bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of 25‐hydroxyvitamin D3 (25OHD3) and 25‐hydroxyvitamin D2 (25OHD2) in human serum using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and its comparison with a radioimmunoassay method
Authors: Chen Huiping , McCoy Leslie F , Schleicher Rosemary L , Pfeiffer Christine M
Published: June 2021

Serum samples (200‐μL) were treated with acetonitrile and centrifuged to remove protein followed by online solid‐phase extraction. Calibration solutions (5–100 ng/mL) of 25OHD2 and 25OHD3 were prepared using 4% albumin in phosphate‐buffered saline. Chromatographic conditions: C18 column, isocratic elution with ethanol/water (83/17, v/v). Mass spectrometric conditions: atmospheric pressure chemical ionization in the positive ion mode; mass transitions: 25OHD3, m/z 401.4→383.4; 25OHD2, m/z 413.4→395.4; and the internal standard hexadeuterated‐25OHD3, m/z 407.7→389.7. The retention time for all analytes was <4 minutes. Detection limits were 0.49 ng/mL (25OHD3) and 1.86 ng/mL (25OHD2). For 25OHD3, intra‐ and inter‐assay coefficients of variation were <4% and <10%, respectively, and for 25OHD2 coefficients of variation were <7% and <12%, respectively. Recovery assessed using a stable isotope‐labeled internal standard averaged (±SD) 94±2%. In a method comparison of 551 non‐representative serum specimens from the National Health and Nutrition Examination Survey, the LC‐MS/MS method gave results that were on average 13% higher (95%CI: 11–15%) than the traditionally used DiaSorin radioimmunoassay results. This high throughput candidate reference method requires minimal sample preparation and is suitable for routine use for analysis of vitamin D status.
Suppression of growth, progression and metastasis of breast cancer xenografts by dietary complex sphingolipids
Authors: Ding Shengli , Simon Kirk , Schmelz Eva M
Published: June 2021

Dietary sphingolipids are hydrolyzed in the intestinal tract to bioactive metabolites. In the present study, we investigated if these bioactive can suppress tumor formation, progression and metastasis in distant organs. Human cells representing early, intermediate, late and metastatic stages of breast cancer were injected into the mammary fat pad of nude mice. 0.1% of complex sphingolipids were added to the diet either before or after tumor implant to test both a preventive and an intervention approach. Orally administered sphingolipids affected tumor number and volume in a stage‐dependent manner, with the less aggressive xenografts responding with both lower tumor incidence and tumor volume while the more advanced xenografts responded with a reduction of tumor size only. This appears to correlate with the reduced rate of proliferation that was more affected in xenografts representing earlier stages of breast cancer. However, sphingolipid supplements significantly reduced metastases of MDA‐MB‐231 xenografts to the lungs (60–73%) and bone marrow (60–84%). These results suggest that sphingolipid supplements suppress less aggressive tumors and metastases while they are relatively ineffective against aggressive tumors, indicating a promising potential as chemopreventive agents.

Supported by grant BCT0503453 from the Susan G. Komen Foundation and NIH/NCI R03 CA101125.
Dietary restriction and induced tumorigenesis in mice with altered hypothalamic appetite regulation
Authors: Minor Robin , Percival Susan , de Cabo Rafael
Published: June 2021

Dietary restriction (DR), the reduction of calorie intake or meal frequency, profoundly effects the health of laboratory animals including cancer incidence. One recurring theme among DR manipulations is the induction of hunger, which may play a critical role in mediating the neuroendocrine response and driving beneficial systemic effects like reduced tumorigenesis. We examined the effects of DR on tumorigenesis under appetite suppression in two mouse models: Neuropeptide‐Y knockout mice (NPY, a major orexigenic peptide) and monosodium glutamate‐injected mice (MSG damages the arcuate nucleus, the ‘satiety center’ of the hypothalamus that coordinates feeding behavior). Calorie restriction (CR) at 30% was compared with every‐other‐day (EOD) feeding, a high‐fiber, low‐calorie chow (LC), and ad libitum (AL) feeding. A two‐stage skin tumor induction model was employed to assess tumorigenesis in the groups. While CR lowered blood leptin levels in both the NPY‐knockout and MSG‐injected mice, tumor latency was not increased by CR in the MSG mice. The LC diet lowered blood leptin in both models and reduced tumor incidence in the MSG mice but not the NPY mice. The EOD diet alone was protective in both models. These data suggest DR's anticancer effects involve both hypothalamus‐dependent and hypothalamus‐independent mechanisms. Funded by the NIH/NIA Intramural Research Program.
The mutagenic activity of heterocyclic amines formed in common foods in Saudi Arabia
Authors: Awney Hala Ahned , Sindi Heba Abas
Published: June 2021

Fried liver, spicy grilled chicken and beef Shawerma are common foods regularly consumed in Saudi Arabia. The aim of present study is measuring the mutagenic activity of heterocyclic amines (HAs) extracted from these foods using Ames test with induced rat microsome S9. All samples were prepared in our laboratory and cooked in different restaurants. HAs were extracted by blue rayon method and determined by reversed‐phase HPLC. In unmarinated foods, the highest amount of HAs was observed in beef Shawerma (15.85 ng/g) which mainly contains 7.46 ng MeIQx/g and 8.39 ng Trp‐P1/g. In marinated foods, HAs was only observed in beef Shawerma (9.2 ng /g) which mainly contains 6.59 ng MeIQx/g and 2.61 ng Trp‐P2/g. Significant levels of mutagenic activity were detected in all samples except unmarinated beef Shawerma before cooking. Our data suggested that the mutagenic activity of these foods may be due to HAs and other cooking toxicants especially polycyclic aromatic hydrocarbons (PAHs) since the cooking methods involved intense heat over direct flame. To the best of our knowledge this is the first study on determining HAs in Saudi foods. Clearly, accurate determination of human exposure to HAs and identification of the key enzyme systems involved will be required for rational assessment of its carcinogenic risk among Saudi population.
Effects of diets supplemented with sphingomyelin and Lactobacillus casei and Bifidobacterium bifidum on colon cancer development
Authors: Florencio Marzo , Elena Urdaneta , Jauregui Patricia , Barrenetxe Jaione , Alfaro Maria , Ibañez Francisco , Santidrian Santiago
Published: June 2021

Functional foods, probiotic and prebiotic, could play a modulating function on colon cancer development. In the present study, we have analysed the possible use of Lactobacillus casei (Lc) and Bifidobacterium bifidum (Bb) (108 ufc/ml) and sphingomyelin (SM) (0.05%), determining its effect on intestinal physiology.

