bluuebunny/crossref_metadata_2025 huggingface dataset:
Cognitive impacts of diet quality depend on socioeconomic position: the NuAge study
Authors: Parrott Matthew , Shatenstein B , Ferland G , Morais JA , Belleville S , Kergoat MJ , Payette H , Greenwood CE
Published: June 2021

We examined whether equivalent adherence to ‘Prudent’ and ‘Western’ dietary patterns, identified by principle components analysis, exerted the same beneficial or detrimental effects on cognitive function (Modified Mini‐Mental State Examination; 3MS) in older adults with different socioeconomic position (SEP). The interaction of dietary pattern adherence with household income, educational attainment, occupational prestige, and a composite indicator of SEP combining all three (n=1099) was examined in multiple‐adjusted mixed models over 3 years of follow‐up. Adherence to the Prudent pattern was related to higher 3MS scores at recruitment only in upper categories of income (P=0.015), education (P=0.017), or composite SEP (P=0.026). High Prudent pattern adherence was associated with slower cognitive decline only in those with low composite SEP (P=0.042). Conversely, high Western pattern adherence was associated with accelerated cognitive decline (P=0.023) only in those with low educational attainment. In summary, we found that the neuropsychological parameters of the diet‐cognition relationship were dependent on SEP. Since individuals with equivalent diet quality and dissimilar SEP performed differently, it may be unrealistic to expect diet to act on cognition in isolation from other dimensions of lifestyle.

Grant Funding Source: Canadian Institutes of Health Research
Food consumption in the Nigerian population: Estimates of energy and micronutrient intakes and main food sources of energy, iron and zinc
Authors: Bermudez Odilia I. , Lividini Keith , Fuster Melissa , Smitz Marc‐ Francois , Fiedler John L.
Published: June 2021

Objective

To estimate baseline intakes and food sources of energy (E), and the micronutrients (MN) iron (Fe) and Zinc (Zn) for the Nigerian population.

Methods

Food sources and apparent intakes of Fe & Zn were estimated with food acquisition data from the 2003–04 Nigeria Living Conditions Monitoring Survey. Applying adult male equivalents (AME), individual intakes and levels of inadequacy were assessed. Intakes of MN were adjusted by bioavailability. All estimates were adjusted with sample weights.

Results

Main sources of E were corn (20%), rice (17%), gari (11%) and pulses & nuts (P&N, 11%). P&N, corn and rice provided 25%, 21% & 8% of Fe plus 21%, 27% and 15% of Zn, respectively. National intake of energy was 1,290 Kcal/AME, while MN intakes were 10.6 mg for Fe and 6.4 mg for Zn, per AME. Dietary recommendations (EARs) for Fe and Zn reached, as mean, 54% and 87% of the EAR, although the median EARs were only 37% and 52%. The population at risk of inadequate intakes was 70% for Fe and 76% for Zn.

Conclusions

We were able to obtain estimates of baseline apparent food intakes using food acquisition data from a national survey from Nigeria, from which we identified elevated proportion of the Nigerian population at risk of inadequate intakes of Fe and Zn.

Funding:

Bill & Melinda Gates Foundation Grand Challenge Grant
VO2 max improves in Indian school children after a feeding trail with iron biofortified pearl millet
Authors: Pompano Laura M. , Przybyszewski Eric M. , Udipi Shobha A. , Ghugre Padmini , Haas Jere D.
Published: June 2021

Objective

To examine the effects of an iron biofortified pearl millet intervention on physical performance in children, measured by VO2max.

Methods

135 children (11–18 y) were enrolled in a randomized trial of an iron biofortified pearl millet intervention (vs. control pearl millet), daily for a 6 month period in India. VO2max at baseline and end line was calculated from oxygen consumption and heart rate during a sub‐maximal exercise test on a cycle ergometer. General linear models were used to evaluate the effect of the intervention on VO2max in children.

Results

At baseline 32% of subjects were iron deficient with serum transferrin receptor (sTfR) values above 8.0 mg/L. The average VO2max at baseline was 36.9 mL/kg/min for girls. After six months, there was a significant relationship between decreasing sTfR (improved iron status) and increased VO2max in the iron biofortified pearl millet group, but not in control group (P<0.01 and P=0.10, respectively).

