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Key Points ChipCytometry was used for multiplex immunophenotyping of human tonsils. It is viable for high-content profiling of cell suspensions and tissue sections. ChipCytometry can identify and localize cell populations and rare cell types., ChipCytometry is a multiplex imaging method that can be used to analyze either cell suspensions or tissue sections. Images are acquired by iterative cycles of immunostaining with fluorescently labeled Abs, followed by photobleaching, which allows the accumulation of multiple markers on a single sample. In this study, we explored the feasibility of using ChipCytometry to identify and phenotype cell subsets, including rare cell types, using a combination of tissue sections and single-cell suspensions. Using ChipCytometry of tissue sections, we successfully demonstrated the architecture of human palatine tonsils, including the B and T cell zones, and characterized subcompartments such as the B cell mantle and germinal center zone, as well as intrafollicular PD1-expressing CD4+ T cells. Additionally, we were able to identify the rare tonsillar T cell subsets, mucosal-associated invariant T (MAIT) and γδ-T cells, within tonsil tissue. Using single-cell suspension ChipCytometry, we further dissected human tonsillar T cell subsets via unsupervised clustering analysis as well as supervised traditional manual gating. We were able to show that PD1+CD4+ T cells are comprised of CXCR5+BCL6high follicular Th cells and CXCR5−BCL6mid pre–follicular Th cells. Both supervised and unsupervised analysis approaches identified MAIT cells in single-cell suspensions, confirming a phenotype similar to that of blood-derived MAIT cells. In this study, we demonstrate that ChipCytometry is a viable method for single-cell suspension cytometry and analysis, with the additional benefit of allowing phenotyping in a spatial context using tissue sections.

Kerri Beckmann, 1 Sudarshan Selva-Nayagam, 2 Ian Olver, 3 Caroline Miller, 4, 5 Elizabeth S Buckley, 1 Kate Powell, 4 Dianne Buranyi-Trevarton, 6 Raghu Gowda, 7 David Roder, 1 Martin K Oehler 8 1Cancer Epidemiology and Population Health Research, University of South Australia, Adelaide, Australia; 2Medical Oncology, Royal Adelaide Hospital Cancer Centre, Adelaide, Australia; 3Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, Australia; 4South Australian Health and Medical Research Institute, Adelaide, Australia; 5School of Public Health, University of Adelaide, Adelaide, Australia; 6Central Adelaide Local Health Network, SA Health, Adelaide, Australia; 7Radiation Oncology, Royal Adelaide Hospital, Adelaide, Australia; 8Gynaecological Oncology, Royal Adelaide Hospital, Adelaide, AustraliaCorrespondence: Kerri BeckmannCancer Epidemiology and Population Health Research, University of South Australia, Level 8 SAHMRI Building, PO Box 2471 North Terrace, Adelaide, South Australia, AustraliaTel +61 8 83027018Email kerri.beckmann@unisa.edu.auBackground: Uncertainties remain about the most effective treatment for uterine carcinosarcoma (UCS), a rare but aggressive uterine cancer, due to the limited scope for randomized trials. This study investigates whether nodal excision or adjuvant therapies after hysterectomy offer a survival benefit, using multi-institutional clinical registry data from South Australia.Methods: Data for all consecutive cases of UCS from 1980 to 2019 were extracted from the Clinical Cancer Registry. Clinical and treatment-related factors associated with disease-specific mortality (DSM) and all-cause mortality (ACM) were determined using multivariable Cox proportional hazards regression, with subgroup analyses by stage.Results: Median follow-up for the 140 eligible cases was 21 months. 94% underwent hysterectomy, and 72% had an additional pelvic lymph node dissection (PLND). Furthermore, 16% received adjuvant chemotherapy; 11% adjuvant radiotherapy and 16% multimodal chemoradiotherapy, with an increase in the latter two modalities over time. DSM was reduced among those who underwent PLND (HR: 0.41; 95%CI: 0.23– 0.74), adjuvant chemotherapy (HR: 0.39; 95%CI: 0.18– 0.84) or multimodality treatment (HR: 0.11; 95%CI: 0.06– 0.30) compared with hysterectomy alone for the whole cohort and for late stage disease (FIGO III/IV) but not for earlier stage disease, except for reduced DSM with multimodal therapy. Findings were similar for ACM.Conclusion: Our findings indicate better survival among those who received PLND, chemotherapy and multimodal adjuvant therapy, with the latter applying to early and late stage disease. However, cautious interpretation is warranted, due to potential “indication bias” and limited power. Further research into effective treatment modalities, ideally using prospective study designs, is needed.Keywords: uterine carcinosarcoma, management, survival, multimodal therapy, adjuvant chemotherapy, adjuvant radiotherapy

Background: We investigated treatment and survival by clinical and sociodemographic characteristics in South Australia for service evaluation using linked data.Method: Data on invasive female breast cancers (n=13494) from the South Australian Cancer Registry (2000-2014 diagnoses) were linked to hospital inpatient, radiotherapy, and universal health insurance data. Treatments <12 months from diagnosis and survival were analysed, using adjusted odds ratios (aORs) from logistic regression, and adjusted sub-hazard ratios (aSHRs) from competing risk regression. Results: Five-year disease-specific survival increased to 91% for 2010-2014. Survival was lower for: ages 70+ years, and lowest for 80+ years (aSHR 2.04, 95%CI 1.69-2.47), compared with ages Conclusions: High survival from breast cancer in South Australia was comparable to the Australia-wide rate and did not differ by residential remoteness and country of birth. Surgical treatment within 12 months after cancer diagnosis increased during the study period and strongly predicted higher survival. Patients aged 70+ years had lower survival and less treatment, and more trial evidence is needed to optimize trade-offs between benefits and harms in this older age range. Systemic therapy was less for residents from most disadvantaged areas, while radiotherapy was less for residents of outer regional and remote areas.

