Normal Pool Research Articles

Histones Induce the Release of Extracellular Hemoglobin and Red Blood Cell-Derived Microvesicles with Procoagulant Activity

BackgroundHistones are nuclear proteins that can be released extracellularly following cell death or certain mechanisms of cell activation. Infusion of high doses of histones into mice causes lethality. Circulating levels of extracellular histones are elevated in several disease states associated with inflammation and thrombosis. The mechanisms by which extracellular histones induce morbidity and mortality are not fully understood. Histones may activate or be toxic to several cell types in vitro. Most of these activities have been shown to be dependent on histone signaling through toll-like receptors (TLRs). Red blood cells (RBC) do not express TLRs. However, previous studies have reported that histones promote RBC aggregation, increase shear-dependent RBC fragility, reduce deformability under flow and cause hemolytic anemia in mice. Histones also induce calcium influx and promote PS expression on RBC membranes thereby supporting the prothrombinase activity and shortening clotting times in plasma. Histones may insert into cell membranes (independently of TLRs), and form pore-like structures similar to ionophore. The role of this process in histone-induced RBC toxicity has not been fully investigated.ObjectiveWe assessed the ability of individual histones to promote the release of extracellular hemoglobin (Ex-Hb) and RBC-derived microvesicles (RBC-MVs), as well as RBC-MVs' ability to initiate or propagate thrombin generation (TG) in MV-free plasma.MethodsRBCs were isolated from citrated whole blood from healthy volunteers, and were re-suspended in buffer with or without 10uM A23187 (calcium ionophore), recombinant histones H2A, H2B, H3 or H4 for 1 hour at 37°C.To assess histone binding, RBCs were washed and dual-stained with fluorophore-conjugated anti-glycophorin A (GPA) and anti-panhistone antibodies. Stained RBCs were analyzed using imaging flow cytometry, and were considered positive for histone binding if the fluorescence intensity was > 95th percentile of RBCs processed in the absence of histones.RBCs were removed by centrifugation at 2,500g for 15 minutes x2. RBC-MVs present in the supernatant were pelleted by centrifugation at 20,000g for 30 min. Isolated RBC-MVs were then re-suspended in buffer to assess PS exposure using the microparticle activity assay (Hyphen Biomed). RBC-MVs were also re-suspended in re-calcified MV-free plasma supplemented with 4uM 41%PC/44%PE/15%PS lipid vesicles to assess their ability to initiate TG by monitoring the activity of thrombin on a fluorogenic substrate over time. To assess their ability to propagate TG, coagulation was initiated by 1pM tissue factor in MV-free plasma containing histone-induced RBC-MVs without addition of synthetic lipid vesicles. In this condition, phospholipids present on the surface of histone-induced RBC-MVs are the limiting factor for TG.The supernatant collected after pelleting RBC-MVs was used to measure Ex-Hb by absorbance at 540 nm.ResultsThe percentage of RBCs positive for histone binding was 21.6%, 2.3%, 76.9% and 98.1% for H2A, H2B, H3 and H4, respectively. Representative images of RBC staining for GPA and histone H4 is shown in Figure 1A.All histone types induced the release of Ex-Hb (Fig. 1B) and RBC-MVs that possess PS activity (Fig. 1C) in a dose-dependent manner, with the highest PS activity observed with H2A. RBC-MVs released by H2A or H2B clearly initiated TG in MV-free normal pool plasma (Fig.1D). They also significantly enhanced TF-initiated TG in the absence of additional lipid vesicles. The release of Ex-Hb and procoagulant RBC-MVs induced by histones were re-capitulated by ionophore to a greater degree, as expected (Fig.1B-E).ConclusionsAll 4 types of core histones bind to RBCs and induce the release of Ex-Hb. Our findings suggest that despite their lower binding to RBCs, histones H2A and H2B may have a greater ability to induce the release of procoagulant RBC-MVs that can initiate and propagate TG in plasma. The reason for the differential toxicity of histone types on RBCs and the discrepancy between the relative effect of histone-induced RBC-MVs on PS equivalents and TG are unknown. Histone-induced RBC toxicity appears to mimic calcium ionophore, supporting the idea that histones may insert and form functional pores in cell membranes. The contribution of Ex-Hb and procoagulant RBC-MVs to the morbidity and mortality of extracellular histones in vivo requires further studies. [Display omitted] DisclosuresMonroe:Novo Nordisk A/S: Honoraria, Research Funding.

