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In May 2017, The University of Texas Hydrate Pressure Coring Expedition Gulf of Mexico 2-1 (UT-GOM2-1) drilled two adjacent holes in Green Canyon Block 955 in the deep-water Gulf of Mexico as part of The University of Texas at Austin and US Department of Energy Deepwater Methane Hydrate Characterization and Scientific Assessment. Expedition operations included testing two configurations of a rotary pressure-coring tool in a gas hydrate–bearing formation. In the first hole, an extended core barrel (cutting shoe) configuration of the Pressure Coring Tool with Ball Valve (PCTB-CS) was deployed, and in the second hole, the PCTB face bit configuration (PCTB-FB) was deployed. The PCTB-CS successfully recovered and maintained pressure for only one core out of eight deployments. A series of incremental modifications were made during and after the PCTB-CS deployment period that impacted the operations of the subsequent PCTB-FB deployments. Thus, in the second hole, the PCTB-FB successfully recovered and maintained pressure within the hydrate stability zone for 11 cores out of 13 deployments. The PCTB cored gas hydrate–bearing sandy silt interbedded with non–hydrate-bearing clayey silt within the main reservoir. The PCTB also recovered long intervals of unbroken, high-quality core with preserved sedimentary structures. We recovered one pressure core 130 m (437 ft) above the main hydrate reservoir in the silty clay. Pressure coring is the only available technology for recovering intact cores from sediment that is normally disturbed by gas expansion, dissolution, or dissociation; this allows a wide range of scientific measurements to be obtained with minimal disturbance to the core sediment fabric. Analysis of pressure cores has the potential to illuminate the in situ properties, gas saturation, and gas composition of a wide range of reservoirs including unconventional shale systems. more...

The University of Texas Hydrate Pressure Coring Expedition (UT-GOM2-1) recovered cores at near in situ formation pressures from a gas hydrate reservoir composed of sandy silt and clayey silt beds in Green Canyon Block 955 in the deep-water Gulf of Mexico. The expedition results are synthesized and linked to other detailed analyses presented in this volume. Millimeter- to meter-scale beds of sandy silt and clayey silt are interbedded on the levee of a turbidite channel. The hydrate saturation (the volume fraction of the pore space occupied by hydrate) in the sandy silts ranges from 79% to 93%, and there is little to no hydrate in the clayey silt. Gas from the hydrates is composed of nearly pure methane (99.99%) with less than 400 ppm of ethane or heavier hydrocarbons. The δ13C values from the methane are depleted (−60‰ to −65‰ Vienna Peedee belemnite), and it is interpreted that the gases were largely generated by primary microbial methanogenesis but that low concentrations of propane or heavier hydrocarbons record at least trace thermogenic components. The in situ pore-water salinity is very close to that of seawater. This suggests that the excess salinity generated during hydrate formation diffused away because the hydrate formed slowly or because it formed long ago. Because the sandy silt deposits have high hydrate concentration and high intrinsic permeability, they may represent a class of reservoir that can be economically developed. Results from this expedition will inform a new generation of reservoir simulation models that will illuminate how these reservoirs might be best produced. more...

The distinction between nevoid melanoma and a mitotically active nevus can be challenging at the microscopic level. In this study, we performed cytogenetic testing on a cohort of 25 mitotically active melanocytic proliferations resembling common melanocytic nevus from 25 patients. Based on cytogenetic findings, the lesions were classified as "nevoid melanoma" (n = 13) or "mitotically active nevus" (n = 12). Subsequently, we compared the clinicopathological features between these 2 groups. Nevoid melanomas occurred in older patients (P = 0.007); however, there were no significant differences in gender, size, or anatomical distribution between the 2 groups. Histologically, deep/marginal mitoses (P = 0.006), lack of maturation with depth (P = 0.036), and pseudo-maturation (P = 0.006) were significantly more common in nevoid melanomas. Immunohistochemically, complete loss of p16 was an important divisive feature (P = 0.0004), seen in 70% of nevoid melanomas, and highly correlated with loss of CDKN2A gene (chromosome 9p21). Our findings suggest that such reproducible immunomorphological differences can be of value in distinguishing nevoid melanoma from mitotically active nevus. Nevoid melanomas demonstrated a spectrum of chromosomal aberrations similar to those seen in common subtypes of melanoma, which can serve as a powerful adjunct diagnostic tool in morphologically challenging lesions. more...

