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Objective To determine whether trace amounts of ethylene glycol (EG), diethylene glycol (DEG), or triethylene glycol (TEG) in PEG 3350 are associated with increased blood levels of EG, DEG, or TEG in children receiving daily PEG 3350 therapy. Study design Blood samples were drawn from 9 children who were being treated for constipation with PEG 3350 (6-12 years old) before and every 30 minutes for 3 hours after receiving 17 g of PEG 3350. PEG 3350, tap water, and blood samples from 18 age- and sex-matched controls also were analyzed. Results Baseline blood levels of EG and TEG did not differ between control and treated groups. DEG levels (median [IQR]) were lower in the PEG 3350 group (40.13 ng/mL [36.69, 63.94] vs 92.83 ng/mL [51.06, 128.93], P = .008). After PEG 3350 dose, levels of EG (390.51 ng/mL [326.06, 624.55]) and TEG (2.21 ng/mL [0, 4.5]) peaked at 90 minutes at 1032.81 ng/mL (826.84, 1486.13) (P = .009) and 35.17 ng/mL (15.81, 45.13) (P = .0005), respectively. DEG levels did not significantly change. Standard 17-g doses of PEG 3350 in 8 oz (237 mL) of water resulted in concentrations (mean ± SD) of EG, DEG, and TEG of 1.32 ± 0.23 µg/mL, 0.18 ± 0.03 µg/mL, and 0.12 ± 0.01 µg/mL, respectively. EG, DEG, and TEG levels in public water supply were 0.07 µg/mL, 0.21 µg/mL, and 0.02 µg/mL, respectively. Conclusions Daily PEG 3350 therapy in children was not associated with sustained elevation of EG, DEG, or TEG blood levels over levels in matched controls. Although EG and TEG levels increased after a standard dose of PEG 3350, their peak values remained well below toxic levels.

The uptake of sorbitol and sodium chloride in slabs of green bell pepper (10 × 10 × 2 mm) osmotically dehydrated at different concentrations of sodium chloride (30, 40, and 110 g/L) and sorbitol (7°Bx, 24°Bx, and 41°Bx) at different pressures (4.05, 44.66, and 85.33 kPa) with infusion times ranging from 6 to 60 min for sorbitol and up to 36 min for NaCl was evaluated. Models that describe the uptake of these solutes were developed under different experimental conditions. A completely randomized 3 × 3 factorial design was used to study the effect of the process variables on uptake of solutes for both sorbitol and NaCl. The uptake kinetic of solutes for both sorbitol and NaCl increased to an asymptotic level with both solution concentration and pressure. A negative exponential model adequately fitted the kinetics for both solutes with R 2 = 0.97 and 0.94. The best conditions for the highest increase in total soluble solids during sorbitol solution infusion were 4.05 kPa of pressure and 16 min of immersion. NaCl uptake in peppers followed an exponential trend without reaching the equilibrium conditions. The fitted models were found adequate to estimate that the increase in total soluble solids and NaCl in bell peppers during osmotic process can be used to establish pretreatment conditions in the drying process.

Polarized gastrointestinal epithelial cells form tight junctions that spatially separate apical and basolateral cell membrane domains. These domains harbor functionally distinct proteins that contribute to cellular homeostasis and morphogenesis. Transforming growth factor beta (TGFbeta) is a critical regulator of gastrointestinal epithelial cell growth and differentiation. Functional assays of vectorial TGFbeta signaling and immunofluorescence techniques were used to determine the localization of TGFbeta receptors and ligand secretion in polarizing Caco-2 cells, a colon cancer cell line. Results were compared to the nontransformed MDCK cell line. In both Caco-2 and MDCK cells, addition of TGFbeta1 to the basolateral medium resulted in phosphorylation of Smad2. No phosphorylation was observed when TGFbeta1 was added to the apical chamber, indicating that receptor signaling is localized at the basolateral membrane. In support of this, immunofluorescence and biotinylation assays show receptor localization along the basolateral membrane. Secretion of TGFbeta1 from MDCK and Caco-2 cells into the apical or basolateral medium was measured by ELISA. Interestingly, secretion was exclusively apical in the nontransformed MDCK line and basolateral in transformed Caco-2 cells. Collectively, these results show basolateral domain specificity in localization of the TGFbeta receptor signaling apparatus. These observations have important implications for understanding the biology of TGFbeta in polarized epithelia, including elements of communication between epithelial and mesenchymal layers, and will prove useful in the design of therapeutics that target TGFbeta function.

Missing data, especially when coupled with noncompliance, are a challenge even in the setting of randomized experiments. Although some existing methods can address each complication, it can be difficult to handle both of them simultaneously. This is true in the example of the New York City School Choice Scholarship Program, where both the covariates and the outcomes were sometimes missing, and there was complicated noncompliance. The authors propose a modified general location model to integrate the ideas of missing data techniques and principal stratification and then analyze the same data as in Barnard, Frangakis, Hill, and Rubin (2003) , where a pattern-mixture model was used. Their results are presented and compared with those in Barnard et al.

Atrial fibrillation (AF) is the most common sustained arrhythmia. Previous studies have identified several genetic loci associated with typical AF. We sought to identify common genetic variants underlying lone AF. This condition affects a subset of individuals without overt heart disease and with an increased heritability of AF. We report a meta-analysis of genome-wide association studies conducted using 1,335 individuals with lone AF (cases) and 12,844 unaffected individuals (referents). Cases were obtained from the German AF Network, Heart and Vascular Health Study, the Atherosclerosis Risk in Communities Study, the Cleveland Clinic and Massachusetts General Hospital. We identified an association on chromosome 1q21 to lone AF (rs13376333, adjusted odds ratio = 1.56; P = 6.3 x 10(-12)), and we replicated this association in two independent cohorts with lone AF (overall combined odds ratio = 1.52, 95% CI 1.40-1.64; P = 1.83 x 10(-21)). rs13376333 is intronic to KCNN3, which encodes a potassium channel protein involved in atrial repolarization.

Perspectives on whether the functions of MAS, a G protein-coupled receptor, are beneficial or deleterious in the heart remain controversial. MAS gene knockout reduces coronary vasodilatation leading to ischemic injury. G protein signaling activated by MAS has been implicated in progression of adaptive cardiac hypertrophy to heart failure and fibrosis. In the present study, we observed increased expression of MAS, connective tissue growth factor (CTGF) and collagen genes in failing (HF) human heart samples when compared to non-failing (NF). Expression levels of MAS are correlated with CTGF in HF and NF leading to our hypothesis that MAS controls CTGF production and the ensuing expression of collagen genes. In support of this hypothesis we show that the non-peptide MAS agonist AR234960 increases both mRNA and protein levels of CTGF via ERK1/2 signaling in HEK293-MAS cells and adult human cardiac fibroblasts. MAS-mediated CTGF expression can be specifically blocked by MAS inverse agonist AR244555 and also by MEK1 inhibition. Expression of CTGF gene was essential for MAS-mediated up-regulation of different collagen subtype genes in HEK293-MAS cells and human cardiac fibroblasts. Knockdown of CTGF by RNAi disrupted collagen gene regulation by the MAS-agonist. Our data indicate that CTGF mediates the profibrotic effects of MAS in cardiac fibroblasts. Blocking MAS-CTGF-collagen pathway should be considered for pharmacological intervention for HF.

