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Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disease of the macula that leads to bilateral loss of central vision and is caused by mutations in the TIMP3 gene. However, the mechanisms by which TIMP3 mutations cause SFD are poorly understood. Here, we generated human induced pluripotent stem cell-derived retinal pigmented epithelial (hiPSC-RPE) cells from three SFD patients carrying TIMP3 p.(Ser204Cys) and three non-affected controls to study disease-related structural and functional differences in the RPE. SFD-hiPSC-RPE exhibited characteristic RPE structure and physiology but showed significantly reduced transepithelial electrical resistance associated with enriched expression of cytoskeletal remodelling proteins. SFD-hiPSC-RPE exhibited basolateral accumulation of TIMP3 monomers, despite no change in TIMP3 gene expression. TIMP3 dimers were observed in both SFD and control hiPSC-RPE, suggesting that mutant TIMP3 dimerisation does not drive SFD pathology. Furthermore, mutant TIMP3 retained matrix metalloproteinase activity. Proteomic profiling showed increased expression of ECM proteins, endothelial cell interactions and angiogenesis-related pathways in SFD-hiPSC-RPE. By contrast, there were no changes in VEGF secretion. However, SFD-hiPSC-RPE secreted higher levels of monocyte chemoattractant protein 1, PDGF and angiogenin. Our findings provide a proof-of-concept that SFD patient-derived hiPSC-RPE mimic mature RPE cells and support the hypothesis that excess accumulation of mutant TIMP3, rather than an absence or deficiency of functional TIMP3, drives ECM and angiogenesis-related changes in SFD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Introduction/Background* Endometrial cancer (EC) patients (Pts) with advanced and recurrent disease relapse despite treatment with combination chemotherapy and have a short progression-free survival (PFS). Nintedanib (N) is a potent, orally available triple receptor tyrosine kinase inhibitor targeting VEGFR 1–3, PDGFR α/β, and FGFR 1–3. This study explored the preliminary efficacy of nintedanib in EC. Methodology The primary objective of this placebo-controlled, randomized study was to evaluate efficacy defined by median PFS of concomitant and maintenance N against placebo (P) in combination with chemotherapy. Patients with histologically confirmed stage IIIC2 or IVA & B or relapsed after adjuvant therapy for stage I-III disease; prior surgery; adjuvant chemotherapy; radiation therapy; hormonal therapy in metastatic setting; with measurable/non-measurable disease were permitted. Pts were randomized 1:1 to receive N 200mg or P, twice daily days 2–21 during chemotherapy (six cycles of Carboplatin (AUC5) and paclitaxel (175mg/m2) every 21 days) and continuously in maintenance phase. N/P was continued until disease progression, unacceptable toxicity, or withdrawal of consent. Stratification by stage of disease, prior adjuvant chemotherapy and measurable/non-measurable disease. This is an ENGOT Model A study. Clinical trial information: NCT02730416. Result(s)* 146 of 148 pts were eligible for PFS: 72N/74P; mean age 66yrs; FIGO stage III 18%, IV 42%, recurrent 40%; follow-up 30 mo. N added to chemotherapy did not improve PFS (119 events) as compared to chemotherapy plus P: median for N 8.3 vs. for P 7.2 mo; hazard ratio (HR) adjusted for stratification factors 1.03; 95% confidence interval (CI),[0.71 to 1.48]; p0.879. Median overall survival (85 events) for N 20 vs. for P 22 mo; HR: 1.10; CI: 0.72–1.69; p0.665. Treatment-emerged grade 3–4 adverse events were higher in N vs P arm: liver function tests 13%/0%; diarrhea 12%/6%; neutropenia 21%/14%; asthenia 4%/1%. Patient-reported outcomes will be reported. Conclusion* Addition of nintedanib to chemotherapy did not improve PFS nor OS. This regimen cannot be recommended to undergo further testing in a phase III trial.

Introduction/Background* The primary objective of this multicentre, phase II study was to evaluate the preliminary efficacy of O (3000mg, IV q2W) in combination with D (1500mg, IV, q4W) in relapsed OC patients with the CD73 expression on intraepithelial inflammatory cells in the TME, defined by disease-control rate (DCR) at 16 weeks. Key eligibility criteria include: histologically confirmed OC with CD73 expression; relapsed disease; measurable disease; no prior immunotherapy; mandatory paired biopsy. 25 patients have been enrolled. Treatment was continued until disease progression, unacceptable toxicity, or withdrawal of consent. Primary endpoint is DCR; secondary endpoints include ORR, DoR, PFS, OS, safety and tolerability. Responses are also corelated to CD8 and PD-L1 positive expression in tumours. Assessment of PD-L1 (≥5%) and CD8 (≥5%) were performed by immunohistochemistry on archival tumour specimens. Clinical trial information: NCT03267589 Methodology The primary objective of this multicentre, phase II study was to evaluate the preliminary efficacy of O (3000mg, IV q2W) in combination with D (1500mg, IV, q4W) in relapsed OC patients with the CD73 expression on intraepithelial inflammatory cells in the TME, defined by disease-control rate (DCR) at 16 weeks. Key eligibility criteria include: histologically confirmed OC with CD73 expression; relapsed disease; measurable disease; no prior immunotherapy; mandatory paired biopsy. 25 patients have been enrolled. Treatment was continued until disease progression, unacceptable toxicity, or withdrawal of consent. Primary endpoint is DCR; secondary endpoints include ORR, DoR, PFS, OS, safety and tolerability. Responses are also corelated to CD8 and PD-L1 positive expression in tumours. Assessment of PD-L1 (≥5%) and CD8 (≥5%) were performed by immunohistochemistry on archival tumour specimens. Clinical trial information: NCT03267589 Result(s)* 80% of patients had received ≥2 lines of therapy for relapse. DCR was 27%, median PFS was 2.7months and median OS was 8.4 months. 74% of patients were CD8-positive, 42% were PD-L1-positive, while 37% were both CD8 & PD-L1-positive. Biomarker positivity was not significantly associated with better DCR (p=0.584). TEAE: 1 thromboembolic event (grade 3), one neutropenia (grade 4), one cardiac arrest (grade5). Conclusion* Combination of Oleclumab-durvalumab is feasible and demonstrate modest preliminary activity in relapsed OC. Further biomarker analysis research to predict response is ongoing.