Air-blood barrier thickening and alterations of alveolar epithelial type 2 cells in mouse lungs with disrupted hepcidin/ferroportin regulatory system.

Iron accumulates in the lungs of patients with common respiratory diseases or transfusion-dependent beta-thalassemia. Based on our previous work, we hypothesized that systemic iron overload affects the alveolar region of the lung and in particular the surfactant producing alveolar epithelial type II (AE2) cells. Mice with a point mutation in the iron exporter ferroportin, a model for human hemochromatosis type 4 were compared to wildtype mice (n = 5 each). Lungs were fixed and prepared for light and electron microscopy (EM) according to state-of-the-art protocols to detect subcellular iron localization by scanning EM/EDX and to perform design-based stereology. Iron was detected as electron dense particles in membrane-bound organelles, likely lysosomes, in AE1 cells. AE2 cells were higher in number but had a lower mean volume in mutated mice. Lamellar body volume per AE2 cell was lower but total volume of lamellar bodies in the lung was comparable to wildtype mice. While the volume of alveoli was lower in mutated mice, the volume of alveolar ducts as well as the surface area, volume and the mean thickness and composition of the septa was similar in both genotypes. The thickness of the air-blood barrier was greater in the mutated than in the WT mice. In conclusion, disruption of systemic iron homeostasis affects the ultrastructure of interalveolar septa which is characterized by membrane-bound iron storage in AE1 cells, thickening of the air-blood barrier and hyperplasia and hypotrophy of AE2 cells despite normal total intracellular surfactant pools. The functional relevance of these findings requires further analysis to better understand the impact of iron on intra-alveolar surfactant function.

Thiol-disulfide homoestasis in pregnancies with fetal growth restriction

Aim: To investigate the role of dynamic thiol-disulfide homeostasis in preeclamptic and idiopathic fetal growth restricted (FGR) pregnancies.Material and method: In this prospective case-control study, a total of 110 singleton pregnancies with FGR (study group) (51 preeclamptic and 59 idiopathic FGR’s cases) were compared with 68 healthy pregnant controls at the same gestational weeks (control group). For serum disulfide-thiol homeostasis, a newly used method described by Erel and Neselioglu was used.Results: Serum native thiol and total thiol levels were lower in FGR pregnancies (285.63 ± 55.92 µmol/L, 324.41 ± 44.18 µmol/L, respectively) than control group (324.41 ± 44.18 µmol/L, 362.98 ± 51.43 µmol/L, p < .001, p = .004, respectively). In subgroup analysis, only preeclamptic FGR’s have lower native and total thiol levels (254.41 ± 59.55, 324.41 ± 44.18 µmol/L, respectively) compare to both idiopathic FGR’s and control’s. There was no difference in native and total thiol levels with idiopathic FGR’s with controls. Idiopathic FGR’s have higher levels of disulfide than preeclamptic FGR’s (21.72 ± 17.72 versus 16.80 ± 11.20 µmol/L). The serum albumin and total protein levels were positively and spot urine protein/creatinine ratio, 24-h urine protein levels were negatively correlated with native thiol and total thiol levels.Conclusion: The balance of thiol-disulfide homeostasis was shifted and native and total thiol levels were decreased only in preeclamptic FGR pregnancies. The serum disulfide level was increased in idiopathic FGR pregnancies compare to preeclamptic FGR pregnancies which may be a sign of oxidative stress in idiopathic FGR pregnancies with normal thiol pool.