Balb/c mice were divided in three groups (n=12) and colon cancer was induced by a dimethilhydrazine injection s.c. for 3 weeks. They were fed for 9 weeks with a control standard diet and milk and standard diet and milk supplemented with the different bacteria strains. Body weight was recorded everyweek. A colon fragment was examined for the presence of aberrant crypt. Jejune was excised and analysed for the expression of SGLT‐1 and PEPT‐1 transporters by western blott. Including Bb/Lc/SM in the diet did affect the final body weight of the animals. In the supplemented diet group the crypt aberrant foci count was smaller (P<0.01) than in other group. SGLT1 expression in the supplemented diet group is significantly higher compared with the cancer colon group and is similar compared with control group. In the expression of PEPT‐1, the supplementation produced a significantly low expression compared with the cancer colon group and it is similar to control group. This data indicate that diets supplemented with symbiotic (Lc, Bb and sphingomyelin) shown beneficial effects related to colon cancer development.
Oral Intake of Genistein Changes DNA Methylation at the Promoters of Rat Colon Genes
Authors: Zheng Shasha , Wang Zuguang , Pan Yuan‐Xiang
Published: June 2021

Colon cancer is the third most frequently diagnosed form of cancer with lethal malignancy as well as the second leading cause of cancer‐related deaths in the western world. The phytoestrogen genistein has been demonstrated to possess antitumor properties by influencing colon cell proliferation. The present study examined the effects of oral intake of genistein at concentrations of 0.5mg and 1mg in sucrose pellets on DNA methylation status. Methylation sensitive PCR was performed to analyze methylation levels of the putative CpG islands at promoter regions of several genes. Preliminary study indicated that genistein of 0.5mg could significantly increase DNA methylation at the CpG island of glucose transporter 4 (GLUT4) companied with lower mRNA expression by quantity real time PCR. But no statistical results observed for methyguanine DNA‐methytransferase (MGMT). Results suggested genistein supplement could change gene expression through DNA methylation that providing novel insights into nutrient effects on epigenetic modifications for cancer invention.
Effects of obesity and 7,12‐dimethylbenz(a)anthracene (DMBA) treatment on hepatic CYP 1A1 expression in ovariectomized obese Zucker rats
Authors: Hakkak Reza , Shaaf Saeid , Dunn Haley , MacLeod Stewart , Fuchs George , Korourian Soheila
Published: June 2021

There is a link between obesity and increased risk of breast cancer among post‐menopausal women. Recently, we have shown that obesity increases the rate of the DMBA‐induced mammary tumor development in intact and ovariectomized Zucker rats. Several data suggest that DMBA require metabolic activation in order to exert their carcinogenic effects. The objective of this study was to investigate the effect of obesity on hepatic expression of CYP1A1 following treatment in obese and lean ovariectomized female Zucker rats. Forty day‐old, ovariectomized obese (n=20) and lean (n=20), female Zucker rats were kept on AIN‐93 G diet and 10 days later were orally gavaged with either with sesame oil (control) or with 65 mg/kg DMBA in sesame oil and were sacrificed 24 hrs post‐DMBA treatment. Liver microsomes were prepared and CYP 1A1 expression were measured by Western blotting using goat anti‐mouse CYP 1A1. DMBA treatment significantly (p<0.001) increased expression of CYP 1A1 in lean and obese ovariectomized rats compared to control group. Obese rats had higher basal CYP 1A1 expression compared to lean rats. These data suggest that obesity can increase expression of enzymes that are responsible for DMBA metabolism using ovariectomized zucker rat model. Also, DMBA treatment will increase the expression of CYP 1A1. (Supported by Susan G. Komen Foundation and ABI to RH).
College Students Dietary and Lifestyle Practices Compared to Recommendations of the American Institute for Cancer Research and World Cancer Research Fund
Authors: Burke Joanne Delaney , Lofgren Ingrid Elizabeth , Morrell Jesse Stabile , Reilly Ruth Ann
Published: June 2021

In fall 2007, World Cancer Research Fund and American Institute for Cancer Research released Food, Nutrition, Physical Activity and the Prevention of Cancer: a Global Perspective. Body mass index (BMI) ranges were further delineated & recommendations to decrease cancer risk proposed. Data from the University of New Hampshire's cross sectional Young Adult Health Risk Screening Initiative were reviewed to evaluate selected criteria. Anthropometric data (measured on site) & dietary records (Diet Analysis +7.0 and 8.0) were analyzed from 1264 students 18–24 yrs (mean age 19.2±1.3). BMI was significantly higher in males than females (p<0.001). No males and 3.3% of females had a BMI of <18.5; only 27.9% males and 44.3% females had a BMI of 18.50–22.99, while 26.7% males and 24.9% of females had a BMI of 23.00–24.99. For overweight, 23.8% of the males and 14.6% of the females had a BMI of 25.00–27.49 while 10.5% of males and 7.1% of females had a BMI of 27.50–29.99. A BMI ≥ 30.00 was found in 11.0% of males and 5.9% of females. Sodium intake was significantly higher in males than females (p<0.00l); it exceeded 2400 mg/day for 92.2% of males and 69.2% of females. For exercise, 21.2% of males and 30.2% of females reported < 30 minutes daily activity. Findings suggest diet & lifestyle practices of college students increase their risk for future cancer development. Supported in part by Office of Sustainability, NH Agr. Exper. Station #11R571.
Geranylgeraniol suppresses the proliferation of murine melanoma cells and human prostate tumor cells
Authors: Katuru Rajasekhar , Fernandes Nicolle , Hynds DiAnna L. , Mo Huanbiao
Published: June 2021