Espresso coffee samples were freshly prepared with 10% sucrose, 0.0143% sucralose (equivalent in sweetness to 10% sucrose), or unsweetened, each with and without nondairy creamer. A sensory panel rated the intensities of “malty,” “caramel,” “roasty,” and “coffee-like.” The concentrations of flavor chemicals associated with the latter three sensations (Furaneol, 2-ethyl-3,5-dimethyl [EDM] pyrazine, and 2-furfuryl thiol, respectively) were determined by gas chromatography, using solid-phase microextraction sampling of coffee headspace. Furaneol and furfuryl thiol were essentially unaffected by creamer addition, but the more nonpolar EDM pyrazine was greatly reduced. The malty, caramel, roasty, and coffee-like flavor intensities were not significantly affected by creamer addition. This appears to be a case of disconnect between the absence of an odorant and perception. Furaneol, furfuryl thiol, and EDM pyrazine concentrations were unaffected by adding either sweetener. The malty sensation was the same with and without added sweetener. The roasty and coffee-like ratings both decreased to similar extents in the samples with the two added sweeteners. The ratings for caramel were considerably increased, again to a similar extent, by both sweeteners. Since the added sweeteners were both nonvolatile, this is clearly a case where taste affected odor perception.

Blends made of wheat flour and bean meal at various levels of moisture and bran content, were subjected to extrusion cooking by varying temperature and screw speed. Extrudates were analysed for expansion index, bulk density, apparent viscosity, water absorption index and water solubility index using a second-order rotatable central composite design. Excepting water absorption index, functional properties were significantly affected ( p

Mass transfer and physical attributes of red bell pepper were evaluated. Pepper strips were placed into brine of constant concentration at different temperatures (25–55 °C) with immersion times ranging from 15 to 480 min with and without ultrasound treatment. Diffusion coefficients were evaluated for calcium, sodium, citric acid, soluble and total solids, impregnation and water loss. Firmness, kinetic coefficients and color were determined for each treatment. Ultrasound increased the uptake of solutes ( p −10 m 2 /s, 7.26 × 10 −10 m 2 /s and 14.42 × 10 −10 m 2 /s, respectively. These increases may be attributed to increased cell wall permeability, facilitating transport of water and solute, as evidenced by product firmness, where ultrasound treatments had a negative effect on firmness.

Further structure–activity relationships of a novel series of fungal efflux pump inhibitors with respect to potentiation of the activity of fluconazole against strains of C. albicans and C. glabrata over-expressing ABC-type efflux pumps are systematically explored. Rat protein binding and pharmacokinetics of selected analogues are reported.

Some patients classified as having lower-risk (LR)-disease by the International Prognostic Scoring System (IPSS) fare more poorly than predicted. We examined the prognostic utility of IPSS, the MD Anderson LR-Prognostic System (LR-PSS), and the revised IPSS (IPSS-R) in a large cohort of patients classified as having IPSS LR-MDS in the MDS Clinical Research Consortium database. Actual overall survival (OS) was assessed in patients with IPSS LR-MDS (i.e. low and intermediate-1) using Kaplan-Meier methods. Harrell's c index (HCI) and Akaike information criteria (AIC) were used to compare the models. Median OS of 1,140 eligible patients was 47 months (95% CI, 44-52). Median follow-up was 62 months. HCI values indicating the discriminatory power of the models (higher is better) were better for LR-PSS (0.74, 95% CI, 0.70-0.78) than IPSS-R (0.64, 95% CI, 0.60-0.67) and IPSS (0.64, 95% CI, 0.60-0.68). Similarly, AIC values indicating the goodness of the fit were better for LR-PSS than IPSS-R and IPSS (8,110, 8,147, and 8,150, respectively, lower is better). LR-PSS assigned 25.1% and 37.4% of patients with IPSS LR-MDS into LR-PSS Category 3 and IPSS-R Categories ≥Intermediate, respectively. Of 291 patients (25.5%) who survived ≤24 months from diagnosis, only 37.1% and 45% were classified as LR-PSS category 3 and IPSS-R categories ≥Intermediate, respectively (P = 0.06). While both LR-PSS and IPSS-R distinguish groups with varied survival outcome among patients with IPSS LR-MDS, both tools fail to identify a significant subset with poor OS. Future studies should assess whether patients identified as at increased risk will benefit from earlier interventions with disease-modifying therapies.

An association between lower educational attainment (EA) and an increased risk for depression has been confirmed in various western countries. This study examines whether pleiotropic genetic effects contribute to this association. Therefore, data were analyzed from a total of 9662 major depressive disorder (MDD) cases and 14 949 controls (with no lifetime MDD diagnosis) from the Psychiatric Genomics Consortium with additional Dutch and Estonian data. The association of EA and MDD was assessed with logistic regression in 15 138 individuals indicating a significantly negative association in our sample with an odds ratio for MDD 0.78 (0.75-0.82) per standard deviation increase in EA. With data of 884 105 autosomal common single-nucleotide polymorphisms (SNPs), three methods were applied to test for pleiotropy between MDD and EA: (i) genetic profile risk scores (GPRS) derived from training data for EA (independent meta-analysis on similar to 120 000 subjects) and MDD (using a 10-fold leave-one-out procedure in the current sample), (ii) bivariate genomic-relationship-matrix restricted maximum likelihood (GREML) and (iii) SNP effect concordance analysis (SECA). With these methods, we found (i) that the EA-GPRS did not predict MDD status, and MDD-GPRS did not predict EA, (ii) a weak negative genetic correlation with bivariate GREML analyses, but this correlation was not consistently significant, (iii) no evidence for concordance of MDD and EA SNP effects with SECA analysis. To conclude, our study confirms an association of lower EA and MDD risk, but this association was not because of measurable pleiotropic genetic effects, which suggests that environmental factors could be involved, for example, socioeconomic status. Refereed/Peer-reviewed

Objective— Apolipoprotein (apo) E-deficient mice are hypercholesterolemic and develop atherosclerosis on low-fat chow diets; however, the genetic background strain has a large effect on atherosclerosis susceptibility. This study aimed to determine the genetic regions associated with strain effects on lesion area. Methods and Results— We performed a strain intercross between atherosclerosis sensitive DBA/2 and atherosclerosis resistant AKR apoE-deficient mice. Aortic root lesion area, total cholesterol, body weights, and complete blood counts were ascertained for 114 male and 95 female F 2 progeny. A high-density genome scan was performed using a mouse single nucleotide polymorphism chip yielding 1967 informative polymorphic markers. Quantitative trait locus (QTL) statistical analyses were performed. Novel loci associated with lesion or log lesion area were identified for the female and male F 2 cohorts. The atherosclerosis QTLs in female mice reside on chromosomes 15, 5, 3, and 13, and in male mice on chromosomes 17, 18, and 2. QTL were also identified for body weight, total cholesterol, and blood count parameters. Conclusions— Loci were identified for atherosclerosis susceptibility in a strain intercross study. The identity of the responsible genes at these loci remains to be determined.