Representation of multiple objects in macaque category-selective areas

Object recognition in the natural world usually occurs in the presence of multiple surrounding objects, but responses of neurons in inferotemporal (IT) cortex, the large brain area responsible for object recognition, have mostly been studied only to isolated objects. We study rules governing responses to multiple objects by cells in two category-selective regions of macaque IT cortex, the middle lateral face patch (ML) and the middle body patch (MB). We find that responses of single ML and MB cells to pairs of objects can be explained by the widely accepted framework of normalization, with one added ingredient: homogeneous category selectivity of neighboring neurons forming the normalization pool. This rule leads to winner-take-all, contralateral-take-all, or weighted averaging behavior in single cells, depending on the category, spatial configuration, and relative contrast of the two objects. The winner-take-all behavior suggests a potential mechanism for clutter-invariant representation of face and bodies under certain conditions.

Estimating the age of Hb G-Coushatta [β22(B4)Glu→Ala] mutation by haplotypes of β-globin gene cluster in Denizli, Turkey.

BackgroundHb G‐Coushatta variant was reported from various populations’ parts of the world such as Thai, Korea, Algeria, Thailand, China, Japan and Turkey. In our study, we aimed to discuss the possible historical relationships of the Hb G‐Coushatta mutation with the possible migration routes of the world. For this purpose, associated haplotypes were determined using polymorphic loci in the beta globin gene cluster of hemoglobin G‐Coushatta and normal populations in Denizli, Turkey.MethodsWe performed statistical analysis such as haplotype analysis, Hardy–Weinberg equilibrium, measurement of genetic diversity and population differentiation parameters, analysis of molecular variance using F‐statistics, historical‐demographic analyses, mismatch distribution analysis of both populations and applied the test statistics in Arlequin ver. 3.5 software program.ResultsThe diversity of haplotypes has been shown to indicate different genetic origins for two populations. However, AMOVA results, molecular diversity parameters and population demographic expansion times showed that the Hb G‐Coushatta mutation develops on the normal population gene pool. Our estimated τ values showed the average time since the demographic expansion for normal and Hb G‐Coushatta populations ranged from approximately 42,000 to 38,000 ybp, respectively.ConclusionOur data suggest that Hb G‐Coushatta population originate in normal population in Denizli, Turkey. These results support the hypothesis that the multiple origin of Hb G‐Coushatta and indicate that mutation may have been triggered the formation of new variants on beta globin haplotypes.

Porosity prediction: Supervised-learning of thermal history for direct laser deposition

The objective of this study is to investigate the relationship between the melt pool characteristics and the defect occurrence in an as-built additive manufacturing part. One of the major detrimental microstructure properties associated with additive manufacturing (AM) is porosity within final parts. State-of-the-art porosity detection methods focus primarily on post-manufacturing approaches that are susceptible to high cost of process, longer process time, and are incapable of characterizing pores during fabrication. A real-time porosity prediction method is developed using morphological characteristics of the melt pool boundary (i.e., features obtained via functional principal component analysis (FPCA)). A thermal monitoring system is used to capture the time-varying melt pool signal, which are labeled as either pores or normal melt pools by X-ray tomography. Supervised learning methods are utilized to identify the patterns of melt pool images and build a black-box model for the probability distribution of class labels (namely, porosity) based on data characteristics of predictors (e.g., melt pool characteristics). The resultant model does not depend on specific design of specimens with varying material properties; and can be effectively developed as long as thermal-porosity data can be obtained. In the current study, multiple supervised machine learning approaches are used to classify melt pools to predict porosity in a part. Two different accuracy measures are used and numerical experiments show that among the classification approaches used (i.e., Decision Tree (DT), K-Nearest Neighbor (KNN), Support Vector Machine (SVM), Linear Discriminant Analysis (LDA), and Quadratic Discriminant Analysis (QDA)), KNN results in the highest rate of accurately classifying melt pools (98.44%). However, DT results in the lowest rate for incorrectly identifying normal melt pools as pores (0.03%). A comparative study is conducted that compares the performance of supervised learning methods leveraging the proposed morphological model and simple metrics of the melt pool. Numerical experiments show that the morphological model combined with supervised learning techniques vastly outperform the simple melt pool metrics combined with supervised learning techniques (approximately 250% better performance for correctly predicting abnormal melt pools). Our approach may potentially be applied to other AM processes that share similar energy-material interactions (e.g., powder bed fusion, electron beam melting).

