bluuebunny/crossref_metadata_2025 huggingface dataset:
Tracking GPCR promiscuity at the source: How receptor conformation is translated to differential function
Authors: Sivaramakrishnan Sivaraj , Malik Rabia U
Published: June 2021

G‐protein coupled receptors (GPCRs) are dynamic sensory proteins that transduce extracellular stimuli into diverse physiological responses. GPCRs are known to adopt multiple structural conformations and differentially activate G‐proteins through direct interactions. However, the molecular mechanisms that translate conformational changes in the GPCR to selective G‐protein activation are poorly understood. In this study, novel FRET sensors for two prototypical GPCRs, opsin and β2 adrenergic receptor (β2‐AR) were used to dissect the GPCR‐G‐protein interaction. The Gα C‐terminus emerged as a selective, nucleotide‐independent sensor of the GPCR active conformation and was used to examine the structural role of intramolecular GPCR interactions. The ionic lock formed by the highly conserved E/DRY motif was necessary and sufficient to stabilize the inactive conformation for β2‐AR but not opsin. Suppression of β2‐AR basal signaling by inverse agonist ICI118,5511 required an intact E/DRY ionic lock. Unlike ICI118,551, another inverse agonist metoprolol was found to switch the β2‐AR interaction from the Gαs to Gαi C‐terminus, resulting in Gαi mediated inhibition of adenylyl cyclase. Together, these findings provide mechanistic insights into the differential function of GPCR ligands along with a novel mechanism to explain GPCR promiscuity.
Ebselen: a covalent inhibitor of the Antigen 85 complex from Mycobacterium tuberculosis
Authors: Favrot Lorenza , Lajiness Daniel H. , Marvin Rachel , Isailovic Dragan , Ronning Donald R.
Published: June 2021

The World Health Organization (WHO) reported 8.7 million new cases of tuberculosis (TB) and 1.4 million deaths in 2011. Co‐infection with Human Immunodeficiency Virus (HIV), as well as the appearance of multidrug and extensively drug‐resistance tuberculosis (MDR‐TB or XDR‐TB), stress the need of discovering new drugs to treat TB. The antigen 85 complex (Ag85) of Mycobacterium tuberculosis (M. tb) includes three mycolyltransferases: Ag85A, Ag85B and Ag85C, involved in the cell wall biosynthesis. The enzymes catalyze the transfer of mycolic acids to trehalose monomycolate (TMM) producing the cord factor trehalose dimycolate (TDM) through a transesterification reaction. The Ag85 complex appears as a valuable drug target to treat TB as inactivation of one of the enzymes affects mycobacterial cell growth in the host; cell wall organization is disrupted and an increase in sensitivity to drugs targeting the cell wall biosynthesis is observed. This study aims to improve methods for inhibitor screening using Ag85C. We report the development of a new high‐throughput assay using resorufin butyrate as an acyl donor. This fluorescence‐based assay provided a Z′ value of 0.82, validating the assay for inhibitor screening. The screening of the NIH clinical collection identified ebselen as a potent inhibitor of M. tb Ag85C. Initial dose‐dependence studies suggested an inhibitory activity in the nanomolar range. However, characterization using mass spectrometry and X‐ray crystallography, gave further insight into the mechanism of action; ebselen binds covalently to the lone cysteine located near the active site of Ag85C, locking the enzyme in an inactive conformation.

Funding from the National Institutes of Health (R15AI089653–01) supported this work.
Effect of Carbamyl (Sevin) on the Activities of Acetyl Cholinesterase and Nitric Acid Synthase in Different Parts of Rat Brain
Authors: Osman Mohamed Yousry Hassan , Osman Hassan M. Y.
Published: June 2021

Carbamyl (sevin) is a wide spectrum carbamate insecticide. Its chemical structure is 1‐naphthalenyl methyl carbamate (C2 H11 NO2), m.w.201. The effect of sevin on both acetyl cholinesterase (AChE) and nitric oxide synthase (NOS) obtained from whole and five different parts of male albino rats namely: frontal cortex, basal ganglia, cerebellum, pons and medulla oblongata were studied. This work was carried out by two experiments: Exp. A: depending on the dose of intraperitoneal infusion of sevin, (0, 20, 30, 40 and 50 mg/kg BW) and decapitated after contstant time 10 min postinfusion, where signs of tremors, shivering, and craving for air. Exp. B:depending on the time course post infusion of a constant dose of sevin 40 mg/kg BW (dose causing 50% inhibition in vivo) The results indicated that sevin inhibited both AChE and NOS in the whole and different parts of the brain, and the degree of the inhibition differs from one part to another. AChE obtained from the pons and the cerebellum were highly inhibited than the other parts. Meanwhile NOS obtained from the pons and frontal cortex were highly inhibited than the other parts. The inhibition of both AChE and NOS was dose and time dependent. The parts which were the most affected by sevin are those responsible for the vital centers and coordination of movement. Medical Research Institute, Alexandria University, EGYPT
Obesity induced fibrinogen formation and its effect on adipocyte inflammation and glucose uptake
Authors: Paton Chad M
Published: June 2021

Obesity and insulin resistance are associated with increased fibrinogen production and inflammation, yet the effect of fibrin degradation products (FDP‐E and FDP‐D) on glucose disposal is completely unknown. We hypothesize that FDP‐E will decrease glucose disposal by increasing inflammation and cytokine production in macrophages.

First, we determined the effect of obesity on fibrinogen expression and fibrin deposition. WT B6 mice fed a high fat diet for 15 weeks increased plasma fibrinogen content along with hepatic fibrinogen expression and FDP‐E content increased 4‐fold. Next, we injected FDP‐E directly into S.C fat which induced inflammation and apoptosis. In fibroblasts, FDP‐E reduced cell viability and induced pro‐fibrotic gene expression. 3T3‐L1 adipocytes were treated with FDP‐E or conditioned media (CM) from FDP‐E treated peritoneal macrophages. We found that FDP‐E and CM suppressed glucose uptake in adipocytes and caused a significant increase in M1 proinflammatory biomarkers such as TNF‐á and IL‐6 in macrophages and mature adipocytes with no appreciable effect on M2 antiinflammatory gene expression.

