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DOE OSTI2018

Evolutionary conservation of transferrin genomic organization and expression characterization in seven freshwater turtles

Highlights: • Transferrin have evolutionary conservation in genomic organization in freshwater turtles. • Transferrin have evolutionary conservation in expression characterization in freshwater turtles. • 12 positive selected sites were detected in freshwater turtles which were mainly located on the surface of Tf proteins. Serum transferrin (tf), encoding an iron-binding glycoprotein, has been revealed to play important roles in iron transportation and immune response, and it also has been demonstrated to be valuable for phylogenetic analysis in vertebrates. However, the evolutionary conservation, expression profiles and positive selection of transferrin genes among freshwater turtle species remain largely unclear. Here, the genomic DNA and coding sequences of transferrin genes were cloned and characterized in seven freshwater turtles including Mauremys mutica, Mauremys sinensis, Cyclemys dentate, Mauremyssi reevesi, Heosemys grandis, Trachemys scripta and Chrysemys picta. The isolated coding sequences of turtles' tf genes were 2118 bp or 2121 bp, encoding 706 or 707 amino acids. The predicted Tf proteins of turtles share high identities with M. mutica Tf, up to 91%–98% and the M. mutica Tf has the highest identity (91%) in amino acid with the Chelomia mydas Tf, the moderate with other reptiles' Tfs (65%–59%), chicken (58%), and Human Tf (∼55%), and the lowest with zebrafish Tf (41%). Additionally, tf genes were consistently composed of 17 exons and 16 introns with the same splicing sites in introns in all the turtles examined. Moreover, 12 positive selected sites were detected in these turtles’ Tf and mainly distributed on the surface of transferrin protein. Importantly, it was found that transferrin genes in all turtles examined were predominantly expressed in adult liver via real-time quantitative PCR. The molecular characterizations and expression profiles of transferrin would shed new insights into understanding the conversations and divergences of transferrin genes in turtles, even in vertebrates.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Bovine lactoferrin reverses programming of epithelial-to-mesenchymal transition to mesenchymal-to-epithelial transition in oral squamous cell carcinoma

Highlights: • bLF reverses EMT phenotype to MET in OSCC cells. • bLF enhances E-cadherin expression and suppresses vimentin in OSCC cells. • bLF inhibits TWIST through ERK pathway to gain E-cadherin. Epithelial-to-mesenchymal transition (EMT) is a biological process of invasion and metastasis in cancers, including in oral squamous cell carcinoma (OSCC). However, an effective anticancer drug that directly targets EMT has not yet been discovered. Therefore, we aimed to investigate the repressive effects of bovine lactoferrin (bLF) on EMT to achieve mesenchymal-to-epithelial transition (MET) in OSCC. OSCC cell lines, HOC313 (EMT-induced) and SCCVII (without EMT induction), were treated with bLF. The effects of bLF on EMT in OSCC were identified histologically by haematoxylin and eosin staining and observed morphologically and immunohistochemically using an anti-E-cadherin antibody. Expression levels of E-cadherin and vimentin were investigated using RT-PCR and western blotting. Immuno-expression of E-cadherin was examined in vivo in tumour tissues of C3H/HeN mice, transplanted with SCCVII cells, with or without bLF administration. We found that bLF changed the spindle-like mesenchymal cells to cuboidal-like epithelial cells and enhanced the affinity of membrane-bound E-cadherin in HOC313 cells. The transformation of EMT-MET in HOC313 cells was confirmed by the upregulation of E-cadherin and suppression of vimentin. Moreover, bLF suppressed TWIST expression through downregulation of ERK1/2 phosphorylation. Additionally, the inhibition tumour cell infiltration and increase in E-cadherin expression were observed in xenografts of the mice orally administered with bLF. Thus, based on the results from in vitro and in vivo studies, we concluded that bLF caused the restoration of epithelial properties through MET. Importantly, this finding is novel and is the first report indicating that bLF inhibited EMT and induced MET in OSCC, suggesting that bLF may provide a novel therapeutic strategy in OSCC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