Myocardial late gadolinium enhancement: a head-to-head comparison of motion-corrected balanced steady-state free precession with segmented turbo fast low angle shot

To evaluate the image quality and diagnostic agreement with a head-to-head comparison of late gadolinium enhancement (LGE) images acquired by the motion-corrected (MOCO) balanced steady-state free precession (bSSFP) phase sensitivity inversion recovery (PSIR) and conventional segmented fast low angle shot (FLASH) PSIR methods15,16 in a patient cohort with a wide spectrum of cardiovascular disease. In 59 consecutive patients, signal-to-noise ratios (SNRs), contrast-to-noise ratios (CNRs) of the normal myocardium (NM), LGE, and blood pool (BP) were pair-wise compared between the two different sequences. A further semi-qualitative score system (graded 1-4)was used to compare the overall image quality (OIQ). The diagnostic agreement of the two techniques were evaluated by both transmural severity and absolutely quantitative size of LGE. The SNRs of the NM, LGE, and BP of MOCO bSSFP were 4.8±3.4, 53.6±35.6 and 43.2±29.3, compared with 3.9±3.6 (p=0.126), 27.7±18.5 (p<0.001) and 24.3±13.4 (p<0.001) of FLASH LGE, respectively. The CNRs of LGE to NM, LGE to BP, and BP to NM were 48.3±33.1 versus 23.8±16.7 (p<0.001), 6.5±21.6 versus 3.8±10.8 (p<0.001), and 38.3±27.2 versus 20.3±10.7 (p=0.448), respectively. The OIQ of MOCO bSSFP was higher than that of segmented FLASH (median 4 versus median 3, p<0.001). For quantification of LGE size, there is good agreement and high correlation (r=0.992, p<0.001) between the two methods. MOCO bSSFP is a feasible, robust sequence for LGE imaging, especially for patients with arrhythmia and those incapable of breath-holding due to severe heart failure.

Establishment of the World Health Organization First International Standard for Factor XII, Plasma, Human.

Until recently, the role of factor XII (FXII) in hemostasis was not considered to be important since patients with FXII deficiency do not present with bleeding. The activation of FXII by agents including mast cells and platelet polyphosphates suggests that it may have a role in thrombogenesis. The inhibition of FXII therefore presents an option for antithrombotic therapy, and antibodies and inhibitors are already in development. Assays for FXII will be required to support these technologies, and an international standard (IS) for FXII would be useful for the development of these methods and for the clinical monitoring of patients. The purpose of this study was to develop an IS for FXII, with values for functional activity (FXII:C) and antigen (FXII:Ag). Double-spun normal plasma was pooled, filled into siliconized glass ampoules, and freeze-dried to prepare the candidate material. Data from 20 laboratories using the one-stage clotting assay were used to assign the functional activity value in units (u). The antigen value was calculated using data from eight laboratories that carried out antigen assays. Each laboratory was requested to collect two local normal plasma pools. Units of activity and antigen were calculated relative to these pools, as is usual for new coagulation factor analytes. The amount of activity or antigen in 1 ml of normal plasma from each pool was taken to be 1 unit. A total of 566 donors were used across the pools for the FXII:C study and 216 donors for the FXII:Ag study. The overall geometric mean per ampoule for FXII:C was 0.86 u and for FXII:Ag was 0.80 u. The inter-laboratory variation was 10 and 11%, respectively (expressed as the geometric coefficient of variation). Based on these data, the candidate was deemed suitable for use as an IS for FXII. In 2017, the candidate was established by the World Health Organization (WHO) Expert Committee on Biological Standardization as the WHO first IS for blood coagulation FXII, Plasma (National Institute for Biological Standards and Control code 15/180). The values assigned were 0.86 international units (IU) of functional activity (FXII:C) per ampoule and 0.80 IU/ampoule of antigen (FXII:Ag).