The rate‐limiting activity of the mevalonate pathway, 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase, provides essential intermediates for the prenylation or dolichylation of growth‐related proteins including nuclear lamins, Ras and growth factor receptors. A diterpene geranylgeraniol accelerates HMG CoA reductase degradation and suppresses the growth of human liver, lung, ovary, pancreas, colon, stomach, and blood tumors. We evaluated geranylgeraniol in murine and human cells. Geranylgeraniol induced dose‐dependent suppression of the cell proliferation following 48 (murine B16 melanoma) or 72 (human DU145 prostate carcinoma) h incubations in 96‐well plates. Cell cycle arrest at the G1 phase, manifested by a dose‐dependent increase in G1/S ratio, and apoptosis detected by fluorescence microscopy following acridine orange and ethidium bromide dual staining, contribute to the growth suppression in DU145 cells. The impact of geranylgeraniol on B16 cell proliferation was attenuated by supplemental mevalonate. Geranylgeraniol also attenuated the growth of human LNCaP prostate carcinoma and PC‐3 prostate adenocarcinoma cells. Mouse 3T3‐L1 fibroblasts were more resistant than B16 cells to geranylgeraniol. The differential sensitivity between neoplastic and normal cells points to the potential application of geranylgeraniol to cancer prevention. TDA, TWU REP & Summer Stipend Award.
Red Bull Energy Drink and its affect on vasomotor stimulation and pain perception
Authors: Ragsdale Frances Ruth , Hooks Tisha L , Wilson Ted
Published: June 2021

The effects of Red Bull Energy Drink (RB) were examined using the cold pressor (CP) test for autonomic nervous system activation and pain determinations in college‐aged volunteers. Volunteers were allowed to relax 15 min prior to testing. Heart rates (HR) and Blood pressures (BP) were taken prior to the CP test (submersion of the hand in 5°C water bath). Pain threshold and pain tolerance were recorded during the test. The intensity of pain felt during the trial was recorded using the visual analogy scale (VAS) for pain assessment. BP was again evaluated immediately after the trial. The volunteers were given a single serving of RB and the tests were repeated 30 minutes postprandially. Preliminary findings suggest that men and women react differently to RB. There was a significant difference in heart rate in women prior to RB and a significant difference in heart rate in men post RB consumption. RB had no significant effect on BP. Likewise, there was no difference in pain threshold; however, there appeared to be a significant increase in pain tolerance after RB consumption. This study suggests that a single serving of RB is not associated with any dramatic physiological change in healthy college‐aged individuals.
Effect of long‐term intake of natural and artificial sweeteners in rats
Authors: Tovar‐Palacio Claudia , Durán‐de‐Bazúa Carmen , Tinajero Claudia Martínez , Gonzalez‐Filomeno Esteban , Garcia‐Gomez Rolando , Constantino‐Casas Fernando , Gracia Isabel
Published: June 2021

The use of sugar substitutes has been increased but its use is controversial. The aim of the present work was to study the effect of different natural and artificial sweeteners on obesity promotion and abnormalities due to lipids in the liver and heart. An experimental design using five nine‐rats groups was established using two natural and two artificial sweeteners during 73d: 1 Fructose 15%; 2 Sucrose 10%; 3 Aspartame 0.3%; 4 Sucralose 0.19%; and 5 Water as control. All animals received a standard diet ad libitum. Body weight, food and water intake were measured three times a week. Rats were sacrificed and heart and liver were removed for histological analysis. Results indicated that long‐term fructose intake in water promoted the highest weight gain in the rats, as well as the highest increase in pericardic tissue. Rats that consumed sucrose, although the ingestion of water volume was the highest, they had the lowest food consumption, and the lowest body weight gain. Animals on artificial sweeteners, showed the highest food intake and the pericardic fat areas were much higher than control groups. These experiments show a direct relationship of gain weight, alterations in food intake and alterations heart and hepatic histology of rats on natural and artificial substances.
Expression and Activity of CYP3A Enzymes in the Liver of Piglets Fed Dairy‐ or Soy‐Based Formula in Comparison to Breast Feeding
Authors: Ronis Martin J , Chen Ying , Badeaux Jamie , Pittman Trae , Ferguson Matt , Helm Ricki , Badger Thomas M
Published: June 2021

We examined induction of CYP3A expression and activity in hepatic microsomes prepared from 21 d old male and female piglets either breast‐fed via the sow or fed dairy‐based or soy‐based formulas. Dairy formula‐feeding reduced CYP3A activity 20–40% (p < 0.05) compared to breast‐feeding. Soy‐formula increased CYP3A activities 2–3‐fold relative to diary‐formula fed piglets. These effects were more pronounced in females. A similar pattern of diet effects was observed on CYP3A protein expression measured in Western blots using antibodies raised against both rat CYP3A1 and human CYP3A4. Examination of expression patterns of mRNAs coded by the 4 known porcine CYP3A genes: CYP3A22, CYP3A29, CYP3A39 and CYP3A46 using quantitative RT‐PCR revealed increases in hepatic CYP3A29 mRNA in soy‐formula‐fed piglets of both sexes relative to dairy formula‐fed piglets and increases in CYP3A22 mRNA in soy‐formula fed females (p < 0.05). However, increases were only 15–20% suggesting diet effects on neonatal porcine CYP3A protein expression and activity are largely post‐transcriptional. No diet effects were observed on polyribosomal distribution of CYP3A29 mRNA, suggesting little effect on mRNA translation. These data suggest that formula feeding has significant effects on hepatic CYP3A activity and may alter clearance of pediatric medications relative to breast feeding. Supported in part by ARS CRIS #6251‐51000‐005‐00D.
DNA damage in HT‐29 colon cancer cells is enhanced by high concentrations of the anthocyanin malvidin
Authors: Patterson Sarah Jane , Fischer Joan G , Dulebohn Rachel V
Published: June 2021