Recent interest in integrated crop-livestock (ICL) systems has prompted numerous investigations to quantify ecosystem service tradeoffs associated with management. However, few investigations have quantified ICL management effects on net global warming potential (GWP), particularly in semiarid regions. Therefore, we determined net GWP for grazed and ungrazed cropland in a long-term ICL study near Mandan, ND USA. Factors evaluated for their contribution to net GWP included carbon dioxide (CO2) emissions associated with production inputs and field operations, methane (CH4) emissions from enteric fermentation by beef cattle, change in soil carbon stocks, and soil-atmosphere CH4 and nitrous oxide (N2O) fluxes. Net GWP was significantly greater for grazed cropland (946 kg CO2equiv. ha-1 yr-1) compared to ungrazed cropland (200 kg CO2equiv. ha-1 yr-1) (P=0.0331). The difference in net GWP between treatments was largely driven by emissions from enteric fermentation (602 kg CO2equiv. ha-1 yr-1). Among other contributing factors, CO2 emissions associated with seed production and field operations were lower under ungrazed cropland (P = 0.0015 and 0.0135, respectively), while soil CH4 uptake was greater under grazed cropland (P = 0.0102). Soil-atmosphere N2O flux from each system negated nearly all the CO2equiv. sink capacity accrued from soil carbon stock change. As both production systems resulted in net greenhouse gas (GHG) emissions to the atmosphere, novel practices that constrain GHG sources and boost GHG sinks under semiarid conditions are recommended.

Early detection and monitoring of cancer progression is key to successful treatment. Therefore, much research is invested in developing technologies, enabling effective and valuable use of non-invasive liquid biopsies. This includes the detection and analysis of circulating tumor cells (CTCs) from blood samples. Recombinant malaria protein VAR2CSA (rVAR2) binds a unique chondroitin sulfate modification present on the vast majority of cancers and thereby holds promise as a near-universal tumor cell-targeting reagent to isolate CTCs from complex blood samples. This study describes a technical approach for optimizing the coupling of rVAR2 to magnetic beads and the development of a CTC isolation platform targeting a range of different cancer cell lines. We investigate both direct and indirect approaches for rVAR2-mediated bead retrieval of cancer cells and conclude that an indirect capture approach is most effective for rVAR2-based cancer cell retrieval.

Simple Summary Circulating tumor cells are cancer cells that have entered blood or lymphatic vessels wherefrom they might get access to distant body parts and form metastases. The presence of cancer cells in a blood sample can be exploited for non-invasive diagnostic purposes. However, as blood consists of a vast number of healthy red and white blood cells the task of identifying the few potential cancer cells in a sample is a technical challenge. In this study we explore strategies for detecting circulating tumor cells after a pre-enrichment through binding to VAR2CSA protein coupled to magnetic beads. We evaluate the performance of a novel workflow that recognizes and detects the cancer cells based on their gene expression and compare this with the more traditional detection strategy using antibodies for cell staining. The highly sensitive assay presented here could potentially provide a novel strategy for early cancer detection. Abstract Analysis of circulating tumor cells (CTCs) from blood samples provides a non-invasive approach for early cancer detection. However, the rarity of CTCs makes it challenging to establish assays with the required sensitivity and specificity. We combine a highly sensitive CTC capture assay exploiting the cancer cell binding recombinant malaria VAR2CSA protein (rVAR2) with the detection of colon-related mRNA transcripts (USH1C and CKMT1A). Cancer cell transcripts are detected by RT-qPCR using proprietary Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) technology. We validate each step of the workflow using colorectal cancer (CRC) cell lines spiked into blood and compare this with antibody-based cell detection. USH1C and CKMT1A are expressed in healthy colon tissue and CRC cell lines, while only low-level expression can be detected in healthy white blood cells (WBCs). The qPCR reaction shows a near-perfect amplification efficiency for all primer targets with minimal interference of WBC cDNA. Spike-in of 10 cancer cells in 3 mL blood can be detected and statistically separated from control blood using the RT-qPCR assay after rVAR2 capture (p < 0.01 for both primer targets, Mann-Whitney test). Our results provide a validated workflow for highly sensitive detection of magnetically enriched cancer cells.

