ER contact sites define the position and timing of endosome fission.

Endocytic cargo and Rab GTPases are segregated to distinct domains of an endosome. These domains maintain their identity until they undergo fission to traffic cargo. It is not fully understood how segregation of cargo or Rab proteins is maintained along the continuous endosomal membrane or what machinery is required for fission. Endosomes form contact sites with the endoplasmic reticulum (ER) that are maintained during trafficking. Here, we show that stable contacts form between the ER and endosome at constricted sorting domains, and free diffusion of cargo is limited at these positions. We demonstrate that the site of constriction and fission for early and late endosomes is spatially and temporally linked to contact sites with the ER. Lastly, we show that altering ER structure and dynamics reduces the efficiency of endosome fission. Together, these data reveal a surprising role for ER contact in defining the timing and position of endosome fission.

Structure and function of ER membrane contact sites with other organelles.

The endoplasmic reticulum (ER) is the largest organelle in the cell, and its functions have been studied for decades. The past several years have provided novel insights into the existence of distinct domains between the ER and other organelles, known as membrane contact sites (MCSs). At these contact sites, organelle membranes are closely apposed and tethered, but do not fuse. Here, various protein complexes can work in concert to perform specialized functions such as binding, sensing and transferring molecules, as well as engaging in organelle biogenesis and dynamics. This Review describes the structure and functions of MCSs, primarily focusing on contacts of the ER with mitochondria and endosomes.

Real world study of sacituzumab govitecan in metastatic triple-negative breast cancer in the United Kingdom.

BACKGROUND: Treatment options for pre-treated patients with metastatic triple-negative breast cancer (mTNBC) remain limited. This is the first study to assess the real-world safety and efficacy of sacituzumab govitecan (SG) in the UK. METHODS: Data was retrospectively collected from 16 tertiary UK cancer centres. Pts had a diagnosis of mTNBC, received at least two prior lines of treatment (with at least one being in the metastatic setting) and received at least one dose of SG. RESULTS: 132 pts were included. Median age was 56 years (28-91). All patients were ECOG performance status (PS) 0-3 (PS0; 39, PS1; 76, PS2; 16, PS3;1). 75% (99/132) of pts had visceral metastases including 18% (24/132) of pts with CNS disease. Median PFS (mPFS) was 5.2 months (95% CI 4.5-6.6) with a median OS (mOS) of 8.7 months (95% CI 6.8-NA). The most common adverse events (AEs) were fatigue (all grade; 82%, G3/4; 14%), neutropenia (all grade; 55%, G3/4; 29%), diarrhoea (all grade; 58%, G3/4, 15%), and nausea (all grade; 38%, G3/4; 3%). SG dose reduction was required in 54% of pts. CONCLUSION: This study supports significant anti-tumour activity in heavily pre-treated pts with mTNBC. Toxicity data aligns with clinical trial experience.

Lincoln Trail District Health Department's Innovative Shift to Public Health 3.0.

The Lincoln Trail District Health Department's (LTDHD) transformation into the Public Health 3.0 model was applied from frameworks established through public health accreditation standards and innovative strategies. The awareness of strengths and weaknesses discovered through strategic planning and a culture of quality improvement built over time has created numerous performance improvement opportunities. Those opportunities established greater collaboration and transparency between departments. The shift to the Public Health 3.0 and focus on Foundational Public Health Services model made for an easier transition into Kentucky's larger plan for public health transformation. LTDHD continues to provide public health protection by preventing the spread of disease, ensuring the safety of food, air, and water quality, supporting maternal and child health, improving access to clinical care services, and preventing chronic disease and injury.

Evolution of reference materials for the determination of organic nutrients in food and dietary supplements-a critical review.