Non‐nutritive phytochemicals such as anthocyanins may reduce risk of chronic disease in part via antioxidant activity. However it is possible that at higher concentrations they may act as prooxidants and thus stimulate oxidative stress and apoptosis. The goal of our study was to determine whether the aglycone anthocyanins malvidin and peonidin act as antioxidants or prooxidants in vitro, with varying doses and cell types. HT‐29 human adenocarcinoma cells were plated in 12 well plates and incubated with 10, 25, or 50 μ mol/L malvidin or peonidin for two hours at 37°C, 95% CO2, 5% air. Cells were then incubated with 75 μ mol/L H2O2 for 5 minutes. The Comet Assay (Trevigen) was performed to determine DNA damage, and apoptosis was assessed using a DNA fragmentation Cell Death Detection ELISA Plus kit (Roche). Neither malvidin nor peonidin decreased H2O2‐induced DNA damage, and malvidin, but not peonidin, at 50 μ mol/L significantly increased (p<.05) H2O2 induced DNA damage (control (8.5 ± 2.7); H2O2 alone (13.7 ± 2.4); Malvidin 50 μ mol/L, (20.4 ± 3.3)). Data thus far suggests that even at concentrations of 10–50 μ mol/L malvidin and peonidin did not function as antioxidants to decrease H2O2 induced DNA damage, and malvidin at 50 μ mol/L increased H2O2‐induced DNA damage in HT‐29 cells. Support: GA Ag Exp Sta.
Change of Isoflavone Composition and Antioxidative Potential during Manufacturing of Cheonggukjang with Bacillus species
Authors: Kim Jong‐Sang , Lim Ji Sun , Seo Ji Yeon , Lee In Ae , Ha Young Ran , Quintos Lesley , Kim Yong Suk , Kim Jeong Hwan , Lim Jinkyu
Published: June 2021

Cheonggukjang, one of the most widely consumed soyfoods in Korea, was manufactured by fermenting steamed soybeans with B. subtilis MB4 and some other strains. Analysis of HPLC of cheonggukjang samples collected at different fermentation times showed diverse conversion rates from glycosides into aglycones of isoflavones from strain to strain. Genistein and daidzein revealed relatively strong antioxidant activity assessed by DPPH (1,1‐diphenyl‐2‐picryl hydrazyl) and DCFDA (2', 7' ‐dichlorofluorescin diacetate:H2) assays. Furthermore, methanolic extract of cheonggukjang showed much higher antioxidative activity compared to that of soybeans or isoflavones, suggesting the production of strong antioxidant(s) during fermentation of cheonggukjang.
DIETARY SOY PROTEIN PROMOTES UNSATURATION OF HEPATIC TRIGLYCERIDES INCREASING SCD‐1 EXPRESSION IN THE LIVER OF MICE
Authors: Gonzalez‐Granillo Marcela , Granados Omar , Torres Nimbe , Tovar Armando R
Published: June 2021

It has been demonstrated that accumulation of triglycerides in the liver leads to the formation of fatty liver. We have previously demonstrated that consumption of a soy protein (SP) diet prevent the accumulation of hepatic triglycerides. It has been shown that a reduction in the expression of the enzyme stearoyl desaturase 1 (SCD‐1) in the liver is associated with low hepatic lipid accumulation. Thus the aim of the present work was to study whether the consumption of SP reduces lipid accumulation in the liver by regulating SCD‐1 expression. Thus, mice were fed casein (C) or SP diets with and without the addition of 1 % cholesterol. The results showed that mice fed SP diet had lower lipid depots than mice fed C diet, and this was associated with a high saturation index (saturated fatty acids/monounsaturated fatty acids), and a low SCD‐1 expression in comparison with mice fed C diet. Expression of SCD‐1 was not parallel with SREBP‐1 expression, and it is possibly mediated via LXR‐alpha. CONACYT grant 62145‐2007.
Dietary soy protein intake reduces ceramide synthesis in liver and adipose tissue of obese rats
Authors: Torre‐Villalvazo Ivan , Gonzalez Fabiola , Tovar Armando R , Torres Nimbe
Published: June 2021

Obesity is characterized by increased lipid synthesis and its accumulation in non‐adipose tissues as the liver and skeletal muscle leading in the formation of ceramides. Ceramides are lipid‐derived metabolite that promotes insulin resistance and apoptotic cell death. The aim of the present study was to determine the effect of dietary soy protein (SP) on ceramide content in liver, skeletal muscle and adipose tissue of diabetic Zucker and diet‐induced obese rats. Zucker fatty rats fed SP had a significant reduction in hepatic ceramide content, associated with reduced serine‐palmitoil transferase 1 (SPT‐1) and dihydroceramide desaturase (DES) mRNA expression. We found no differences in ceramide content in muscle or adipose tissue. Diet‐induced obese rats fed SP showed a reduction in ceramide content in liver and adipose tissue but not in skeletal muscle. Gene expression of SPT‐1 and DES were only reduced in adipose tissue of obese rats. These results indicate that in Zucker diabetic rats, SP reduced ceramide content in liver by reducing directly hepatic synthesis. On the other hand, the effect of dietary SP in diet‐induced obese rats was primarily in reducing adipose tissue ceramide synthesis, that may lead to a lesser release of ceramide to circulation and hence hepatic uptake and accumulation.
Iodine deficiency in mothers and children in a rural community of Wondogenet in southern Ethiopia
Authors: Bogale Alemtsehay , Stoecker Barbara J. , Abebe Yewelsew , Abuye Cherinet , Gurmu Kassu , Hambidge K. Michael
Published: June 2021