Background Health professionals and ancillary staff have a unique opportunity to make a positive difference to the end of life (EOL) experiences of their patients and those who are important to them. In order to do so, they need to be equipped with a range of skills, knowledge, attitudes and behaviours. The End of Life Care Core Skills Education and Training Framework sets out the skills and knowledge to achieve this. This framework was used to benchmark the current provision of EOL training and education at a London-based NHS Trust and to develop a novel approach to meeting identified gaps. Methods A task and finish group consisting of members of the Trust’s specialist palliative care team and the EOL care clinical leads reviewed the education mapping exercise and identified key areas of focus for the Trust’s EOL education and training programme. These were triangulated with other national EOL frameworks, audits and quality standards. Results The key education themes identified were: 1) personalisation of end of life care (finding out ‘what matters most’ to each patient); 2) every staff member has a role in EOL care. Non-clinical staff involved with patients at the end of life were identified as one of the key target groups in the EOL education gap analysis. A short video was agreed as the most innovative educational resource with a focus on the core themes for a non-clinical target audience. In collaboration with an external production team, the narratives of ten Trust employees from a diverse range of clinical and non-clinical roles were recorded. Conclusion This quality improvement initiative sought to innovatively address a gap in EOL education through the development of an educational video with contributions from a diverse range of clinical and non-clinical staff. The next steps are to implement and evaluate the impact of this project.

Balanitis is an inflammatory condition typically affecting the glans penis but is commonly associated with inflammation of the foreskin and is consequentially known as ‘balanoposthitis’. It is an illustrative term of which there are numerous causes and risk factors. Among young men, up to 6% are shown to suffer from balanitis.1 Prevalence is higher in uncircumcised men2 due to the accumulation of microorganisms, secretions and epithelial debris between the penis and foreskin.3 In October 2018, the National Institute for Health and Care Excellence (NICE) produced a Clinical Knowledge Summary (CKS) of current evidence. This review will focus on the aspects of the CKS that relate to the paediatric population.4 In NICE CKS, there is little …

Macular degeneration is the leading cause of blindness in the developed world. Whilst most patients lose sight owing to atrophic changes, no treatments currently exist that improve the vision deficit due to atrophy. Here, we identify loss of lateral inhibition as a specific mechanism by which photoreceptor degeneration reduces visual function beyond the atrophic area. We find that this inhibition is adaptive, and that if modulated can improve visual function, making inhibitory circuits an unexpected therapeutic target for age related macular degeneration and related disorders.

Objective: To investigate treatment and survival over three decades. Methods: Clinical registry data from three major public hospitals analysed using Kaplan–Meier product-limit estimates and multivariate proportional hazard regression to determine disease-specific survival. Results: Five-year survival increased from 75% to 84%. The adjusted hazard ratio (HR, 95% CI) was 0.56 (0.41, 0.77) for 2010–2016 compared with 1984–1989 and was higher for: ages 80+ years; more advanced stages; poorly differentiated tumours; and complex mixed epithelial and mesenchymal tumours and sarcomas. Treatment was by surgery (92%), radiotherapy (33%), chemotherapy (12%) and hormone therapy (10%). Adjusted analyses showed radiotherapy and hormone therapy were less common from 1990 and chemotherapy more common for 2010–2016. Treatment likelihood was lower for ages ≥80 years, mixed epithelial and mesenchymal tumours receiving surgery and chemotherapy, but higher for radiotherapy. Advanced cancers (FIGO stage IV) had less surgery but more non-surgical treatments. Marginal evidence presented of more hormone therapy for high socio-economic areas. Conclusions: Survival was equivalent to national figures for Australia and the United States, but potentially higher than for England and Wales. Cases aged 80+ years had less care and poorer survival. Findings illustrate the complementary roles of hospital and population-based registries in local service evaluation Refereed/Peer-reviewed

Background: The value of hospital registries for describing treatment and survival outcomes for vulval cancer was investigated. Hospital registry data from four major public hospitals in 1984-2016 were used because population-based data lacked required treatment and outcomes data. Unlike population registries, the hospital registries had recorded FIGO stage, grade and treatment. Methods: Unadjusted and adjusted disease-specific survival and multiple logistic regression were used. Disease-specific survivals were explored using Kaplan-Meier product-limit estimates. Hazards ratios (HRs) were obtained from proportional hazards regression for 1984-1999 and 2000-2016. Repeat analyses were undertaken using competing risk regression. Results: Five-year disease-specific survival was 70%, broadly equivalent to the five-year relative survivals reported for Australia overall (70%), the United Kingdom (70%), USA (72%), Holland (70%), and Germany (Munich) (68%). Unadjusted five-year survival tended to be lower for cancers diagnosed in 2000-2016 than 1984-1999, consistent with survival trends reported for the USA and Canada, but higher for 2000-2016 than 1984-1999 after adjusting for stage and other covariates, although differences were small and did not approach statistical significance (p≥0.40). Surgery was provided as part of the primary course of treatment for 94% of patients and radiotherapy for 26%, whereas chemotherapy was provided for only 6%. Less extensive surgical procedures applied in 2000-2016 than 1984-1999 and the use of chemotherapy increased over these periods. Surgery was more common for early FIGO stages, and radiotherapy for later stages with a peak for stage III. Differences in treatment by surgery and radiotherapy were not found by geographic measures of remoteness and socioeconomic status in adjusted analyses, suggesting equity in service delivery. Conclusions: The data illustrate the complementary value of hospital-registry data to population-registry data for informing local providers and health administrations of trends in management and outcomes, in this instance for a comparatively rare cancer that is under-represented in trials and under-reported in national statistics. Hospital registries can fill an evidence gap when clinical data are lacking in population-based registries. Refereed/Peer-reviewed

Background: Clinical registry data from major South Australian public hospitals were used to investigate trends in invasive breast-cancer treatment and survival by age. Methods: Disease-specific survival was calculated for the 1980 to 2013 diagnostic period using Kaplan-Meier product-limit estimates, with a censoring of live cases on December 31, 2014. Cox proportional hazards regression was used to examine differences in survival by age and tumour characteristic. First-round treatments following diagnosis were analysed, using multiple logistic regression to adjust for confounding. Results: Five-year survival increased from 75% in the 1980s to 87% in 2000 to 2013, consistent with national trends, and with increases occurring irrespective of age. There was an increased use of breast conserving surgery, radiotherapy, chemotherapy, and hormone treatments. Five-year survival was lower for women aged 80+ years, increasing from 65% in the 1980s to 74% in 2000 to 2013. Lower survival in these older women persisted after adjusting for TNM stage, other clinical variables, and diagnostic year, without evidence of a reduced disparity over time. Older women were less likely to have surgery, radiotherapy, and chemotherapy throughout 1980 to 2013. By comparison, their use of hormone therapy was elevated. The adjusted relative odds of mastectomy (as opposed to breast conserving surgery) were lower for the 80+ year age range. Conclusions: Breast-cancer survival increases applied to all ages, including 80+ years, but poorer outcomes persisted in this older group and the gap did not reduce. A key question is whether the best trade-off now exists between optimally therapeutic cancer treatment and accommodations for frailty and co-morbidity in the aged, or whether opportunities exist for better trade-offs and better survival. Local registry data are important for describing local service activity and outcomes by age for local service providers, health administrations and consumer groups; monitoring disparities; and indicating effects of local initiatives. Refereed/Peer-reviewed