Increasing evidence points to the presence of low-level de novo T790M mutations in patients with non-small cell lung carcinoma (NSCLC) harboring activating EGFR mutations. We utilized digital PCR (dPCR), a highly sensitive gene mutation detection method, to detect pre-treatment T790M mutations in NSCLC tumor samples and correlated the T790M status with clinical features and patient outcomes. DNA extracted from pre-treatment NSCLC tumor tissue with known activating EGFR mutations, diagnosed between October 2010 and May 2017 at PathWest laboratory, was used to perform targeted dPCR for quantitative detection of T790M mutations. T790M was detected in 42 of 109 pre-treatment samples (38.5%). Median variant allele frequency was 0.14% (range 0.02–28.5%). Overall response rate to first generation EGFR tyrosine kinase inhibitors (TKI) was 67% regardless of T790M status. The median progression free survival was 10.7 (IQR 5.6–19.9) versus 6.7 (IQR 3.5–20.8) months in T790M negative and positive patients respectively. T790M positivity correlated with increased rate of early disease progression. It also correlated with increased mortality (HR 3.1 95%CI 1.2–8.1, p = 0.022) in patients who did not respond to TKI treatment. We detected a significant rate of low-level pre-treatment T790M mutations in NSCLC using highly sensitive dPCR. Low-level pre-treatment T790M did not impact treatment response rate or overall survival, but was associated with increased rate of early progression on TKI therapy. more...

ContextPatient safety is a priority for healthcare organisations worldwide and is a key factor in providing high quality healthcare. Prescribing medications correctly is critical to ensuring safety, especially in the setting of a Paediatric Intensive Care Unit (PICU) where patients are vulnerable to being exposed to incidents due to highly complex care and illness severity. In our 21 bedded PICU, any prescribing errors detected by critical care pharmacists are recorded on a prescribing error database each day (Microsoft Access). Information inputted includes the drug involved in the error, the route of administration, prescriber identifier number, type of error and category of error based on the NCC MERP1classification system. Information is extracted monthly from this database to further populate a prescribing errors dashboard, highlighting the total number of prescribing errors each month and sub-categorising the number of errors according to drug cause and error type.Data collected in 2021 was analysed by our Trust’s Quality Improvement (QI) Team who generated pareto charts for the highest reported prescribing errors according to drug and error type. Although pharmacist data showed that many drugs were responsible for prescribing errors, pareto analysis by the QI team identified that Teicoplanin, Heparin, Fentanyl, Chloral Hydrate and Octenisan® were the drugs associated with the most frequent number of errors and causing the biggest cumulative impact on our prescribing error data. In terms of error type, pareto analysis identified that 80% of our cumulative errors were attributed to the wrong route, wrong dose or missing route of administration.ConclusionA pareto chart is a graph that indicates the frequency of defects as well as their cumulative impact. By applying this statistical control process to PICU prescribing error data for 2021, we were able to identify the drugs and error types responsible for the majority of our cumulative errors. Using the ‘Brilliant Basics’ methodology,2we followed a two-step approach in dissecting our data. For step one, we analysed the data that we had and then in step two, using this analysis, we were able to agree and introduce measures to our prescribing systems in order to mitigate the risk of the errors re-occurring. These measures have included redesigning our PICU prescription to add or adapt prescribing recommendations for Teicoplanin, Heparin and Fentanyl, updating prescribing advice in our PICU electronic drugs formulary for Chloral Hydrate and placing an additional daily task on our nurses’ electronic task list to ensure Octenisan® is used. In terms of error type, we have raised awareness of the prevalence of the errors causing the biggest impact on reported prescribing errors, through the medium of pharmacy newsletters, which are disseminated to all PICU staff and by educating new PICU prescribers as part of their induction to the unit. To assess whether the above changes have contributed to an improvement in our reported errors by drug and type, we will continue to perform statistical analysis on prescribing data collected throughout 2022.ReferencesNational Coordinating Council for Medication Error Reporting and Prevention. 2001. http://www.nccmerp.org/types-medication-errors Accessed 14 June 2022.A Catalyst for Better Health Strategy 2019/22. NHS Business Services Authority (V1) 03 2019. https://www.nhsbsa.nhs.uk/sites/default/files/2019-04/Strategy%202019-22%20%28V1%29%2003.2019.pdf more...