Fibrinogen expression and deposition is increased with obesity. The increase in fibrinogen expression and fibrin deposition leads to increased adipocyte inflammation and macrophage infiltration which suppresses glucose uptake and may promote adipose tissue fibrosis.
Ultra‐Sensitive Detection of Breast Cancer Stem Cell Specific Surface Marker CD44 based on a Polyvalent Directed Peptide Polymer
Authors: Yoon Moon‐Young , Cho Jun‐Haeng , Lee Sang‐Choon
Published: June 2021

Early detection of the cancer biomarkers have important clinical applications, improves the chances of efficient treatment and cure. CD44, a cancer‐associated membrane glycoprotein involved in cell adhesion and tumor progression, has been implicated as a cancer stem cell antigen in several cancers including breast cancer and thus early detection is crucial. Herein, we screened a peptide library and identified two novel diverse peptides that bind with high affinity to CD44 (KD of ~100 – 200 nM). Further, a polyvalent directed peptide polymer (PDPP), a flexible poly‐D‐lysine polymer conjugated with different target‐binding peptides through N‐succinimidyl‐3‐(2‐pyridyldithio)‐propionate linker, was designed to achieve higher binding affinity and sensitivity than the single peptides. The PDPP constructed with both of the identified peptides shown increased binding affinity (KD of ~18 nM) to CD44 than PDPPs constructed with individual peptides and detects a lower limit of 100 pg/ml. Our work demonstrates a basis to develop novel ultra‐sensitive PDPP as diagnostic probe which may replace antibodies as CD44 detection probes.
An exploration of functional domains in the zebrafish ortholog of human Usher syndrome gene USH2A
Authors: Drake Rachel Rae , Phillips Jennifer , Westerfield Monte
Published: June 2021

Usher syndrome is the most prevalent cause of hereditary deafblindness. Usher type 2A is the most widespread form of the syndrome, caused by mutations in the USH2A gene. USH2A patients experience congenital hearing impairment due to defects in the mechanosensory hair cells of the inner ear and progressive retinal degeneration. USH2A consists of 72 exons encoding the protein Usherin. Several functional domains have been identified and human mutations causing Usher Syndrome have been located in multiple regions of the protein. Determining the relative importance of these functional domains is crucial to the development of effective therapies.

We designed morpholino oligonucleotides to interfere with pre‐mRNA splicing at the 5′ and 3′ regions of the transcript. Protein levels in sensory cells were visualized with antibody staining. Both morpholinos achieved a similar degree of Usherin depletion, suggesting that altered splicing at either the 5′ or 3′ region can impact Usherin production or stability. We noted circle‐swimming and failure to inflate the swim bladder in morphant larvae, both common phenotypes indicating hair cell defects. Because of the retinal degeneration seen in human Usher syndrome, we analyzed retinal cells in morphants and saw significant increases in photoreceptor death. These results will enable further functional analyses of zebrafish Usher proteins.
Binding Site Selection and Cleavage Activity of Zinc Finger Nucleases Targeting Gaucher Mutation
Authors: Dahal Sanjeev , Rowland Elizabeth , Oakes Benjamin , Noyes Marcus
Published: June 2021

Zinc Finger Nucleases (ZFNs) are one of the most promising tools of gene editing as they can create double stranded breaks (DSBs) at specific sites in the genome. Since ZFNs can be used for producing DSBs, they can be used for targeting specific mutations. In this project, the targeted mutation was a G→A point mutation. This change in the GBA gene (G1226) produces a mutant Glucosidase that is unable convert glucosylceramide into glucose and ceramide. Consequently, the substrate accumulates in the macrophages of liver and spleen manifesting into Gaucher Disease. The goal of this project is to design and assess zinc finger nucleases that specifically target the G1226 mutation. First of all, a target sequence that included the zinc finger binding sites (left and right) flanking a 6 bp spacer (that contained the mutation) was designed and integrated into the yeast genome. Zinc fingers were designed in two rounds of PCR and cloned. These fingers were tested for their binding specificity using Bacterial One Hybrid (B1H) Assay; the cleavage activity of the nucleases was determined using Yeast Nuclease Assay (YNA). The results for both of these assays are inconclusive and inconsistent at this moment. If this project is successful, these nucleases can be used in mammalian models for further investigation. The success in mammalian models will then encourage engineering of novel nucleases that can treat diseases like Gaucher Disease.
Production and characterization of Polylactic acid (PLA) nanoparticles containing drugs applied in cancer therapy
Authors: Conceição João Bosco F , Gomes Anderson J
Published: June 2021

Polymeric nanoparticles can be considered systems with increased therapeutic performance, since they are able to low the toxicity and side‐effects, increase bioavailability and efficacy of different drug classes. The aim is developing PLA nanoparticles containing tamoxifen (TMX‐PLA NPs) as promise to application in cancer therapy. The TMX‐PLA nanoparticles were prepared by the method of emulsion and solvent evaporation, presenting nanometer diameter (345.5 ± 3.0 nm), suitable zeta potential of − 8.73 ± 0.43 mV and high entrapping efficiency of 85%. Some variables for TMX‐PLA nanoparticles production process were evaluated obtaining nanoparticles with diameter below 250 nm and monomodal size distribution. Our system showed a sustained release profile in vitro. TMX‐PLA nanoparticles stability was performed in 15 days and demonstrated unsatisfactory stability due to zeta potential near zero. The TMX‐PLA nanoparticles have suitable physical, chemical and morphological characteristics, dealing for future tests delivery system for TMX as an alternative cancer therapy. Financial support: CNPq, CAPES, FAPDF, FINATEC, DPP‐UnB
Optically invisible properties of membrane organization are revealed by TIRF‐FCS on sphingolipid‐interacting probes
Authors: Kraut Rachel , Manna Manoj , Sankaran Jagadish , Wohland Thorsten
Published: June 2021