Malaria research has led to the discovery of oncofetal chondroitin sulfate, which appears to be shared between placental trophoblasts and cancer cells and can be detected by the evolutionary refined malaria protein VAR2CSA. Interestingly, using recombinant VAR2CSA to target oncofetal chondroitin sulfate shows promise for novel cancer diagnostics and therapeutics.

We report on the upgraded One Degree Imager (ODI) at the WIYN 3.5 meter telescope at the Kitt Peak Observatory after the focal plane was expanded by an additional seventeen detectors in spring 2015. The now thirty Orthogonal Transfer Array CCD detectors provide a total field of view of 40' x 48' on the sky. The newly added detectors underwent a design revision to mitigate reduced charge transfer efficiency under low light conditions. We discuss the performance of the focal plane and challenges in the photometric calibration of the wide field of view, helped by the addition of telescope baffles. In a parallel project, we upgraded the instrument's three filter arm mechanisms, where a degrading worm-gear mechanism was replaced by a chain drive that is operating faster and with high reliability. Three more filters, a u' band and two narrow band filters were added to the instrument's complement, with two additional narrow band filters currently in procurement (including an H-alpha filter). We review the lessons learned during nearly three years of operating the instrument in the observatory environment and discuss infrastructure upgrades that were driven by ODI's needs., Comment: submitted to SPIE Astronomical Telescopes + Instrumentation conference

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular dystrophy with an estimated prevalence of 1 in 220,000 and an onset of disease around the 4th to 6th decade of life. Similar to age-related macular degeneration (AMD), ophthalmoscopy reveals accumulation of protein/lipid deposits under the retinal pigment epithelium (RPE), referred to as drusen, in the eyes of patients with SFD.SFD is caused by variants in the gene for tissue inhibitor of metalloproteinases-3 (TIMP3), which has been found in drusen-like deposits of SFD patients. TIMP3 is constitutively expressed by RPE cells and, in healthy eyes, resides in Bruch's membrane. Most SFD-associated TIMP3 variants involve the gain or loss of a cysteine residue. This suggests the protein aberrantly forms intermolecular disulphide bonds, resulting in the formation of TIMP3 dimers. It has been demonstrated that SFD-associated TIMP3 variants are more resistant to turnover, which is thought to be a result of dimerisation and thought to explain the accumulation of TIMP3 in drusen-like deposits at the level of Bruch's membrane.An important function of TIMP3 within the outer retina is to regulate the thickness of Bruch's membrane. TIMP3 performs this function by inhibiting the activity of matrix metalloproteinases (MMPs), which have the function of catalysing breakdown of the extracellular matrix. TIMP3 has an additional function to inhibit vascular endothelial growth factor (VEGF) signalling and thereby to inhibit angiogenesis. However, it is unclear whether SFD-associated TIMP3 variant proteins retain these functions. In this review, we discuss the current understanding of the potential mechanisms underlying development of SFD and summarise all known SFD-associated TIMP3 variants.Cell culture models provide an invaluable way to study disease and identify potential treatments. These allow a greater understanding of RPE physiology and pathophysiology, including the ability to study the blood-retinal barrier as well as other RPE functions such as phagocytosis of photoreceptor outer segments. This review describes some examples of such recent in vitro studies and how they might provide new insights into degenerative diseases like SFD.Thus far, most studies on SFD have been performed using ARPE-19 cells or other, less suitable, cell-types. Now, induced pluripotent stem cell (iPSC) technologies allow the possibility to non-invasively collect somatic cells, such as dermal fibroblast cells and reprogram those to produce iPSCs. Subsequent differentiation of iPSCs can generate patient-derived RPE cells that carry the same disease-associated variant as RPE cells in the eyes of the patient. Use of these patient-derived RPE cells in novel cell culture systems should increase our understanding of how SFD and similar macular dystrophies develop.

ABO hemolytic disease occurs among neonates with blood groups A or B delivered to group O women. Extreme neonatal hyperbilirubinemia due to ABO disease has been reported, but its frequency is not well known. We sought to determine the odds of developing severe ABO hemolytic disease in the 13 years since adopting universal bilirubin screening/management in the Intermountain Healthcare system.We conducted a retrospective analysis of neonates born between 2004 and 2016, defining "severe hemolytic disease" as; (1) total serum bilirubin (TSB)25 mg/dL, or (2) hospital readmission for jaundice, or (3) bilirubin encephalopathy. Neonates born to group O (+) mothers were included and considered either; (1) Controls (not at risk for ABO disease because they were group O), (2) Study subjects (at risk for ABO disease because they were group A or B).Of 400,531 live births, 47% were to group O women; 86% of whom were group O (+). Overall, 42,529 (27%) neonates born to group O (+) women had their blood group determined; 29,729 (68%) were O, 10,682 (25%) A, and 3109 (7%) B. Peak TSBs during the first 10 days were higher in group A (11.0 ± 4.2 mg/dL) and B (11.5 ± 4.3) than group O neonates (10.3 ± 4.1). However the relative risks of a TSB ≥25 mg/dL, readmission for jaundice, or kernicterus, were the same in the control vs. study groups.In our health system, severe hemolytic disease in neonates born to group O (+) woman is not more likely in group A or B neonates than in controls (group O). We recognize that in other practices, particularly those who do not have a universal bilirubin screening/management program, ABO hemolytic disease severity might be different than in our system.