Summary Ki-67 proliferative index (PI) has prognostic and predictive value in invasive breast carcinoma (IBC), but clinical uptake has been hampered by suboptimal accuracy, reproducibility and standardisation. Published guidelines have addressed pre-analytical and analytical factors to improve Ki-67 PI utility; however, practicalities of ongoing monitoring of Ki-67 PI quality in IBC reporting have not been established. We aimed to evaluate the internal and external quality of our established digital Ki-67 PI IBC reporting practice at a tertiary institution. In the 5 years since initial validation work, we've completed a series of internal and external quality assurance (QA) projects: (1) an interobserver agreement study, (2) a two site interlaboratory agreement study, (3) determination of the error of our Ki-67 results, (4) an audit of the year-to-year Ki-67 values, (5) an audit of Ki-67 in neoadjuvant chemotherapy (NAC) treated cases, and (6) comparison of our Ki-67 datasets with similar published datasets. There was excellent concordance (intra-class correlation = 0.98) and good agreement [kappa (κ) = 0.76–0.96] between pathologists, excellent concordance [Pearson correlation (R) = 0.94] and very good agreement (κ = 0.80) between laboratories and excellent concordance (R = 0.92–0.95) and good agreement (κ = 0.67–1.0) over time for our Ki-67 results. No significant difference was observed in Ki-67 data from year-to-year. Expected associations with clinico-pathological prognosticators, pathological complete response following NAC and mitotic index were evident. The median Ki-67 values from the overall and NAC treated datasets were within the range reported in other studies, and our data could be separated into similarly proportioned ‘high’ and ‘low’ Ki-67 PI groups when dichotomised as per protocols in other studies. Collectively, our work provides evidence of adequate internal and external quality control for our digital Ki-67 PI IBC reporting protocols. Given the paucity of formal Ki-67 QA programs, our approach could be emulated, and results compared between laboratories as a framework for internal and external Ki-67 QA. more...

Increasing evidence points to the presence of low-level de novo T790M mutations in patients with non-small cell lung carcinoma (NSCLC) harboring activating EGFR mutations. We utilized digital PCR (dPCR), a highly sensitive gene mutation detection method, to detect pre-treatment T790M mutations in NSCLC tumor samples and correlated the T790M status with clinical features and patient outcomes. DNA extracted from pre-treatment NSCLC tumor tissue with known activating EGFR mutations, diagnosed between October 2010 and May 2017 at PathWest laboratory, was used to perform targeted dPCR for quantitative detection of T790M mutations. T790M was detected in 42 of 109 pre-treatment samples (38.5%). Median variant allele frequency was 0.14% (range 0.02-28.5%). Overall response rate to first generation EGFR tyrosine kinase inhibitors (TKI) was 67% regardless of T790M status. The median progression free survival was 10.7 (IQR 5.6-19.9) versus 6.7 (IQR 3.5-20.8) months in T790M negative and positive patients respectively. T790M positivity correlated with increased rate of early disease progression. It also correlated with increased mortality (HR 3.1 95%CI 1.2-8.1, p = 0.022) in patients who did not respond to TKI treatment. We detected a significant rate of low-level pre-treatment T790M mutations in NSCLC using highly sensitive dPCR. Low-level pre-treatment T790M did not impact treatment response rate or overall survival, but was associated with increased rate of early progression on TKI therapy. more...

Abstract. Objective: Fecal incontinence and constipation are common disorders in childhood. The enteric nervous system and the central nervous system are highly interactive along the brain-gut axis. The interaction is mainly afferent. These afferent pathways include centers that are involved in the central nervous processing of emotions as the mid/posterior insula and the anterior cingulate cortex. A previous study revealed altered processing of emotions in children with fecal incontinence. The present study replicates these results. Methods: In order to analyze the processing of emotions, we compared the event-related potentials of 25 children with fecal incontinence and constipation to those of 15 control children during the presentation of positive, negative, and neutral pictures. Results: Children with fecal incontinence and constipation showed altered processing of emotions, especially in the parietal and central cortical regions. Conclusions: The main study results of the previous study were replicated, increasing the certainty and validity of the findings. more...