The objective of this study was to observe sphingolipid behavior in cell membranes. For this, we generated the ganglioside‐binding probes SBD (Sphingolipid Binding Domain), derived from Alzheimer's Aß peptide, and TeNT46, from Tetanus toxin. Both these peptides display slow, bimodal diffusion behavior at membranes typical for ordered domain (lo) probes, but show different lipid requirements for binding and slow diffusion. Fluorescence correlation spectroscopy (FCS) has sufficient time resolution to follow dynamics of these membrane domain‐associated probes, but it cannot discern features of lateral organization. We therefore measured a parameter called the ΔCCF (for difference in the cross‐correlation function), which detects anisotropic distributions of diffusion behaviors at the membrane, from correlations between neighboring pixels in a field obtained by TIRF‐FCS. ΔCCF shows that the high anisotropy of lo probes, like diffusion, is sensitive to domain disruption. Notably, heterogeneity revealed by ΔCCF responds differently from diffusion after various perturbations, and appears to detect changes to which traditional FCS is blind. Thus, two levels of organization at the plasma membrane are distinguishable by these methods: single point diffusion, vs. longer‐scale heterogeneity. Financial support provided by the Ministry of Education, and Nanyang Technological University, Singapore.
Biofilms and Light Signaling in Synechocystis sp. Strain PCC 6803
Authors: Dong Zihan , Allen Mary , Huang Jean , Christianson Rebecca
Published: June 2021

Biofilms are how bacteria naturally exist‐enclosed in an extracellular matrix of proteins, nucleic acids, and polysaccharides. This state protects bacteria against environmental stresses, such as antibiotics and biocides. Two strains of Synechocystis sp. Strain 6803, a wildtype strain and an extremely motile mutant strain, were grown in flow cells under identical conditions and their biofilms analyzed using confocal microscopy. A significant difference was found in terms of their biofilm structures and growth. Wildtype biofilms were significantly lower in height compared to the motile strain, and formed uniform biofilms as opposed to dense clusters. We also explored the ability of the motile mutant to move towards light, known as positive phototaxis. Both positive and negative phototaxis have been found in cyanobacteria in response to light signaling. Motile cyanobacteria move via twitching motility, which has been traced to surface appendages known as Type IV pili (Mattick, J. 2002). Our goal was to find the physical conditions required for maximal positive phototaxis and to determine the wavelength of light that induces it. We have found that the motile strain moves at a greater rate under incandescent than fluorescent conditions, and are trying to identify the specific wavelength to which the cyanobacteria respond. This research is supported by the Wellesley College Biological Sciences Department.
KHCO3 Prevents Increase in Bone Resorption with High Protein in Bed Rest (MEP Study)
Authors: Heer Martina , Buehlmeier Judith , Smith Scott M. , Baecker Natalie , Frings‐Meuthen Petra
Published: June 2021

Protein supplementation in bed rest may increase protein synthesis, but it also increases bone resorption, likely mediated by low‐grade metabolic acidosis. To test the hypothesis that providing KHCO3 with whey protein (WP) will prevent the increase in bone resorption that results from WP alone, we conducted a crossover study with 9 healthy male subjects (age 31±6 y, body mass [BM] 77±7 kg). In both study phases, following a 7‐d ambulatory period, subjects participated in 21‐d of ‐6° head‐down tilt bed rest. Diet was standardized across phases. In the control phase, subjects received no supplements. In the intervention phase, they received 6 doses per day of 0.6 g WP/kg BM together with 15 mmol KHCO3. WP was added to the nominal dietary protein, isocalorically (i.e., fat and CHO reduced). HDBR led to an increase in excretion of bone resorption markers C‐ (CTX) and N‐telopeptide (NTX) by 85±28% (p<0.001) and 35±19% (p<0.001), respectively. WP/KHCO3 prevented an additional rise of these markers. The bone formation marker procollagen‐I‐N‐terminal propeptide was increased (p=0.02) with WP/KHCO3, while bone alkaline phosphatase was unchanged. We conclude that increased bone resorption from high protein intake during bed rest can be mitigated by KHCO3. Funded by the DLR Space Program.
Validation of Toddler NutriSTEP®
Authors: Simpson Janis Randall , Whyte Kylie , Lac Jane , Rysdale Lee , Keller Heather , Beyers Joanne
Published: June 2021

The objective was to establish criterion and construct validity for Toddler NutriSTEP®, a 17‐item, community‐based parent‐administered nutrition risk screening questionnaire (total score = 68), adapted from the original preschooler version. Parents of 200 toddlers (18–35 mo), recruited from community settings, completed Toddler NutriSTEP® and a demographic questionnaire; an assessment by a registered dietitian (RD) included medical and nutritional history, weight, height, 3‐d food records. The RD rated nutritional risk on a scale of 1 (low) to 10 (high). Receiver operating curves (ROC) were used to determine sensitivity and specificity for various cut‐points on the scale. Differences in Toddler NutriSTEP® scores for selected demographic variables were determined by ANOVA.

Scores on Toddler NutriSTEP® and the RD rating were correlated (r=0.66, p<0.000). Areas under the ROC curves for the moderate risk (score 5–7) and the high risk (score 8+) RD ratings were 0.87 and 0.82 with cut points of >;20 & >;25, respectively. Scores on Toddler NutriSTEP® were significantly higher (more risk) for parents with low incomes, low education and for immigrants to Canada.