Conclusions

Supplementation with iron biofortified pearl millet significantly improved VO2max in children.

Grant Funding Source: HarvestPlus
Plasma zinc response to supplementation in apparently healthy women from rural Sidama in southern Ethiopia
Authors: Bogale Alemtsehay , Peterson Sandra , Stoecker Barbara J.
Published: June 2021

This placebo‐controlled trial in apparently healthy, reproductive age women evaluated effects of supplementation of 20 mg Zn as zinc sulfate for 23 days on plasma zinc concentrations. The study was conducted in June/July, 2012. Participants were randomly selected from the list of non‐pregnant, non‐lactating women residing in the village. Additional exclusion criteria were body mass index (BMI) outside of the 18.5 to 24.9 range and selfreported recent illness. Thirty‐five women were included for this analysis. Demographic as well as household dietary diversity data were collected. Anthropometric measurements were taken, women were randomized to placebo or treatment groups and blood samples were collected by venipuncture at baseline, midpoint and the end of the 23‐day supplementation period. Plasma zinc was measured by inductively coupled plasma mass spectrometry (ICPMS). Mean ± SD age of participants was 32.7 ± 5 and family size was 6.8 ± 2.7. Education of the participants ranged from 0 to 6 yr of school. Household dietary diversity score was 4.5. None of the participants reported consuming meat, fish or egg in the 24 hr prior to the survey. The mean plasma zinc concentration at base line was 651 ± 80 μg/L for the zinc group and 721 ± 86 μg/L for the placebo group. At endpoint plasma zinc for placebo (713 ± 112 μg/L) did not change significantly from baseline. However, in the zinc‐supplemented group, plasma zinc at 735 ± 84 μg/L was significantly higher than baseline (p<0.002). [This study was supported by the Oklahoma Agricultural Experiment Station].
Consumption of flavored waters, sports and energy drinks is potentially underestimated: trends in sales, purchases and intake in the US, 2000–11
Authors: Stern Dalia , Piernas Carmen , Popkin Barry M
Published: June 2021

New caloric beverage options are increasingly being introduced. We investigated the dynamics of consumption of flavored waters (FW), sports (SD) and energy drinks (ED) in the US.

We analyzed per capita volume (mL/day) of FW, SD and ED using three sources of data for the US population: NHANES 2003–10 dietary intake data; sales data from Euromonitor (EM) 2000–11; purchases from the Homescan Longitudinal (HM) dataset 2000–11. This analysis was restricted to consumer‐packaged goods from convenience/grocery stores, excluding vending and away‐fromhome sources. Trends for each beverage group were explored in sales (EM 2000–10) and intake (NHANES 2003–10). Total intake (NHANES) with total purchases (HM) for 2007–08 (later all 11y) were examined. Survey commands were used to account for survey design (STATA 12).

SD ranked the highest among intake, sales and purchasing data, followed by FW and ED. Overall, we found increasing trends in sales (EM) of SD, FW and ED whereas trends in intake (NHANES) declined for these beverage groups. In addition, the absolute per capita volume (mL/day) of sales (EM) was significantly higher compared to intake (NHANES). Purchase and intake patterns are comparable.

These different trends and intake levels between commercial data and NHANES reported intake suggest potential underreporting of these new beverages, which represent important new sources of added sugars in the US diets.

Grant Funding Source: Robert Wood Johnson Foundation Grant 70017; National Institutes of Health R01‐HD030880 R01‐ HL104580 R01‐HL114091 R01‐HL108427; Conacyt Grant 309902
Determinants of milk volume consumed at a single morning feed by healthy, rural Polish infants
Authors: Klein Laura Danielle , Galbarczyk Andrzej , Klimek Magdalena , Nenko Ilona , Jasienska Grazyna , Hinde Katherine
Published: June 2021