For over 40 years, food-matrix certified reference materials (CRMs) have been available for determination of trace element content, and a wide variety of materials are available from most producers of CRMs. However, the availability of food-matrix CRMs for organic nutrients has been more limited. The European Commission (EC) Bureau Communautaire de Référence (BCR) and the National Institute of Standards and Technology (NIST) introduced food-matrix CRMs with values assigned for vitamins and other organic nutrients such as fatty acids and carotenoids in the 1990s. The number of organic nutrients for which values were assigned has increased significantly in the past decade, and the approach and analytical methods used for assignment of the certified values have also evolved. Recently, dietary supplement-matrix CRMs such as multivitamin tablets with values assigned for vitamins and carotenoids, and fish and plant oils with values assigned for fatty acids have appeared. The development, evolution, and improvement of food- and dietary supplement-matrix CRMs for determination of vitamins, carotenoids, and fatty acids are described, with emphasis on CRMs made available in the past 10 years. Recent food and dietary supplement CRMs for the determination of organic nutrients include infant formula, multivitamin tablets, milk and egg powders, breakfast cereal, meat homogenate, blueberries, soy flour, fish and plant oils, dry cat food, and protein drink powder. Many of these food- and supplement-matrix CRMs have values assigned for over 80 organic and inorganic nutrients, toxic elements, proximates, and contaminants. The review provides a critical assessment of the challenges and evolving improvements in the production and the analytical methods used for value assignment of these CRMs. The current status and future needs for additional food- and dietary supplement-matrix CRMs for organic nutrients are also discussed. Graphical abstract Food Composition Triangle with currently-available food-matrix certified reference materials (CRMs) for the determination of organic nutrients positioned according to fat, protein, and carbohydrate composition.

Challenges in Developing Analytically Validated Laboratory-Derived Dietary Supplement Databases.

The Dietary Supplement Label Database (DSLD) is sponsored by the Office of Dietary Supplements (ODS) and the National Library of Medicine (NLM). It provides a searchable, free database of the contents of ∼65,000 supplement labels. A companion database of analytically verified product labels [the Dietary Supplement Ingredient Database (DSID)] was created by ODS, NLM, and the USDA. There are considerable challenges to populating both databases, but the DSID faces unique analytic chemistry challenges. This article describes the challenges to creating analytically verified marketplace surveys of dietary supplement (DS) product content claims for inclusion in public databases. Nutritionists and public health scientists require information on actual exposures to DS constituents because labeled content may not match labeled product content. Analytic verification of composition of DSs provides a link to actual exposure. A public database of analytically derived DS content was developed to provide more accurate estimates of dietary intake in population-based epidemiologic studies. The DSID has conducted surveys of several types of vitamin- and mineral-containing DSs. Results showing label content claims as analytically derived values are available in the current DSID. A recent pilot project explored the feasibility of adding botanical DS products to the DSID. Candidates for future botanical DSID studies will be based on sales volume, potential public health impacts, and the availability of validated analytic methods and reference materials. Databases like DSID and the DSLD are essential for researchers and clinicians to evaluate dietary ingredient intakes in population-based epidemiologic studies. Together, these databases provide a picture of the DS marketplace. The DSID provides an analytic survey of marketed DSs. However, selection of future botanical supplements for DSID evaluation involves analytic challenges. Even when appropriate resources are available, method selection and data evaluation are resource- and time-consuming.

Liquid chromatography with absorbance detection and with isotope-dilution mass spectrometry for determination of isoflavones in soy standard reference materials.

Two independent analytical approaches, based on liquid chromatography with absorbance detection and liquid chromatography with mass spectrometric detection, have been developed for determination of isoflavones in soy materials. These two methods yield comparable results for a variety of soy-based foods and dietary supplements. Four Standard Reference Materials (SRMs) have been produced by the National Institute of Standards and Technology to assist the food and dietary supplement community in method validation and have been assigned values for isoflavone content using both methods. These SRMs include SRM 3234 Soy Flour, SRM 3236 Soy Protein Isolate, SRM 3237 Soy Protein Concentrate, and SRM 3238 Soy-Containing Solid Oral Dosage Form. A fifth material, SRM 3235 Soy Milk, was evaluated using the methods and found to be inhomogeneous for isoflavones and unsuitable for value assignment. Graphical Abstract Separation of six isoflavone aglycones and glycosides found in Standard Reference Material (SRM) 3236 Soy Protein Isolate.

Liquid chromatography with isotope-dilution mass spectrometry for determination of water-soluble vitamins in foods.