The Toddler NutriSTEP® questionnaire demonstrates both criterion and construct validity in children 18 – 35 months. Funding: Canadian Institutes of Health Research
Relationship between household structure, maternal autonomy and undernutrition in Brazilian children
Authors: Barrios Pamela L. , Hoffman Daniel J.
Published: June 2021

The prevalence of child malnutrition is high in many regions of the world, but especially in developing countries. A woman's autonomy may be an important social factor responsible for influencing child nutritional status. Therefore, the objective of this study was to determine the relationship between maternal autonomy and stunting in Brazil. A cross‐sectional analysis of the Women's National Demographic Health Survey 2006 was conducted. Demographic, health and anthropometric data for 3,399 mothers and their youngest child <60 months were used. Maternal autonomy was estimated by the decision‐making power of each woman in their household. Using logistic regression analyses, women with high autonomy, indicated by the final say on what items are to be cooked, were more likely to have a stunted child compared to women with low autonomy [(OR) = 1.856; 95% (CI) 1.004, 3.433], independent of individual and household level factors. Maternal autonomy for own health care, child's health care, daily purchases, visits to family/relatives and making large household purchases did not have a statistically significant association with stunting. In conclusion, based on the results of this study, we found no significant relationship between maternal autonomy and child stunting, possibly due to the low number of stunted children of low‐income households in this national represented sample.
Early Intervention and Reduction of Patient Default Improve Cost Effectiveness of a Supplementary Feeding Intervention for HIV+ Malnourished Patients in Ethiopia
Authors: Rogers Beatrice Lorge , Coates Jennifer , Passarelli Simone , Bontrager Elizabeth , Suri Devika , Ghosh Shibani , Kidane Yitbarek , Sadler Kate
Published: June 2021

Objective

assess marginal cost and cost‐effectiveness of adding a nutritional supplement to standard treatment of wasted HIV+ patients to raise BMI.

Methods

Supplement was provided in 8 clinics; 8 non‐randomly assigned comparison clinics provided standard care: ART, counseling. Costs included: supplement, staff time; administrative cost. Patients’ time in clinic visits was tracked.

Results

Recovery was defined as BMI=18.5 for 2 visits. Treatment patients spent more time per visit. Marginal cost per recovered patient was $12,192 for those entering with severe acute malnutrition (SAM=BMI<= 16) and $1980 for those with moderate acute malnutrition (MAM=BMI >; 16 < 18.5) due to low recovery in SAM patients. Default (failure to complete treatment) was high (70.6% treatment; 59.6% comparison). Three models assessed potential effects of reducing default. Reducing default substantially increased recovery rates and reduced marginal cost per recovered patient; costs were much lower for patients entering as MAM than SAM.

Conclusions

Supplementation of wasted HIV+ patients should start early for maximum cost‐effectiveness; reducing default greatly increases cost‐effectiveness.

No conflict of interest.
Prevalence of Vitamin A Deficiency and Associations with Socioeconomic Status in Lactating HIV‐infected Malawian Mothers
Authors: Patel K. D. , Bentley M. E. , Flax V. L. , Shahab‐Ferdows S. , Allen L. H. , Chasela C. S. , Kayira D. , Tegha G. , Jamieson D. J. , van der Horst C. M. , Adair L. S.
Published: June 2021

Vitamin A deficiency (VAD) is a concern in HIV+ women because it lowers epithelial integrity, the immune response, and red blood cell production, leading to greater risk of negative health outcomes. A subsample of Malawian lactating HIV+ mothers enrolled in the Breastfeeding, Antiretrovirals, and Nutrition Study, with demographic and plasma retinol binding protein (RBP) data (n=623), was evaluated to understand prevalence of VAD (serum RBP ≤1.17 μmol/L) and the correlation between socioeconomic status (SES) and VAD. The Thurnham method was used to adjust RBP values for inflammation, using C‐reactive protein>;5 mg/L and α‐1‐acid glycoprotein>;1 g/L. The prevalence of VAD was 71% at 2 or 6 weeks postpartum and 77% at 24 weeks postpartum and did not change when adjusted for inflammation. Logistic regression models were used to calculate odds of deficiency for each SES indicator. Lower maternal age and having no paid work were associated with higher levels of VAD (p<0.05), but VAD was unrelated to other SES indicators. The high level of VAD in this population, especially among younger and poorer mothers, suggests the need for vitamin A supplementation, but concerns that supplementation is associated with increased viral shedding and a higher risk of mother‐to‐child HIV transmission need to be weighed in settings with high HIV prevalence.

Funding: CDC(U48‐DP000059–01); Gates Foundation(OPP53107)
Nutritional factors and c‐peptide preservation in type 1 diabetes
Authors: Mayer‐Davis Elizabeth Jane , Dabelea Dana , Crandell Jamie L , Crume Tessa , D'Agostino Ralph B , Dolan Lawrence , King Irena B , Lawrence Jean M , Norris Jill M , Pihoker Catherine , The Natalie
Published: June 2021

Preservation of endogenous insulin production in youth with recent onset type 1 diabetes (T1D) is associated with better outcomes. We tested the novel hypothesis that selected nutritional factors are prospectively associated with fasting c‐peptide (FCP). Included were 656 youth with T1D (mean diagnosis age 9.8 yrs [SD 3.9], T1D duration 9.9 mo [SD 6.3] at baseline study visit) who had preserved beta‐cell function at the baseline visit (FCP >; 0.23 ng/ml) and who participated in a follow‐up (mean time to follow up, 24.3 mo [SD 8.2]). Nutritional exposures assessed at the baseline visit included infant feeding, plasma eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), vitamins D and E; and, from a food frequency questionnaire, intake of leucine and carbohydrate. Longitudinal multiple linear regression models adjusted for baseline FCP value, time, demographics, T1D duration at baseline and other disease‐related factors and potential confounders. Baseline EPA (p=0.02), EPA + DHA (p=0.03), and leucine (p=0.03) were each positively and significantly associated with follow‐up ln(FCP) while vitamin D was unexpectedly inversely associated with FCP (p=0.002). Among youth with T1D, increased intake of branched chain amino acids and long‐chain n‐3 fatty acids may support preservation of beta‐cell function. This represents a new direction for future research to improve prognosis for T1D.
Research on nutritional knowledge, preference and intake of Korean traditional foods and fast foods by middle school students in Incheon, Korea
Authors: Chung Hyun‐Jung , Yang In‐Sun
Published: June 2021

