently reported during the menopausal transition. There is lack of standardized scales available to measure brain fog across the menopausal transition. This study aimed to evaluate the factor structure of the Everyday Memory Questionnaire-Revised (EMQ-R) and to determine the most commonly reported everyday cognitive symptoms in a menopausal population. METHODS: Four hundred seventeen eligible women aged from 40 to 60 years (107 premenopausal, 149 perimenopausal, and 161 early postmenopausal) were recruited from the general community and were included in the analyses. Confirmatory factor analysis was conducted to test the model fit of the bifactor structure (ie, 4-item attentional subscale ranged 0-16, 7-item retrieval subscale ranged 0-28) of the 13-item EMQ-R (ranged 0-52) in a menopausal populations. Typical items in the retrieval subscale include "difficulty finding words," the attentional subscale include "difficulty following the thread of a story," and analysis of variance and multivariate analysis of covariance were used to investigate the group differences of individual items and two subscales. RESULTS: Confirmatory factor analysis indicated the bifactor structure of the EMQ-R has a good fit in all three groups. A significant difference was identified in the mean retrieval scores (pre: 11.8, peri: 13.6, early post: 11.7) but not in the mean attentional scores (pre: 4.53, peri: 5.01, early post: 4.65). CONCLUSIONS: The EMQ-R retrieval subscale may serve as a potential instrument to assess memory retrieval symptoms that contribute to "brain fog" in menopause. Increased memory retrieval complaints reported by the perimenopausal group suggests a transition-related memory retrieval dysfunction during menopausal transition.

Perceived Discrimination in Australia During the COVID-19 Pandemic: a Longitudinal Study.

OBJECTIVES: There have been global reports of increased discrimination during the COVID-19 pandemic relative to the pre-pandemic era, though this has not been well explored within Australia. The aim of the study was to characterise discriminatory behaviour experienced by groups previously identified as most at risk of experiencing discrimination (i.e. those of Asian descent or healthcare workers) both from pre-pandemic to pandemic and during the pandemic era in Australia. METHODS: From April 2020 to September 2021, 1479 Australian adults completed the everyday discrimination scale (EDS). Initially, participants were asked to retrospectively consider discrimination experienced pre-COVID-19 pandemic and then to consider experiences in the past month during the pandemic. Participants were invited to repeat the latter every 2 months. RESULTS: Collectively, there was a reduction in EDS scores from pre-pandemic to pandemic. Within the pandemic era, mean trajectory graphs across time revealed that changes in EDS scores in the 'non-Asian healthcare workers' and 'Asian healthcare workers' subgroups tended to mirror onto fluctuations in Australian COVID-19 case numbers. CONCLUSION: Our findings suggest social solidarity amongst the general Australian population during the pandemic, but still highlight a need to dedicate resources towards groups with heightened risk of experiencing discrimination during future public health threats.

Real or fake? Sourcing and marketing of non-prescribed benzodiazepines amongst two samples of people who regularly use illicit drugs in Australia.

INTRODUCTION: There is concern around non-prescribed benzodiazepine use, particularly with increasing detections of counterfeit products containing high-risk novel compounds. The aims of this study were to investigate how and which non-prescribed benzodiazepines are being sourced; forms, appearance and packaging; and awareness of risks associated with non-prescribed benzodiazepines. METHODS: Data were collected from a sample of Australians who inject drugs or use ecstasy and/or other illicit stimulants on a monthly or more frequent basis, and who reported past 6-month use of non-prescribed benzodiazepines (n = 235 and n = 250, respectively). Data were collected on source, diversion from a known/trusted prescription, product name and aesthetic characteristics for the last non-prescribed benzodiazepine obtained. RESULTS: Amongst participants who injected drugs, 71% reported that their last non-prescribed benzodiazepines were diverted from a known/trusted prescription, compared to 59% of participants who used ecstasy/other stimulants. Sourcing via cryptomarkets was rare. Across both samples, the majority reported last obtaining substances sold/marketed as diazepam or alprazolam. Participants sourcing via non-diverted means were twice as likely to obtain alprazolam. Known sourcing of novel compounds was rare. Amongst participants who used ecstasy/other stimulants, 36% reported confidence in the content/dose of non-prescribed benzodiazepines even when the source is unknown. DISCUSSION AND CONCLUSIONS: Most participants obtained substances sold as classic/registered benzodiazepines, mostly via diverted prescriptions, with a substantial minority potentially unaware of counterfeits circulating. While diverted use undeniably presents risks, tightening of prescriptions in Australia could inadvertently lead to greater supply of novel benzodiazepines as seen internationally, reinforcing prioritisation of demand and harm reduction strategies.

Systematic review and narrative synthesis of cognition in perimenopause: The role of risk factors and menopausal symptoms.