Vitamins are essential for improving and maintaining human health, and the main source of vitamins is the diet. Measurement of the quantities of water-soluble vitamins in common food materials is important to understand the impact of vitamin intake on human health, and also to provide necessary information for regulators to determine adequate intakes. Liquid chromatography (LC) and mass spectrometry (MS) based methods for water-soluble vitamin analysis are abundant in the literature, but most focus on only fortified foods or dietary supplements or allow determination of only a single vitamin. In this work, a method based on LC/MS and LC/MS/MS has been developed to allow simultaneous quantitation of eight water-soluble vitamins, including multiple forms of vitamins B3 and B6, in a variety of fortified and unfortified food-matrix Standard Reference Materials (SRMs). Optimization of extraction of unbound vitamin forms and confirmation using data from external laboratories ensured accuracy in the assigned values, and addition of stable isotope labeled internal standards for each of the vitamins allowed for increased precision.

Peroral endoscopic myotomy (POEM) leads to similar results in patients with and without prior endoscopic or surgical therapy.

INTRODUCTION: Traditional treatment for the esophageal motility disorder, achalasia, ranges from endoscopic botulinum toxin (Botox) injections or balloon dilatation, to laparoscopic or open surgical myotomy. Recent advances in endoscopic therapy have led to peroral endoscopic myotomy (POEM) as a viable alternative to traditional techniques for myotomy. Uncertainty exists as to whether the procedure is feasible for patients who have already received prior endoscopic or surgical procedures for therapy, as these groups experience higher failure rates as well as intraoperative mucosal perforations and technical difficulty during Heller myotomy. We describe our first 40 patients who have undergone POEM and compare outcomes between patients who have or have not received previous treatment for achalasia. METHODS AND PROCEDURES: We evaluated our prospectively collected database of POEM procedures performed by two surgeons (JLP and JMM) at a single institution. Perioperative data was collected for operative and hospital outcomes. Patients completed pre- and postoperative GERD-Health-Related Quality of Life Questionnaires (GERD-HRQL) and SF-12 surveys for symptom scoring. RESULTS: Forty patients received a POEM procedure between 2011 and 2013. Of these, 40% (n = 16) had had at least one prior endoscopic or surgical procedure. Nine had prior Botox injections, 7 had balloon dilations, 3 had both Botox and dilations, and 3 received prior laparoscopic Heller myotomy (two with Dor fundoplication). Mean operative time was 102 min for patients with prior procedures (Prior Tx) and 118 min for patients without any prior procedure (No Tx) (p = 0.07). Intraoperative complication rates for the Prior Tx group were 12.5 versus 16.7% for the No Tx group. Mean follow-up was 10 months. Both groups independently demonstrated clinical improvement in both the GERD-HRQL and SF-12 scores following POEM. There were no statistical differences between the two groups for GERD-HRQL reflux and dysphagia subset scores, or SF-12 mental component summary. CONCLUSION: We found favorable outcomes following POEM in patients who have had prior endoscopic or surgical treatments for achalasia, as well as for patients without prior intervention. There were no significant differences between these two groups with regards to operative times, GERD-HRQL scores, and mental component SF-12 scores. One complication requiring intervention occurred in a patient that had received multiple prior Botox injections and balloon dilatations. POEM appears to be a viable alternative for treatment of achalasia compared to traditional techniques, however, long-term data are needed to establish the durability of this technique and to determine whether symptoms will recur necessitating re-intervention.

Determination of Isoflavone Content in SRM 3238 Using Liquid Chromatography-Particle Beam/Electron Ionization Mass Spectrometry.

The characterization of marker components in botanical materials is a challenging task, and the increased consumption of botanicals and dietary supplements demands a greater understanding of the associated health benefits and risks. In order to successfully acquire and compare clinical results and correlate health trends, accurate, precise, and validated methods of analysis must be developed. Presented here is the development of a quantitative method for the determination of soy isoflavones (daidzin, glycitin, genistin, daidzein, and genistein) using LC-particle beam/electron ionization-MS (LC-PB/EIMS). An internal standard (IS) approach for quantitation with 7-hydroxy-4- chromone as the IS compound was used, with response factors for each individual isoflavone obtained from calibrant solutions. The results from this method were compared with the certified and reference values for National Institute of Standards and Technology (NIST) SRM 3238 Soy-Containing Solid Oral Dosage Form to demonstrate that the method was in control. Results obtained using LC-PB/EIMS were consistent with the NIST certified or reference values and their uncertainties for all five isoflavones, demonstrating that the LC-PB/EIMS approach is both accurate and precise when used for the determination of the target isoflavones in soy-containing dietary supplement finished products while simultaneously providing structural information.