Summary Distinction between melanocytic naevi and melanoma occasionally poses a diagnostic challenge in ambiguous cases showing overlapping histological features. Melanomas are characterised by the presence of multiple genomic copy number variants (CNVs), while this is not a feature of naevi. We assessed the feasibility and utility of array-based comparative genomic hybridisation (aCGH) to assess CNVs in melanocytic lesions. DNA was extracted from formalin fixed, paraffin embedded (FFPE) sections of unambiguous naevi ( n = 19) and melanomas ( n = 19). The test DNA and gender mismatched human reference DNA were differentially labelled with fluorophores. Equal quantities of the two DNA samples were mixed and co-hybridised to a SurePrint G3 Human CGH 8x60K array, and digitally scanned to capture and quantify the relative fluorescence intensities. The ratio of the fluorescence intensities was analysed by Cytogenomics software (Agilent). Frequent large CNVs were identified in 94.7% of melanoma samples, including losses of 9p (73.6%), 9q (52.6%), 10q (36.8%), 11q (36.8%), 3p (21%), and 10p (21%), and gains of 6p (42.1%), 7p (42.1%), 1q (36.8%), 8q (31.5%) and 20q (21%). Only one naevus showed two large copy number changes. Overall aCGH showed a specificity and sensitivity of 94.7% in separating naevi from melanomas. Based on our results, aCGH can be successfully used to analyse CNVs of melanocytic lesions utilising FFPE derived biopsy samples, providing a potentially useful adjunctive test for the classification of diagnostically challenging melanocytic proliferations. more...

Invasive lobular carcinoma (ILC) is almost always classified as Nottingham histological grade 2. Despite this, prognosis is markedly varied, with some ILCs behaving more akin to grade 3 invasive ductal carcinoma (IDC). Methods to separate these aggressive ILCs are needed. Digital image analysis (DIA) of the Ki-67 biomarker has potential in this regard; thus, we sought to determine the feasibility of its use for automated evaluation of ILC. An initial pilot study demonstrated no ILC specific changes were required to our Ki-67 DIA algorithm for reproducible results. Subsequently, 42 consecutive cases of ILC were evaluated by visual mitosis counting in HE p more...

Fecal incontinence and constipation are common disorders in childhood. The enteric nervous system and the central nervous system are highly interactive along the brain-gut axis. The interaction is mainly afferent. These afferent pathways include centers that are involved in the central nervous processing of emotions as the mid/posterior insula and the anterior cingulate cortex. A previous study revealed altered processing of emotions in children with fecal incontinence. The present study replicates these results.In order to analyze the processing of emotions, we compared the event-related potentials of 25 children with fecal incontinence and constipation to those of 15 control children during the presentation of positive, negative, and neutral pictures.Children with fecal incontinence and constipation showed altered processing of emotions, especially in the parietal and central cortical regions.The main study results of the previous study were replicated, increasing the certainty and validity of the findings. more...

Mitosis counting in HE stained sections is the most informative constituent of the Nottingham histological grade in breast carcinoma prognosis. Phosphohistone H3 (PHH3) immunohistochemistry (IHC) is a highly specific marker of mitoses, with practical application in identifying mitoses in poorly fixed or distorted tissue and is of prognostic significance in breast carcinoma. Our aim was to assess methods of PHH3 IHC mitosis counting in a tissue microarray (TMA) of 2 mm cores from 36 resected breast carcinomas. Mitoses in HE and PHH3 stained slides were manually scored by pathologist consensus and expressed as counts/2 mm. PHH3 stained cores were also evaluated by automated digital image analysis (DIA). Results were compared using Spearman correlation. A strong and significant correlation was observed between manual PHH3 and manual HE mitotic counts (correlation = 0.81; p 0.0001) and between automated PHH3 DIA and manual HE mitotic counts (correlation = 0.79; p 0.0001). More mitoses were identified with PHH3 IHC than with HE. Manual and DIA PHH3 counts were strongly and significantly correlated (correlation = 0.83; p 0.0001) and of similar absolute values. PHH3 DIA is a valid alternative to manual counting with potential application in breast cancer reporting and prognostication. more...

Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the BRAF p.Val600Glu (V600E) mutation in HCL makes it a valuable molecular diagnostic marker. We compared the ability of Sanger sequencing, fluorescent single-strand conformational polymorphism (F-SSCP) and high resolution melting (HRM) analysis to detect BRAF mutations in 20 cases of HCL consisting of four archival Romanowsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) splenectomy samples and one fresh peripheral blood sample. DNA was amplified and BRAF mutation status determined by the three methods above. V600E mutation was identified in 94%, 89% and 72% of HCL cases by F-SSCP, HRM and Sanger sequencing, respectively. In one case, in addition to the p.Val600Glu mutation, a p.Lys601Thr (K601T) mutation was identified. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity, especially using HRM/F-SSCP. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL and the presence of the mutation may provide a target for therapy. more...