Tissue-resident memory T (TRM) cells provide key adaptive immune responses in infection, cancer, and autoimmunity. However transcriptional heterogeneity of human intestinal TRM cells remains undefined, and definitive markers of CD103-TRM cells are lacking. Here, we investigated transcriptional and functional heterogeneity of human TRM cells through the study of donor-derived intestinal TRM cells from intestinal transplant recipients. Single-cell transcriptional profiling identified four conventional TRM populations, with two distinct transcriptional states of CD8+ TRM cells, delineated by ITGAE and ITGB2 expression. We defined a transcriptional signature discriminating the two CD8+ populations, including differential expression of key residency-associated genes and cytotoxic molecules. Flow cytometry of recipient-derived cells infiltrating the graft and intestinal lymphocytes from healthy gut confirmed the two CD8+ TRM phenotypes, with β2-integrin acting as a CD103-CD8+ TRM marker. CD103+ CD8+ TRM cells produced IL-2, and demonstrated greater polyfunctional cytokine production, while β2-integrin+ CD69+ CD103-TRM cells had higher granzyme expression. Phenotypic and functional analysis of intestinal CD4+ T cells identified many parallels, including a distinct β2-integrin+ population. Together, these results describe the transcriptional, phenotypic, and functional heterogeneity of human intestinal TRM cells, and suggest a role for β2-integrin in TRM development.SummaryHeterogeneity within human tissue-resident memory T (TRM) cells is poorly understood. We show that transcriptionally, phenotypically, and functionally distinct CD4+ and CD8+ TRM subsets exist in the human intestine, and that β2-integrin expression identifies a distinct population of CD8+ TRM cells.

Background To investigate the association between pre-diagnostic colonoscopy and colorectal cancer mortality in South Australia. Methods Colonoscopy histories were obtained for colorectal cancer patients diagnosed in 2003–2013 using linked Medical Benefits Schedule (MBS) claims, hospital-inpatient and cancer-registry data. Colonoscopy histories included the year of colonoscopy, numbers of examinations, and the time from first colonoscopy to diagnosis. Histories of multiple exposures to colonoscopies, and exposures of greater than a year from initial colonoscopy to diagnosis, were regarded as indicators of screening or surveillance activity. Colonoscopies occurring within one year of diagnosis were regarded as more likely to be a response to cancer symptoms than those occurring > 1 year before diagnosis. Associations between colonoscopy history and post-diagnostic survival were analysed using sub-hazard ratios (SHRs) from competing risk regression adjusted for socio-demographic and cancer characteristics. Results Having pre-diagnostic colonoscopy was associated with an unadjusted reduction in risk of colorectal cancer death of 17% (SHR: 0.83, 95% CI 0.78–0.89). After adjusting for time period and sociodemographic characteristics, the risk of colorectal cancer death reduced by 17% for one pre-diagnostic colonoscopy examination; 27% for two pre-diagnostic colonoscopy examinations; and 45% for three or more pre-diagnostic colonoscopy examinations. Those with a time of over one year from first colonoscopy in the study window to diagnosis, when compared with less than one year, had a 17% lower risk of colorectal cancer death in this adjusted analysis. These reductions were substantially reduced or eliminated when also adjusting for less advanced stage. Conclusions Pre-diagnostic colonoscopy, and more so, multiple colonoscopies and first colonoscopy occurring over one year from initial colonoscopy to diagnosis, were associated with longer survival post diagnosis. This was largely explained by less advanced cancer stage at the time of diagnosis. Electronic supplementary material The online version of this article (10.1186/s12885-019-6092-4) contains supplementary material, which is available to authorized users.

OBJECTIVE: Autosomal dominant optic atrophy (ADOA) starts in early childhood with loss of visual acuity and color vision deficits. OPA1 mutations are responsible for the majority of cases, but in a portion of patients with a clinical diagnosis of ADOA, the cause remains unknown. This study aimed to identify novel ADOA-associated genes and explore their causality. METHODS: Linkage analysis and sequencing were performed in multigeneration families and unrelated patients to identify disease-causing variants. Functional consequences were investigated in silico and confirmed experimentally using the zebrafish model. RESULTS: We defined a new ADOA locus on 7q33-q35 and identified 3 different missense variants in SSBP1 (NM_001256510.1; c.113G>A [p.(Arg38Gln)], c.320G>A [p.(Arg107Gln)] and c.422G>A [p.(Ser141Asn)]) in affected individuals from 2 families and 2 singletons with ADOA and variable retinal degeneration. The mutated arginine residues are part of a basic patch that is essential for single-strand DNA binding. The loss of a positive charge at these positions is very likely to lower the affinity of SSBP1 for single-strand DNA. Antisense-mediated knockdown of endogenous ssbp1 messenger RNA (mRNA) in zebrafish resulted in compromised differentiation of retinal ganglion cells. A similar effect was achieved when mutated mRNAs were administered. These findings point toward an essential role of ssbp1 in retinal development and the dominant-negative nature of the identified human variants, which is consistent with the segregation pattern observed in 2 multigeneration families studied. INTERPRETATION: SSBP1 is an essential protein for mitochondrial DNA replication and maintenance. Our data have established pathogenic variants in SSBP1 as a cause of ADOA and variable retinal degeneration. ANN NEUROL 2019;86:368-383.