LncRNA LINC00319 accelerates ovarian cancer progression through miR-423-5p/NACC1 pathway

Highlights: • LINC00319 is upregulated in ovarian cancer. • LINC00319 promotes ovarian cancer proliferation, migration and invasion. • LINC00319 promotes NACC1 expression by interacting with miR-423-5p. • LINC00319 promotes ovarian cancer progression relying on miR-423-5p/NACC1 pathway. Long noncoding RNA (lncRNA) LINC00319 has been reported to promote carcinogenesis of lung cancer and cutaneous squamous cell carcinoma. However, the role and mechanism of LINC00319 in ovarian cancer progression is unclear. In this study, LINC00319 expression was found to be upregulated in ovarian cancer tissues and cell lines. And our evidence showed that LINC00319 could be a potential prognostic biomarker for patients with ovarian cancer. Cell Counting Kit-8 (CCK-8), colony formation and transwell assays indicated that LINC00319 upregulation promoted proliferation, migration and invasion of ovarian cancer cells. Bioinformatics analysis and luciferase reporter assay revealed that LINC00319 worked as the sponge for miR-423-5p. Furthermore, miR-423-5p directly targeted NACC1. qRT-PCR and western blot results demonstrated that LINC00319 upregulates NACC1 expression through inhibiting miR-423-5p in ovarian cancer cells. Moreover, we observed an inverse expression correlation between miR-423-5p and LINC00319 or between miR-423-5p and NACC1 in ovarian cancer tissues. Finally, rescue assay showed that NACC1 restoration rescued the potentials of proliferation, migration and invasion in LINC00319-depleted ovarian cancer cells. In conclusion, our findings demonstrated that LINC00319 promotes ovarian cancer progression through upregulating NACC1 expression by restraining miR-423-5p.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Short-term administration of iso-α-acids increases transthyretin transcription in the hippocampus

Cognitive decline and dementia are currently recognized as major problems in the aging population; however, there is still no promising treatment for these conditions. Previously, our group reported that iso-α-acids (IAAs), which are hop-derived bitter components present in beer, prevent inflammation and cognitive impairment in an Alzheimer's disease model mice (5xFAD mice) and yield significant reduction in amyloid β (Αβ) in the brain. However, data on the molecular mechanisms underlying these physiological effects of IAAs remain limited. Here, we used transcriptome analysis and found that oral administration of IAAs to 5xFAD mice for 7 days induces a 58.9-fold increase in the expression of transthyretin (TTR; Ttr) in the hippocampus compared with controls. In addition, real-time quantitative PCR showed that oral administration of IAAs significantly increased Ttr transcription in the hippocampi of wild type C57BL/6J mice but not in the cerebral cortex. TTR is an Αβ protein scavenger; thus, an increase in its expression could prevent amyloid aggregate formation. These results indicate that IAAs reduce Αβ in the brain by elevating TTR levels.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Reduction and bacterial adsorption of dissolved mercuric ion by indigenous bacteria at the Oak Ridge Reservation site

Mercury exists in various forms in the environment and the indigenous bacteria mediated processes have the potential to be used for mercury remediation. In this study, two mixed cultures of indigenous bacteria at the Oak Ridge Reservation site (i.e., ORR soil culture and ORR sediment culture) were selected to study the microbial mediated mercuric reduction under an aerobic condition as well as mercury adsorption onto bacterial surfaces. PCR analysis was performed to provide insights into the microbial community. The mercuric volatilizing experiment demonstrated the mercuric reducing capacity for both ORR cultures, in which the Pseudomonas genus was the dominating Hg 0 producer. The investigation of the impact of the sole carbon source revealed the energy-dependent characteristics of the mercuric reduction in this study. Namely, the mercuric reduction was nearly not impacted by the type of carbon source but positively related to the energy that a unit amount of substrate could provide. The study also indicated that the mercury adsorption competed with the reduction. According to the fitting of the Langmuir isotherm, the ORR soil culture was found to have a higher mercury adsorption capacity (i.e., 67.5 mg Hg/g dry biomass) than the ORR sediment culture (i.e., 53.1 mg Hg/g dry biomass). The negative correlation between the reduced mercury mass and adsorbed mercury mass was identified for both ORR cultures.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2016

Environmental product declarations in accordance with EN 15804 and EN 16485 — How to account for primary energy of secondary resources?