Magainins, short peptides with broad-spectrum antimicrobial activity in vitro, were assayed for their ability to confer resistance to pathogens in transgenic grapevines. Embryogenic cell suspensions of 'Chardonnay' (Vitis vinifera L.) were co-transformed by microprojectile bombardment with a plasmid carrying the npt-II gene and a second plasmid harboring either a natural magainin-2 (mag2) or a synthetic derivative (MSI99) gene. Magainin genes and the marker gene were driven by Arabidopsis ubiquitin-3 and ubiquitin-11 promoters, respectively. A total of 10 mag2 and 9 MSI99 regenerated lines were studied by Southern blot hybridization, which showed 1-6 transgene integration events into the plant genome. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed a variable range in transcription levels among mag2 and MSI99 lines. A positive correlation between number of integration events and transcription level was observed (p

Background— Prior transcriptional studies of atrial fibrillation (AF) have been limited to specific transcripts, animal models, chronic AF, right atria, or small samples. We sought to characterize the left atrial transcriptome in human AF to distinguish changes related to AF susceptibility and persistence. Methods and Results— Left atrial appendages from 239 patients stratified by coronary artery disease, valve disease, and AF history (no history of AF, AF history in sinus rhythm at surgery, and AF history in AF at surgery) were selected for genome-wide mRNA microarray profiling. Transcripts were examined for differential expression with AF phenotype group. Enrichment in differentially expressed genes was examined in 3 gene set collections: a transcription factor collection, defined by shared conserved cis -regulatory motifs, a miRNA collection, defined by shared 3′ untranslated region motifs, and a molecular function collection, defined by shared Gene Ontology molecular function. AF susceptibility was associated with decreased expression of the targets of CREB/ATF family, heat-shock factor 1, ATF6, SRF, and E2F1 transcription factors. Persistent AF activity was associated with decreased expression in genes and gene sets related to ion channel function consistent with reported functional changes. Conclusions— AF susceptibility was associated with decreased expression of targets of several transcription factors related to inflammation, oxidation, and cellular stress responses. In contrast, changes in ion channel expression were associated with AF activity but were limited in AF susceptibility. Our results suggest that significant transcriptional remodeling marks susceptibility to AF, whereas remodeling of ion channel expression occurs later in the progression or as a consequence of AF.

To determine signaling pathways responsible for modulation of COX-2 expression in nontransformed and transformed epithelial cells, we studied a rat intestinal epithelial (RIE) cell line expressing constitutively active Ras and RhoA. Expression of COX-2 protein was higher in RIE-RhoA(63L) (four-fold) and RIE-Ras(12V) (seven-fold) cells than in parental cells. Prior work suggests that Ras hyperactivity induces the expression of transforming growth factor (TGF) β and increases epidermal growth factor (EGF)-related peptide signaling—possible mechanisms for increased COX-2 expression. Expression of COX-2 was stimulated by TGFβ and TGFα in RIE and RIE-Rho(63L) cells, but not further stimulated in RIE-Ras(12V) cells. PD153035, an inhibitor of EGF receptor tyrosine kinase, and PD98059, an inhibitor of Erk, attenuated COX-2 expression in RIE and RIE-RhoA(63L). However, the high levels of COX-2 expression in RIE-Ras(12V) cells were not inhibited by either compound. Titration with a panneutralizing anti-TGFβ antibody did not decrease COX-2 in RIE-Ras(12V) cells, even with concurrent EGFR inhibition. Thus, stimulation of the EGF receptor is important in the modulation of COX-2 expression in nontransformed RIE and RIE-RhoA(63L) cells. In Ras-transformed cells, signaling by additional Ras effector pathways, perhaps the RhoA pathway, must be invoked. Identification of these pathways is critical for therapeutic manipulation of COX-2 expression.

Anthers and ovaries of six grapevine cultivars (three Vitis vinifera L., two V × Labruscana L. H. Bailey, and one complex hybrid) were extracted from flower buds over 2 yr and cultured on three media reported to promote somatic embryogenesis in Vitis tissues. The highest percent embryogenesis from the hybrid ‘Chancellor’ and V. vinifera ‘Chardonnay’, ‘Merlot’, and ‘Pinot Noir’ occurred on medium C [Nitsch and Nitsch, 1969, basal medium with 3.0% (w/v) sucrose, 0.01% (w/v) inositol. 0.3% (w/v) Phytagel, 2.5 μM 2.4-dichlorophenoxyacetic acid, 2.5μM β-naphthoxyacetic acid, 5.0μM N-(2-chloro-4-pyridyl)-N′-phenylurea, and 0.05% (w/v) glutamine]. Regardless of the media, the labrusca cultivars ‘Concord’ and ‘Niagara’ produced soft non-embryogenic callus that was sometimes mixed with well-developed somatic embryos. Nine vinifera genotypes were further tested for several different years on medium C. Embryogenic cultures suitable for transformation were obtained from all genotypes in more than 1 yr. The average percent embryogenesis from ovaries was 7-fold higher than from anthers. There was significant annual variation in percent embryogenesis, demonstrating the need for media comparisons to be replicated for more than one season. Suspension cultures suitable for use in genetic transformation were initiated from ‘Chardonnay’, ‘Merlot,’ and ‘Pinot Noir’ pro-embryogenic masses. ‘Chardonnay’ suspension cultures plated and grown under conditions developed for recovery of plants after biolistic transformation yielded approximately 500 non-transformed embryos per plate after 4 mo. of culture, with 68.6% of the embryos converting to plants. This is the first reported protocol for embryogenesis from ‘Concord,’ ‘Cabernet Franc,’ and ‘Pinot Noir’ grapevines.

Summary The effects of low temperature blanching in calcium chloride solution of jalapeno peppers prior to freezing, on firmness retention, pH, methanol, colour and calcium ions of the product, were evaluated by response surface methodology. Texture, methanol content and pH were affected by all of the variables studied (P = 0.05). The optimum response was obtained at temperatures of 63.3–66 °C, calcium chloride concentrations of 0.17–0.21 m, immersion time of 11.6–14.4 min, with a holding time after blanching of 56.6–66.1 min. Microscopic evaluation of the thawed pepper showed that blanching in CaCl2 solution provided a protective effect in maintaining cell wall integrity.

Intergenerational mixing of DNA through meiotic recombinations of homologous chromosomes during gametogenesis is a major event that generates diversity in the eukaryotic genome. We examined genome-wide microsatellite data for 23,066 individuals, providing information on recombination events of 14,140 maternal and paternal meioses each, and found a positive correlation between maternal recombination counts of an offspring and maternal age. We postulated that the recombination rate of eggs does not increase with maternal age, but that the apparent increase is the consequence of selection. Specifically, a high recombination count increased the chance of a gamete becoming a live birth, and this effect became more pronounced with advancing maternal age. Further support for this hypothesis came from our observation that mothers with high oocyte recombination rate tend to have more children. Hence, not only do recombinations have a role in evolution by yielding diverse combinations of gene variants for natural selection, but they are also under selection themselves.

Structure-activity relationships of a novel series of fungal efflux pump inhibitors with respect to potentiation of the activity of fluconazole against strains of Candida albicans and Candida glabrata over-expressing ABC-type efflux pumps are systematically explored.

This study examined the effects and interactions of UV light dose (1,800 to 20,331 microJ/cm2) and apple cider pH (2.99 to 4.41) on the inactivation of Escherichia coli ATCC 25922, a surrogate for E. coli O157:H7. A predictive model was developed to relate the log reduction factor of E. coli ATCC 25922 to the UV dose. Bacterial populations for treated and untreated samples were enumerated with the use of nonselective media. The results revealed that UV dose was highly significant in the inactivation of E. coli, whereas pH showed no significant effect at higher UV doses. Doses of 6,500 microJ/cm2 or more were sufficient to achieve a greater than 5-log reduction of E. coli. Experimental inactivation data were fitted adequately by a logistic regression model. UV irradiation is an attractive alternative to conventional methods for reducing bacteria in unpasteurized apple cider.