Acquired Tracheo-Esophageal Fistula : A Rare Case Report of the Neglected Denture

Introduction: Dentures are common accidental ingested foreign body (FB) especially among the elderly. It is frequent to have foreign body impacted at esophagus in adults however it is very unusual to have Tracheoesophageal fistula (TEF) caused by the denture. The diagnosis of TEF is challenging due to two reasons. Firstly, most of the dental prosthesis is radiolucent and not visible in the routine radiological investigation. Secondly, a patient with a history of swallowed dentures prosthesis may be asymptomatic initially and develops symptoms over time. On the contrary, the prolonged history of FB in the esophagus with TEF has a higher risk of developing serious complications such as pneumonia and lung abscess.Case Presentation: We report a case of a 62-year-old gentleman with a background history of hypertension and temporal lobe epilepsy presented with a history of choking on taking solid and liquid associated with significant weight loss past 2 months. He had lost his denture for almost 1 year during sleep. Endoscopic examination of the larynx showed normal anatomy but pooling of saliva. CT thorax showed a foreign body within a tracheoesophageal fistula. OGDS showed denture within a well-formed tracheoesophageal fistula. He had acquired TEF secondary to the dentures. Conclusion: Symptomatic elderly who lose their denture during sleep should not be neglected. They need immediate medical assessment thus will reduce further debilitating complications. Failing to identify and treat this condition urgently, the patient will suffer acquired trachea-oesophageal fistula on which the treatment is challenging and the morbidity and mortality are high.

Development of Unrecoverable Displacements of the Dam of the Sayano-Shushenskaya Hydroelectric Power Plant While in Use Under Design Load Parameters (1990 – 2016)

Questions regarding the adaption of the dam of the Sayano-Shushenskaya hydroelectric power plant once the designed normal pool level had been reached in 1990 are considered. The impact of natural and technogenic factors on this process at different periods of operation of the structure (prior to repair of the dam and foundation (1990 – 1996), during repair (1996 – 2003), and following repairs (2004 – 2012) are investigated. Causes for amplification of asymmetry in operations between the left-bank and right-bank parts of the dam are established. Data attesting to the existence of a stabilized state of the structure in 2013 – 2016 are presented and conditions that will prevent the development of unrecoverable processes in the near future are formulated.

Differential expression levels of plasma microRNA in Hashimoto's disease

BackgroundThe altered expression of circulating miRNAs has been discovered in many autoimmune diseases (ADs). With rare existing research, it is still unclear in Hashimoto's thyroiditis (HT). We detected plasma miRNA expression of HT patients in this three-stage designed study. MethodsDifferently expressed miRNAs (4 HT pools vs. 1 normal control pool) were identified using quantitative reverse transcription polymerase chain reaction (qRT-PCR) based Exiqon panel (miRCURY-Ready-to-Use- PCR-Human- panel-I+II-V1.M) in the initial discovery stage. These miRNAs were then confirmed in the training stage and further validated in the testing stage using qRT-PCR with 64 (32 HT vs. 32 NCs) and 136 samples (68 HT vs. 68 NCs), respectively. ResultsA total of 10 miRNAs showed differential expression through the training stage. For further validation in the testing stage, expression of 6 miRNAs (miR-205, miR-20a-3p, miR-375, miR-296, miR-451, miR-500a) were consistent with those in the training stage. Combination results showed that these 6 miRNAs were significantly up-regulated in peripheral plasma of HT patients compared with normal controls (P<0.05). In addition, the six-miRNA signature was evaluated to be a potential diagnostic marker of HT. The areas under the receiver operating characteristic curve of the signature were 0.80, 0.75 and 0.69 for the training, testing and the combined stages, respectively. Three miRNAs were associated with TSH levels in HT patients (miR-451, P=0.043; miR-375, P=0.043; miR-500a, P=0.043). Additionally, miR-20a-3p was related with TgAb level (P=0.046). ConclusionsWe identified a miRNA signature including six dysregulated plasma miRNAs which could act as a diagnostic marker in plasma of HT, providing more evidence and better understanding for the association between circulating miRNAs and autoimmune diseases.