As a core product category rule (PCR), EN 15804 defines rules for conducting the life cycle assessment (LCA) of building products in the context of environmental product declarations (EPDs). This European standard is complemented by EN 16485, which provides further guidance for specific aspects for the LCA of wood and wood-based construction products. For all life cycle stages under consideration, the renewable and non-renewable primary energy employed for energy generation or material use is accounted for. Furthermore, the inputs and outputs of secondary materials (SM), renewable secondary fuels (RSF) and non-renewable secondary fuels (NRSF) have to be reported. Especially in the end-of life stage as well as in the production stage, the standards do not exactly rule the accounting method of the primary energy contained in SM, RSF and NRSF. As both standards leave room for interpretation, we wrote this discussion article to introduce this issue to the LCA community and to present our developed accounting specifications. In general, we consider EN 15804 and EN 16485 as helpful tools for the LCA of building products. We hope that our ideas on certain aspects contribute to a better understanding of the standards, possibly leading to further improvement in the course of the standardization process.

09 BIOMASS FUELS↗

DOE OSTI2014

Cytotoxicity of TiO{sub 2} nanoparticles towards freshwater sediment microorganisms at low exposure concentrations

There is a persistent need to assess the effects of TiO{sub 2} nanoparticles on the aquatic ecosystem owing to their increasing usage in consumer products and risk of environmental release. The current study is focused on TiO{sub 2} nanoparticle-induced acute toxicity at sub-ppm level (≤1 ppm) on the three different freshwater sediment bacterial isolates and their consortium under two different irradiation (visible light and dark) conditions. The consortium of the bacterial isolates was found to be less affected by the exposure to the nanoparticles compared to the individual cells. The oxidative stress contributed considerably towards the cytotoxicity under both light and dark conditions. A statistically significant increase in membrane permeability was noted under the dark conditions as compared to the light conditions. The optical and fluorescence microscopic images showed aggregation and chain formation of the bacterial cells, when exposed to the nanoparticles. The electron microscopic (SEM, TEM) observations suggested considerable damage of cells and bio-uptake of nanoparticles. The exopolysaccrides (EPS) production and biofilm formation were noted to increase in the presence of the nanoparticles, and expression of the key genes involved in biofilm formation was studied by RT-PCR. - Highlights: • Toxicity of NPs towards freshwater sediment bacteria at sub-ppm concentrations. • Decreased toxicity of the nanoparticles in the consortium of microorganisms. • Enhanced bacterial resistance through EPS and biofilm formation in the presence of NPs. • Considerable surface damage of cells and internalization of NPs. • Gene expression analyses related to biofilm formation in the presence of NPs.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2015

Evaluation of the effect of divalent metal transporter 1 gene polymorphism on blood iron, lead and cadmium levels