ObjectivesSome early studies indicated lower survival with longer time from diagnosis to cancer treatment, but others showed the reverse. We investigated time to treatment of colorectal cancer and associations with survival.Setting and participantsClinical registry data for colorectal cancer cases diagnosed in 2000–2010 at four major public hospitals in South Australia and treated by surgery (n=1675), radiotherapy (n=616) and/or systemic therapy (n=1556).DesignA historic cohort design, with rank-order tests for ordinal clinical and sociodemographic predictors and multiple logistic regression for comparing time from diagnosis to treatment. Unadjusted Kaplan-Meier estimates and adjusted Cox proportional hazards regression were used to investigate disease-specific survival by time to treatment.Outcome measuresTime to treatment and survival from diagnosis to death from colorectal cancer.ResultsTreatment (any type) commenced for 87% of surgical cases<60 days of diagnosis, with 80% having surgery within this period. Of those receiving radiotherapy, 59% began this treatment<60 days, and of those receiving systemic therapy, the corresponding proportion was 56%. Adjusted analyses showed treatment delay >60 days was more likely for rectal cancers, 2006–2010 diagnoses, residents of northern than other metropolitan regions and for surgery, younger ages <2 years from diagnosis for time to treatment >30 days. Survival in the 3–10 years postdiagnosis generally did not differ by time to treatment, except for lower survival for any treatment >90 days for surgical cases.ConclusionsThe lower survival<2 years from diagnosis for treatment<30 days of diagnosis is consistent with other studies attributed to preferencing more complicated cases for earlier care. Lower 3–10 years survival for surgical cases first treated >90 days from diagnosis is consistent with previously reported U-shaped relationships.

Summary Age-related macular degeneration and other macular diseases result in the loss of light-sensing cone photoreceptors, causing irreversible sight impairment. Photoreceptor replacement may restore vision by transplanting healthy cells, which must form new synaptic connections with the recipient retina. Despite recent advances, convincing evidence of functional connectivity arising from transplanted human cone photoreceptors in advanced retinal degeneration is lacking. Here, we show restoration of visual function after transplantation of purified human pluripotent stem cell-derived cones into a mouse model of advanced degeneration. Transplanted human cones elaborate nascent outer segments and make putative synapses with recipient murine bipolar cells (BCs), which themselves undergo significant remodeling. Electrophysiological and behavioral assessments demonstrate restoration of surprisingly complex light-evoked retinal ganglion cell responses and improved light-evoked behaviors in treated animals. Stringent controls exclude alternative explanations, including material transfer and neuroprotection. These data provide crucial validation for photoreceptor replacement therapy and for the potential to rescue cone-mediated vision., Graphical abstract, Highlights • Rescue of cone-mediated function by transplantation of purified human cones • Restoration of complex retinal responses and behavior in advanced degeneration • Formation of human-murine putative synaptic connections • Relevant controls exclude material transfer and trophic support, Cone photoreceptor death and associated central vision loss are common to many retinal dystrophies. Ribeiro et al. show that transplantation of purified human pluripotent stem cell-derived cones into a mouse model of end-stage disease allows the formation of functional connections with the underlying retina and restores cone-mediated visual function.

Rationale Screening has been found to reduce breast cancer mortality at a population level in Australia, but these studies did not address local settings where numbers of deaths would generally have been too low for evaluation. Clinicians, administrators, and consumer groups are also interested in local service outcomes. We therefore use more common prognostic and treatment measures and survivals to gain evidence of screening effects among patients attending 4 local hospitals for treatment. Aims and objectives To compare prognostic, treatment, and survival measures by screening history to determine whether expected screening effects are occurring. Methods Employing routine clinical registry and linked screening data to investigate associations of screening history with these measures, using unadjusted and adjusted analyses. Results Screened women had a 10-year survival from breast cancer of 92%, compared with 78% for unscreened women; and 79% of screened surgical cases had breast conserving surgery compared with 64% in unscreened women. Unadjusted analyses indicated that recently screened cases had earlier tumor node metastasis stages, smaller diameters, less nodal involvement, better tumor differentiation, more oestrogen and progesterone receptor positive lesions, more hormone therapy, and less chemotherapy. Radiotherapy tended to be more common in screening participants. More frequent use of adjunctive radiotherapy applied when breast conserving surgery was used. Conclusions Results confirm the screening effects expected from the scientific literature and demonstrate the value of opportunistic use of available registry and linked screening data for indicating to local health administrations, practitioners, and consumers whether local screening services are having the effects expected.

ObjectiveUsing linked cancer registry and administrative data to monitor, tumour, node and metastases (TNM) stage and survival from female breast cancer in Australia.MethodAnalysis of 2000–2014 diagnoses with linked population-based data to investigate: (1) sociodemographic predictors of advanced stage (stages III and IV), using unadjusted and adjusted logistic regression; and (2) sociodemographic factors and stage as predictors of breast cancer survival using competing risk regression.DesignPopulation-based registry cohort.Setting and participants14 759 South Australian women diagnosed in 2000–2014.Primary and secondary outcome measuresStage and survival.ResultsAt diagnosis, 46% of women were classified as stage I, 39% as stage II, 12% as stage III and 4% as stage IV. After adjusting for sociodemographic factors, advanced stage was more common: (1) for ages ConclusionsStage varied by age, diagnostic period and socioeconomic status, and was a stronger predictor of survival than other statistically significant sociodemographic predictors. Achieving earlier diagnosis outside the original BreastScreen target of 50–69 years (as applying

Registry data on invasive cervical cancers (n = 1,274) from four major hospitals (1984-2012) were analysed to determine their value for informing local service delivery in Australia. The methodology comprised disease-specific survival analyses using Kaplan-Meier product-limit estimates and Cox proportional hazards models and treatment analyses using logistic regression. Five- and 10-year survivals were 72% and 68%, respectively, equating with relative survival estimates for Australia and the USA. Most common treatments were surgery and radiotherapy. Systemic therapies increased in recent years, generally with radiotherapy, but were less common for residents from less accessible areas. Surgery was more common for younger women and early-stage disease, and radiotherapy for older women and regional and more advanced disease. The proportion of glandular cancers increased in-step with national trends. Little evidence of variation in risk-adjusted survival presented over time or by Local Health District. The study illustrates the value of local registry data for describing local treatment and outcomes. They show the lower use of systemic therapies among residents of less accessible areas which warrants further investigation. Risk-adjusted treatment and outcomes did not vary by socio-economic status, suggesting equity in service delivery. These data are important for local evaluation and were not available from other sources. Refereed/Peer-reviewed

Classical feed-forward inhibition involves an excitation-inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses.