Analytical comparison of three different versions of a high-sensitivity cardiac troponin I assay over 10 years

BackgroundConcerns have been raised on the long-term analytical performance of high-sensitivity cardiac troponin (hs-cTn) assays with respect to different reagent formulations, lots and instrumentation. Our goal for the present study was to compare three different versions of an hs-cTnI assay in two different study populations to evaluate if assay re-formulation over 10years has also affected the analytical results. MethodsBeckman Coulter's CE marked hs-cTnI assay (Access hsTnI, 2017) was tested in 100 lithium heparin plasma samples first tested in 2007 with their prototype hs-cTnI assay and in 100 serum samples tested with their enhanced hs-cTnI assay in 2011 with comparison performed by Passing-Bablok regression. The Beckman Coulter hs-cTnI results from 2017 and 2011 from the serum samples were also compared to the Abbott ARCHITECT i1000 hs-cTnI results (2013) with 3-fold differences used to identify possible outliers. Freeze/thaw stability testing (−20°C) was also performed on normal cTnI (Beckman=4.0ng/L; Abbott=5.3ng/L) and high cTnI concentration (Beckman=77.6ng/L; Abbott=126.1ng/L) lithium heparin plasma pools for both hs-cTnI assays. ResultsAfter 3 freeze-thaws the Beckman hs-cTnI assay yielded minor decreases in concentrations (normal pool −0.7ng/L and high pool −12.6ng/L lower). Regression analyses yielded the following relationship between the Beckman hs-cTnI versions: 2017 hs-cTnI=2.0*(2007 prototype hs-cTnI)-5.1ng/L and 2017 hs-cTnI=1.04∗(2011 enhanced hs-cTnI)-2.5ng/L. Compared to the Abbott 2013 hs-cTnI results, the 2011 Beckman enhanced version had 8 results 3-fold higher, with the 2017 Beckman version yielding 6 results 3-fold lower.Conclusions: The 2017 Beckman hs-cTnI version (Access hsTnI) is closely aligned with the previous enhanced hs-cTnI assay and appears to have reduced the frequency of aberrantly high results.

Deletion Mutagenesis and Identification of Causative Mutations in Maize.

We describe a method for gamma-irradiation of mature maize seeds to generate mutants with opaque endosperm and reduced kernel fill phenotypes. We also describe methods for mapping mutants and identifying causal gene mutations. Using this method, a population of 1788M2 families and 47 Mo17×F2s showing stable, segregating, and viable kernel phenotypes was developed. For molecular characterization of the mutants, we utilized a novel functional genomics platform that combines separate Bulked Segregant RNA and exome sequencing data sets (BSREx-seq) to map causative mutations and identify candidate genes within mapping intervals. We also describe the use of exome capture sequencing of F2 mutant and normal pools to perform mapping and candidate gene identification without the need for separate RNA-seq (BSEx-seq). To exemplify the utility of the deletion mutants for functional genomics and provide proof-of-concept for the bioinformatics platform, we summarize the identification of the causative deletion in two mutants. Mutant 937, which was characterized by BSREx-seq, harbors a 6203-bp in-frame deletion covering six exons within the Opaque-1 gene on chromosome 4. Preliminary investigation of opaque mutant 1486 with BSEx-seq shows a tight mapping interval and associated deletion on chromosome 10.

Analytical sensitivity and diagnostic performance of serum protein electrophoresis on the HYDRAGEL 30 PROTEIN(E) β1-β2 Sebia Hydrasys system