Although unregulated activation of the Ras/Raf/mitogen-activated protein kinase kinase/Erk signaling pathway is believed to be a central mechanism by which many cell types undergo oncogenic transformation, recent studies indicate that activation of Raf kinase by oncogenic Ras is not sufficient to cause tumorigenic transformation in intestinal epithelial cells. Thus, identification of signaling proteins and pathways that interact with Raf to transform intestinal epithelial cells may be critical for understanding aberrant growth control in the intestinal epithelium. Functional interactions between Raf and the small GTPase RhoA were studied in RIE-1 cells overexpressing both activated Raf(22W) and activated RhoA(63L). Double transfectants were morphologically transformed, formed colonies in soft agar, grew in nude mice, overexpressed cyclin D1 and cyclooxygenase-2 (COX-2), and were resistant to growth inhibition by transforming growth factor (TGF) β. RIE-Raf and RIE-RhoA single transfectants showed none of these characteristics. Expression of a dominant-negative RhoA(N19) construct in RIE-Ras(12V) cells was associated with markedly reduced COX-2 mRNA, COX-2 protein, and prostaglandin E2 levels when compared with RIE-Ras(12V) cells transfected with vector alone. However, no change in transformed morphology, growth in soft agar, cyclin D1 expression, TGFα expression, or TGFβ sensitivity was observed. In summary, coexpression of activated Raf and RhoA induces transformation and TGFβ resistance in intestinal epithelial cells. Although blockade of RhoA signaling reverses certain well-described characteristics of RIE-Ras cells, it is insufficient to reverse the transformed phenotype and restore TGFβ sensitivity. Blockade of additional Rho family members or alternate Ras effector pathways may be necessary to fully reverse the Ras phenotype.

Although gastrointestinal polyps are more common in the first decade of life than during adolescence, underlying genetic polyposis syndromes are more likely in adolescents. In the past decade, the discovery of gene defects associated with polyposis syndromes has improved classification of these disorders, assisted in the stratification of cancer risk, and permitted more precise diagnosis. Genetic testing is now clinically available for the gene defects that occur in familial adenomatous polyposis coli, Peutz-Jeghers syndrome, Cowden syndrome, and juvenile polyposis syndrome. This review outlines clinical features, genetics, and management strategies for the major polyposis syndromes that affect adolescents.

Colletotrichum graminicola, like many plant pathogenic fungi develop appressoria on germling apices, to facilitate penetration of their host. Induction of these structures occurs after contact with the host surface has been established by the germling. Surface contact and subsequent development of appressoria by germlings of C. graminicola was assessed using interference-reflection microscopy (IRM) and microfabricated pillared silicon substrata. Observations with IRM revealed that under low nutrient conditions, 90% of the germlings developed appressoria once they established 4.5 microm of continuous contact with the substratum. Substrata bearing pillars < or =5 microm in width supported < or =10% appressoria; however, as pillar width was increased the percentage of appressoria formed increased in a sigmoid fashion to a maximum of 80%. The percentage of appressoria produced experimentally on these surfaces was compared to data sets generated from a model designed to calculate the probability of appressorium development on similar pillar arrays at various germ tube contact lengths. These results indicate that germ tubes of C. graminicola require more than 4microm of continuous contact with a hydrophobic substratum for induction of appressoria.

The effect of low-temperature blanching and drying processes on the ultrastructural and physical properties of Anaheim chilli pepper was studied and optimum conditions to provide a final product with maximum firmness were determined. Lots of Anaheim pepper were blanched in water for 4min at 48, 55, 65, 75 and 82 ◦ C and maintained for hold times of 35, 45, 60, 75 and 85min, blanched again for 4min at 96 ◦ C and dehydrated at 53, 60, 70, 80 and 87 ◦ C. After treatment the samples were rehydrated in water at 30 ◦ C. Rehydration ratio, texture and structural changes were evaluated. Optimisation used a second-order rotatable central composite design. Texture and rehydration ratio were affected by blanching temperature and the interaction of blanching temperature with hold time (p ≤ 0.05); drying temperature did not show a significant effect. The best results, ie those which gave greatest firmness, were obtained by blanching at 64 ◦ C for 4min, holding for 55min after blanching, followed by a second blanching at 96 ◦ C for 4min and then drying at 70 ◦ C. Evaluation of the rehydrated dried pepper by microscopy showed that low-temperature blanching close to the optimum conditions provided a protective effect in maintaining cell wall integrity. The results of processing increased firmness in the rehydrated product by a factor of 1.97.  2003 Society of Chemical Industry

Calcium absorption in sliced carrots was evaluated by immersion in calcium chloride solution (0.3, 0.6 and 0.9 M) at 65C and 100C. Absorption kinetics were obtained for each of the experimental combinations using linear and quadratic statistical models. The apparent diffusion coefficient was estimated using Fick's second law for infinite slabs. Linear models of calcium absorption versus temperature and concentration were adequate for both 65C and 100C conditions (P ≤ 0.05). Diffusion coefficients of calcium ions into carrot tissue showed no significant increase with selected concentrations of calcium chloride. A significant increase (P ≤ 0.05) in the average diffusion coefficient (0.918-2.32 x 10 -9 m 2 /s) was observed with a rise in temperature from 65C to 100C.

The precarious state of the educational system in the inner cities of the United States, as well as its potential causes and solutions, have been popular topics of debate in recent years. Part of the difficulty in resolving this debate is the lack of solid empirical evidence regarding the true impact of educational initiatives. The efficacy of so-called “school choice” programs has been a particularly contentious issue. A current multimillion dollar program, the School Choice Scholarship Foundation Program in New York, randomized the distribution of vouchers in an attempt to shed some light on this issue. This is an important time for school choice, because on June 27, 2002 the U.S. Supreme Court upheld the constitutionality of a voucher program in Cleveland that provides scholarships both to secular and religious private schools. Although this study benefits immensely from a randomized design, it suffers from complications common to such research with human subjects: noncompliance with assigned “treatmen...

The objective of this study was to evaluate the effect of low temperature blanching in calcium chloride solutions to increase firmness retention of frozen carrot slices and to determine optimum conditions to provide a final product with maximum firmness. Low temperature (65C) blanching in CaCl 2 solutions (0.05-0.6 molar) for holding times (5-90 min) was performed on carrots Apache' cultivar. Calcium absorption was affected (P≤0.05) by the CaCl 2 concentration. Behavior of this variable was adequately fitted by a second order model (R 2 =0.99). Firmness was affected (P≤0.05) by the CaCl 2 solutions and holding times. Optimum conditions gave extrusion forces of 1693-2095 N and calcium levels of 69-144 mg/100 g. These were verified experimentally and agreed closely with predicted calcium absorption and firmness values.

This study reports evidence that schizophrenia patients are significantly impaired in both spatial and object (shape) working memory. A 3-s delay between exposure and recall of targets was used and Bayesian item-response theory was applied to compensate for the tasks' differential difficulty while simultaneously taking account of missing data from participant attrition. Weaker evidence was found that in schizophrenia both domains are equally impaired on average, that spatial and object working memory appear to be more highly correlated with each other in the schizophrenia population than in the normal population, and that schizophrenia patients show greater variability in spatial than object working memory performance.

We applied partial least squares (PLS) as a novel multivariate statistical technique to examine neuropsychological correlates of magnetic resonance imaging (MRI) measures of brain volumes in a well studied sample of 15 male patients with chronic schizophrenia. In the current study, because the total number of measures far surpassed the total number of subjects, extant multivariate techniques such as canonical correlation could not be used to examine relationships among simultaneous measures of MRI and neuropsychology. Moreover, because MRI measures were expected to be highly inter-correlated, as would be neuropsychological test scores, extant multivariate statistical techniques would be substantially limited because they typically assume statistical independence among sets of measures. PLS, on the other hand, proved to be especially well suited to examining the relationships among function and anatomy measures in this sample, where statistically significant relationships were demonstrated that were entirely consistent with prior studies using univariate correlation techniques. In particular, statistically significant relationships emerged among sets of MRI temporal lobe measures and neuropsychological tests of verbal memory and categorization as well as among MRI frontal measures and neuropsychological tests of working memory.