Here we present a preliminary report of milk production from an agriculturalist population at the Mogielica Human Ecology Study Site in rural Poland. While a modern population in many respects, the local mountain geography prohibits the use of modern farm equipment and most households utilize physically demanding traditional methods. As women engage fully in harvesting, this site allows for investigating lactation among women with highly variable seasonal energy expenditure. Non‐contracepting women (n=24, 19–44 years old) with infants up to one year old provided a morning, mid‐feed milk sample. Infants were weighed before and after the feeding bout to obtain milk volume. The only significant predictor of milk consumption was delivery mode. Infants delivered via cesarean section (n=4) consumed less milk than infants born vaginally (37.4±5.5g vs. 57.1±6.5g, t=−2.312, p=0.03). Maternal parity, breast‐feeding exclusivity, and maternal mass did not predict milk sample volume. Although derived from a small sample, these results motivate further investigation into lactation among diverse human populations. This research was supported by a Harvard University Human Evolutionary Biology Summer Research Grant.
Does thawing and warming affect the integrity of human milk?
Authors: Handa Deepali , Ahrabi Ali Faraghi , Codipilly Champa N , Shah Syed , Potak Debra , Schanler Richard J
Published: June 2021

Objective

As most neonates in the NICU are fed defrosted human milk (HM), we evaluated the integrity of the HM after freezer storage, thawing, warming, and waiting to be fed.

Methods

NICU mothers (n=41) donated 100 ml of milk. Baseline aliquot was stored at −80°C, and the remaining milk was stored 7 days at −20°C. Subsequently, milk was thawed and warmed using tepid water or waterless warmer. A separate sample of thawed milk was maintained in refrigerator for 24 h and warmed. Warmed milk was maintained at room temperature (RT) for 4 h. Milk integrity was described as the effect on pH, protein, sIgA, lactoferrin, and bacterial colony counts (total, TBCC; Gram positive, GPCC; Gram negative, GNCC). Data analyzed by ANOVA and paired t test.

Results

There were no differences in milk integrity between the two warming methods. There were no changes in protein, sIgA, and lactoferrin with processing. There was a significant change in pH between baseline, thawing, and warming, p < 0.001. When thawed milk was warmed after refrigeration, there was a further decline in pH. TBCC, GPCC, GNCC did not change significantly with processing but when warmed milk was maintained at RT all counts increased, p < 0.01.

Conclusion

Thawing and warming do not change the integrity of previously frozen human milk adversely. The integrity of the milk is affected similarly by the two methods. Concerns about maintaining warmed milk at RT need to be explored.
EFFECTS OF DIFFERENT CALORIE RESTRICTION DIETS IN OBESE WOMEN
Authors: de Toledo Triffoni‐Melo Andresa , Jordão Alceu Afonso , Dick‐de‐Paula Ingrid , Ovídio Paula Payão , Diez‐Garcia Rosa Wanda
Published: June 2021

The aim of this study was to compare the short‐term effects of two different calorie restriction (CR) diets on anthropometric and biochemical parameters in obese women. Participants were 30 women, BMI 37.3±4.4kg/m2, and 39±6.3 years of age. This study was conducted in 3 phases with a follow‐up at one month from the end of phase 3. In Phases 1 and 3 participants were on an ad libitum diet at home for 2 days. In Phase 2, participants were on a 1000 kcal/d CR diet for 3 days and were randomized to either the experimental group (EG n=15), which received a high‐fiber diet or the control group (CG n=15), which received the conventional diet. Anthropometric and biochemical data were collected at the beginning and end of each phase for a total of 5 measurements including the 1month follow up period. There was no significant reduction within groups in body weight (EG, p=0.92; CG, p=0.95) and waist circumference (WC: EG, p=0.97; CG, p=0.98). No significant between groups difference in weight (p=0.92) or WC (p=0.85) were observed. A significant reduction in fasting glucose (p=0.01) was observed within the EG during CR. There was also a reduction of triglycerides (p=0.03) in EG compared to CG. The results of this short‐term study indicate that a high‐fiber diet results in a greater decrease in fasting glucose and triglycerides, compared to a conventional diet.

This research was supported by The State of São Paulo Foundation (FAPESP).
Interactions between physical performance, body‐composition and nutrition in individuals one to three years after stroke
Authors: Vahlberg Birgit Maria , Lindmark Birgitta , Zetterberg Lena , Hellström Karin , Cederholm Tommy
Published: June 2021

Interactions between physical performance, body composition and nutrition in individuals 1–3 years after stroke.