Background/aims: human embryonic stem cells (hESCs) are a potential source of cells for treatment of many degenerative diseases, but in culture have a propensity to spontaneously differentiate, possibly due to suboptimal conditions. Culture at low oxygen tensions improves hESC maintenance and regulates carbohydrate metabolism. Hence, a greater understanding of the nutrient requirements of hESCs will allow production of more appropriate culture media. This study aims to investigate the effect of environmental oxygen tension on the amino acid metabolism of hESCs. Methods: the production or depletion of amino acids by hESCs cultured at 5% or 20% oxygen in the presence or absence of FGF2 was measured by reversephase HPLC. Results: atmospheric oxygen, or removal of FGF2 from hESCs cultured at 5% oxygen, perturbed the uptake or release of individual amino acids and the total amino acid turnover compared to hESCs cultured at 5% oxygen. In particular, serine uptake was reduced at 20% oxygen and by removal of FGF2. Conclusions: highly pluripotent hESCs, cultured at 5% oxygen, demonstrate a greater amino acid turnover than hESCs cultured at 20% oxygen, or without FGF2. These data suggest that amino acid turnover could be used as a measure of the self-renewal capacity of hESCs

Summary Background Several drug classes are known to be associated with serious upper gastrointestinal bleeding (UGIB), among others NSAID, low-dose acetylsalicylic acid (ASA), vitamin K antagonists (VKA), clopidogrel and selective serotonin reuptake inhibitors (SSRIs). There are few data on how and to what extent these drugs are reintroduced in patients who have been discharged after a bleeding episode related to any of them. Aim To assess if physicians re-prescribed potential causative drugs after an episode of UGIB and to explore whether drugs with antihaemostatic action (DAHA) are re-prescribed without a gastro-protective agent. Methods By use of the Kaplan–Meyer method, we estimated the time from UGIB to re-prescribing for 3652 cases who were all admitted to hospital with a diagnosis of serious upper gastrointestinal bleeding from 1995 to 2006. Data on drug exposure were retrieved from a Danish prescription database, a recent study on drug-related UGIB, and The National Board of Health in Denmark. Results One-year rates of re-prescribing after UGIB were; 82%, 25%, 43%, 68%, 55%, 71% for SSRIs, NSAID, low-dose ASA, VKA, clopidogrel and dipyridamol, respectively. However, re-prescribing rates without proton pump inhibitors (PPIs) were markedly lower 25%, 3%, 5%, 1%, 17% and 6%, respectively. Non-users of DAHA had a prevalence of PPI use of about 30% a few months after an UGIB. Conclusions Drugs with antihaemostatic action are re-prescribed to a large extent after an episode of upper gastrointestinal bleeding, but usually covered by PPIs. This use of PPI is specific for users of drugs with antihaemostatic action.

Metastasis is the leading cause of cancer-related deaths. The metastatic process requires malignant cells to break out of the primary tumor and travel to distant sites through the vasculatory system. Studies have shown that blood circulating tumor cells (CTCs) can be used for patient management as CTC enumeration provides a non-invasive tool for stratification and monitoring disease status. Furthermore, there is a great interest in fully characterizing this highly metastatic subfraction of the tumor cells. Various assays have been developed to enable magnetic enrichment of CTCs from patient blood. Most depend on antibodies targeting the epithelial marker, EpCAM, and are therefore limited to tumors of epithelial origin. Consequently, there is a need for technologies that enable broad capture of CTCs from all types of cancers. We have recently shown that placental and cancer cells share a unique form of oncofetal chondroitin sulfate (ofCS), not present on normal tissue. We have further shown that ofCS is an acquired trait for cancer cells to enable migration and to potentiate the metastatic potential of cancer cells. Most importantly, we showed that VAR2CSA, a malaria protein involved in placental malaria, binds specifically to ofCS on a wide range of cancer patient-derived cell lines and tissues of both epithelial and mesenchymal origin. Since ofCS seems to be universally expressed across most cancer types and is heavily involved in cancer cell migration, we hypothesized that the recombinant VAR2CSA (rVAR2) protein could enable an efficient isolation of CTCs from blood samples from a broad spectrum of cancer patients. Here, we show that we can use rVAR2-coated magnetic beads to capture ofCS-positive CTCs. The method was validated using blood samples spiked with cancer cells, followed by clinical feasibility testing in diverse types of cancers to demonstrate versatility. We show the presence of CTCs in blood from more than a hundred cancer patients having pancreatic, hepatic, lung, colorectal, melanoma or prostate cancer. CTCs were even found at very early stage disease. No CTCs were detected in samples from healthy subjects. This method not only enabled the efficient isolation of CTC from non-epithelial cancers, but also showed a dramatic increase in CTC capture compared to EpCAM-based CTC isolation. Furthermore, the method allows for downstream single cell analyses as well as for culturing of the CTCs. This method could potentially prove beneficial in cancer diagnosis, patient stratification, monitoring response to therapy, and in future molecular characterization of this highly metastatic subpopulation of cancer cells. Citation Format: Mette Ø. Agerbæk, Sara R. Bang-Christensen, Ming-Hsin Yang, Thomas M. Clausen, Sisse B. Ditlev, Marina A. Pereira, Morten A. Nielsen, Swati Choudhary, Tobias Gustavsson, Poul H. Sorensen, Tim Meyer, David Propper, Jonathan Shamash, Thor G. Theander, Alexandra Aicher, Mads Daugaard, Christopher Heeschen, Ali Salanti. The VAR2CSA malaria protein efficiently retrieves CTCs from a broad spectrum of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4595.