Elevated levels of endothelial cell (EC)-derived extracellular vesicles (EVs) circulate in patients with antiphospholipid antibodies (APLAs), and APLAs, particularly those against β2 -glycoprotein I (β2 GPI), stimulate EV release from ECs. However, the effects of EC-derived EVs have not been characterized.To determine the mechanism by which EVs released from ECs by anti-β2 GPI antibodies activate unstimulated ECs.We used interleukin (IL)-1 receptor inhibitors, small interfering RNA (siRNA) against Toll-like receptors (TLRs) and microRNA (miRNA) profiling to assess the mechanism(s) by which EVs released from ECs exposed to anti-β2 GPI antibodies activated unstimulated ECs.Anti-β2 GPI antibodies caused formation of an EC inflammasome and the release of EVs that were enriched in mature IL-1β, had a distinct miRNA profile, and caused endothelial activation. However, activation was not inhibited by an IL-1β antibody, an IL-1 receptor antagonist, or IL-1 receptor siRNA. EC activation by EVs required IL-1 receptor-associated kinase 4 phosphorylation, and was inhibited by pretreatment of cells with TLR7 siRNA or RNase A, which degrades ssRNA. Profiling of miRNA in EVs released from ECs incubated with β2 GPI and either control IgG or anti-β2 GPI antibodies revealed numerous differences in the content of specific miRNAs, including a significant decrease in mIR126. These observations demonstrate that, although anti-β2 GPI-derived endothelial EVs contain IL-1β, they activate unstimulated ECs through a TLR7-dependent and ssRNA-dependent pathway. Alterations in miRNA content may contribute to the ability of EVs derived from ECs exposed to anti-β2 GPI antibodies to activate unstimulated ECs in an autocrine or paracrine manner.

Poor response to antiplatelet drugs is associated with adverse outcomes. We assessed platelet inhibition and its stability and tested correlation and agreement between platelet function assays.Peripheral blood from 58 patients on both aspirin and clopidogrel who underwent percutaneous coronary intervention (PCI) was collected at hospital discharge (visit-1) and at 30-90 days (visit-2). Platelet function was measured using light transmission aggregometry (LTA-AA and LTA-ADP), VerifyNow® (Aspirin; ARU and P2Y12; PRU), ex vivo TxB2, urinary 11dhTxB2, and VASP (PRI) assays. Data were analyzed as continuous, quartiles and binary. Patients were defined as aspirin poor responder (PR) with ARU ≥ 550, LTA-AA maximum ≥ 20%, TxB2 ≥ 1 ng/mL or 11dhTxB2 ≥ 1,500 pg/mg of creatinine and as clopidogrel PR with PRU ≥ 240, PRU ≥ 208, LTA-ADP maximum ≥ 40%, PRI ≥ 50%, or PRI ≥66%.Aspirin PR was 3-33% and clopidogrel PR was 10-35% in visit-1. LTA-AA, 11dhTxB2, and all clopidogrel-response measures showed correlation and agreement between visit-1 and visit-2. The highest agreement between two visits was revealed by PRU ≥ 240 and PRI ≥ 66% (PRU-κ=0.7, 95% CI=0.47, 0.93; PRI-κ=0.69, 95% CI=0.42, 0.95, p-values0.001). Comparison of platelet function assays in a single visit (visit-1) revealed a poor correlation between LTA-AA and 11dhTxB2 assays and no agreement among aspirin-response assays. The highest correlation and agreement were obtained between VerifyNow® P2Y12 and VASP assays (rho=0.7, p-value0.001 and PRU ≥ 208-PRI-κ=0.41-0.42, 95% CI=0.13, 0.69, p-values0.001).Platelet inhibition is stable during aspirin and clopidogrel treatment. Clopidogrel-response assays correlate and agree with each other better than aspirin-response assays.

Atrial Fibrillation (AF), the most common sustained arrhythmia, has a strong genetic component, but the mechanism by which common genetic variants lead to increased AF susceptibility is unknown. Genome-wide association studies (GWAS) have identified that the single nucleotide polymorphisms (SNPs) most strongly associated with AF are located on chromosome 4q25 in an intergenic region distal to the PITX2 gene. Our objective was to determine whether the AF-associated SNPs on chromosome 4q25 were associated with PITX2c expression in adult human left atrial appendages. Analysis of a lone AF GWAS identified four independent AF risk SNPs at chromosome 4q25. Human adult left atrial appendage tissue was obtained from 239 subjects of European Ancestry and used for SNP analysis of genomic DNA and determination of PITX2c RNA expression levels by quantitative PCR. Subjects were divided into three groups based on their history of AF and pre-operative rhythm. AF rhythm subjects had higher PITX2c expression than those with history of AF but in sinus rhythm. PITX2c expression was not associated with the AF risk SNPs in human adult left atrial appendages in all subjects combined or in each of the three subgroups. However, we identified seven SNPs modestly associated with PITX2c expression located in the introns of the ENPEP gene, ∼54 kb proximal to PITX2. PITX2c expression in human adult left atrial appendages is not associated with the chromosome 4q25 AF risk SNPs; thus, the mechanism by which these SNPs are associated with AF remains enigmatic.

Diced green bell pepper was blanched twice, once at 51-79 °C for 19-61 min, and once at 95 °C for 3 min, and dried. The firmness of rehydrated samples was measured by puncture, and optimum conditions assessed by response surface methodology. The optimized model showed that, blanching at 65 °C for 49 min gave a 64% increase in puncture force over the control. The optimum temperature was used to evaluate the effect of adding CaCl 2 . The dices were blanched twice, once at 65 °C for 3 min in either 0 or 4% CaCl 2 , secondly in either 0 or 2% CaCl 2 solution at 95 °C for 3 min. In the second case the dices had been held at room temperature for 0-30 min before treatment. Adding CaCl 2 increased puncture force significantly (P ≤ 0.05). The best results, those which gave greatest firmness, were obtained by blanching at 65 °C for 3 min in 4% CaCl 2 , holding for 16 min after blanching, followed by a secondary blanching at 95 °C in 2% CaCl 2 . Du poivron vert en cube a ete blanchi deux fois puis seche. La fermete du poivron rehydrate a ete mesure et les conditions optimales ont ete determinees: le blanchiment a 65° pendant 49 min donne les meilleurs resultats. Le poivron en cube a ensuite ete blanchi a 65° pendant 3 min.puis 95° pendant 3 min. en presence de chlorure de calcium a concentration variable, a chaque fois. Les meilleurs resultats sont obtenus pour CaCl 2 a 4% puis CaCl 2 a 2% pour le deuxieme blanchiment.