Iodine deficiency is common worldwide and is a major cause of preventable mental retardation. In our study demographic characteristics, food consumption patterns, anthropometric measurements, and iodine status were assessed for ninety‐five women of child‐bearing age and their five‐year‐old children. Mean (±SD) age of the mothers was 29 (6) y and family size was 7.0 (2.6). Mean maternal weight was 53.0 (8.9) kg and height was 159.4 (5.9) cm. Maternal BMI ranged from 15.3 to 29.0 with 14% of the mothers <18.5. Prevalence of goiter was 86.6% in the women. Anthropometric assessment of children revealed 29.7% to be stunted (<−2 Z‐score) and 10% to be underweight (<–2 Z‐score). Goiter prevalence in children was 31.3%. Urinary iodine for all participants was <49 μg/L indicating moderate to severe iodine deficiency. Bioavailability of iodine can be reduced by plant foods such as cabbage and kale, which are among the more frequently consumed foods in the study area that could have been a source of goitrogens. Furthermore, only 2% consumed iodized salt and none consumed fish. The high prevalence of goiter and the low urinary iodine demonstrate serious long and short‐term iodine deficiency in the study area. Efficient and cost effective intervention methods to secure iodine availability are urgently needed. (Supported by NIH Grant R21 TW06729 (Fogarty International Center & the Office of Dietary Supplements) and Hawassa University.
Effect of delayed processing of whole blood on the stability of vitamin B12, folate and transferrin receptor in human serum
Authors: Zhang Mindy , Schleicher Rosemary L , Drammeh Bakary S , Pao Ching‐I , Pfeiffer Christine M
Published: June 2021

As part of a comprehensive assessment of analyte stability in support of international nutrition studies, we compared several analytical methods that use different principles. Vitamin B12: protein binding (Biorad) and immunoassay (Axsym). Folate: protein binding (Biorad) and microbiological (MA) assay. TfR: immuno‐turbidimetry (Hitachi 912) and ELISA (Ramco). To evaluate stability under harsh field conditions, whole blood (WB) collected from healthy volunteers was stored at 32°C for up to 3 days (d) before serum separation. Specimen treatment effects were calculated using relative changes (%) compared with immediately processed WB. Differences > ± 5% were considered significant.

Serum B12 levels (n=27) were unchanged using Axsym; an apparent artifact of incubation of WB at 32°C was measured using the Biorad B12 assay. Significant folate (n=16) losses were measured using either method (greater folate losses with MA versus Biorad). Hitachi serum TfR levels (n=35) were unchanged. Using Ramco, TfR levels were slightly more variable. We conclude that apparent analyte ‘stability’ may be method‐dependent, thus multiple methods should be used to verify findings.
Neurodevelopmental Toxins Deplete Glutathione and Inhibit Folate and Vitamin B12‐Dependent Methionine Synthase Activity: A Link between Oxidative Stress and Autism
Authors: WALY MOSTAFA IBRAHIM‐AHMED , DETH RICHARD
Published: June 2021

Ethanol, arsenic, lead, mercury, aluminum and the vaccine preservative thimerosal are suspected to be etiological factors for neurodegenerative and neurodevelopmental disorders. Autism is a neurodevelopmental disorder characterized by oxidative stress and impaired methylation status, including decreased activity of the folate and vitamin B12‐dependent enzyme methionine synthase (MS). MS‐mediated conversion of homocysteine to methionine is crucial for neurons and all mammalian cells to sustain normal methylation status, involving more than 100 different reactions. Glutathione (GSH) protects MS from oxidative inactivation by reactive oxygen species, while MS inactivation increases GSH synthesis by augmenting transsulfuration. Utilizing SH‐SY5Y cultured human neural cells, we found that a 1 hour pre‐incubation of cells with arsenic, lead, mercury, aluminum and thimerosal potently decreased both hydroxocobalamin (OHCbl) and methylcobalamin (MeCbl)‐based MS activity, although OHCbl exhibited greater sensitivity than MeCbl. At a concentration of 100 nmol, each of these neurodevelopmental toxins caused a 60–70% reduction of intracellular GSH levels. 22 mM (0.1%) ethanol caused a similar inhibition of OHCbl‐ and MeCbl‐based MS activity and a similar decrease in GSH levels. Our findings suggest that heavy metals and ethanol may contribute to the occurrence of neurodevelopmental disorders such as autism via a mechanism that involves oxidative stress and inhibition of MS activity.
Lymphocyte DNA strand breakage declines in Mexican American men with the MTHFR 677TT genotype in a folate deplete environment
Authors: Shin William , Vasquez Marie , Moriarty David , Caudill Marie A
Published: June 2021

This study sought to ascertain the influences of declining folate stores, MTHFR C677T genotype, and varying choline intakes on biomarkers of lymphocyte DNA damage in Mexican American men (n=30) with the MTHFR 677CC (n=11) or TT (n=19) genotype under conditions of controlled nutrient intake. For each subject, lymphocyte DNA damage was assessed at wk 0 and 12 by use of the Comet Assay (single cell gel electrophoresis) and four derived parameters including percentage tail DNA, tail length, extent tail moment and olive tail moment. Although no main effects of the independent variables were detected on metrics of DNA damage, the response of the tail moments to declining folate status tended to differ between the MTHFR genotypes (time × genotype, P=0.08–0.10). Upon stratifying the data by MTHFR C677T genotype, declines (P< 0.05) in all four Comet‐derived DNA damage parameters were observed in men with the 677TT genotype whereas no changes (P>0.73) were observed in the 677CC genotype. These data suggest that a reduction in MTHFR activity in Mexican American men with the MTHFR 677TT genotype poses an advantage of decreased DNA strand breakage possibly by directing more folate to participate in nucleotide biosynthesis reactions. Supported by NIH #S06GM053933 and funds from the California Agricultural Research Initiative.
Standard antenatal care regimen, with or without multivitamins and additional deworming, improve anemia outcomes in pregnant Zanzibari women
Authors: Stoltzfus Rebecca J , Farag Tamer H , Kavle Justine , Khalfan Sabra Said , Khatib Mzee Rajab , Tielsch James
Published: June 2021