This study was performed to gain knowledge on the recognition over the korean foods and fast foods, nutrient knowledge, preference and eating habits among middle school students. This study was conducted over 287 middle school students (155 boys, 132 girls) in Incheon, Korea. The research on the recognition of Korean foods and fast foods, 63.8% of the students recognize the Korean foods as the food with original historic background and cultural characteristics. However 49.5% of the students have recognized the fast food as the promptly served foods with less cooking. Regarding the nutritional knowledge on korean foods and fast foods, Korean food was 7.03 and fast food was 7.00 out of 10 in scale and the male students showed higher rate of recognition but not significantly different. The frequency and preference for the Korean foods were higher with static relevance among middle school students (p<.001). Concerning the level of the nutritional knowledge, students had less knowledge and more frequencies of eating fast foods with statistical significance (p<.05). This study can be used to build the base information for the nutrient education to establish proper eating habits and to make students choose nutritious and balanced foods.
Changes in the food expenditure of Korean household
Authors: Chung Hyun‐Jung , Lee MiYoung
Published: June 2021

This research examined the level and changes in household food expenditure in Korea using socio‐economic and demographic factors. The data from the Family Income and Expenditure Survey between 1994 to 2009 produced by Korean National Statistical Office were used. The proportion of the food expenditure was decreased during this period of time, while that of eating‐out expenses were increased. Food, clothing, and housing expenditures were decreased, while expenditure for education, health care, and communication were increased. Economic factors including economic crisis (e.g., 1997 economic crisis, 2008 global financial crisis) affects food expenditure. The relative price of food increased about 12% during this period of time and the price is a significant factor affecting food expenditure. It was also found that demographic factors including the age of household head, household income, household size, household type, number of children were significant factors affecting food expenditure in Korean household. Findings on these food consumption analyses are expected to provide useful information understanding Korean consumers¡ expenditure patterns and developing appropriate policy.
BLOOD GLUTAMINE CONCENTRATION INCREASE AFTER BLOOD DONATION IN HEALTHY DOGS
Authors: Cordeiro Manso Helena Emilia C. C. , Manso‐Filho Helio C. , Vaz Simone Gutman , da Silva Rodrigues Armele Karina , Melo Stephania Kartuchi , de Almeida Telga L.C. , da Silva Elizabeth Regina Rodrigues , D'Paula Joyci Torres
Published: June 2021

Blood transfusion is an important practice in canine medicine because many diseases are associated with anemia and blood loss. Glutamine and glutamate are major carriers of nitrogen, carbon and energy in mammalian blood. The aim of this research was to evaluate possible changes in blood GLN and GLU in dogs after blood donation. Twelve Grayhound dogs (~5yo) were sampled before (pre‐test) and 1, 7, 14 and 30 days after donating blood. The dogs were drawn 450 mL of blood per donation. The [GLN] and [GLU] were measured by enzimatic assays and data were analyzed by ANOVA, for repeated measurements, and Tukey's test, with P<0.05, to compare all results. Results showed that [GLU] were not different in any samples. In contrast, [GLN] increased from pre‐test levels, 0.603±0.040mol/mL, to 0.728±0.036 mol/mL after 1 day, and to (0.755±0.041mol/mL) after 14days. Even after 30 days the blood [GLN] (0.671+0.043mol/mL) did not return to pre‐blood donation concentrations. Results indicate that either glutamine synthesis has increased or glutamine utilization has decreased and indicate that further research is required to determine the significance of these findings.
Caging Influences Tissue Concentrations of Vitamin K in Rodents
Authors: Shen Xiaohua , Shea M. Kyla , Booth Sarah , Callahan Michael F , Loeser Richard , Fu Xueyan
Published: June 2021

Animal models used in vitamin K (VK) studies require VK tissue manipulation while maintaining adequate coagulation. To investigate this in a rodent model used for the study of VK on cartilage, male Sprague Dawley rats (3mo, n=20) were fed either a VK‐deficient diet [21.9±3.9 μg phylloquinone (PK)/kg, D group] or control diet (1035±131μg PK/kg, C group) following destabilization of the medial meniscus knee surgery. Bleeding (in feces) was observed in D group only when housed in wire bottomed cages for 3–5 d. The bleeding stopped once moved to conventional cages. Fecal samples (n=5 group) were obtained at 3.5 wk. In D group, PK concentrations were only 2% of that in C group. No menaquinone‐4 (MK4) was detected in feces in either group whereas MK6–13 concentrations were present in both (p=0.49). At 6 wk, no MK4 was detected in either D or C group in serum or liver; PK was detected in C group only. Kidney PK and MK4 concentrations were 3.4±2.6 and 39.1±3.7nmol/kg, respectively in D group, which were 24% and 50% of their respective values in C group. Our data suggest that conventional cages used with a VK deficient diet reduces PK and MK‐4 concentrations in tissues while avoiding abnormal bleeding. The biological availability of long chain MKs from feces is not known.
Resistant Starch Promotes Vitamin D Balance in Type 1 Diabetic Mice
Authors: Anderegg Alysse S. , Schalinske Kevin L.
Published: June 2021