Introduction Recurrent somatic mutations in SF3B1, a gene encoding a spliceosome component, have been identified in patients (pts) with myelodysplastic syndromes (MDS). SF3B1 mutations (MT) are more commonly detected in pts with ring sideroblast (RS) morphology and are associated with favorable outcome. The proposed 2016 World Health Organization (WHO) MDS classification categorizes pts with >5% RS and SF3B1 MT as MDS with RS, in contrast to prior WHO classifications which required ≥15% RS regardless of genotype. In this study, we explored the prognostic value of RS and SF3B1 MT and assessed the validity of the new proposal. Methods We identified 471 pts with MDS and known SF3B1 mutational status from MDS CRC institutions. RS were assessed as present or absent (RS +/-) based on bone marrow aspirate reports (n=157); in cases where quantitative data on RS% were available (n=41), pts were grouped in the 5-15% RS group or > 15% RS group. Survival was calculated from time of diagnosis. Cox regression analysis was used to estimate hazard ratios for overall survival (OS) and AML free survival (AFS), which was defined as time to death or AML transformation, respectively. Chi-squared and Wilcoxon tests were used to test for differences in categorical and continuous distributions, respectively. Results: Among 471 pts with known SF3B1 mutational status, 76 (16%) had MT. Pts with MT had lower-risk International Prognostic Scoring System (IPSS) scores compared to SF3B1 wild-type (WT; 79% vs 57%, p < .001). Among pts with MT, 50% had RS + compared to 19% RS + in the WT group (p < .001). MT were independently associated with better OS (HR 0.48, p= .001) and longer AFS (HR 0.5, p We compared outcomes of four groups: WT/RS-, MT/RS-, WT/RS+, and MT/ RS +. Adjusting for age and IPSS, pts with MT/RS + had the best outcome, with hazard ratios for AFS of 4.2 for WT/RS- vs. MT/RS+ (p =.018), 4.1 for MT/RS- vs. MT/RS+ (p =.045), and 5.1 for WT/RS+ vs. MT/RS+ (p= .01). We compared 7 pts with 5-15% RS to 22 pts with >15% RS. Among patients with RS 5-15%, 4/7 pts (57%) were classified as MDS with excess blasts compared to 24% for those RS >15% (p=.09). Pts with 5-15% RS were more likely to be thrombocytopenic (5/7, 71%) compared to >15% RS (29%, p=.04). One patient (14%) with 5-15% RS had MT compared to 12 (55%) pts with > 15% RS, p= .06. In Cox regression analysis using the RS 5-15% group as the reference, the hazard ratio for RS > 15% for AFS was 0.26 (p = .034) and the hazard ratio for MT for AFS was 0.08 (p= .002). Conclusions SF3B1 somatic mutations in MDS are commonly associated with RS, better OS and longer AML-free survival. Patients with RS and MT had a significantly better outcome than those with either isolated RS or MT, or neither. These data support incorporation of SF3B1 mutation status into the WHO classification regardless of RS percent, though with differentiation for those with RS and MT. Disclosures Komrokji: Novartis: Consultancy, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy; Boehringer-Ingelheim: Research Funding. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy.

Background Lower-risk (LR) MDS (Low/Int-1 per International Prognostic Scoring System (IPSS)) are a heterogeneous group of disorders characterized mainly by refractory anemia and transfusion dependency. As survival of this patient (pt) population is measured in years. Goals of therapy focus on decreasing blood transfusions, improving quality of life, while minimizing treatment toxicities. While achieving complete remission (CR) in higher-risk MDS correlates with improved overall survival (OS), its relationship to OS in LR MDS is not well defined. We evaluated the impact of achieving CR on OS in LR MDS and defined the clinical characteristics that may predict for this response in this pt population. Method Included pts were diagnosed with MDS (per 2008 WHO criteria) and had LR disease with clinical and pathologic data collected from MDS Clinical Research Consortium institutions. Only pts with bone marrow blasts of 5-9% who would thus qualify both as having LR MDS and for being eligible to assess CR were included. Responses (including CR, PR, HI, stable disease and progressive disease) were defined per International Working Group 2006 criteria. OS was calculated from the time of achievement of best response to time of death or last follow-up. Cox proportional hazard analysis that included all clinical variables and treatment characteristics was used to identify independent prognostic factors. Results Of 1470 pts included in the database, 999 identified with LR disease, and 174 had bone marrow blasts of 5-9%. The median age was 60 years (range, 22-87), and 37% were female. Median neutrophil count was 1.25 X 109\L (range, .10-51.0), hemoglobin was 9.8 g/dl, platelets were 109 k/ml (range, 18-562), and bone marrow blasts were 6% (range, 5-9%). Best responses to therapy included: 26 pts (15%) with CR, 10 pts (6%) with PR, and 13 pts (7%) with HI. Among pts who achieved CR/PR/HI, 27 received HMA (25 with azacidtine +/- combination and 2 with single agent decitabine), 16 intensive chemotherapy, 2 lenalidomide, and 4 received other therapies. With a median follow up from diagnosis of 31.2 months, the median time from diagnosis to best response was 11.9 months (range, .69-81.0) and was similar in pts who achieved CR compared to PR/HI (11.5 vs. 12.4 months, respectively, p = .74). The median OS from time of CR/PR/HI for the entire cohort was 21.3 months. The median OS for pts who achieved a CR was longer compared to pts with PR/HI (46.5 vs. 18.5 months, respectively, p = .03). In multivariate analyses that included clinical variables and treatment history, achieving CR remained an independent prognostic factor for longer OS (HR .32, p = .03) but no individual demographic, clinical or treatment variables were predictive of CR. Conclusions Similar to pts with higher-risk MDS, LR MDS pts who achieve CR to therapy have improved OS compared to those with PR or HI. CR is thus an important endpoint in LR MDS, though is difficult to predict. As OS is measured in years in LR MDS, CR may be used as a surrogate endpoint for OS in clinical trials in this pt population. Disclosures Komrokji: Novartis: Consultancy, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Introduction While lenalidomide (LEN) is the standard of care for treatment of red blood cell (RBC) transfusion-dependent (TD) lower-risk myelodysplastic syndromes (LR-MDS) with chromosome 5q deletion (del 5q), it is widely used off-label in the non-del5q setting. In the MDS-002 and MDS-005 studies, 26% of TD non-del5q LR-MDS patients became RBC transfusion independent after LEN treatment. National Comprehensive Cancer Network (NCCN) clinical guidelines list LEN as a second line treatment option for TD anemia in lower-risk non-del 5q MDS after hypomethylating agents (HMAs). Thatrecommendationhas led to wide use of HMAs as frontline therapy after erythroid stimulating agents (ESA) failure in LR-MDS. The response rate to LEN after HMA failure, however, is not known, as MDS-002 and MDS-005 excluded patients previously treated with HMAs. To assess the best order of LEN and HMA in optimizing response potential in lower-risk MDS, we examined response rates to each drug when treatment order (LEN followed by HMA or HMA followed by LEN) differed. Methods We identified patients with LR- MDS (International Prognostic Scoring System (IPSS) low or intermediate-1 (int-1) risk groups) within the MDS Clinical Research Consortium database who received both LEN and HMA as first or second line therapy after ESA failure or patients who had low chance of response to ESA. We excluded patients with isolated del5q or del q + one additional cytogenetic abnormality. The primary objective was to compare rates of erythroid hematological improvement (HI-E), defined using 2006 International Working Group criteria (IWG 2006), between patients who received LEN as first line therapy followed by HMA as second line (LEN 1st line group) versus those who received LEN as second line therapy after HMA (LEN 2nd line group). Results We identified 144 patients who received both HMA and LEN as first and second line therapies: 80 patients were in group 1 (LEN 1st line) and 64 patients were in group 2 (LEN 2nd line). Baseline characteristics between the two groups are summarized in Table 1. There were no statistically significant differences between the 2 groups. The rate of HI-E was 20% (16/80) for the LEN 1st line group compared to 11% (7/64) in the LEN 2nd line group. (p=.046). There were no differences in response rates to HMA between the two groups: In the LEN 1st line group, response to 2nd line HMA was 30% (24/78) compared to 39% (25/64) for those who received HMA as first line (p=.2). There was no difference in overall survival (OS) between the two groups, The median OS was 79 months (mo) the LEN 1st line group compared to 61 mo in Len 2nd line group (p= .4). The rate of AML transformation was 9% in Len 1st line group compared to 22% in Len 2nd line group rate (p=.03). There was no difference in AML free survival (78mo versus 61mo respectively, p=0.4). In multivariable analyses adjusting for age, sex, and IPSS-R order of treatment still did not impact overall survival. Conclusion LEN yields a higher rate of HI-E in LR- MDS when used as first line therapy, but responses to HMAs were similar when used before or after LEN. The rate of AML transformation was lower when LEN was used as first line. The order of treatment does not impact overall survival. Lenalidomide should be used prior to HMA if it is to be considered for treatment of anemia in non-del5q LR-MDS. Table 1 Baseline characteristics Table 1. Baseline characteristics Disclosures Komrokji: Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy; Boehringer-Ingelheim: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Roboz:Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy; Cellectis: Research Funding.