Serosal pathologies including malignant mesothelioma (MM) can show features of osseous and/or cartilaginous differentiation although the mechanism for its formation is unknown. Mesothelial cells have the capacity to differentiate into cells with myofibroblast, smooth muscle and endothelial cell characteristics. Whether they can differentiate into other cell types is unclear. This study tests the hypothesis that mesothelial cells can differentiate into cell lineages of the embryonic mesoderm including osteoblasts and adipocytes. To examine this, a functional assay of bone formation and an adipogenic assay were performed in vitro with primary rat and human mesothelial cells maintained in osteogenic or adipogenic medium (AM) for 0–26 days. Mesothelial cells expressed increasing levels of alkaline phosphatase, an early marker of the osteoblast phenotype, and formed mineralized bone-like nodules. Mesothelial cells also accumulated lipid indicative of a mature adipocyte phenotype when cultured in AM. All cells expressed several key osteoblast and adipocyte markers, including osteoblast-specific runt-related transcription factor 2, and demonstrated changes in mRNA expression consistent with epithelial-to-mesenchymal transition. In conclusion, these studies confirm that mesothelial cells have the capacity to differentiate into osteoblast- and adipocyte-like cells, providing definitive evidence of their multipotential nature. These data strongly support mesothelial cell differentiation as the potential source of different tissue types in MM tumours and other serosal pathologies, and add support for the use of mesothelial cells in regenerative therapies. more...

Prospective surveillance programmes to monitor the incidence of surgical-site infection (SSI) in patients who have had orthopaedic implant surgery can be difficult to implement due to limited human and technical resources. In addition, prolonged patient follow-up, up to one year, may be required. Traditional methods of surveillance can be enhanced by using administrative databases to assist in case finding and facilitate overall surveillance activities. The aim of this study was to identify the incidence of SSI in patients who had undergone total hip replacement (THR) or total knee replacement (TKR) surgery in all Western Australian (WA) hospitals during 1999 using the Western Australian Data Linkage System. The WA Data Linkage System links several population-based administrative health datasets within the state, including the Hospital Morbidity Data System (HMDS), containing International Classification of Disease-coded discharge information, and mortality records. A total of 1476 THR and 1875 TKR procedures was identified from 21 WA hospitals during 1999. The incidence of SSI after these procedures was 5% (95% CI 4.3-5.7) [THR (4.86%, 95% CI 3.77-5.95) and TKR (5.15%, 95% CI 4.15-6.15)]. The incidence was 33.72 infections per 1000 person-years. Patients aged over 80 years experienced a significantly higher rate of infection after THR compared with patients aged 80 or less (z-test, z = 2.56, P = 0.015), but not for TKR. No patients with an SSI died during follow-up. The WA Data Linkage System provided a unique opportunity to review the incidence of SSIs in patients undergoing THR or TKR surgery in WA hospitals. more...

Absorption of non-haeme iron occurs mainly in the duodenum. It involves the divalent metal transporter 1 (DMT1) in the uptake of ferrous Fe(II) iron and the basolateral transporter ferroportin/IREG-1/MTP-1/SLC40A1 in its release. Whether ferroportin functions in this process at other sites in the enterocyte is unknown. In this study the effect of a blocking antibody to ferroportin on the uptake and release of iron was evaluated in enterocyte-like cells (IEC-6 and Caco-2) and in freshly isolated duodenal enterocytes from rats.Uptake of 1 microM Fe(II) and its release by cells was studied in the presence of the antibody. Ferroportin expression was determined by western blot analysis of duodenal mucosa enriched microvillus membranes, Caco-2 cells, IEC-6 cells, and freshly isolated enterocytes. Immunofluorescent detection of ferroporitn was performed on frozen sections of duodenum from rats with variations in body iron stores.Ferroportin was expressed in all cell types. In these cells, the antibody significantly reduced (p0.05) uptake of Fe(II) by 40-50% but had no effect on the release of iron. In Caco-2 cells, Fe(II) uptake was reduced only when the antibody was in contact with the apical membrane. Ferroportin protein was enriched in microvillus membrane preparations. In enterocytes from iron deficient rats, ferroportin was expressed along the brush border where it colocalised with lactase. Ferroportin was seen in the basal cytoplasm and along the basolateral membranes. Iron loading markedly reduced intracellular expression of ferroportin. In Caco-2 cells, ferroportin also localised to the microvillus and lateral and basal membranes.In addition to release, ferroportin functions in the uptake of iron at the apical membrane, possibly by modulating the activity of DMT1. more...