Type 1 diabetes mellitus (T1DM) is a chronic disease characterized by abnormal carbohydrate metabolism and hyperglycemia owing to beta cell dysfunction and insulin deficiency. A serious complication of T1DM is diabetic nephropathy, which can result in vitamin D imbalance, characterized by the loss of 25‐OH‐vitamin D in the urine. Using streptozotocin (STZ)‐treated rats as a model of T1DM, we have demonstrated that cooked dietary resistant starch (RS) attenuates the weight loss associated with T1DM and prevents the loss of vitamin D binding protein and 25‐OH‐vitamin D in the urine. The focus of this study was to determine if uncooked dietary RS would produce similar effects in a STZ‐induced mouse model of T1DM. In this study, 18 female mice were randomly assigned to one of 3 treatment groups: control, T1DM, and T1DM + uncooked dietary RS. Control and T1DM mice were fed a standard cornstarch diet during the 6‐wk treatment period, whereas T1DM + RS group were fed a diet containing high amylose cornstarch (36% resistant to digestion). To date, we have observed similar results in the mouse model, including diminished weight loss owing to the positive effect of dietary RS. Future research will be directed toward understanding the mechanism underlying the impact of RS on maintaining vitamin D balance, owing to the potential prevention of diabetic nephropathy in type 1 diabetic mice.

Support: Plant Sciences Institute, ISU.

Grant Funding Source: Plant Sciences Institute, ISU
Associations of Age, Sex, and Vitamin K Status with Vitamin K Absorption
Authors: Faherty Meghan , Dallal Gerard E , Shen Xiaohua , Dolnikowski Gregory , Grusak Michael A , Booth Sarah L , Fu Xueyan
Published: June 2021

We assessed associations of age, sex, and vitamin K (VK) status with VK absorption to evaluate dietary and non‐dietary determinants of VK absorption. Younger (18–40y; n=21) and older (55–80y; n=21) adults were fed deuterium labeled (d)‐collard greens to assess absorption of d‐VK by monitoring d‐VK in plasma and triglyceride rich lipoprotein (TRL) fractions. Participants were pre‐conditioned to either a VK depleted (510 μg VK/d for 28d) or VK repleted (10 μg VK/d for 28d) state. After adjusting for triglycerides (TG), %body fat and BMI, median plasma d‐VK and median TRL d‐VK were not significantly associated with VK status. BMI was significant for the models of median plasma d‐VK and median TRL d‐VK for older adults (p= 0.01, p=0.01) and for younger men (p=0.03, p=0.015). TG were significant for the models of median plasma d‐VK and median TRL d‐VK for older adults (p<0.0001, p<0.0001) and younger adults (p=0.023, p=0.005). The model adjusting for age, sex, VK status, and anthropometrics accounted for approximately 20% variability in VK absorption. Large inter‐individual differences in VK absorption exist, the determinants of which still need to be identified.
Cranberry bioactives induce TLR signaling and promote a state of immune preparedness in THP‐1 monocytes/macrophages
Authors: Creasy Rebecca A , Khoo Christina , Percival Susan S
Published: June 2021

Cranberries are a rich source of proanthocyanidins (PAC). Because of the unique structure of PAC, we hypothesized that cranberry interacts with pathogen recognition receptors on innate immune cells. Our objective was to evaluate Toll‐like receptor (TLR)‐4 PAC recognition and resulting changes in cytokine mRNA and protein. HEK‐Blue‐hTLR4 cells, containing inducible SEAP reporter genes, were treated with cranberry fractions, LPS, or vehicle. Cranberry fraction containing primarily PAC significantly increased SEAP activity in cell supernatant relative to vehicle. To determine if cranberry primes innate immune cells, THP‐1 cells were differentiated into macrophages and incubated with cranberry or vehicle. Cranberry treatment resulted in significantly higher MIP‐1α mRNA levels without TNFα and IL‐ 1β mRNA increases. An LPS challenge of cranberry‐treated macrophages induced a subtle increase in MIP‐1α protein secretion. In a second experiment, THP‐1 macrophages were treated with cranberry fractions, LPS, or DMSO, then treated with actinomycin D and harvested at times up to 3 hr. Cranberry treatment stabilized MIP‐1α transcripts to a greater extent than did LPS or vehicle. This data suggest that cranberry activates a TLR signaling pathway and primes THP‐1 cells for enhanced response to pathogen challenge via transcript stabilization. Funding provided by Ocean Spray Cranberries, Inc
A Comparative Study of the Antiproliferative Effect of Kohlrabi and Green Cabbage on Colorectal Cancer Cell Lines In Vitro
Authors: Rizk Sandra , Zarzour valia
Published: June 2021

Experimental studies showed that phytochemicals from Brassicaceae Cruciferae vegetables possess anticarcinogenic properties. Two members of this family, namely cabbage (Brassica oleracea, group Capitata) and kohlrabi (Brassica oleracea, group Gongylodes) seem to share similar anticarcinogenic characteristics. The aim of this study is to compare the effect of the ethanolic extracts of the edible parts of these two plants on the proliferation and apoptosis of HT‐29 and Caco‐2 colorectal cancer cell lines. The cytotoxicity of the extracts was assayed using the Trypan Blue Exclusion method and the IC50 was determined to be higher in kohlrabi compared to cabbage. Consequently, WST‐1 assay was used to assess the effect of these plant extracts on the proliferation of colorectal cancer cell lines: both extracts exhibited a sharp anti‐proliferative effect in a dose and time dependent manner. Analysis of cell DNA content by flow cytomtery revealed the ability of both kohlrabi and cabbage extracts to induce cell cycle arrest in HT‐29 cells at the G phase and S phase in similar patterns. Furthermore both extracts promoted apoptosis in colorectal cancer cell lines. The apoptotic pathway seems to involve the pro‐apoptotic protein Bax and the anti‐apoptotic protein Bcl‐2. As a conclusion, kohlrabi should be considered, like cabbage, among the potent anti‐carcinogenic cruciferous vegetables.
Chronic alcohol consumption has greater impact on hepatic DNA hydroxymethylation in young mice relative to old
Authors: Tammen Stephanie A. , Dolnikowski Gregory G. , Ausman Lynne M. , Sauer Julia , Friso Simonetta , Choi Sang‐Woon
Published: June 2021