Current bioavailability models, such as the free ion activity model and biotic ligand model, explicitly consider that metal exposure will be mainly to the dissolved metal in ionic form. With the rise of nanotechnology products and the increasing release of metal-based nanoparticles (NPs) to the environment, such models may increasingly be applied to support risk assessment. It is not immediately clear, however, whether the assumption of metal ion exposure will be relevant for NPs. Using an established approach of oral gluing, a toxicokinetics study was conducted to investigate the routes of silver nanoparticles (AgNPs) and Ag+ ion uptake in the soil-dwelling earthworm Lumbricus rubellus. The results indicated that a significant part of the Ag uptake in the earthworms is through oral/gut uptake for both Ag+ ions and NPs. Thus, sealing the mouth reduced Ag uptake by between 40% and 75%. An X-ray analysis of the internal distribution of Ag in transverse sections confirmed the presence of increased Ag concentrations in exposed earthworm tissues. For the AgNPs but not the Ag+ ions, high concentrations were associated with the gut wall, liver-like chloragogenous tissue, and nephridia, which suggest a pathway for AgNP uptake, detoxification, and excretion via these organs. Overall, the results indicate that Ag in the ionic and NP forms is assimilated and internally distributed in earthworms and that this uptake occurs predominantly via the gut epithelium and less so via the body wall. The importance of oral exposure questions the application of current metal bioavailability models, which implicitly consider that the dominant route of exposure is via the soil solution, for bioavailability assessment and modeling of metal-based NPs.

In chronic respiratory disease, the formation of dense, 3-dimensional “microcolonies” by Pseudomonas aeruginosa within the airway plays an important role in contributing to resistance to treatment. An in vitro biofilm model of pseudomonal microcolony formation using artificial-sputum (AS) medium was established to study the effects of low-molecular-weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation, and the efficacy of colistin. The studies employed clinical cystic fibrosis (CF) isolates ( n = 3) and reference nonmucoid and mucoid multidrug-resistant (MDR) CF isolates ( n = 7). Bacterial growth and biofilm development and disruption were studied using cell viability assays and image analysis with scanning electron and confocal laser scanning microscopy. Pseudomonal growth in AS medium was associated with increased ATP production ( P < 0.05) and the formation (at 48 h) of discrete (>10-μm) microcolonies. In conventional growth medium, colistin retained an ability to inhibit growth of planktonic bacteria, although the MIC was increased (0.1 to 0.4 μg/ml) in AS medium compared to Mueller-Hinton (MH) medium. In contrast, in an established-biofilm model in AS medium, the efficacy of colistin was decreased. OligoG CF-5/20 (≥2%) treatment, however, induced dose-dependent biofilm disruption ( P < 0.05) and led to colistin retaining its antimicrobial activity ( P < 0.05). While circular dichroism indicated that OligoG CF-5/20 did not change the orientation of the alginate carboxyl groups, mass spectrometry demonstrated that the oligomers induced dose-dependent (>0.2%; P < 0.05) reductions in pseudomonal quorum-sensing signaling. These findings reinforce the potential clinical significance of microcolony formation in the CF lung and highlight a novel approach to treat MDR pseudomonal infections.

Monitoring screening mammography effects in small areas is often limited by small numbers of deaths and delayed effects. We developed a risk score for breast cancer death to circumvent these limitations. Screening, if effective, would increase post-diagnostic survivals through lead-time and related effects, as well as mortality reductions. Linked cancer and BreastScreen data at four hospitals (n = 2,039) were used to investigate whether screened cases had higher recorded survivals in 13 small areas, using breast cancer deaths as the outcome (M1), and a risk of death score derived from TNM stage, grade, histology type, hormone receptor status, and related variables (M2). M1 indicated lower risk of death in screened cases in 12 of the 13 areas, achieving statistical significance (p

Initiatives to support the sustainable development of the nanotechnology sector have led to rapid growth in research on the environmental fate, hazards and risk of engineered nanoparticles (ENP). As the field has matured over the last 10 years, a detailed picture of the best methods to track potential forms of exposure, their uptake routes and best methods to identify and track internal fate and distributions following assimilation into organisms has begun to emerge. Here we summarise the current state of the field, focussing particularly on metal and metal oxide ENPs. Studies to date have shown that ENPs undergo a range of physical and chemical transformations in the environment to the extent that exposures to pristine well dispersed materials will occur only rarely in nature. Methods to track assimilation and internal distributions must, therefore, be capable of detecting these modified forms. The uptake mechanisms involved in ENP assimilation may include a range of trans-cellular trafficking and distribution pathways, which can be followed by passage to intracellular compartments. To trace toxicokinetics and distributions, analytical and imaging approaches are available to determine rates, states and forms. When used hierarchically, these tools can map ENP distributions to specific target organs, cell types and organelles, such as endosomes, caveolae and lysosomes and assess speciation states. The first decade of ENP ecotoxicology research, thus, points to an emerging paradigm where exposure is to transformed materials transported into tissues and cells via passive and active pathways within which they can be assimilated and therein identified using a tiered analytical and imaging approach.

Screening has been found to reduce breast cancer mortality at a population level in Australia, but these studies did not address local settings where numbers of deaths would generally have been too low for evaluation. Clinicians, administrators, and consumer groups are also interested in local service outcomes. We therefore use more common prognostic and treatment measures and survivals to gain evidence of screening effects among patients attending 4 local hospitals for treatment.To compare prognostic, treatment, and survival measures by screening history to determine whether expected screening effects are occurring.Employing routine clinical registry and linked screening data to investigate associations of screening history with these measures, using unadjusted and adjusted analyses.Screened women had a 10-year survival from breast cancer of 92%, compared with 78% for unscreened women; and 79% of screened surgical cases had breast conserving surgery compared with 64% in unscreened women. Unadjusted analyses indicated that recently screened cases had earlier tumor node metastasis stages, smaller diameters, less nodal involvement, better tumor differentiation, more oestrogen and progesterone receptor positive lesions, more hormone therapy, and less chemotherapy. Radiotherapy tended to be more common in screening participants. More frequent use of adjunctive radiotherapy applied when breast conserving surgery was used.Results confirm the screening effects expected from the scientific literature and demonstrate the value of opportunistic use of available registry and linked screening data for indicating to local health administrations, practitioners, and consumers whether local screening services are having the effects expected.