Introduction: Somatic mutations are common in MDS and mutation profiling is becoming increasingly standard as part of the initial diagnostic evaluation. The value of serial monitoring of mutation profiles in MDS patients at multiple disease course time points is unknown. We evaluated the impact of serial mutation monitoring in a cohort of MDS patients. Methods: MDS patients from participating institutions of the MDS CRC with molecular genetic data by next-generation sequencing available from at least two distinct time points were included in analyses. Mutations present at the first time point were compared to those present at subsequent time points and correlated with age, International Prognostic Scoring System (IPSS) score, and overall survival, measured from diagnosis. Cox proportional hazards model of time from diagnosis to death or loss of follow up with time varying number of mutated genes, gained genes and lost genes adjusted for sex, age at diagnosis, and IPSS category at/near diagnosis was applied. Results: Of 737 MDS patients with molecular genetic data available from 1994 to 2016, 94 had information on the 18 genes presented in Table 1 from at least two distinct time points. Patients had a median of 2 (range 2-9) myeloid mutation panel assessments, with a median interval of 3.3 months (range 0.1-39) between studies. Mutation testing was repeated at the investigator's discretion either at time of suspected disease progression or change of therapy. Clinical characteristics of the patients are summarized in Table 2. Almost half (46%) had an initial mutation profile performed within 1 month of diagnosis, 23 patients (24%) within 1-12 months, and 28 patients (30%) more than 12 months after diagnosis. At the time of initial evaluation, patients had a median of 1.5 mutations (range 0-4), with the most common being TP53 (28%), RUNX1 (20%) TET2 (18%), DNMT3A (17%), and ASXL1 (17%). Independent of age or IPSS score at baseline, the number of mutations initially present in a gene set consisting of ASXL1, EZH2, ETV6, RUNX1, TP53 was associated with poor overall survival (HR 2.19, p=0.003, 95% CI, 1.30-3.69). Overall, 32 patients (34%) acquired at least one additional mutation at subsequent testing, including ASXL1 (10%), RUNX1 (7%), TP53 (5%), SETBP1 (5%), and DNMT3A (4%). The acquisition of additional mutations in any gene in subsequent testing was associated with worse overall survival (HR 1.93, p=0.0002, Cl, 1.36-2.75), irrespective of age, IPSS score, and the number of mutations initially identified among the core group of five prognostically significant mutations. Of note, treatment was similar for patients who were found to have acquired additional mutations compared to those who did not: hypomethylation agents (93% vs 80%, p=0.11), lenalidomide (10% vs 11%, p=0.94), and chemotherapy (38% vs 49%, p=0.33). Patients who acquired at least one additional mutation beyond those identified at diagnosis had a higher rate of acute myeloid leukemia transformation (62% vs 37%, p=0.02). Additional acquired mutations were more likely to be observed in serial testing after AML transformation, compared to patients without AML transformation (36% vs 17%, p=0.01). In this model, acquisition of a mutation in DNMT3A, IDH1, IDH2, SETBP1, or TET2 was associated with poor overall survival (HR 3.88, p=0.006, CI, 1.46-10.3). Gain of mutations in CBL, MPL, NPM1, or PHF6 was also associated with poor survival (HR 8.15, p=0.0008, CI, 2.38-27.86). Loss of mutation in serial testing was observed in 33% of patients was and did not correlate with overall survival (HR 0.66, p=0.25, CI, 0.32-1.36). A small subset of patients who underwent allogeneic stem cell transplantation were found to lose their diagnostic mutation(s) at the time of transplant. Conclusions: The acquisition of new mutations in MDS patients undergoing serial monitoring is associated with poor overall survival independent of age, sex, and IPSS, in some part related to the indication for testing. Serial mutation profiling identified a small number of patients who acquired "actionable" mutations for which clinical trials were potentially available, but did not substantively alter treatment choices. Further investigation of the relationships between disease biology, treatment effects, and acquisition/loss of mutations should be conducted in the context of clinical trials. The role of serial mutation profiling in MDS warrants further investigation. Disclosures Lee: Boehringer Ingelheim: Consultancy; Baxalta: Consultancy; Amgen: Consultancy; Alexion Pharmaceuticals: Consultancy; Pfizer Inc: Consultancy. Steensma:Genoptix: Consultancy; Janssen: Consultancy; Millenium/Takeda: Consultancy; Ariad: Equity Ownership; Celgene: Consultancy; Amgen: Consultancy. Ritchie:Arian: Speakers Bureau; Novartis: Honoraria; Incyte: Speakers Bureau; Pfizer: Honoraria; Celgene: Speakers Bureau. Desai:Boehringer Ingelheim: Consultancy. Komrokji:Boehringer-Ingelheim: Research Funding; Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy.