The Idaho National Laboratory (INL) has been at the forefront of developing methods to reduce the cost and schedule of deactivating spent fuel pools (SFP). Several pools have been deactivated at the INL using an underwater approach with divers. These projects provided a basis for the INL cooperation with the Dresden Nuclear Power Station Unit 1 SFP (Exelon Generation Company) deactivation. It represents the first time that a commercial nuclear power plant (NPP) SFP was decommissioned using this underwater coating process. This approach has advantages in many aspects, particularly in reducing airborne contamination and allowing safer, more cost effective deactivation. The INL pioneered underwater coating process was used to decommission three SFPs with a total combined pool volume of over 900,000 gallons. INL provided engineering support and shared project plans to successfully initiate the Dresden project. This report outlines the steps taken by INL and Exelon to decommission SFPs using the underwater coating process. The rationale used to select the underwater coating process and the advantages and disadvantages are described. Special circumstances are also discussed, such as the use of a remotely-operated underwater vehicle to visually and radiologically map the pool areas that were not readily accessible. A larger project, the INTEC-603 SFP in-situ (grouting) deactivation, is reviewed. Several specific areas where special equipment was employed are discussed and a Lessons Learned evaluation is included.

Since the ALMA North America Prototype Antenna was awarded to the Smithsonian Astrophysical Observatory (SAO), SAO and the Academia Sinica Institute of Astronomy & Astrophysics (ASIAA) are working jointly to relocate the antenna to Greenland. This paper shows the status of the antenna retrofit and the work carried out after the recommissioning and subsequent disassembly of the antenna at the VLA has taken place. The next coming months will see the start of the antenna reassembly at Thule Air Base. These activities are expected to last until the fall of 2017 when commissioning should take place. In parallel, design, fabrication and testing of the last components are taking place in Taiwan.

Human embryonic stem cells (hESCs) have the capacity to differentiate into all cell types and thus have great potential for regenerative medicine. hESCs cultured at low oxygen tensions are more pluripotent and display an increased glycolytic rate but how this is regulated is unknown. This study therefore aimed to investigate the regulation of glucose metabolism in hESCs and whether this might impact OCT4 expression. In contrast to the glucose transporter GLUT1, GLUT3 was regulated by environmental oxygen and localised to hESC membranes. Silencing GLUT3 caused a reduction in glucose uptake and lactate production as well as OCT4 expression. GLUT3 and OCT4 expression were correlated suggesting that hESC self-renewal is regulated by the rate of glucose uptake. Surprisingly, PKM2, a rate limiting enzyme of glycolysis displayed a nuclear localisation in hESCs and silencing PKM2 did not alter glucose metabolism suggesting a role other than as a glycolytic enzyme. PKM2 expression was increased in hESCs cultured at 5% oxygen compared to 20% oxygen and silencing PKM2 reduced OCT4 expression highlighting a transcriptional role for PKM2 in hESCs. Together, these data demonstrate two separate mechanisms by which genes regulating glucose uptake and metabolism are involved in the hypoxic support of pluripotency in hESCs.

The traditional sample preparation workflow for mass spectrometry (MS)-based phosphoproteomics is time consuming and usually requires multiple steps, e.g., lysis, protein precipitation, reduction, alkylation, digestion, fractionation, and phosphopeptide enrichment. Each step can introduce chemical artifacts, in vitro protein and peptide modifications, and contaminations. Those often result in sample loss and affect the sensitivity, dynamic range and accuracy of the mass spectrometric analysis. Here we describe a simple and reproducible phosphoproteomics protocol, where lysis, denaturation, reduction, and alkylation are performed in a single step, thus reducing sample loss and increasing reproducibility. Moreover, unlike standard cell lysis procedures the cell harvesting is performed at high temperatures (99 °C) and without detergents and subsequent need for protein precipitation. Phosphopeptides are enriched using TiO2 beads and the orbitrap mass spectrometer is operated in a sensitive mode with higher energy collisional dissociation (HCD).