This paper presents the results of two studies investigating the incidence of “double voting” by respondents in multi‐wave mail surveys. Two separate surveys with a total sample in excess of 1300 were conducted. In both cases these were genuine studies, but dealt with innocuous topics. The results appear to indicate that the tendency for respondents to register their opinions more than once is limited. This, however, may not be the case in surveys involving topics of a somewhat controversial nature. more...

Regulation of iron absorption, which is the primary mechanism for maintaining body iron stores, occurs primarily in the proximal small intestine. Recent identification of proteins that are involved in iron absorption such as the uptake transporter-divalent metal transporter (DMT1), the basolateral transporter, IREG1, and the ferroxidase-hephaestin provide new opportunities to study this process. We evaluated the rat intestinal cell line, IEC-6, as a model of intestinal iron transport. This involved measuring the expression of DMT1 and IREG1 by Western blot analysis and confocal microscopy, and hephaestin by protein-dependent copper oxidase activity. DMT1 and IREG1 were expressed in IEC-6 cells. The uptake of 1 micromol/L ferrous iron [Fe(II)]:ascorbate and its efflux also was associated with the expression of DMT1 under different levels of iron loading. The expression of DMT1 changed inversely with iron levels as did the uptake of Fe(II). However, with different levels of cellular iron, IREG1 expression remained constant, as did the release of iron from the cells, suggesting that they could be related. Ceruloplasmin and apotransferrin did not enhance the rate or extent of iron release. Copper oxidase activity, considered to indicate hephaestin activity, was detected only intracellularly. Confocal microscopy showed DMT1 and IREG1 on the cell membrane of IEC-6 cells at 4 degrees C but at intracellular locations at 37 degrees C, indicating that these proteins can function at the cell membrane and intracellularly. In terms of iron absorption, IEC-6 cells have a villous enterocyte phenotype and are regulated by iron stores as occurs in vivo; therefore, they represent an appropriate cell model with which to study this process. more...

The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications. more...

Regulation of iron absorption is thought to be mediated by the amount of iron taken up by duodenal crypt cells via the transferrin receptor (TfR)-transferrin cycle and the activity of the divalent metal transporter (DMT1), although DMT1 cannot be detected morphologically in crypt cells. We investigated the uptake of transferrin-bound iron by duodenal enterocytes in Wistar rats fed different levels of iron and Belgrade (b/b) rats in which iron uptake by the transferrin cycle is defective because of a mutation in DMT1. We showed that DMT1 in our colony of b/b rats contains the G185R mutation, which in enterocytes results in reduced cellular iron content and increased DMT1 gene expression similar to levels in iron deficiency of normal rats. In all groups the uptake of transferrin-bound iron by crypt cells was directly proportional to plasma iron concentration, being highest in iron-loaded Wistar rats and b/b rats. We conclude that the uptake of transferrin-bound iron by developing enterocytes is largely independent of DMT1. more...

BACKGROUND—The mechanism of iron absorption by the intestine and its transfer to the main iron storage site, the liver, is poorly understood. Recently an iron carrier was cloned and named DMT1 (divalent metal transporter 1). AIMS—To determine the level of DMT1 gene expression and protein distribution in duodenum and liver. METHODS—A DMT1 cRNA and antibody were produced and used in in situ hybridisation and immunohistochemistry, respectively, in rats in which the iron stores were altered by feeding diets with normal, low, and high iron content. RESULTS—Duodenal DMT1 mRNA was low in crypts and increased at the crypt-villus junction in iron deficient and control rats; it fell in the iron loaded state. Staining for DMT1 protein was not detected in crypts. In villus enterocytes, protein staining was localised to the microvillus membrane in iron deficiency, in the cytoplasm and to a lesser extent in the membrane in controls, and entirely in the cytoplasm of iron loaded animals. Liver DMT1 mRNA was distributed evenly across hepatocytes. DMT1 protein staining was observed on hepatocyte plasma membranes, with highest values in the iron loaded state, lower values in control animals, and none after iron depletion. CONCLUSIONS—Results are consistent with a role for DMT1 in the transmembrane transport of non-transferrin bound iron from the intestinal lumen and from the portal blood. Keywords: DMT1; iron transporters; iron absorption; duodenum; liver more...

A majority of forced marriage victims are young people who are still at school and teachers and school nurses have a key role in helping to spot the warning signs. Carla Thomas offers her advice and signposts some resources. more...