Where anemia and parasitic infections are endemic, iron + folic acid supplementation (IFA), single‐dose deworming (DW), and antimalarial treatment (IPT) are recommended in pregnancy, but the benefits of multivitamins (MV) and more deworming are unknown. We studied these enhancements to antenatal care in Zanzibar, where anemia, hookworms and malaria are common. We conducted an open‐label cluster‐randomized trial of standard care (SC=IFA + IPT + single‐dose DW) vs enhanced care (EC= IFA + IPT + MV and multi‐dose DW). In both groups, women with severe anemia [SA, hemoglobin (Hb) < 70 g/L] were given double dose of IFA. 8 antenatal clinics were allocated to SC vs. EC, and 2062 women with fundal height <32 cm were enrolled. Outcomes measured at 32–36 wk gestation were: Hb, incidence of SA in women without SA (n=1939), and cure of SA in those with SA (n=77). Cure was defined as final Hb ≥ 90 g/L or ΔHb > 25 g/L. Baseline Hb was 100 ± 14 g/L. AHb was 9 ± 15 g/L in SC vs. 11 ± 15 g/L in EC (NS). Incidence of SA was 4/855 in EC vs. 25/955 in SC (RR =0.18, p=0.11). In women with SA, the cure rate was 76% and did not differ by treatment. In both groups for all outcomes, longer duration of treatment was associated with better outcome. In sum, EC was not significantly better than SC, but our study lacked power. The large Hb increase, high cure rate for SA, low incidence of SA, and dose‐response effects suggest that both regimens were effective. (Funded by Gates Fndn.)
Effect of a nutrition education program and diet modification in iron deficient anemic boarding school adolescent girls from Southern Benin
Authors: Alaofè Halimatou , Zee John , Dossa Romain , O'Brien Huguette Turgeon
Published: June 2021

The purpose of this study was to evaluate the impact of a multi‐dietary strategy to treat IDA in adolescent girls. A quasi‐experimental design involving 34 intervention and 34 control girls aged 12 to 17 years and suffering from mild IDA was carried out in two boarding schools from southern Benin. IDA was defined as haemoglobin (Hb) between 100–120 g/L and serum ferritin (SF) <20 μg/L or serum ferritin between 20–50 μg/L plus two of the following parameters: serum iron <11 μmol/L, total iron binding capacity >68 μmol/L or transferrin saturation <20%. A 26‐week intervention including 4 weeks of nutrition education combined with an increase in the content and bioavailability of dietary iron in the cafeteria menu for 22 weeks was implemented in the intervention school, but not in the control school. After controlling for baseline variables, Hb and SF were higher (122 vs. 113g/L; P=0.0002; 32 vs. 19μg/L; P=0.04), whereas incidence of anemia (32 vs. 85%; P=0.0003) and of IDA (26 vs. 56%; P=0.04) was lower in the intervention group compared to the control group. Also, nutrition knowledge scores were significantly higher in intervention girls compared to control girls at the end of the study. No significant differences were observed in IPI and malaria status of both groups post‐intervention. These findings indicate that dietary changes to improve available dietary iron can reduce iron deficiency anemia.
Utility of Automated Protocols, Qdots and Quantitative Tissue Image Analysis for in‐situ Biomarker Studies
Authors: Pestano Gary Anthony , Carpenter Anne , Ashworth‐Sharpe Julia , Vanpatten Kristie , Haubein Ned , LeFever Mark , Palting John , Riley Janice , Kosmeder Jerome , Lett Scott , Bieniarz Chris , Miller Philip
Published: June 2021

Analyses of biomarkers in tissues represent a critical component in the development of therapeutics. In oncology drug development, entire signaling pathways may be profiled with a variety of high‐throughput techniques. However, in the field of histology, tissue‐based diagnostics (IHC and ISH) are generally dominated by single‐ or dual‐analyte tests. In addition, manual methods for slide processing promote inconsistency, are labor demanding and time‐consuming. We have developed automated platforms for conducting IHC and ISH in widely available formalin‐fixed paraffin‐embedded (FFPE) tissues. These instruments enable rapid evaluation of gene and protein biomarkers in a high‐throughput format. Further, advances in detection, including Quantum dots (Qdots®), and hyper‐spectral imaging, provide a growing set of tools that complement biomarker studies. We have applied these methods to monitor markers that may be therapeutic targets, prognostic of disease, or surrogates of drug response. This report demonstrates the utility of the platform in the simultaneous detection and quantitation of breast cancer markers including, HER2, ER, PR, and Ki67. In addition to automation, spectral imaging and deconvolution algorithms were critical to the reproducible determination of multiplexed protein expression levels in FFPE samples.
AFPep Enters Breast Cancer Cells via the Endocytic/Lysosomal Pathway
Authors: Kanzaria Mitul , Barroso Margarida , Andersen Thomas
Published: June 2021