BackgroundSerum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are used in the diagnosis and monitoring of plasma cell dyscrasias. IFE is considered the most sensitive method for the detection of monoclonal proteins (M-proteins), but it is not quantitative. The goal of this study was to establish the analytical sensitivity and diagnostic performance of SPE on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) β1-β2. MethodologyPatient sera with a previously identified M-protein (IgG, IgA or IgM) were serially diluted with a normal serum pool and electrophoresed on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) β1-β2. The SPE gels were individually interpreted by five independent observers and IFE was performed on selected samples. Limit of detection was determined as the lowest concentration of M-protein band visible on the gel. SPE diagnostic performance was evaluated against the “gold standard” IFE according CLSI EP12-A2 guidelines. ResultsDetection limit was comparable among all M-proteins migrating in the gamma region, IgG-κ (0.18±0.08g/L; n=6), IgG-λ (0.36±0.25g/L; n=8), IgA-κ (0.40±0.13g/L; n=7), IgA-λ (0.37±0.23g/L; n=4), IgM-κ (0.47±0.20g/L; n=13) and IgM-λ (0.29±0.24g/L; n=6). Percentage agreement with IFE for IgG and IgA in the gamma region ranged from 65% to 100%, whereas IgM migrating in the gamma region and immunoglobulins co-migrating with alpha or beta globulins, showed poor (0–38%) agreement. ConclusionsThis study evaluates the analytical sensitivity and diagnostic performance of SPE on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) β1-β2. There was acceptable agreement between SPE and IFE for IgG-κ/λ and IgA-κ/λ migrating in the gamma region, suggesting that repeating IFE for samples with these isotypes, when the previous IFE and second SPE are both negative, may not be necessary.

Establishment of the World Health Organization 2nd International Standard for Factor XI, Plasma, Human.

The 1st International Standard (IS) for blood coagulation factor XI (FXI), plasma, has been successfully used for potency labeling of FXI therapeutics and for diagnosis of FXI deficiency in patients. With stocks of the 1st IS near depletion, a replacement is required. In addition to the functional activity value, assignment of an antigen value to the 2nd IS would allow harmonization of antigen assay methods and differentiation of patients who have low functional activity but normal antigen FXI levels from patients who have both low functional and antigen FXI levels. The aims of this study were, therefore, to assign FXI functional activity to the 2nd IS for FXI, plasma, and to additionally assign a new analyte, FXI antigen, to the same International Standard. The candidate material was prepared from double-spun, virology negative, normal plasma, which was pooled and filled into siliconized glass ampoules and subsequently freeze-dried. Assignment of the functional activity (FXI:C) value in International Units (IUs) was performed by one-stage clotting assay by 29 laboratories, relative to the 1st IS. The overall geometric mean (GM) was 0.71 IU/amp with extremely low inter-laboratory variability (expressed as geometric coefficient of variation) of 1.8%. The antigen value assignment was performed by 11 laboratories and was calculated relative to normal plasma pools, as is customary with new coagulation factor analytes. The amount of antigen present in 1 ml of normal plasma was taken to be 1 U. The overall GM for the antigen assays was 0.78 IU/amp with an inter-laboratory variation of 10%. The candidate (National Institute for Biological Standards and Control code, 15/180) was established by the World Health Organization (WHO) Expert Committee on Biological Standardization in 2016 as the WHO 2nd IS for blood coagulation FXI, plasma, with a functional activity value (FXI:C) of 0.71 IU/amp and an antigen value (FXI:Ag) of 0.78 IU/amp.

Bug mapping and fitness testing of chemically synthesized chromosome X.

Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in FIP1, and a loxPsym site affecting promoter function of ATP2 PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping.

Intra-image referencing for simplified assessment of HER2-expression in breast cancer metastases using the Affibody molecule ABY-025 with PET and SPECT

PurposeIn phase I/II-studies radiolabelled ABY-025 Affibody molecules identified human epidermal growth factor receptor 2 (HER2) expression in breast cancer metastases using PET and SPECT imaging. Here, we wanted to investigate the utility of a simple intra-image normalization using tumour-to-reference tissue-ratio (T/R) as a HER2 status discrimination strategy to overcome potential issues related to cross-calibration of scanning devices.MethodsTwenty-three women with pre-diagnosed HER2-positive/negative metastasized breast cancer were scanned with [111In]-ABY-025 SPECT/CT (n = 7) or [68Ga]-ABY-025 PET/CT (n = 16). Uptake was measured in all metastases and in normal spleen, lung, liver, muscle, and blood pool. Normal tissue uptake variation and T/R-ratios were established for various time points and for two different doses of injected peptide from a total of 94 whole-body image acquisitions. Immunohistochemistry (IHC) was used to verify HER2 expression in 28 biopsied metastases. T/R-ratios were compared to IHC findings to establish the best reference tissue for each modality and each imaging time-point. The impact of shed HER2 in serum was investigated.ResultsSpleen was the best reference tissue across modalities, followed by blood pool and lung. Spleen-T/R was highly correlated to PET SUV in metastases after 2 h (r = 0.96, P < 0.001) and reached an accuracy of 100% for discriminating IHC HER2-positive and negative metastases at 4 h (PET) and 24 h (SPECT) after injection. In a single case, shed HER2 resulted in intense tracer retention in blood. In the remaining patients shed HER2 was elevated, but without significant impact on ABY-025 biodistribution.ConclusionT/R-ratios using spleen as reference tissue accurately quantify HER2 expression with radiolabelled ABY-025 imaging in breast cancer metastases with SPECT and PET. Tracer binding to shed HER2 in serum might affect quantification in the extreme case.