The real-time measurement of electrically evoked dopamine was established in brain extracellular fluid of freely moving rats. Dopamine was monitored by fast-scan cyclic voltammetry at carbon fiber microelectrodes lowered into the striatum by means of a detachable micromanipulator. A stimulating electrode, previously implanted in the substantia nigra, was used to evoke striatal dopamine efflux. Evoked extracellular dopamine was both current and frequency dependent. When low current intensities (+/-125 microA) and frequencies (10-20 Hz) were applied, detectable levels of dopamine were elicited without a perceptible behavioral response. Reproducible concentrations of extracellular dopamine could be evoked in the same rat for at least 2 months. These concentrations, moreover, were significantly higher in freely moving rats compared with rats anesthetized with Equithesin. Analysis of measured curves for dopamine uptake and release rates revealed that anesthesia inhibits release but does not affect uptake. It is concluded that (a) fast-scan cyclic voltammetry at carbon fiber microelectrodes is a viable technique for the measurement of electrically evoked dopamine in brain extracellular fluid of freely moving rats, (b) it is possible to determine in situ rate constants for dopamine release and uptake from these temporally and spatially resolved measurements of levels of dopamine, and (c) transient changes in extracellular dopamine levels elicited by electrical stimulation are affected by anesthesia.

The effects of phospholipid acyl chain length (nc) and cholesterol on Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. The optimal hydrophobic thickness of the lipid bilayer decreases from nc = 22 to 18 in the presence of 40 mol% cholesterol. Hydrophobic matching as well as specific interactions of cholesterol with the phosphorylation/dephosphorylation reactions is found to be important. A novel regulatory protein has been identified in Na,K-ATPase membrane preparations from the shark (phospholemmanlike protein from shark, PLMS) with significant homology to phospholemman (PLM), the major protein kinase substrate in myocardium. Both are members of the FXYD gene family. Another member of this family is the Na,K-ATPase γ subunit indicating that these proteins may be specific regulators of the Na,K-ATPase. A regulatory mechanism is described in which association/dissociation of PLMS with the Na,K-ATPase is governed by its phosphorylation by protein kinases.

Bubble collapse in a binary solution with simultaneous heat and mass transfer is studied under a nonspherically symmetrical condition. A numerical technique is presented in this paper that is suitable for solving the axisymmetric moving boundary problem. Making use of the boundary-fitted curvilinear coordinate system and the Lagrangian method, this finite difference technique is able to dynamically track the evolving bubble shape and size during the collapsing process through adaptive grid regeneration. The grid moving velocity is incorporated as an integral part of the governing equations. The numerical results demonstrate that the combined effect of zero shear stress condition, the decreasing curvature, and short life span results in the absence of a wake behind a collapsing bubble.

A relatively high level of extracellular ascorbate in the striatum, which is known to modulate impulse flow in striatal neurons, originates primarily from glutamate-containing corticostriatal afferents. Increasing evidence suggests that ascorbate release from these fibers is regulated by a multisynaptic loop that includes gamma-aminobutyric acid (GABA) mechanisms in the substantia nigra. To assess the role that nigral GABA plays in striatal ascorbate release, extracellular ascorbate was monitored voltammetrically in the striatum during infusions of GABA into the substantia nigra pars reticulata (SNr) of awake, unrestrained rats. Compared to vehicle infusions, intranigral GABA lowered striatal ascorbate by50%. In contrast, intranigral application of picrotoxin, a GABA antagonist, had the opposite effect. Neither GABA nor picrotoxin altered striatal 3,4-dihydroxyphenylacetic acid (DOPAC), a major dopamine metabolite. Collectively, these results indicate that intranigral GABA exerts a tonic inhibitory influence on ascorbate release in the striatum.

Species-sensitivity distribution methods assemble single-species toxicity data to predict hazardous concentrations (HCps) affecting a certain percentage (p) of species in a community. The fit of the lognormal model and required number of individual species values were evaluated with 30 published data sets. The increasingly common assumption that a lognormal model best fits these data was not supported. Fifteen data sets failed a formal test of conformity to a lognormal distribution; other distributions often provided better fit to the data than the lognormal distribution. An alternate bootstrap method provided accurate estimates of HCp without the assumption of a specific distribution. Approximate sample sizes producing HC5 estimates with minimal variance ranged from 15 to 55, and had a median of 30 species-sensitivity values. These sample sizes are higher than those suggested in recent regulatory documents. A bootstrap method is recommended that predicts with 95% confidence the concentration affecting 5% or fewer species.

Angiopoietin-1 is a unique growth factor which induces Tie2 receptor autophosphorylation and interaction with signal transduction molecules, GRB2 and p85 subunit of PI 3-kinase, but no detectable mitogenic response. Here we show that PI 3-kinase-dependent activation of Akt and attachment to extracellular matrix are required for angiopoietin-1-mediated endothelial cell survival. Apoptosis of growth factor-deprived cells grown in monolayer was decreased by angiopoietin-1 and correlated with Akt activation. In contrast, angiopoietin-1, bFGF or VEGF failed to protect cells in suspension culture. Ceramide, an intermediate of several apoptotic pathways, interferes with growth factor-mediated Akt activation. Ceramide induced endothelial cell death and abolished angiopoietin-1-mediated activation of Akt and the effect on cell survival. In addition, we found that PI 3-kinase activity is necessary for migration of endothelial cells in response to Angiopoietin-1. A transient activation of MAPK/ERKs was also detected within 10 min after stimulation with angiopoietin-1. In contrast to VEGF-mediated biological effects, inhibition of MAPK/ERKs by PD98059 in endothelial cells did not affect angiopoietin-1 mediated survival or migration. These findings indicate significant differences in intracellular signaling between VEGF and angiopoietin-1 and that PI 3-kinase lipid products are key mediators of the biological effects of angiopoietin-1.