Purpose: A challenge in ocular preventive medicine is identification of patients with early pathological retinal damage that might benefit from nutritional intervention. The purpose of this study is to evaluate retinal thinning (RT) in early atrophic age-related macular degeneration (AMD) against visual function data from the Zeaxanthin and Visual Function (ZVF) randomized double masked placebo controlled clinical trial (FDA IND #78973). Methods: Retrospective, observational case series of medical center veterans with minimal visible AMD retinopathy (AREDS Report #18 simplified grading 1.4/4.0 bilateral retinopathy). Foveal and extra-foveal four quadrant SDOCT RT measurements were evaluated in n = 54 clinical and ZVF AMD patients. RT by age was determined and compared to the OptoVue SD OCT normative database. RT by quadrant in a subset of n = 29 ZVF patients was correlated with contrast sensitivity and parafoveal blue cone increment thresholds. Results: Foveal RT in AMD patients and non-AMD patients was preserved with age. Extrafoveal regions, however, showed significant slope differences between AMD patients and non-AMD patients, with the superior and nasal quadrants most vulnerable to retinal thinning (sup quad: −5.5 μm/decade thinning vs. Non-AMD: −1.1 μm/decade, P < 0.02; nasal quad: −5.0 μm/decade thinning vs. Non-AMD: −1.0 μm/decade, P < 0.04). Two measures of extrafoveal visual deterioration were correlated: A significant inverse correlation between % RT and contrast sensitivity (r = −0.33, P = 0.01, 2 Tailed Paired T) and an elevated extrafoveal increment blue cone threshold (r = +0.34, P = 0.01, 2 Tailed T). Additional SD OCT RT data for the non-AMD oldest age group (ages 82–91) is needed to fully substantiate the model. Conclusion: A simple new SD OCT clinical metric called “% extra-foveal RT” correlates well with functional visual loss in early AMD patients having minimal visible retinopathy. This metric can be used to follow the effect of repleting ocular nutrients, such as zinc, antioxidants, carotenoids, n-3 essential fats, resveratrol and vitamin D. more...

Background: The risk of injury or fatality (driver, passenger or pedestrian) associated with motor vehicle accidents has been determined to increase with age, as a result of age-related declines in vision, motor and cognitive functioning. Elderly drivers with age related macular degeneration are particularly vulnerable to sensory visual impairment when driving at night, as they suffer declines in both Contrast sensitivity (CS) and Glare recovery (GR). Objective: This study evaluates the relationship between carotenoid supplementation, CS and GR and the relationship between driving ability and retinal macular pigmentation. Methods: Self-described driving performance is the basis of this report, with data derived from the Zeaxanthin and Vision Function (ZVF) Study (FDA IND #78,973), a 1 year, n=60, 4 visit, prospective randomized controlled clinical trial (RCT) of predominantly elderly male veterans (74.9 SD 10 y) with mild / moderate Age related Macular Degeneration (AMD). The twenty five question - National Eye Institute Visual Functioning Questionnaire (VFQ-25) v. January 2000, Rand Corporation® includes 3 questions assessing driving performance and were completed at baseline and 1 year after nutritional supplementation with approximately equal daily doses of the retinal macular carotenoids: lutein (9 mg) or zeaxanthin (8 mg). Replicate measures of foveal 1 degree estimated retinal macular pigment optical density were evaluated with the QuantifEye® (ZeaVision, Chesterfield, MO) heterochromic flicker photometer. Results: VFQ25 self-described driving ability was notably associated with baseline pre-supplementation macular pigmentation. Linear regression modeling suggests that self-described ability to safely drive a car was strongly associated with final macular re-pigmentation post supplementation (P=0.02). Moreover, the greatest effect was found with zeaxanthin (ns but P = 0.057 for trend) even though lutein had greater effects than zeaxanthin with respect to CS and GR, suggesting that unique zeaxanthin post-receptorial processes may be at play. Discussion: Carotenoid supplementation and subsequent macular repigmentation improved the self-described driving ability of patients with macular degeneration. Older male drivers with AMD should be encouraged to have their foveal macular MP measured at yearly eye examinations. If low macular pigment is found, these patients should attempt macular re-pigmentation via diet and/or supplementation to improve the sensory aspect of driving. more...