Health care justice for temporary migrant workers on 457 visas in Australia: a case study of internationally qualified nurses.

Workers and their families in Australia under the Temporary Work (Skilled) Visa (subclass 457) scheme have no access to publicly funded health care. Rather, they are required by the Commonwealth government to purchase costly private health insurance. Our empirical study revealed the serious negative effects of the government's policy on the ability of internationally qualified nurses on 457 visas to meet their basic health care needs and to settle effectively into Australian society This article argues that the current policy is unjust and evaluates three options for reform which would accord more fully with the government's obligations to minimise harm to people's health and to ensure that all people in society have their health care needs met in a fair manner.

Ochratoxin A case study: Establish metrological traceability of mycotoxin measurements using certified reference materials.

For the US Food and Drug Administration's (FDA) mycotoxin program, the desired application of certified reference materials (CRMs) is primarily for validating methods for the determination of mycotoxins in foods and establishing metrological traceability of measurement results generated by such validated methods. The latter has been an important but insufficiently addressed challenge due to the lack of appropriate protocols and CRMs. Taking advantage of two recently available mycotoxin CRMs, OTAN-1 and Standard Reference Material (SRM) 1565, a protocol was developed for systematically examining uncertainty and establishing metrological traceability of measurement results of ochratoxin A (OTA) in corn through a series of calibration operations using the two CRMs. Instrument and method calibrations were performed using OTAN-1 and SRM 1565. The OTA value and its standard and expanded (k = 2, approximately 95% confidence) uncertainties were estimated from the calibration data and documented. These results demonstrate that the major contributing source of uncertainty is the sample matrix, highlighting the important role of the certified matrix reference material in method calibration. The value of OTA (38.5 ± 7.2 µg/g; 95% confidence interval) in the incurred sample was metrologically traceable to the International System of Units through two CRMs using the multi-laboratory validated liquid chromatography-mass spectrometry FDA compendial method. In addition, an alternative estimation of uncertainty was conducted using a one-point calibration, resulting in comparable uncertainty.

Carboplatin AUC 10 for IGCCCG good prognosis metastatic seminoma.

OBJECTIVE: Metastatic seminoma is a highly curable disease. Standard treatment comprises of combination chemotherapy. The short- and long-term toxicities of this treatment are increasingly recognised and the possibility of over treatment in such a curable disease should be considered. We have therefore assessed the use of single agent carboplatin at a dose of AUC 10 in patients with good prognosis metastatic seminoma. MATERIALS AND METHODS: Patients with good prognosis metastatic seminoma treated with carboplatin (AUC 10) were identified at our institution and affiliated institutions. Treatment was three weekly for a total of three or four cycles. Outcome and toxicities were analysed. RESULTS: With a median follow-up of 36 months, 61 patients in total were treated with carboplatin AUC 10, all good prognosis by the IGCCCG criteria. Forty-eight percent had stage IIA/IIB disease and 52% had greater than stage IIB disease. Thirty-one patients (51%) had a complete response following treatment. Three-year survival was 96.3% with a three-year progression free survival of 93.2%. The main treatment toxicity was haematological with 46% having grade 3, 24% having grade 4 neutropenia and 54% experiencing grade 3/4 thrombocytopenia. There were no treatment related deaths. CONCLUSION: Single agent carboplatin at a dose of AUC 10 is an effective treatment for good prognosis metastatic seminoma. The outcome compares favourably to previously published outcomes of combination chemotherapy. Although haematological toxicity is a concern, single agent carboplatin treatment for good prognosis metastatic seminoma could be considered a treatment option and is associated with less toxicity than combination regimens currently used.

Developing qualitative LC-MS methods for characterization of Vaccinium berry Standard Reference Materials.