Woodlice efficiently sequester copper (Cu) in ‘cuprosomes' within hepatopancreatic ‘S' cells. Binuclear ‘B’ cells in the hepatopancreas form iron (Fe) deposits; these cells apparently undergo an apocrine secretory diurnal cycle linked to nocturnal feeding. Synchrotron-based µ-focus X-ray spectroscopy undertaken on thin sections was used to characterize the ligands binding Cu and Fe in S and B cells of Oniscus asellus (Isopoda). Main findings were: (i) morphometry confirmed a diurnal B-cell apocrine cycle; (ii) X-ray fluorescence (XRF) mapping indicated that Cu was co-distributed with sulfur (mainly in S cells), and Fe was co-distributed with phosphate (mainly in B cells); (iii) XRF mapping revealed an intimate morphological relationship between the basal regions of adjacent S and B cells; (iv) molecular modelling and Fourier transform analyses indicated that Cu in the reduced Cu+ state is mainly coordinated to thiol-rich ligands (Cu–S bond length 2.3 Å) in both cell types, while Fe in the oxidized Fe3+ state is predominantly oxygen coordinated (estimated Fe–O bond length of approx. 2 Å), with an outer shell of Fe scatterers at approximately 3.05 Å; and (v) no significant differences occur in Cu or Fe speciation at key nodes in the apocrine cycle. Findings imply that S and B cells form integrated unit-pairs; a functional role for secretions from these cellular units in the digestion of recalcitrant dietary components is hypothesized.

Data from registries at four major public hospitals in South Australia indicate increased 5-year disease-specific survivals for colorectal cancer from 48% to 63% between 1980-1986 and 2005-2010. For 80+ year olds, the increase was smaller, from 47% to 52%. Risk of case fatality halved overall, adjusting for age, gender, stage, differentiation and sub-site. Patients aged 80+ years had a lower risk reduction of about a third (hazards ratio: 0.69; 95% confidence limits, 0.52-0.92). Percentages having surgery and other specified treatments were lower for 80+ year olds than younger cases, although increases in treatment intensity occurred in this age range during 1980-2010, as seen in younger ages, in accordance with guidelines. The study illustrates the important feedback clinical registries can provide to clinicians on care patterns and outcomes in their hospital settings. Feedback can be the subject of local deliberations on how to achieve the best outcomes, including in the elderly by considering the best trade-offs between optimal cancer care and accommodations for co-morbidity and frailty. Clinical registry data can be used in comparative effectiveness research in local settings where there are sufficient case numbers. Refereed/Peer-reviewed

In primary visual cortex (V1), connectivity between layer 2/3 (L2/3) excitatory neurons undergoes extensive reorganization after the onset of visual experience whereby neurons with similar feature selectivity form functional microcircuits (Ko et al., 2011, 2013). It remains unknown whether visual experience is required for the developmental refinement of intracortical circuitry or whether this maturation is guided intrinsically. Here, we correlated the connectivity between V1 L2/3 neurons assayed by simultaneous whole-cell recordings in vitro to their response properties measured by two-photon calcium imaging in vivo in dark-reared mice. We found that neurons with similar responses to oriented gratings or natural movies became preferentially connected in the absence of visual experience. However, the relationship between connectivity and similarity of visual responses to natural movies was not as strong in dark-reared as in normally reared mice. Moreover, dark rearing prevented the normally occurring loss of connections between visually nonresponsive neurons after eye opening (Ko et al., 2013). Therefore, our data suggest that the absence of visual input does not prevent the emergence of functionally specific recurrent connectivity in cortical circuits; however, visual experience is required for complete microcircuit maturation.

The ‘NEET’ population has recently become a national focus for concern. The term refers to young people, aged 16 to 19, who are ‘not in education, employment or training’. The nature of the NEET population is recognised as very diverse, and the size is estimated as between 13.2 per cent to 14.5 per cent of all 16- to 19-year-olds. Routes into the population can be explained through psychological theories such as developmental contextualism, the focal model and resiliency theory. Several interventions and preventative strategies are already in place, with varying degrees of success. It is proposed that EPs can play an important role in reducing the NEET population through application of their five core functions.

The cerebellum plays a crucial role in the regulation of locomotion, but how movement is represented at the synaptic level is not known. Here, we use in vivo patch-clamp recordings to show that locomotion can be directly read out from mossy fiber synaptic input and spike output in single granule cells. The increase in granule cell spiking during locomotion is enhanced by glutamate spillover currents recruited during movement. Surprisingly, the entire step sequence can be predicted from input EPSCs and output spikes of a single granule cell, suggesting that a robust gait code is present already at the cerebellar input layer and transmitted via the granule cell pathway to downstream Purkinje cells. Thus, synaptic input delivers remarkably rich information to single neurons during locomotion. DOI: http://dx.doi.org/10.7554/eLife.07290.001, eLife digest Our voluntary movements, such as shaking hands and walking, are controlled by a region of the brain called the cerebellum. Inside this region is a layer of cells called granule cells, which are the smallest and also the most numerous type of neuron in the brains of mammals. Granule cells receive information from many other parts of the brain and respond by producing electrical signals that influence the motor system, which tells our muscles how to move. However, it is not clear how the granule cells interpret the information they receive and ensure that the right muscles are stimulated at the right time by the motor system. Powell et al. have now used ‘patch-clamp electrodes’ to measure the electrical activity of individual granule cells in the cerebellum of mice, both at rest and as they walked. This is a powerful approach as it enables the recording of both the information received by each granule cell (input) and the electrical signals produced by it in response (output). Each mouse was placed on a treadmill with its head held still and given the choice to either rest or walk. These experiments show that when the mouse is resting, the granule cells are mostly inactive, producing only very low levels of fast electrical signals called ‘spikes’. When the mouse starts walking, the input to the granule cells triggers a strong increase in spiking in the granule cells. Powell et al. used a computer model to understand how the granule cells represent movement. Remarkably, this model could be used to predict walking patterns of the mouse based on the activity of a single granule cell and its inputs. These findings suggest that even single neurons in the cerebellum contain rich information about the movement of the animal. The next challenge is to understand how this code interacts with the rest of the motor system to produce precisely coordinated movements. Furthermore, it will be important to determine whether a similar code is used in other parts of the brain that control movement. DOI: http://dx.doi.org/10.7554/eLife.07290.002