Divalent metal transporter 1 (DMT1), a member of the proton-coupled metal ion transporter family, mediates transport of ferrous iron from the lumen of the intestine into the enterocyte and export of iron from endocytic vesicles. It has an affinity not only for iron but also for other divalent cations including manganese, cobalt, nickel, cadmium, lead, copper, and zinc. DMT1 is encoded by the SLC11a2 gene that is located on chromosome 12q13 in humans and express four major mammalian isoforms (1A/+IRE, 1A/-IRE, 2/+IRE and 2/-IRE). Mutations or polymorphisms of DMT1 gene may have an impact on human health by disturbing metal trafficking. To study the possible association of DMT1 gene with the blood levels of some divalent cations such as iron, lead and cadmium, a single nucleotide polymorphism (SNP) (IVS4+44C/A) in DMT1 gene was investigated in 486 unrelated and healthy individuals in a Turkish population by method of polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). The genotype frequencies were found as 49.8% homozygote typical (CC), 38.3% heterozygote (CA) and 11.9% homozygote atypical (AA). Metal levels were analyzed by dual atomic absorption spectrometer system and the average levels of iron, lead and cadmium in the blood samples were 446.01±81.87 ppm, 35.59±17.72 ppb and 1.25±0.87 ppb, respectively. Individuals with the CC genotype had higher blood iron, lead and cadmium levels than those with AA and CA genotypes. Highly statistically significant associations were detected between IVS4+44 C/A polymorphism in the DMT1 gene and iron and lead levels (p=0.001 and p=0.036, respectively), but no association was found with cadmium level (p=0.344). This study suggested that DMT1 IVS4+44 C/A polymorphism is associated with inter-individual variations in blood iron, lead and cadmium levels. - Highlights: • DMT1 IVS4+44 C/A polymorphism is associated with inter-individual variations in blood iron, cadmium and lead levels. • Individuals with the DMT1 IVS4+44 CC genotype had higher blood iron, lead and cadmium levels. • Individuals with DMT1 IVS4+44 CC genotype may be more susceptible for metal toxicity.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2015

Maternal hemochromatosis gene H63D single-nucleotide polymorphism and lead levels of placental tissue, maternal and umbilical cord blood

Human hemochromatosis protein (HFE), a major histocompatibility complex class I-like integral membrane protein, participates in the down regulation of intestinal iron absorption by binding to transferrin receptor (TR). HFE competes with transferrin-bound iron for the TR and thus reduces uptake of iron into cells. On the other hand, a lack of HFE increases the intestinal absorption of iron similarly to iron deficiency associated with increasing in absorption and deposition of lead. During pregnancy, placenta cannot prevent transfer lead to the fetus; even low-level lead poisoning causes neurodevelopmental toxicity in children. The aim of this study was to determine the association between the maternal HFE H63D single-nucleotide polymorphism and lead levels in placental tissue, maternal blood and umbilical cord bloods. The study population comprised 93 mother–placenta pairs. Venous blood from mother was collected to investigate lead levels and HFE polymorphism that was detected by standard PCR–RFLP technique. Cord bloods and placentas were collected for lead levels which were analyzed by dual atomic absorption spectrometer system. The HFE H63D genotype frequencies of mothers were found as 75.3% homozygote typical (HH), 23.6% heterozygote (HD) and 1.1% homozygote atypical (DD). Our study results showed that the placental tissue, umbilical cord and maternal blood lead levels of mothers with HD+DD genotypes were significantly higher than those with HH genotype (p<0.05). The present study indicated for the first time that mothers with H63D gene variants have higher lead levels of their newborn's placentas and umbilical cord bloods. - Highlights: • Mothers with H63D gene variants have higher lead levels of their newborn's umbilical cord blood. • Unborn child of women with HD+DD genotypes may be at increased risk of internal exposure to lead. • Maternal HFE status may have an effect on increased placenta, maternal and cord blood lead levels. • Maternal HFE status may have an effect on lead transfer from maternal to fetal circulation. • Placental, maternal and cord blood lead levels were not correlated with mothers' age.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

Identifying risk factors for exposure to culturable allergenic moulds in energy efficient homes by using highly specific monoclonal antibodies