Aging and chronic alcohol consumption affects DNA methylation, changing gene transcription. It is not yet known whether these factors also alter DNA hydroxymethylation (hmC), an epigenetic mark produced by oxidation of methylcytosine. Our objective was to determine changes in hmC caused by aging and alcohol. Young (4 months; n=20) and old (18 months; n=18) male C57BL/6 mice were pair‐fed a Lieber‐DeCarli liquid diet with or without alcohol (18% of energy) for 5 weeks. Global hmC was analyzed from hepatic DNA using an LC/MS‐MS method and calculated as percent of total cytosine. Differentially hydroxymethylated regions (DhMRs) were determined by microarray‐based hydroxymethyl‐DNA immunoprecipitation (hmeDIP‐chip). Relative to the controls, chronic alcohol consumption did not alter global hmC in old mice, but significantly reduced global hmC in young mice (0.22%±0.01% vs 0.29±0.06%, p=0.004). Old mice had significantly lower global DNA hmC than young mice (0.24±0.02% vs 0.29±0.06%, p=0.04). This model also suggests an interaction between aging and alcohol in determining global hmC levels (pint.=0.01). Genomic hmC patterns followed a similar trend; the alcohol diet resulted in more DhMRs in young mice compared to old (431 vs 196 DhMRs). This is the first study on the effects of alcohol consumption and aging on hmC levels and posits that chronic alcohol consumption differently affects hmC in young and old mice.

Grant Funding Source: NIH grant R01 AG025834
The Influence of Type II Diabetes on the Immune Response of Obese Adults
Authors: Paich Heather A. , Sheridan Patricia A. , Noah Terry L. , Weir Samuel S. , Beck Melinda A.
Published: June 2021

We have previously shown that obesity impairs expression of CD4+ and CD8+ T cell markers of activation and function. Obesity is associated with type II diabetes; however, not all obese individuals develop type II diabetes. Type II diabetes is associated with low levels of chronic inflammation and a higher prevalence of infections. The objective of our study was to elucidate the differences in the human cellular immune response between obese individuals with type II diabetes and obese individuals without type II diabetes. We stimulated peripheral blood mononuclear cells, obtained from obese individuals with and without type II diabetes, ex vivo with live pandemic influenza virus A and anti‐CD3/anti‐CD28 beads. Markers of activation, function, and signaling were measured using flow cytometry. We also measured three parameters of cellular glycolysis, including glycolytic rate, glycolytic capacity, and glycolytic reserve capacity, in T cells stimulated with or without anti‐CD3/anti‐CD28 bead stimulation and with or without insulin stimulation. We found significant differences between obese individuals with and without type II diabetes, demonstrating that metabolic disruptions in insulin signaling further increase immune dysfunction in obese adults.

Grant Funding Source: NIH ROI AI078090 and P30DK056350
Identification of endogenous pro‐resolving mechanisms in a murine model of cryptococcal pneumonia
Authors: Colby Jennifer Kay , Gott Katherine , Wilder Julie , Levy Bruce D.
Published: June 2021

Lipoxins (LX) are potent anti‐inflammatory and pro‐resolving lipid mediators that augment host defense. We hypothesized that established mouse strain‐dependent differences in clearance of the fungal pathogen Cryptococcus neoformans (Cne) were linked to alterations in LX synthesis and signaling. C.B‐17 and C57BL/6 (B6) mice were inoculated intranasally with 5000 cfu Cne (day 0). C.B‐17 but not B6 mice effectively cleared Cne from the lungs. Leukocyte counts revealed fewer lung macrophages, but increased eosinophils and activated CD4+ T cells in lung and bronchoalveolar lavage fluids (BALFs) at d7, 14, 21 and 28 after infection. BALF levels of LXA4 as measured by ELISA were highest at d14 and d28 in B6 and d18 and 21 in C.B‐17 (p≤0.025 vs. uninfected). Similar patterns were observed for 15‐epi‐LXA4. Comparative qPCR analysis of naive and d14 lungs showed Cne‐induced increases in expression of Alox15 (15‐lipoxygenase‐1) at d14 (both strains), Alox5 (5‐lipoxygenase) (B6 only), and Fpr2 (lipoxin receptor, ALX) (C.B‐17 only). In summary, altered cellular immunity in B6 mice is associated with differences in LX synthesis and LX receptor expression. This experimental system provides a window into regulation of pathogen‐mediated inflammation and clearance and highlights important relationships between lipoxins and lung host defense.
CYTOKINE PROFILE IN CELIAC DISEASE: A LASER CAPTURE MICRODISSECTION APPROACH
Authors: Iaquinto Gaetano , Aufiero Vera Rotondi , Preziuso Marco , Marano Angela , Stillitano Ilaria , Giardullo Nicola , Bevilacqua Rosa Maria , Iacomino Pino , Mazzarella Giuseppe
Published: June 2021

Background

Different approaches have been used to study the pattern of cytokines in celiac disease (CD), which have led sometimes to contradictory results. Laser capture microdissection (LCM) is a powerful tool for the isolation of specific cell populations from stained sections.

Aim

Characterize the pattern of cytokines within the tissue compartments of the small intestinal mucosa of CD patients, dissected by LCM.

Methods

Frozen sections of jejunal biopsies were obtained from 7 untreated CD patients. Epithelium and lamina propria were isolated separately by LCM, and the mRNA levels for IFN‐γ, IL‐21, IL‐15, IL‐10 and TGF‐β, were determined by using real‐time PCR.