Background: Registry data from four major public hospitals indicate trends in clinical care and survival from colorectal cancer over three decades, from 1980 to 2010. Materials and Methods: Kaplan-Meier product-limit estimates and Cox proportional hazards models were used to investigate disease-specific survival and multiple logistic regression analyses to explore first-round treatment trends. Results: Five-year survivals increased from 48% for 1980-1986 to 63% for 2005-2010 diagnoses. Survival increases applied to each ACPS stage (Australian Clinico-Pathological Stage), and particularly stage C (an increase from 38% to 68%). Risk of death from colorectal cancer halved (hazards ratio: 0.50 (0.45, 0.56)) over the study period after adjusting for age, sex, stage, differentiation, primary sub-site, health administrative region, and measures of socioeconomic status and geographic remoteness. Decreases in stage were not observed. Survivals did not vary by sex or place of residence, suggesting reasonable equity in service access and outcomes. Of staged cases, 91% were treated surgically with lower surgical rates for older ages and more advanced stage. Proportions of surgical cases having adjuvant therapy during primary courses of treatment increased for all stages and were highest for stage C (an increase from 5% in 1980-1986 to 63% for 2005-2010). Radiotherapy was more common for rectal than colonic cases. Proportions of rectal cases receiving radiotherapy increased, particularly for stage C where the increase was from 8% in 1980-1986 to 60% in 2005-2010. The percentage of stage C colorectal cases less than 70 years of age having systemic therapy as part of their first treatment round increased from 3% in 1980-1986 to 81% by 1995-2010. Based on survey data on uptake of adjuvant therapy among those offered this care, it is likely that all these younger patients were offered systemic treatment. Conclusions: We conclude that pronounced increases in survivals from colorectal cancer have occurred at major public hospitals in South Australia due to increases in stage-specific survivals. Use of adjuvant therapies has increased and the patterns of change accord with clinical guideline recommendations. Reasons for sub-optimal use of radiotherapy for rectal cases warrant further investigation, including the potential for limited rural access to impede uptake of treatments at metropolitan-based radiotherapy centres. Refereed/Peer-reviewed

Polychaetes are frequented in toxicological studies, one reason being that some members occupy shallow burrows in sediments and are maximally exposed to the contaminants that accumulate within them. We have been studying one population of the polychaete Nereis (Hediste) diversicolor exhibiting inheritable tolerance to extreme copper contamination in estuarine sediment. Using transcriptome sequencing data we have identified a suite of genes with putative roles in metal detoxification and tolerance, and measured their regulation. Copper tolerant individuals display significantly different gene expression profiles compared to animals from a nearby population living without remarkable copper levels. Gene transcripts encoding principle copper homeostasis proteins including membrane copper ion transporters, copper ion chaperones and putative metallothionein-like proteins were significantly more abundant in tolerant animals occupying contaminated sediment. In contrast, those encoding antioxidants and cellular repair pathways were unchanged. Nontolerant animals living in contaminated sediment showed no difference in copper homeostasis-related gene expression but did have significantly elevated levels of mRNAs encoding Glutathione Peroxidase enzymes. This study represents the first use of functional genomics to investigate the copper tolerance trait in this species and provides insight into the mechanism used by these individuals to survive and flourish in conditions which are lethal to their conspecifics.

The rd1 mouse with a mutation in the Pde6b gene was the first strain of mice identified with a retinal degeneration. However, AAV-mediated gene supplementation of rd1 mice only results in structural preservation of photoreceptors, and restoration of the photoreceptor-mediated a-wave, but not in restoration of the bipolar cell-mediated b-wave. Here we show that a mutation in Gpr179 prevents the full restoration of vision in rd1 mice. Backcrossing rd1 with C57BL6 mice reveals the complete lack of b-wave in a subset of mice, consistent with an autosomal recessive Mendelian inheritance pattern. We identify a mutation in the Gpr179 gene, which encodes for a G-protein coupled receptor localized to the dendrites of ON-bipolar cells. Gene replacement in rd1 mice that are devoid of the mutation in Gpr179 successfully restores the function of both photoreceptors and bipolar cells, which is maintained for up to 13 months. Our discovery may explain the failure of previous gene therapy attempts in rd1 mice, and we propose that Grp179 mutation status should be taken into account in future studies involving rd1 mice., The rd1 mouse is the most widely used model to study retinal degeneration. Here, the authors identify a wide-spread mutation in these mice that may explain the failure of previous gene therapeutic approaches and show that long-lasting restoration of vision is possible in rd1 mice without this mutation.

Current bioavailability models, such as the free ion activity model and biotic ligand model, explicitly consider that metal exposure will be mainly to the dissolved metal in ionic form. With the rise of nanotechnology products and the increasing release of metal-based nanoparticles (NPs) to the environment, such models may increasingly be applied to support risk assessment. It is not immediately clear, however, whether the assumption of metal ion exposure will be relevant for NPs. Using an established approach of oral gluing, a toxicokinetics study was conducted to investigate the routes of silver nanoparticles (AgNPs) and Ag(+) ion uptake in the soil-dwelling earthworm Lumbricus rubellus. The results indicated that a significant part of the Ag uptake in the earthworms is through oral/gut uptake for both Ag(+) ions and NPs. Thus, sealing the mouth reduced Ag uptake by between 40% and 75%. An X-ray analysis of the internal distribution of Ag in transverse sections confirmed the presence of increased Ag concentrations in exposed earthworm tissues. For the AgNPs but not the Ag(+) ions, high concentrations were associated with the gut wall, liver-like chloragogenous tissue, and nephridia, which suggest a pathway for AgNP uptake, detoxification, and excretion via these organs. Overall, the results indicate that Ag in the ionic and NP forms is assimilated and internally distributed in earthworms and that this uptake occurs predominantly via the gut epithelium and less so via the body wall. The importance of oral exposure questions the application of current metal bioavailability models, which implicitly consider that the dominant route of exposure is via the soil solution, for bioavailability assessment and modeling of metal-based NPs.

Optical coherence tomography (OCT) has revolutionises the diagnosis of retinal disease based on the detection of microscopic rather than subcellular changes in retinal anatomy. However, currently the technique is limited to the detection of microscopic rather than subcellular changes in retinal anatomy. However, coherence based imaging is extremely sensitive to both changes in optical contrast and cellular events at the micrometer scale, and can generate subtle changes in the spectral content of the OCT image. Here we test the hypothesis that OCT image speckle (image texture) contains information regarding otherwise unresolvable features such as organelle changes arising in the early stages of neuronal degeneration. Using ultrahigh resolution (UHR) OCT imaging at 800 nm (spectral width 140 nm) we developed a robust method of OCT image analyses, based on spatial wavelet and texture-based parameterisation of the image speckle pattern. For the first time we show that this approach allows the non-invasive detection and quantification of early apoptotic changes in neurons within 30 min of neuronal trauma sufficient to result in apoptosis. We show a positive correlation between immunofluorescent labelling of mitochondria (a potential source of changes in cellular optical contrast) with changes in the texture of the OCT images of cultured neurons. Moreover, similar changes in optical contrast were also seen in the retinal ganglion cell- inner plexiform layer in retinal explants following optic nerve transection. The optical clarity of the explants was maintained throughout in the absence of histologically detectable change. Our data suggest that UHR OCT can be used for the non-invasive quantitative assessment of neuronal health, with a particular application to the assessment of early retinal disease.