The aim of this study was to determine the accuracy of monoclonal antibodies (mAbs) in identifying culturable allergenic fungi present in visible mould growth in energy efficient homes, and to identify risk factors for exposure to these known allergenic fungi. Swabs were taken from fungal contaminated surfaces and culturable yeasts and moulds isolated by using mycological culture. Soluble antigens from cultures were tested by ELISA using mAbs specific to the culturable allergenic fungi Aspergillus and Penicillium spp., Ulocladium, Alternaria, and Epicoccum spp., Cladosporium spp., Fusarium spp., and Trichoderma spp. Diagnostic accuracies of the ELISA tests were determined by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2-encoding regions of recovered fungi following ELISA. There was 100% concordance between the two methods, with ELISAs providing genus-level identity and ITS sequencing providing species-level identities (210 out of 210 tested). Species of Aspergillus/Penicillium, Cladosporium, Ulocladium/Alternaria/Epicoccum, Fusarium and Trichoderma were detected in 82% of the samples. The presence of condensation was associated with an increased risk of surfaces being contaminated by Aspergillus/Penicillium spp. and Cladosporium spp., whereas moisture within the building fabric (water ingress/rising damp) was only associated with increased risk of Aspergillus/Penicillium spp. Property type and energy efficiency levels were found to moderate the risk of indoor surfaces becoming contaminated with Aspergillus/Penicillium and Cladosporium which in turn was modified by the presence of condensation, water ingress and rising damp, consistent with previous literature. - Highlights: • Monoclonal antibodies were used to track culturable allergenic moulds in homes. • Allergenic moulds were recovered from 82% of swabs from contaminated surfaces. • The mAbs were highly specific with 100% agreement to PCR of recovered fungi. • Improvements to energy efficiency lowered risk of exposure to allergenic fungi.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2016

Hypermethylation of gene promoters in peripheral blood leukocytes in humans long term after radiation exposure

Some human genes known to undergo age-related promoter hypermethylation. These epigenetic modifications are similar to those occurring in the course of certain diseases, e.g. some types of cancer, which in turn may also associate with age. Given external genotoxic factors may additionally contribute to hypermethylation, this study was designed to analyzes, using methylation-sensitive polymerase chain reaction (PCR), the CpG island hypermethylation in RASSF1A, CDKN2A (including p16/INK4A and p14/ARF) and GSTP1 promoters in peripheral blood leukocytes of individuals exposed to ionizing radiation long time ago. One hundred and twenty-four irradiated subjects (24–77 years old at sampling: 83 Chernobyl Nuclear Power Plant clean-up workers, 21 nuclear workers, 20 residents of territories with radioactive contamination) and 208 unirradiated volunteers (19–77 years old at sampling) were enrolled. In addition, 74 non-exposed offspring (2–51 years old at sampling) born to irradiated parents were examined. The frequency of individuals displaying promoter methylation of at least one gene in exposed group was significantly higher as compared to the control group (OR=5.44, 95% CI=2.62–11.76, p=3.9×10{sup −7}). No significant difference was found between the frequency of subjects with the revealed promoter methylation in the group of offspring born to irradiated parents and in the control group. The increase in the number of methylated loci of RASSF1A and p14/ARF was associated with age (β=0.242; p=1.7×10{sup −5}). In contrast, hypermethylation of p16/INK4A and GSTP1 genes correlated with the fact of radiation exposure only (β=0.290; p=1.7×10{sup −7}). The latter finding demonstrates that methylation changes in blood leukocytes of healthy subjects exposed to radiation resemble those reported in human malignancies. Additional studies are required to identify the dose-response of epigenetic markers specifically associating with radiation-induced premature aging and/or with the development of age-associated cancer and non-cancer diseases. - Highlights: • Hypermethylation of genes was found in blood leukocytes in irradiated humans. • Hypermethylation of genes was revealed long term after radiation exposure. • Age- and radiation-related methylation changes were identified. • Revealed methylation changes resemble those described in malignant cells.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗

DOE OSTI2016

Occurrence of vancomycin-resistant and -susceptible Enterococcus spp. in reclaimed water used for spray irrigation