AFPep, a therapeutic agent that has been shown to inhibit the growth of human breast cancer xenografts in mice and prevent the development of carcinogen‐induced mammary cancers in rats, is a cyclic 9‐amino acid peptide derived from the active site of alpha‐fetoprotein. However, the mechanism of action for AFPep is currently unknown. Understanding how this peptide enters breast cancer cells is critical to characterize its mechanism of action. We hypothesize that AFPep is internalized into human breast cancer cells via the endocytic pathway. We characterized the ability of AFPep‐Alexa conjugates to be internalized into T47D cells using confocal microscopy. Internalization of AFPep‐Alexa was tested by competition with unconjugated AFPep and co‐localization with differently labeled holo‐transferrin or dextran. AFPep‐Alexa internalization was significantly reduced in the presence of competing amounts of unconjugated AFPep, demonstrating a specific pathway of entry into breast cancer cells. AFPep‐Alexa colocalized with dextran, a fluid‐phase lysosomal marker, but not with holo‐transferrin, an early endosome marker. When T47D cells were treated with BafilomycinA1, a drug that disrupts lysosomal trafficking, AFPep‐Alexa appeared to be trapped in the early endosomes. We conclude that AFPep enters T47D cells via a specific endocytic pathway and is delivered to lysosomes, confirming our hypothesis.
Regulation of the Plasminogen Activator System in the MDA‐MB‐231 Breast Cancer Cell Line
Authors: Gramling Mark Wilson , Hawkins Leah K , Sandhu Emir S , Church Frank C.
Published: June 2021

The objective of this study was to determine the contribution of the urokinase‐type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI‐1) to invasiveness of the MDA‐MB‐231 breast cancer cell line, and to study control of uPA and PAI‐1 expression. Amiloride, a small molecule inhibitor of uPA, and a uPA blocking antibody inhibited invasion of the 231 cells by 30% while a PAI‐1 blocking antibody increased invasion by 50%. Treatment with the PI3K inhibitor LY294002 increased PAI‐1 protein levels in conditioned media while differences in uPA were not detected. However, another PI3K inhibitor, wortmannin, decreased uPA in conditioned media. Semi‐quantitative RT‐PCR indicated LY294002 and wortmannin increased PAI‐1 mRNA levels with a concomitant decrease in uPA mRNA. Amiloride treatment decreased PI3K activity resulting in increased PAI‐1 in conditioned media. Treatment with the receptor associated peptide (RAP), which blocks activity of the low density like lipoprotein receptor responsible for binding and clearing uPA‐PAI‐1 complexes from the cell surface, decreased uPA mRNA and protein levels. These results confirm uPA and PAI‐1 are important contributors to MDA‐MB‐231 invasiveness, PI3K positively regulates uPA while negatively regulating PAI‐1, and the PI3K pathway which regulates uPA and PAI‐1 expression may be activated by the recognition of uPA‐PAI‐1 complexes at the cell surface.
Combined targeting of c‐myb, c‐myc and cdc2 in human colorectal cancer cells
Authors: Abaza Mohamed Salah I , Bahman Abdul Majeed
Published: June 2021

Targeting of growth‐promoting genes with antisense oligonucleotides might provide a useful approach to control the abnormal proliferation of cancer cells. In this study, we examined the combined targeting of c‐myb, c‐myc and cdc2 using antisense oligonucleotides (AS[S]ODNs) in colorectal cancer cell lines. mRNA and protein expression of c‐myb, c‐myc and cdc2 were drastically reduced following treatment with the corresponding AS[S]ODNs. However, combination antigene therapies targeting c‐myb/c‐myc, c‐myb/cdc2, c‐myc/cdc2 or c‐myb/c‐myc/cdc2 demonstrated greater additive and/or synergistic growth inhibitory effects compared to single antigene therapies. The same combined treatment in the presence of cytotoxic drugs produced a marked inhibition of cancer cell growth compared to treatment with either the combination of AS[S]ODNs alone or cytotoxic drugs alone. Combined targeting of c‐myb, c‐myc and cdc2 produced a marked apoptotic effect, downregulation of mRNAs of cdk1, cyclin B1, cdk2, cyclin E1, Bcl2 and BclxL and upregulation of mRNAs of p21, Bax and caspase‐3 compared to single treatments. These results suggest that combination antigene therapy may provide a promising approach for cancer therapy.
Induction of apoptosis in human chondrosarcoma, synovial sarcoma, liposarcoma and fibrosarcoma cell lines by a novel nutrient mixture
Authors: Roomi M. Waheed , Roomi Nusrath , Ivanov Vadim , Niedzwiecki Aleksandra , Rath Matthias
Published: June 2021

Sarcomas of soft tissues account for more than 10% of diagnosed cancer in adults. Once the sarcomas have metastasized, current treatment methods are generally ineffective. We have identified a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract that demonstrates a broad spectrum of antitumor activity. We investigated whether the antitumor effect of a NM on sarcoma cell lines was due to apoptosis.

Chondrosarcoma (SW‐1353), synovial sarcoma (SW‐982), liposarcoma (SW‐872) and fibrosarcoma (HT‐1080) cell lines (ATCC) were cultured in the recommended media and treated with NM in different doses: 0, 100, 500 and 1000 μg/ml. Cell proliferation was measured by MTT assay, morphology by H&E staining, and apoptosis by Live Green Caspase Detection Kit.

The sarcoma cell lines studied showed no toxicity at 100 μg/ml NM, moderate at 500 μg/ml and significant at 1000 μg/ml. When treated with NM at 500 and 1000 μg/ml, obvious apoptotic cells were observed by H&E. The apoptotic cells showed shrinkage with condensed and darkly stained nuclei and strongly acidophilic cytoplasm. Slight apoptosis was observed at 100 μg, moderate at 500 μg/ml and significant at 1000 μg/ml NM for all sarcoma cell lines.