Effects of climate variation and water levels on reservoir recreation

ABSTRACTBoyer TA, Melstrom RT, Sanders LD. 2017. Effects of climate variation and water levels on reservoir recreation. Lake Reserve Manage. 00:1–11.We combined information on valuation, visitation, and time-varying site quality to measure the effects of climate variation and reservoir water levels on recreational demand at Fort Cobb Reservoir, Oklahoma. A time series model of monthly visitation was estimated to measure the effect of water levels, rainfall, wind speed, and air temperature fluctuations on visitation to the reservoir. The results provide no evidence that visitation is affected by rainfall and wind speed but strong evidence that visitation is affected by water levels and air temperature, even after controlling for seasonality in demand. The results suggest annual reservoir visitation will increase by 2.3% for a 0.5 C (1 F) increase in average annual temperature, but that this increase will vary depending on the month and baseline temperatures; managers can expect an increase of 3.5% to occur in October through May, with essentially no increase in June through September. Visitation increases due to temperatures could potentially be offset with slight changes in water levels away from normal pool heights, however. To illustrate the potential use of these predictions in benefit and damage assessments of environmental change, a nonmarket valuation method was employed to value recreational trips to the reservoir. The results indicate a trip is worth about $60 per person. With explicit values for recreation, lake and reservoir managers may find it easier to gauge the economic benefits of managing lake levels for nonconsumptive uses.

Spatial Distribution of Attentional Modulation at Columnar Resolution in Macaque Area V4.

Attention to a location in a visual scene affects neuronal responses in visual cortical areas in a retinotopically specific manner. Optical imaging studies have revealed that cortical responses consist of two components of different sizes: the stimulus-nonspecific global signal and the stimulus-specific mapping signal (domain activity). It remains unclear whether either or both of these components are modulated by spatial attention. In this study, to determine the spatial distribution of attentional modulation at columnar resolution, we performed cerebral blood volume (CBV)-based optical imaging in area V4 of monkeys performing a color change detection task in which spatial attention was manipulated. We found that spatial attention enhanced global signals of the hemodynamic responses, but did not affect stimulus-selective domain activities. These results indicate the involvement of global signals in neural processing of spatial attention. We propose that global signals reflect the neural substrate of the normalization pool in normalization models of attention.

The Power Function for Representing the Reservoir Rating Curve: Morphological Meaning and Suitability for Dam Breach Modelling

In this paper the authors demonstrate that the simple power function gives a good description of the reservoir morphology. This function proved to be suitable for application on 97 reservoirs taken from three different geographical regions in the world. The power function is suitable to obtain an accurate fitting of the reservoir rating curve using a very limited number of surveyed elevations and volumes or areas. In particular it is shown that two points are enough for a good fitting of the curve, or even only one if volume and surface for an elevation close to normal or maximum pool are both available. The results obtained for dam breach calculations using this equation, have the same quality of those achieved using the elevation-volume table. The paper stresses that the exponent of power equation can be expressed by a formula which has a precise morphological meaning, as it represents the ratio between the volume which the reservoir would have if it were a cylinder with its base area and height equal to the respective maximum values of the actual reservoir, and the real volume of the reservoir.