Mercury and other heavy metal poisoning, represent a problem that is more common than most physicians recognize. Such poisoning can also have far reaching import in the diagnosis and understanding of the patient's hormone status, especially in the aging individual. The diagnosis and treatment of mercury poisoning is a major key to the reversal of hormonal dysregulation, often seen in patients of any age. This paper reviews the important clinical connection between hormone dysregulation and mercury toxicity and offers suggestions for diagnosis and treatment.

It is difficult to obtain an accurate and precise assessment of specific nutrient deficiency metabolism. The cases presented here are those of two middle-aged and unrelated women whose completely non-specific symptoms were those seen every day in the offices of physicians. Because of one patient's conviction that she may be suffering from pernicious anemia, a search was made to attempt to prove or disprove it. Although methylmalonic acid was in the normal range, homocysteine was reported slightly increased. There was no urinary homocystine present. The most important discovery in the urinary amino acid profile was an extremely high concentration of ethanolamine The second patient had symptoms that are traditionally considered to be psychologic in character. A single injection of vitamin B12, given elsewhere, had produced temporary relief and suggested further investigation into possible mechanisms involving transmethylation. Both patients were found to have defects in their ability to control the process of transmethylation.

Low O2 tension is beneficial for human embryonic stem cell (hESC) maintenance but the mechanism of regulation is unknown. HIF-2α was found to bind directly to predicted hypoxic response elements (HREs) in the proximal promoter of OCT4, NANOG and SOX2 only in hESCs cultured under hypoxia (5% O2). This binding induced an array of histone modifications associated with gene transcription while a heterochromatic state existed at atmospheric O2. Interestingly, an enhanced euchromatic state was found when hESCs were exposed to hypoxia followed by 72 hours reoxygenation. This was sustained by HIF-2α which enhanced stemness by binding to an oct-sox cis-regulatory element in the NANOG promoter. Thus, these data have uncovered a novel role of HIF-2α as a direct regulator of key transcription factors controlling self-renewal in hESCs but also in the induction of epigenetic modifications ensuring a euchromatic conformation which enhances the regenerative potential of these cells.

The disappearance and reappearance of outer zone energetic electrons during the November 3{endash}4, 1993, magnetic storm is examined utilizing data from the Solar, Anomalous, and Magnetospheric Particle Explorer (SAMPEX), the Global Positioning System (GPS) series, and the Los Alamos National Laboratory (LANL) sensors onboard geosynchronous satellites. The relativistic electron flux drops during the main phase of the magnetic storm in association with the large negative interplanetary B{sub z} and rapid solar wind pressure increase late on November 3. Outer zone electrons with E{gt}3MeV measured by SAMPEX disappear for over 12 hours at the beginning of November 4. This represents a 3 orders of magnitude decrease down to the cosmic ray background of the detector. GPS and LANL sensors show similar effects, confirming that the flux drop of the energetic electrons occurs near the magnetic equator and at all pitch angles. Enhanced electron precipitation was measured by SAMPEX at L{ge}3.5. The outer zone electron fluxes then recover and exceed prestorm levels within one day of the storm onset and the inner boundary of the outer zone moves inward to smaller L ({lt}3). These measurements provide a data set which is examined in detail and used to determine the mechanisms contributing tomore » the loss and recovery of the outer zone electron flux. The loss of the inner part of the outer zone electrons is partly due to the adiabatic effects associated with the decrease of Dst, while the loss of most of the outer part (those electrons initially at L{ge}4.0) is due to either precipitation into the atmosphere or drift to the magnetopause because of the strong compression of the magnetosphere by the solar wind. The recovery of the energetic electron flux is due to the adiabatic effects associated with the increase in Dst, and at lower energies ({lt}0.5MeV) due to rapid radial diffusion driven by the strong magnetic activity during the recovery phase of the storm. (Abstract Truncated)« less

Energy metabolism is intrinsic to cell viability but surprisingly has been little studied in human embryonic stem cells (hESCs). The current study aims to investigate the effect of environmental O2 tension on carbohydrate utilisation of hESCs. Highly pluripotent hESCs cultured at 5% O2 consumed significantly more glucose, less pyruvate and produced more lactate compared to those maintained at 20% O2. Moreover, hESCs cultured at atmospheric O2 levels expressed significantly less OCT4, SOX2 and NANOG than those maintained at 5% O2. To determine whether this difference in metabolism was a reflection of the pluripotent state, hESCs were cultured at 5% O2 in the absence of FGF2 for 16 hours leading to a significant reduction in the expression of SOX2. In addition, these cells consumed less glucose and produced significantly less lactate compared to those cultured in the presence of FGF2. hESCs maintained at 5% O2 were found to consume significantly less O2 than those cultured in the absence of FGF2, or at 20% O2. GLUT1 expression correlated with glucose consumption and using siRNA and chromatin immunoprecipitation was found to be directly regulated by hypoxia inducible factor (HIF)-2α at 5% O2. In conclusion, highly pluripotent cells associated with hypoxic culture consume low levels of O2, high levels of glucose and produce large amounts of lactate, while at atmospheric conditions glucose consumption and lactate production are reduced and there is an increase in oxidative metabolism. These data suggest that environmental O2 regulates energy metabolism and is intrinsic to the self-renewal of hESCs.