Background: While treatment with the hypomethylating agents (HMAs) azacitidine (AZA) and decitabine (DAC) improves cytopenias and prolongs survival in MDS patients (pts), only 30-40% of pts respond. Genomic and/or clinical models that can predict which pts will respond could prevent prolonged exposure to ineffective therapy, avoid toxicities and decrease unnecessary treatment costs. Machine learning (ML), a field of artificial intelligence, is an advanced computational analysis of complex data sets that can overcome some of the limitations of standard statistical methods. ML uses computational algorithms to automatically extract hidden information from a dataset by learning from relationships, patterns, and trends in the data. Thus, ML can produce powerful, reliable and reproducible predictive models based on large and complex datasets. The aim of this project is to build a geno-clinical model that uses ML algorthims to predict responses to HMAs. Methods: We screened a cohort of 433 pts with MDS who received HMAs at multiple academic institutions for the presence of common myeloid somatic mutations in 29 genes. Responses were assessed per International Working Group 2006 criteria. Five popular supervised classification ML algorithms including: random forest (RF), tree ensemble (TE), naive bayes (NB), decision tree (DT), and support vector machine (SVM) algorithms were used individually and in combination to enhance the accuracy of the proposed model (bag of model approach). For each iteration, the dataset was divided randomly into training and validation cohorts. The partition of the dataset was repeated multiple times randomly to minimize biases in pt selection. A 10-fold cross validation was also used on the entire dataset to assure data reproducibility. Important variables were selected using backward feature elimination and tree depth scores. Performance was evaluated according to the area under curve (AUC) and accuracy matrix. All analyses were done using KNIME (an open analytic platform for ML). Results: Among 433 pts, 193 (45%) received AZA, 176 (40%) DAC, and 64 (14%) received HMA +/- combination. The median age was 70 years (range, 31-100) and 28% were females. Responses included: 95 (58%) complete remission (CR), 14 (3%) marrow CR, 16 (4%) partial remission (PR), and 59 (14%) hematologic improvement (HI). For the purpose of this analysis, pts with CR/PR/HI were considered as responders. The most commonly mutated genes were: ASXL1 (31%), TET2 (22%), SRSF2 (17%), RUNX1 (15%), and DNMT3A (14%). In univariate analyses, no single mutation was more prevalent in responders compared to non-responders except NF1 (more common in non-responders, p = .04). A logistic regression multivariate analysis did not produce a reliable and reproducible model. When applying ML algorithms on learner (80% randomly selected pts) and predictor cohorts, the accuracy rate in predicting responses for RF was 64%, for TE 60%, for NB 60%, for DT 66%, and for SVM 51%. When results from each model were combined (a bag of models approach), the accuracy increased to 69%. Backward feature elimination and tree depth scores identified the following factors as predictors of response: hemoglobin 69 years, TP53 with variant allelic frequency (VAF) >15%, CBL VAF >30%, and RUNX1 VAF > 25%. Only ASXL1mutations at any VAF were predictive of HMA resistance. Interestingly, none of the mutations were selected for response or resistance when the models did not include VAF. Neither treatment modality with azacitidine vs. decitabine vs. combination nor treatment center impacted response. When the analysis was restricted to pts with higher-risk disease by IPSS, the accuracy rate in predicting responses improved: for RF it became 71%, for TE 65%, for NB 60%, for DT 64%, and for SVM 76%. When the analysis was focused on pts who achieved CR vs. No CR, the models predicted the response differently. The RF and TE models were able to predict No CR with an accuracy rate of 75% and 76% respectively. Other models were able to predict CR and No CR with lower accuracy. Conclusion: We propose a novel geno-clinical model that uses machine intelligence to predict HMA response/resistance in pts with MDS. The model has a higher accuracy rate in higher-risk MDS pts. ML can open opportunities in translating genomic data into reliable predictive models that can aid physicians in clinical decision making. Disclosures Bejar: Celgene: Consultancy, Honoraria; Foundation Medicine: Consultancy; Genoptix: Consultancy, Honoraria, Patents & Royalties: No royalties.

Autophagy is a conserved mechanism of protein degradation that plays a physiologic role in iron homeostasis. The RARS subtype of MDS is characterized by inefficient erythropoiesis and the presence of erythroblasts with iron-laden mitochondria (ring sideroblasts, RS). Patients with RARS exhibit an accumulation of iron due to the inability of their erythroid precursors to properly process iron intended for hemoglobin synthesis. RARS erythroid cells display increased autophagy compared to cells from healthy subjects and mice lacking the essential autophagy genes Atg1 and Atg7 develop progressive anemia due to the inefficient removal of defective mitochondria. We previously showed that the presence of SF3B1 mutations, which are frequent in RARS patients, are associated with the presence of increased mitochondrial iron content and low levels of apoptosis. This finding suggests that cell death-independent mechanisms may temper excess iron by triggering the clearance of mitochondria during erythroid maturation. RNA sequencing analyses have shown that the autophagy pathway is upregulated in SF3B1MUT cells. We investigated the role of mitochondrial autophagy in the alleviation of iron-related damage in RARS erythroid cells and the potential therapeutic benefit of autophagy-stimulating agents for the selective improvement of erythropoiesis and iron homeostasis in SF3B1MUT RARS cells. Transmission electron microscopy (TEM) demonstrated an accumulation of iron in the mitochondria of SF3B1MUT (K700E, n=4) compared to SF3B1WT RARS erythroblasts. Flow cytometry confirmed increased mitochondrial iron in SF3B1MUT (n=10) compared to SF3B1WT (n=10) RARS cells (82% ± 10 vs. 24% ± 5; P = 0.004). Autophagic vacuolization of the cytoplasm and an increased number of autophagosomes were found in SF3B1MUT (K700E) compared to SF3B1WT erythroblasts (n=2). The mRNA expression levels of numerous autophagy genes were elevated in SF3B1MUT vs. SF3B1WT RARS cells: ATG complexes (ATG2A/B, FC=2; ATG4A, FC=2; ATG9A, FC=5; ATG4C, P=0.05; ATG18 (FC=4.8; P= 0.02), autophagy-initiating kinases (ULK1, FC=2; ULK3, FC=3.9; P=0.05), and cathepsins involved in late stage autophagy (CTSL1, FC=20; CTSD, FC=5.8; P=0.05; CTSB, FC=2.1; CTSE, FC=5.9; CTSD, FC=2; P=0.01). qRT-PCR confirmed that SF3B1MUT cells expressed elevated mRNA levels of selected genes. We next administered 3 FDA-approved drugs with established autophagy-stimulating properties [temsirolimus, metformin, arsenic trioxide (ATO)] to a transgenic SF3B1 mouse model (SF3B1+/-) that exhibits anemia, dysplasia, and RS (Visconte, J Hematol Oncol 2014) to evaluate drug-mediated improvement of erythropoiesis and autophagic clearance of excess iron. Eight-month-old SF3B1+/- mice (n = 10 per group) were treated with vehicle control and the following: temsirolimus (10 mg/kg i.p. 5d/week for 2 wk), metformin (250 mg/kg/d, gavage for 2 wk), and ATO (10 mg/kg i.p. 5 d/wk for 2 wk). All agents were well tolerated and triggered morphologic features of autophagy including increased autophagosome-like structures by TEM. No effects on BM cellularity and/or dysplasia were noted, although changes in the morphology of myeloid cells (numerous swollen nuclei) were detected by Wright stain. Mitochondria that were engulfed in autophagosomes were frequently seen. After 2 weeks, temsirolimus-treated SF3B1+/- mice showed an incremental increase in hemoglobin by 1.2 g/dL (7.9 vs. 6.6 g/dL; P=0.07) and in mean corpuscular volume (43.5 vs. 42.4 fL; P=0.08). Erythropoiesis was improved as shown by increased levels of CD71-positive cells by immunohistochemistry in cells post-temsirolimus treatment compared to pre-treatment (10-20% vs. 5%). Increased dividing erythroid cells with binucleation and budding were also observed in cells following temsirolimus treatment. These hematologic changes were not detected with ATO or metformin. In sum, our data support a role for activated autophagy in the pathogenesis of RARS and indicate that stimulating autophagy with approved existing drugs or novel investigational drugs may yield therapeutic benefit in SF3B1MUT RARS patients. Disclosures Kelly: Pharmacyclics: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Speakers Bureau. Advani:Pfizer: Consultancy, Research Funding. Carraway:Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Research Funding, Speakers Bureau.

An appealing feature of multiple imputation is the simplicity of the rules for combining the multiple complete-data inferences into a final inference, the repeated-imputation inference (Rubin, 1987). This inference is based on a t distribution and is derived from a Bayesian paradigm under the assumption that the complete-data degrees of freedom, ν com , are infinite, but the number of imputations, m, is finite. When ν com is small and there is only a modest proportion of missing data, the calculated repeated-imputation degrees of freedom, ν m , for the t reference distribution can be much larger than ν com , which is clearly inappropriate. Following the Bayesian paradigm, we derive an adjusted degrees of freedom, ν m , with the following three properties: for fixed m and estimated fraction of missing information, ν m monotonically increases in ν com ; ν m is always less than or equal to ν com ; and ν m equals ν m when ν com is infinite. A small simulation study demonstrates the superior frequentist performance when using ν m rather than ν m .