The purpose of this study is to evaluate whether dietary supplementation with the carotenoid zeaxanthin (Zx) raises macula pigment optical density (MPOD) and has unique visual benefits for patients with early atrophic macular degeneration having visual symptoms but lower-risk National Institute of Health/National Eye Institute/Age-Related Eye Disease Study characteristics.This was a 1-year, n = 60 (57 men, 3 women), 4-visit, intention-to-treat, prospective, randomized controlled clinical trial of patients (74.9 years, standard deviation [SD] 10) with mild-to-moderate age-related macular degeneration (AMD) randomly assigned to 1 of 2 dietary supplement carotenoid pigment intervention groups: 8 mg Zx (n = 25) and 8 mg Zx plus 9 mg lutein (L) (n = 25) or 9 mg L ("Faux Placebo," control group, n = 10). Analysis was by Bartlett's test for equal variance, 3-way repeated factors analysis of variance, independent t test (P0.05) for variance and between/within group differences, and post-hoc Scheffé's tests. Estimated foveal heterochromic flicker photometry, 1° macular pigment optical density (MPOD QuantifEye(®)), low- and high-contrast visual acuity, foveal shape discrimination (Retina Foundation of the Southwest), 10° yellow kinetic visual fields (KVF), glare recovery, contrast sensitivity function (CSF), and 6° blue cone ChromaTest(®) color thresholds were obtained serially at 4, 8, and 12 months.Ninety percent of subjects completed ≥ 2 visits with an initial Age-Related Eye Disease Study report #18 retinopathy score of 1.4 (1.0 SD)/4.0 and pill intake compliance of 96% with no adverse effects. There were no intergroup differences in 3 major AMD risk factors: age, smoking, and body mass index as well as disease duration and Visual Function Questionnaire 25 composite score differences. Randomization resulted in equal MPOD variance and MPOD increasing in each of the 3 groups from 0.33 density units (du) (0.17 SD) baseline to 0.51 du (0.18 SD) at 12 m, (P = 0.03), but no between-group differences (Analysis of Variance; P = 0.47). In the Zx group, detailed high-contrast visual acuity improved by 1.5 lines, Retina Foundation of the Southwest shape discrimination sharpened from 0.97 to 0.57 (P = 0.06, 1-tail), and a larger percentage of Zx patients experienced clearing of their KVF central scotomas (P = 0.057). The "Faux Placebo" L group was superior in terms of low-contrast visual acuity, CSF, and glare recovery, whereas Zx showed a trend toward significance.In older male patients with AMD, Zx-induced foveal MPOD elevation mirrored that of L and provided complementary distinct visual benefits by improving foveal cone-based visual parameters, whereas L enhanced those parameters associated with gross detailed rod-based vision, with considerable overlap between the 2 carotenoids. The equally dosed (atypical dietary ratio) Zx plus L group fared worse in terms of raising MPOD, presumably because of duodenal, hepatic-lipoprotein or retinal carotenoid competition. These results make biological sense based on retinal distribution and Zx foveal predominance. more...

Background Lipofuscin is the most consistent and phylogenically constant morphologic marker of cellular aging. Autofluorescence of the A2E fluorophore within retinal pigment epithelial (RPE) lipofuscin affords the opportunity for noninvasive evaluation of age- and disease-related pathophysiological changes in the human retina. It is being used in National Eye Institute/Age-Related Eye Disease Study II to evaluate age-related macular degeneration (AMD) geographic atrophy expansion. Experiments show lipofuscin can be reversed in cell culture and animal models in heart, brain, spinal cord, and retinal tissues, using an array of antioxidants and iron chelators. Methods An 80-year-old man with a gastric resection presented with complaints of unremitting night driving difficulty despite treatment with lutein and omega III fatty acids. Notable parafoveal deposition of retinal lipofuscin by 50° fundus auto-fluorescence (580 nm excitation/660 barrier filters) and concurrent abnormalities in non-Snellen measures of visual function–Contrast Sensitivity Function, 6.5° large field tritan threshold, 10° threshold visual fields, and deficits in the National Institutes of Health/National Eye Institute Visual Function Questionnaire (VFQ) 25 subjective night driving/mental health subscale questionnaire were obtained. The patient was placed on an over-the-counter daily oral polyphenolic mixture containing resveratrol and re-evaluated 5 months later. Results The data reveal improvements in all measures of visual function, subjective improvement in vision and mental functioning on the VFQ 25, and visible clearing of RPE lipofuscin. Conclusion To our knowledge, we believe this to be the first reported human clinical case of lipofuscin reversal in the human eye correlated with measured clinical and subjective improvement in visual and mental function after nutraceutical intervention. more...