Standard Reference Materials (SRMs) offer the scientific community a stable and homogenous source of material that holds countless application possibilities. Traditionally, the National Institute of Standards and Technology (NIST) has provided SRMs with associated quantitative information (certified values) for a select group of targeted analytes as measured in a solution or complex matrix. While the current needs of the SRM community are expanding to include non-quantitative data, NIST is attempting to broaden the scope of how and what information is offered to the SRM community by providing qualitative information about biomaterials, such as chromatographic fingerprints and profiles of untargeted identifications. In this work, metabolomic and proteomic profiling efforts were employed to characterize a suite of six Vaccinium berry SRMs. In the discovery phase, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data was matched to mass spectral libraries; a subsequent validation phase based on multiple-reaction monitoring LC-MS/MS relied on both retention time matching of authentic standards along with fragmentation data for a qualitative overview of the most prominent organic compounds present. Definitive and putative identifications were determined for over 70 metabolites based on reporting guidelines set forth by the Metabolomics Standards Initiative (Metabolomics 3(3):211-221, 2007), and the capability of electrospray ionization mass spectrometry (ESI-MS) to profile untargeted metabolites within a complex matrix using mass spectral matching is demonstrated. Bottom-up proteomic analyses were possible using peptide databases translated from expressed sequence tags (ESTs). Homology searches provided identification of novel Vaccinium proteins based on homology to related genera. Chromatographic fingerprints of these berry materials were acquired for supplemental qualitative information to be provided to users of these SRMs. An unbounded set of qualitative data about a biomaterial is a valuable complement to quantitative information traditionally provided in NIST Certificates of Analysis.

Breakfast cereal sampling study for nutritional elements.

The National Institute of Standards and Technology (NIST) has established a Dietary Supplement Laboratory Quality Assurance Program (DSQAP) in collaboration with the National Institutes of Health Office of Dietary Supplements (NIH-ODS). The DSQAP invites laboratories twice annually to participate in interlaboratory studies where participants elect to measure concentrations of nutritional and/or toxic elements as well as active and/or marker compounds. One of these studies was designed to determine the effects of material granularity and sample processing techniques on measurement variability (precision) as well as to provide participating laboratories information on their performance relative to the NIST assigned values (bias) and to the other participants (concordance). Participants were asked to determine the mass fractions of Ca, Fe, and Zn, in mg/kg, in six breakfast cereal samples. Cereal samples consisted of three ground materials (homogenized wheat, wheat, and rice), two flake materials (wheat and rice) and a partially crushed material (a wheat/rice mixture). In general, approximately 25% of the laboratories processed and analyzed the suite of six cereal materials with adequate to exemplary measurement precision. Over half of the laboratories (60%) experienced measurement issues related to only a particular type of cereal matrix or for only a single element. A small number (15%) of laboratories experienced significant sample processing or measurement problems. Future studies planned by the DSQAP may be designed to use commercial products to aid laboratories with their sampling and analytical techniques.

Self expandable metal stents for anastomotic stricture following liver transplant.

BACKGROUND: Endoscopic treatment for biliary strictures with plastic stent placement has been used widely. The use of covered self-expandable metal stents (CSEMS) has been reported in anastomotic strictures post liver transplant. The aim of this study was to evaluate the efficacy of different CSEMS in these subjects. METHODS: A total of 55 patients with anastomotic stricture received CSEMS, which were removed after 3-4 months. There were 19 patients in group A (partially covered SEMS), 21 patients in group B (fully covered SEMS with fins) and 15 patients in group C (fully covered SEMS with flared ends). Technical success, stricture resolution, follows up, and complications were documented. RESULTS: CSEMS were successfully deployed in all 55 cases. There was no evidence of significant difference with regards to stricture resolution (14 [74%] vs. 15 [71%] vs. 9 [60%] p=0.6630, df=2) or complications between groups. Stent-related complications were as follows: three in group A (2 migration, 1 occlusion), five in group B (4 occlusions, 1 migration), and one proximal migration in group C (p=0.3894, df=2). Three cases required surgery (hepatico-jejunostomy) due to refractory strictures. CONCLUSIONS: The observed clinical success rate of CSEMS (70.4%) proved to be below the reported one for multiple plastic stents, while no significant differences between CSEMS types were observed.