OBJECTIVE: Many women report cognitive concerns during the menopausal transition, and cognitive testing supports objective declines in some cognitive domains for some women. Identifying risk and protective factors that mediate cognitive difficulties would help women gain better insight into how they can manage cognitive difficulties during the menopause transition. The purpose of this review was to synthesize the studies that examine the relationships between cognition, menopausal symptoms and risk/protective factors during menopause transition. METHODS: A search of the literature examining cognition and risk factors in perimenopausal women between 2010 and September 2020 was performed using Ovid MEDLINE, PsychINFO, and PubMed. The results were synthesized narratively. Studies were categorized into clusters and conceptual mapping was used to illustrate the findings. RESULTS: 33 studies were included in this review and divided into three clusters. Factors associated with cognitive and other menopausal symptoms were grouped into demographic, socio-economic, lifestyle and reproductive factors. CONCLUSION: The current review identified a broad range of demographic, reproductive, socio-economic, and lifestyle risk/protective factors that are associated with cognition and menopausal symptoms. Relationships were also observed between vasomotor, affective and sleep symptoms with cognition, suggesting a complex relationship, including direct and indirect effects of risk/protective factors on cognition during menopause.

Systematic Review of NMR-Based Metabolomics Practices in Human Disease Research.

Nuclear magnetic resonance (NMR) spectroscopy is one of the principal analytical techniques for metabolomics. It has the advantages of minimal sample preparation and high reproducibility, making it an ideal technique for generating large amounts of metabolomics data for biobanks and large-scale studies. Metabolomics is a popular "omics" technology and has established itself as a comprehensive exploratory biomarker tool; however, it has yet to reach its collaborative potential in data collation due to the lack of standardisation of the metabolomics workflow seen across small-scale studies. This systematic review compiles the different NMR metabolomics methods used for serum, plasma, and urine studies, from sample collection to data analysis, that were most popularly employed over a two-year period in 2019 and 2020. It also outlines how these methods influence the raw data and the downstream interpretations, and the importance of reporting for reproducibility and result validation. This review can act as a valuable summary of NMR metabolomic workflows that are actively used in human biofluid research and will help guide the workflow choice for future research.

Changes in illicit drug use and markets with the COVID-19 pandemic and associated restrictions: findings from the Ecstasy and Related Drugs Reporting System, 2016-20.

AIMS: To describe (i) self-reported changes in drug use and (ii) trends in price, perceived availability, and perceived purity of illicit drugs, among people who regularly use ecstasy/ 3,4-methylenedioxymethamphetamine (MDMA) and other illicit stimulants in Australia following COVID-19 and associated restrictions. DESIGN: Annual interviews with cross-sectional sentinel samples conducted face-to-face in 2016-19 and via video conferencing or telephone in 2020. Data were collected via an interviewer-administered structured questionnaire. SETTING: Australian capital cities. PARTICIPANTS: Australians aged 16 years or older who used ecstasy/MDMA and other illicit stimulants on a monthly or more frequent basis and resided in a capital city, recruited via social media and word-of-mouth (n ~ 800 each year). MEASUREMENTS: Key outcome measures were self-reported illicit drug market indicators (price, purity and availability) and, in 2020 only, perceived change in drug use (including alcohol and tobacco) since March 2020 and reasons for this change. FINDINGS: For most drugs, participants reported either no change or a reduction in their use since COVID-19 restrictions were introduced. Ecstasy/MDMA was the drug most frequently cited as reduced in use (n = 552, 70% of those reporting recent use), mainly due to reduced opportunities for socialization. While market indicators were largely stable across most drugs, the odds of perceiving MDMA capsules as 'high' in purity decreased compared with 2016-19 [adjusted odds ratio (aOR) = 0.72, 95% confidence interval (CI) = 0.53-0.99], as did perceiving them as 'easy' to obtain (aOR = 0.42, CI = 0.26-0.67). The odds of perceiving cocaine and methamphetamine crystal as 'easy' to obtain also decreased (aOR = 0.67, CI = 0.46-0.96 and aOR = 0.12, CI = 0.04-0.41, respectively). CONCLUSIONS: After COVID-19-related restrictions were introduced in Australia, use of ecstasy/MDMA, related stimulants and other licit and illicit drugs mainly appeared to remain stable or decrease, primarily due to impediments to socialization.

ABC transporters do not contribute to extracellular translocation of hyaluronan in human breast cancer in vitro.

Extracellular translocation of the polysaccharide, hyaluronan (HA) has been thought to be mediated via its transmembrane synthetic enzyme, hyaluronan synthase (HAS) but recent studies have indicated that the ATP-Binding-Cassette (ABC) transporter, MRP5 contributes to this process. Liberated and cell-associated HA contributes to breast cancer initiation and progression, and therefore the inhibition of ABC transporters and consequently HA transport could provide therapeutic benefit in the treatment of breast cancer. Quantitation of ABC transporter genes, MRP1-5, BCRP and MDR1 were determined in six breast cancer cell lines selected for their differential HA synthetic rates. Low endogenous expression of transporters was detected but no significant correlation existed between ABC transporter and HAS gene expression or HA production. A dose titration of up to ten times the IC(50) of ten small molecule ABC transporter inhibitors did not significantly inhibit HA export in four breast cancer cell lines. Unlike the changes observed after inhibition of HA synthesis by the characterised inhibitor 4-MU, inhibition of ABC transporters did not alter the cell morphology, HA glycocalyx or the intracellular quantity or localisation of HA. Collectively these data indicate that ABC transporters do not contribute to the extracellular transport of HA in breast cancer, supporting a role for the hyaluronan synthase in translocation.