Results

We observed a higher expression of IFN‐γ and IL‐21 mRNA in the lamina propria than in the epithelium and a higher expression of IL‐15 mRNA in the epithelium than in the lamina propria. Same levels of IL‐10 and TGF‐β transcripts were detected in both the compartments of untreated celiac mucosa.

Conclusion

Our data suggest that the epithelium produce cytokines involved in the innate immunity while lamina propria cells produce cytokines involved in the adaptive immune response. Both epithelial and lamina propria compartments produce immunosuppressive cytokines. This work underlines the importance of LCM as a valuable tool to determine inflammatory components in specific intestinal compartments enhancing understanding of CD pathogenesis.
Omega‐3 derived Resolvin D1 reduces the severity of inflammatory arthritis
Authors: Norling Lucy Victoria , Perretti Mauro
Published: June 2021

Dietary supplementation with omega‐3 PUFA is efficacious in reducing joint pain, morning stiffness and NSAID usage in Rheumatoid arthritis patients. However, the mechanisms by which omega‐3 PUFA exert their beneficial effects has not been fully explored. Recent discoveries have alluded that omega‐3 PUFA can be enzymatically converted in vivo to novel bioactive lipid mediators, which promote the resolution of inflammation and are log‐orders more potent than their precursors, termed resolvins. We recently showed that resolvin D1 (RvD1) limits PMN recruitment in experimental peritonitis via its receptor Fpr2, yet whether RvD1 exhibits anti‐arthritic effects remains to be elucidated. We performed the K/BxN serum transfer model of arthritis (50μl serum i.p. on day 0 and day 2) on mice fed control or 10% salmon oil‐enriched diet, in comparison with mice administered daily 17R‐RvD1 (100ng, i.p.) or vehicle (0.1% EtOH), and graded arthritis severity (max. 12). Omega‐3 enriched diet mildly reduced disease severity, yet RvD1‐treated mice exhibited a significantly lower clinical score, and reduced hind paw oedema compared to vehicle‐treated mice. These studies indicate that the endogenous pro‐resolving mediator RvD1 is more efficacious than an omega‐3 enriched diet at reducing the severity of inflammatory arthritis and that omega‐3 PUFA and derivatives are a viable approach to novel anti‐arthritic therapies.

Supported by Arthritis Research UK Fellowship 19909 to LVN.
The emerging role of the GPR109A (HCA2/PUMA‐G) receptor in regulating macrophage function
Authors: Zandi‐Nejad Kambiz , Takakura Ayumi , Jurewicz Mollie , Chandraker Anil , Offermanns Stefan , Mount David Bruce , Abdi Reza
Published: June 2021

The objective of this study is to investigate the novel role of GPR109A (HCA2/PUMA‐G) and its ligand nicotinic acid in regulating macrophage function. Gpr109a expression in the RAW264.7 murine macrophage cell line is strongly induced by lipopolysaccharide (LPS) treatment and correlates with the expression of TNF‐α□ Treatment with 300 μM nicotinic acid (reported EC50 3 μM, peak plasma concentration 50–300 μM), significantly inhibited TNF‐α, IL‐6, IL‐12p40, and IL‐1β production (P<0.05) in LPS (1ng/ml)‐stimulated wild‐type murine bone marrow‐derived macrophages (BMM) but failed to do so in Gpr109a−/− BMM. Treatment with nicotinic acid reduced nuclear factor κB (NF‐κB) activation levels by 30% in wild‐type BMM 6 hours after LPS stimulation but not in Gpr109a−/− BMM. Nicotinic acid significantly inhibited wild‐type BMM chemotaxis (P<0.001), but had no effect on the chemotaxis of Gpr109a−/−BMM. A significant increase in low‐density lipoprotein (LDL) uptake by both wild‐type (P<0.006) and Gpr109a−/− BMM (P<0.03) in response to LPS was observed, which was significantly suppressed by nicotinic acid in wild‐type BMM (P<0.04) but not in Gpr109a−/− BMM. Our results suggest that the nicotinic acid‐GPR109A axis is a negative regulator of macrophage activation.
Diabetes/high glucose induced arginase increases arterial smooth muscle cell proliferation and collagen synthesis/fibrosis through ornithine decarboxylase and ornithine aminotransferase pathways
Authors: Bhatta Anil , Toque Haroldo A , Caldwell Ruth B , Caldwell R. William
Published: June 2021

Arginase can compete with nitric oxide (NO) synthase for their common substrate, L‐arginine, and its increased activity can decrease NO production. Arginase also provides ornithine for synthesis of polyamines via ornithine decarboxylase (ODC) and proline/collagen via ornithine aminotransferase (OAT). We hypothesize that diabetes/high glucose (HG) induce vascular wall thickening and fibrosis through increased level of arginase‐derived ornithine available for ODC and OAT. We exposed rat aortic smooth muscle cells to HG (25 mM), which resulted in increased arginase activity (57%), cell proliferation (22%), and ODC mRNA (25%). Treatment with arginase inhibitor BEC (S‐(2‐boronoethyl)‐l‐cysteine) prevented these changes. We observed increased perivascular fibrosis (68%) in coronary arteries of 8‐week wild‐type (WT) diabetic mice compared to arginase I partial knockout (AI+/−) diabetic mice. Aorta from diabetic WT mice also exhibited impaired endothelium‐dependent relaxation (EDR) compared to control (maximum EDR: 32.9% vs 61.8%). EDR was largely maintained in aorta from diabetic AI+/− mice. Furthermore, adenoviral overexpression of arginase I in isolated aorta concomitantly increased ODC expression, implying an effect of arginase on ODC. In conclusion, arginase appears to have important roles in HG/diabetes‐induced vascular dysfunction, proliferation and fibrosis through ODC and OAT pathway.