BACKGROUND: As new methods for corneal curvature measurement have evolved, users of videokeratscopes need to know the practical limitations of these instruments. We assessed agreement between keratometry and videokeratography in measuring highly astigmatic corneas. METHODS: Two independent examiners made three keratometric and videokeratographic measurements on each of 33 corneas after penetrating keratoplasty. The non-orthogonal keratometric readings obtained with a Zeiss 10 SIVO keratometer (Carl Zeiss Ltd.) were compared to the non-orthogonal si m K readings (maxK, minK) calculated by the algorithms of a TMS-I videokeratoscope (Tomey). Measurement agreement was evaluated for steep and flat meridian power and location, and astigmatism magnitude (D). RESULTS: A systematic bias of the TMS-I in measuring steeper than keratometry for the steep meridian was demonstrated (95% confidence interval: -0.34 to -1.20 D). The limits of agreement (d-2SD to d+2SD) between the two instruments were found to be unacceptable for clinical purposes in measuring steep meridian power (-3.17 to +1.63 D), flat meridian power (-4.92 to +4.48 D) and astigmatism magnitude (-5.84 to +4.87 D). Clinically acceptable differences were observed in identification of steep and flat meridian location. CONCLUSIONS: The Zeiss 10 SL/O keratometer and the TMS-I videokeratoscope showed poor measurement agreement for irregular corneal surfaces, despite the good correlation previously shown between keratometry and videokeratography in calibrated spheres and regular corneas. The TMS-I showed a systematic bias, measuring a greater power in the steeper meridian than the Zeiss 10 SL/O keratometer. It is suggested that the two instruments cannot be used interchangeably in comparing the curvature of corneas after penetrating keratoplasty. [J Refract Surg 1998;14:420-426]

Visualization of cell migration during chemotaxis using spectral domain optical coherence tomography (OCT) requires non-standard processing techniques. Stripe artefacts and camera noise floor present in OCT data prevent detailed computer-assisted reconstruction and quantification of cell locomotion. Furthermore, imaging artefacts lead to unreliable results in automated texture based cell analysis. Here we characterize three pronounced artefacts that become visible when imaging sample structures with high dynamic range, e.g. cultured cells: (i) time-varying fixed-pattern noise; (ii) stripe artefacts generated by background estimation using tomogram averaging; (iii) image modulations due to spectral shaping. We evaluate techniques to minimize the above mentioned artefacts using an 800 nm optical coherence microscope. Effect of artefact reduction is shown exemplarily on two cell cultures, i.e. Dictyostelium on nitrocellulose substrate, and retinal ganglion cells (RGC-5) cultured on a glass coverslip. Retinal imaging also profits from the proposed processing techniques. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Regulator of ubiquitous kinase/Cbl-interacting protein of 85 kDa (Ruk/CIN85) and CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS) comprise a family of vertebrate adaptor proteins involved in several important cellular processes, including downregulation of activated receptor tyrosine kinases, regulation of cytoskeletal rearrangements, phosphatidylinositol 3-kinase (PI 3-kinase) signalling and apoptosis. The role of Ruk/CIN85 as a scaffold protein involved in membrane trafficking processes has been demonstrated in model cell systems. However, intracellular localization of endogenous Ruk/CIN85 has never been comprehensively assessed. We carried out detailed studies of subcellular distribution of Ruk/CIN85 in adherent cultured human cells using antibodies that recognize distinct epitopes of the protein and revealed a punctate immunostaining pattern, common for proteins involved in intracellular trafficking processes. Our data indicate that Ruk/CIN85 is distributed between several different membrane trafficking compartments, but the major pool of Ruk/CIN85 is associated with the Golgi complex, mainly with a subpopulation of COPI-coated vesicles involved in retrograde endoplasmic reticulum-Golgi and intra-Golgi transport. This localization pattern is dependent on the integrity of Golgi complex and intact microtubular network. Only a small pool of Ruk/CIN85 is present in compartments involved in clathrin-mediated endocytosis and sorting. These results suggest that endogenous Ruk/CIN85 may be involved in regulation of specific membrane trafficking processes.

Depolarization-induced Ca2+ influx into rat brain synaptosomes induces dephosphorylation of dephosphin, a 96-94-kDa protein kinase C (PKC) substrate recently identified as dynamin I, a protein associated with endocytosis. We characterized purified dynamin I to better understand regulation of its phosphorylation in nerve terminals. Purified dynamin I possessed a very high affinity for PKC but did not fit Michaelis-Menten kinetics. It had an optimum phosphorylation rate of 1.42 +/- 0.02 mumol/mg/min and a concentration giving half-maximal activity (S0.5) of 0.14 +/- 0.02 microM, the highest affinity reported for a PKC substrate protein. Concentrations of dynamin greater than 0.5 microM inhibited phosphorylation. The stoichiometry was 1.5, indicating more than one phosphorylation site. Dynamin was predominantly associated with the brain particulate fraction under conditions of low ionic strength, and this prevented its phosphorylation by PKC until released by moderate increases in ionic strength (Na+, K+, and Mg2+) or by GTP or ATP. In intact synaptosomes the largest dynamin pool was associated with the particulate fraction, while a smaller pool was cytosolic or extracted with 150 nM NaCl and contained all the phosphorylated protein. Purified dynamin also bound to phospholipid-coated controlled-pore glass beads, but poorly in the presence of NaCl, Mg2+, GTP, or ATP. Ca2+ induced a reversible translocation from the cytosol to the particulate fraction (50% at 183 microM Ca2+) in brain homogenates, and the purified protein also underwent Ca(2+)-sensitive translocation to phospholipid-coated controlled-pore glass beads. We conclude that dynamin I is a nerve terminal Ca(2+)-sensitive phospholipid-binding protein with very high substrate affinity for PKC. We propose that phosphorylation by PKC occurs in the nerve terminal soluble compartment and that Ca2+ may mediate its binding to the particulate fraction, thereby blocking the PKC phosphorylation sites. These properties may contribute to the lack of PKC phosphorylation during depolarization, despite the presence of activated PKC.