The results suggest that NM induces apoptosis in sarcoma cell lines and raises the possibility of its use as a new therapeutic agent.
EFFECTS OF RESVERATROL ON TRIPLE NEGATIVE (ER‐, PR‐, HER2‐) BREAST CANCER CELLS
Authors: Mangum Carla , Cameron Joseph A. , Herbert Brittney‐Shea
Published: June 2021

Triple negative breast cancer is a subtype of cancer which lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). There is no specific targeted therapy for triple negative breast cancers. Resveratrol, found in the skin of red grapes, is associated with a number of health benefits. Therefore, in order to investigate potential new treatment or chemopreventive options for triple negative breast cancer patients, we investigated the effects of resveratrol on triple negative cell lines. We show that resveratrol inhibits cell proliferation in a dose‐dependent fashion. Results also show that BRCA1 mutant cells are more sensitive to the resveratrol than the wild‐type BRCA1 cells, suggesting that BRCA1 may play a role. Non‐malignant breast cells were not as sensitive to resveratrol as the breast cancer cells lines. Our data also suggest that low concentrations of resveratrol (1–10 nM) may increase cell growth and telomerase activity. These studies provide insight into the dose dependent effects of resveratrol on breast cancer cells and the potential of resveratrol to be included in breast cancer treatment or chemoprevention regimens. NIGMS‐NIH Grant No. R25GM67592
Molecular characterization of aggressive basal cell carcinoma
Authors: Malliah Rajit , Fernandes Neil , Bhattacharya Sid , Seth Anjali , Chen Wen , Mirani Neena , Hameed Meera , Lambert Clark W. , Fernandes Helen
Published: June 2021

A subset of Basal Cell Carcinoma (BCC) tumors characterized by rapid growth, recurrence, deep local invasiveness including dura and/or bone is classified as aggressive. These tumors are histologically similar to the non‐aggressive BCC. We have compared molecular signatures of these tumors.

21 BCC specimens, 6 aggressive and 15 non‐aggressive were used in the study. DNA was extracted from formalin fixed paraffin embedded sections of 21 pairs of normal and tumor tissue. The specimens were subjected to Loss of Heterozygosity (LOH) analysis in the PATCHED gene using capillary electrophoresis, single nucleotide polymorphisms (SNP's) in cytokine promoter genes by real‐time PCR and melt curve analysis, UV specific mutations in the Sonic Hedgehog (SHH) gene and the V600E mutation in the BRAF kinase gene by sequencing and allele specific Taqman probes.

Significant LOH was observed in 54.5% of the aggressive BCC compared to 17.9% of the non‐aggressive BCC. The TNF‐a ‐238 SNP was more prevalent in aggressive (3/6) compared to non‐aggressive BCC (3/12). There was a greater incidence of the IL‐10 ‐1082 SNP ‐ 4/6 in aggressive versus 6/16 in the non‐aggressive BCC. No mutation in the BRAF kinase gene was detected and none of the specimens had mutations in the SHH gene.

Our results indicate an increase in LOH in aggressive BCC compared to non‐aggressive specimens and suggest that aggressive BCC prefer a pro‐inflammatory environment.
Expression of Leukemia/Lymphoma Related Factor (LRF) in Human Breast Carcinoma and Comparison with other Cancers
Authors: Agrawal Anshu , Hunter William J. , Silva Edibaldo D. , Mary Davey S. , Murphy Richard F. , Agrawal Devendra K.
Published: June 2021

Leukemia/lymphoma‐related factor (LRF), a member of the POK family, has a vital function in cellular transformation. Apart from its role in lymphomas, very little is known about its expression in most solid tumors, including breast carcinoma. In this study, LRF mRNA transcripts and protein expression was quantified in benign and malignant breast, colon, liver, lung, renal, and mesothelioma tissues by performing RT‐PCR, Western blot, immunohistochemistry, and immunocytochemistry. All malignant breast tissues expressed significantly more LRF mRNA transcripts and protein than the benign breast tissues. LRF was expressed more strongly in fibroadenoma, invasive lobular carcinoma and ductal carcinoma in situ than in other breast diseases. The ducts in ductal hyperplasia tissues of the breast were weakly positive. In general, a significantly higher expression of LRF was seen in malignant tissue than in the corresponding benign or normal tissue. LRF expression in the carcinoma of the colon, liver, and lung was higher than in the corresponding normal or benign tissues. A strong expression of LRF was observed in normal prostate but not in prostate cancer. These data suggest that LRF could be a marker for malignancy or proliferation of the cancers.

(Supported by the Claire Booth Luce Fellowship to A.A.)
Exacerbation of DSS induced intestinal inflammation following inoculation of BALB/c mice with Mycobacterium avium subsp. paratuberculosis
Authors: johnson charles scott , hostetter jesse micheal
Published: June 2021

Mycobacterium avium subsp. paratuberculosis (M. ptb) DNA has been detected in lesions of human patients with Crohn's disease, a distinct clinical and histologic form of inflammatory bowel disease. However, the significance of this observation remains unresolved. The purpose of the following study was to determine if infection of mice with M. ptb would influence the development of DSS induced intestinal inflammation. BALB/c mice were inoculated intraperitoneally with 1x109 CFU of either live or heat‐killed (HK) M. ptb or saline. At 4–7 months post inoculation, the mice were administered dextran sulfate sodium (DSS) at a suboptimal concentration of 2% in the drinking water for 6 days. After addition of DSS to the drinking water, mouse weights were recorded daily. When the mice were euthanized, sections of small intestine, colon, cecum, liver, and spleen, were collected for histopathology, M. ptb isolation, acid‐fast staining, and for cytokine analysis via qRT‐PCR. Histologic lesion scores were generally more severe and the cecal lengths were generally shorter in the BALB/c mice inoculated with HK M. ptb relative to mice inoculated with live M. ptb or saline. Mice inoculated with either HK or live M. ptb expressed higher levels of IL‐6 and IL‐1 alpha. This data suggests that prior exposure of mice in this model system to M. ptb enhances the colitis that develops after acute exposure to DSS.

This study was funded by an Iowa State University research grant.