Reclaiming municipal wastewater for agricultural, environmental, and industrial purposes is increasing in the United States to combat dwindling freshwater supplies. However, there is a lack of data regarding the microbial quality of reclaimed water. In particular, no previous studies have evaluated the occurrence of vancomycin-resistant enterococci (VRE) in reclaimed water used at spray irrigation sites in the United States. To address this knowledge gap, we investigated the occurrence, concentration, and antimicrobial resistance patterns of VRE and vancomycin-susceptible enterococci at three U.S. spray irrigation sites that use reclaimed water. We collected 48 reclaimed water samples from one Mid-Atlantic and two Midwest spray irrigation sites, as well as their respective wastewater treatment plants, in 2009 and 2010. Samples were analyzed for total enterococci and VRE using standard membrane filtration. Isolates were purified and then confirmed using biochemical tests and PCR. Antimicrobial susceptibility testing was conducted using the Sensititre® microbroth dilution system. Data were analyzed by two-sample proportion tests and one-way analysis of variance. We detected total enterococci and VRE in 71% (34/48) and 4% (2/48) of reclaimed water samples, respectively. Enterococcus faecalis was the most common species identified. At the Mid-Atlantic spray irrigation site, UV radiation decreased total enterococci to undetectable levels; however, subsequent storage in an open-air pond at this site resulted in increased concentrations of enterococci. E. faecalis isolates recovered from the Mid-Atlantic spray irrigation site expressed intrinsic resistance to quinupristin/dalfopristin; however, non-E. faecalis isolates expressed resistance to quinupristin/dalfopristin (52% of isolates), vancomycin (4%), tetracycline (13%), penicillin (4%) and ciprofloxacin (17%). Our findings show that VRE are present in low numbers in reclaimed water at point-of-use at the sampled spray irrigation sites; however, resistance to other antimicrobial classes is more prevalent, particularly among non-E. faecalis isolates. - Highlights: • Enterococci were recovered in 71% of reclaimed water samples. • Vancomycin-resistant enterococci were detected in 4% of reclaimed water samples. • UV radiation at irrigation sites reduced enterococci to undetectable levels. • Storage of reclaimed water in open-air ponds increased levels of enterococci.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Cecropin P1 antimicrobial peptide modulates differential expression of immune relevant genes in rainbow trout ( Oncorhynchus mykiss ) gill cell line, RTgill-W1

Accumulated evidence indicates that antimicrobial peptides modulate immune activities in fish. In this study, we profiled the differential expression patterns of representative immune relevant genes in an epithelial-like cell line of rainbow trout gill, RTgill-W1, in response to exposure of cecropin P1 antimicrobial peptide. RTgill-W1 cells were treated with synthetic cecropin P1 over time (0, 2, 4 and 24 h) with or without the present of lipopolysaccharide (LPS) or polyinosinic polycytidylic acid (PolyI:C). The relative abundances of each mRNA were measured by real-time quantitative PCR. The dose-response study revealed significant perturbations of mRNA levels of genes related to pro-inflammation, acute phase, surface proteins and transcription factors at 30 μM of cecropin P1. In addition, cecropin P1 altered the differential expression patterns that were induced by LPS or PolyI:C, at different time points in RTgill-W1. Overall, our results indicate that cecropin P1 exhibits pro-inflammation activity, modulate cell-cell interaction and cytokine signal transduction in rainbow trout gill cell, and may suggest a potential application of this peptide as an immune adjuvant for disease control in aquaculture.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Fluorescence In Situ mRNA Hybridization for Gene Expression Detection in a Wood Decay Fungus

Wood-degrading fungi efficiently decompose wood using a series of steps that are regulated across time and space. RNA sequencing with “bulk” sampling method has indicated processes controlled at the transcription level at different decay stages and locations along hyphal networks. To further understand these regulatory mechanisms, a high-resolution technique for gene expression determination is required. In this work, Fluorescence In Situ Hybridization (FISH) of mRNA was tested and optimized in the ‘brown rot’-type (carbohydrate-selective; lignin-rich residues) model fungus Postia (Rhodonia) placenta to image gene expression at the hyphal scale, <10 micrometers. The mRNA of one housekeeping gene Tef1a and three wood-decaying genes Cel28a, Cel5b and Qrd1 were tested in hyphae grown in liquid nutrients and wood substrate. The 20 bp-long DNA probes were successfully delivered intracellularly to give sufficient hybridization signal for imaging. Ahead of hybridization, permeabilization by lyticase and proteinase K treatments proved to be the critical step to increase probe and mRNA accessibility. Tyramide signal amplification was used after hybridization to enhance signal. This gave qualitative measures of mRNA of the four target genes, which were generally in line with previous expression patterns generated from coarser-scale RNA-seq and qRT-PCR studies. These techniques were not without challenges, and future optimization using multiple probe fragments for each mRNA molecule is proposed.