Studies using high dose testosterone (T) administration in normal men as a male contraceptive have resulted in azoospermia rates of only 50-70%. Previous studies of T and progestogen combinations have shown comparable rates of azoospermia, but have been uncontrolled or used T in doses less than that associated with maximal suppression of sperm production. We conducted a randomized, placebo-controlled, single blind trial comparing 6 months of T enanthate administration (100 mg, im, weekly) with the same dose of T enanthate in conjunction with the progestogen levonorgestrel (LNG; 500 micrograms, orally, daily) in 36 normal men, aged 20-42 yr (n = 18 in each group). The primary end points were induction of azoospermia or severe oligospermia (3 million sperm/mL). The combination of T plus LNG was much more effective in suppressing sperm production than T alone. Sixty-seven percent of the T plus LNG group (12 of 18) and 33% of the T alone group (6 of 18) achieved azoospermia by 6 months (P = 0.06). Severe oligospermia or azoospermia developed in 94% of the T plus LNG (17 of 18) group compared to 61% of the T alone group (11 of 18; P0.05). T plus LNG also suppressed sperm production more rapidly than T alone. Time to azoospermia was 9.9 +/- 1.0 vs. 15.3 +/- 1.9 weeks in the T plus LNG and T alone groups, respectively (mean +/- SEM; P0.05). Serum high density lipoprotein cholesterol decreased 21.7 +/- 3.6% in men given T plus LNG (P0.05), compared to only a 1.8 +/- 3.8% decrease in men in the T alone group. Average weight gain was 5.3 +/- 0.8 kg in the T plus LNG group and 2.3 +/- 0.9 kg in the T alone group (P0.05). Acne and increase in hemoglobin were similar in the two groups. We conclude that combination hormonal therapy with T plus a progestogen might offer a reversible male contraceptive approach with a more rapid onset of action and more reliable induction of both azoospermia and severe oligospermia than T alone.In Washington State, 36 men aged 20-42 years were randomly allocated to either the group receiving intramuscular injection of 100 mg/week testosterone (T) enanthate alone or to the group receiving the same dose T plus oral 500 mcg/day levonorgestrel (LNG). This 6-month, placebo-controlled, single blind trial aimed to compare the effectiveness of both regimes in suppressing sperm production. The combination of T plus LNG was more likely than T alone to achieve severe oligospermia or azoospermia in 6 months (94% vs. 61%; p .05). It also achieved azoospermia more quickly than T alone (9.9 vs. 15.3 weeks; p .05). Men in the T plus LNG group experienced greater reduction in serum high density lipoprotein than those in the T alone group (21.7% vs. 1.8%; p .05). They also gained more weight than those in the T alone group (5.3 vs. 2.3 kg; p .05). The two groups experienced a similar rate for acne and increase in hemoglobin. The increase in hemoglobin was significant for both groups. In conclusion, T plus LNG treatment over a 6-month period is a more effective and quicker acting method than T alone for suppressing sperm production to levels low enough to prevent pregnancy.

We describe the design and performance of the digital servo and motion control system for the 6-meter diameter parabolic antennas of the Submillimeter Array (SMA) on Mauna Kea, Hawaii. The system is divided into three nested layers operating at a different, appropriate bandwidth. (1) A rack-mounted, real-time Unix system runs the position loop which reads the high resolution azimuth and elevation encoders and sends velocity and acceleration commands at 100 Hz to a custom-designed servo control board (SCB). (2) The microcontroller-based SCB reads the motor axis tachometers and implements the velocity loop by sending torque commands to the motor amplifiers at 558 Hz. (3) The motor amplifiers implement the torque loop by monitoring and sending current to the three-phase brushless drive motors at 20 kHz. The velocity loop uses a traditional proportional-integral-derivative (PID) control algorithm, while the position loop uses only a proportional term and implements a command shaper based on the Gauss error function. Calibration factors and software filters are applied to the tachometer feedback prior to the application of the servo gains in the torque computations. All of these parameters are remotely adjustable in software. The three layers of the control system monitor each other and are capable of shutting down the system safely if a failure or anomaly occurs. The Unix system continuously relays antenna status to the central observatory computer via reflective memory. In each antenna, a Palm Vx hand controller displays system status and allows full local control of the drives in an intuitive touchscreen user interface. It can also be connected outside the cabin for convenience during antenna reconfigurations. Excellent tracking performance (0.3 arcsec rms) is achieved with this system. It has been in reliable operation on 8 antennas for over 10 years and has required minimal maintenance., Comment: Accepted for publication in the Journal for Astronomical Instrumentation, 31 pages, 9 figures