Brown rot fungi, decomposition, FISH, gene express↗

DOE OSTI2017

Levan promotes antiproliferative and pro-apoptotic effects in MCF-7 breast cancer cells mediated by oxidative stress

Exopolysaccharides are high-valued bio-products produced by various microbial species and have been described to possess biological response modifying activities. These bio-products have been effective as therapeutic agents in various human disease conditions. The objective of this study was to examine the effects of levan (a (2 → 6)-β-d-fructan) produced on sucrose by the halophilic bacterium, Halomonas smyrnensis AAD6T, in human breast cancer MCF-7 cells. MCF-7 cells were exposed to levan for 24 and 48 h. The antiproliferative activity was analyzed by the MTT assay. Oxidative stress was measured by the CM-H2DCFDA assay, and cell apoptosis was analyzed by the caspase-3/7 assay. Cell cycle was analyzed by flow cytometry and gene expression was determined by RT-PCR. Levan showed a time- and concentration-dependent antiproliferative activity, and this effect was associated with an increase in cell apoptosis and oxidative stress. In addition, levan increased the gene expression of p53 and p27. Here we demonstrated that levan exhibited an antiproliferative effect that was mediated by an increase in apoptosis and oxidative stress.

Queiroz, Eveline A. I. F.↗

DOE OSTI2014

Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA is composed of a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work a pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the β-ketosynthase and acyl-transferase domains of the OTA PKSs which had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both the two domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger, and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A qRT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed with AcOTApks which reached its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production.

fungi, Aspergillus, mycotoxin, ochratoxin, carbona↗

DOE OSTI2013

Hypoxia-Independent Downregulation of Hypoxia-Inducible Factor 1 Targets by Androgen Deprivation Therapy in Prostate Cancer

Purpose: We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred. Methods and Materials: Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvant ADT was assessed by immunohistochemistry. Results: A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT (P<.001). Conclusions: AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to tumor regression. HIF1α expression is probably not a useful hypoxia biomarker during ADT in prostate cancer.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

DOE OSTI2014

Long-Term Results of a Randomized Trial in Locally Advanced Rectal Cancer: No Benefit From Adding a Brachytherapy Boost

Purpose/Objective(s): Mature data on tumor control and survival are presented from a randomized trial of the addition of a brachytherapy boost to long-course neoadjuvant chemoradiation therapy (CRT) for locally advanced rectal cancer. Methods and Materials: Between March 2005 and November 2008, 248 patients with T3-4N0-2M0 rectal cancer were prospectively randomized to either long-course preoperative CRT (50.4 Gy in 28 fractions, per oral tegafur-uracil and L-leucovorin) alone or the same CRT schedule plus a brachytherapy boost (10 Gy in 2 fractions). The primary trial endpoint was pathologic complete response (pCR) at the time of surgery; secondary endpoints included overall survival (OS), progression-free survival (PFS), and freedom from locoregional failure. Results: Results for the primary endpoint have previously been reported. This analysis presents survival data for the 224 patients in the Danish part of the trial. In all, 221 patients (111 control arm, 110 brachytherapy boost arm) had data available for analysis, with a median follow-up time of 5.4 years. Despite a significant increase in tumor response at the time of surgery, no differences in 5-year OS (70.6% vs 63.6%, hazard ratio [HR] = 1.24, P=.34) and PFS (63.9% vs 52.0%, HR=1.22, P=.32) were observed. Freedom from locoregional failure at 5 years were 93.9% and 85.7% (HR=2.60, P=.06) in the standard and in the brachytherapy arms, respectively. There was no difference in the prevalence of stoma. Explorative analysis based on stratification for tumor regression grade and resection margin status indicated the presence of response migration. Conclusions: Despite increased pathologic tumor regression at the time of surgery, we observed no benefit on late outcome. Improved tumor regression does not necessarily lead to a relevant clinical benefit when the neoadjuvant treatment is followed by high-quality surgery.

62 RADIOLOGY AND NUCLEAR MEDICINE↗