Objective

To explore muscle mass, physical activity and nutritional status and association to mobility in stroke survivors.

Methods

134 community living individuals (74±5 y, 69% men) participated. Mobility was measured by Short Physical Performance Battery (SPPB, 0–12 points). Bio‐impedance analysis (BIA) measured fat free mass index (FFMI; kg/m2), fat mass index (FMI; kg/m2) and FM%. Nutritional status was measured by Mini Nutritional Assessment‐Short Form (0–14 p), gait speed by 10 m walking test (m/s) and the self‐reported Physical Activity Scale for the Elderly (PASE, ≥0 points) were recorded.

Results

BMI showed that 71% were overweight or obese. 14% were at risk for malnutrition. FM% was >;20 in 86% of men, and >;30 in 90% of women (related to a ref population). 28% were considered as sarcopenic obese, i.e. FFMI <25th perc + FMI>;50th perc of a ref population. Sarcopenia, i.e. the combination of FFMI<25th perc of normal + gait speed <1 m/s, was observed in 10%. Walking speed was slow (1.1±0.25 m/s), and PASE was low (mean 108±65 p). SPPB was reduced (median 10 p), but neither associated to FFMI nor FMI. Logistic regression indicated that SPPB ≤9 p was significantly related to risk for malnutrition; OR 5.8, reduced physical activity; OR 8.2, and high age; OR 2.8 for each 10 y.

Conclusions

Obesity was observed in ~2/3 subjects 1–3 y after stroke. More than 1/4 was classified as sarcopenic obese. FFM was not related to physical performance, whereas risk for malnutrition, physical activity and age were. Thus, nutrition and physical activity may represent opportunities for rehabilitation after stroke.
The effects of high fiber noodle on metabolic syndrome
Authors: Chiang Pei‐Chin , Yang Ji‐Hua , Chen Pei‐Tung , Chiu Wan‐Chun , Chien Yi‐Wen
Published: June 2021

In view of metabolic syndrome is a global health issue, the aim of the study was to investigate the effect of high fiber noodle (HFN) and fibersol‐2 on its major indicators ‐ hyperlipidemic and diabetic animals. In hyperlipidemic model, male Syrian Hamsters were divided into 4 groups and fed a high fat with 0.2% cholesterol‐enriched diet. Animals were fed the diet without HFN (C) or with 1.2% fibersol‐2 and 14.8% HFN (1x), with 2.4% fibersol‐2 and 13.6% HFN (2x), with 6% fibersol‐2 and 10% HFN (5x), respectively. In diabetic model, male Wistar rats were divided into 5 groups and fed AIN‐93M diet, and 4 groups of rats were induced diabetes by streptozotocin. Normal rats were fed the diet without HFN (N), and diabetic rats were fed AIN‐93M with C, 1×, 2×, 5× diet, respectively. After 8 weeks, we analyzed blood lipids and glycemic parameters to determine whether HFN can reduce the lipid and glucose levels. The results in hamsters showed serum total cholesterol level and low‐density lipoprotein cholesterol, 1×, 2× and 5× were all lower than those in C group (p<0.05). Otherwise, the results in rats showed fasting plasma glucose and area under the blood glucose curve, 5× was lower than C group (p<0.05). In summary, the study demonstrated that intake HFN with fibersol‐2 can regulate blood lipids, fasting blood glucose and postprandial blood glucose to improve metabolic syndrome.

Grant Funding Source: Taipei Medical University
Regulation of the proteasome by ubiquitin chain editing
Authors: Finley Daniel
Published: June 2021

Error‐correction mechanisms in protein synthesis are well‐known. In an interesting analogy, the proteasome contains activities that can remodel, or edit, multiubiquitin chains on substrates. These activities regulate degradation rates in yeast and mammals. The effects are powerful but vary in strength from substrate to substrate. The main editing factors are the deubiquitinating enzyme Ubp6 and the ubiquitin ligase Hul5 (in yeast). Ubp6 suppresses degradation while Hul5 promotes it. Regulation of degradation may reflect that more heavily ubiquitinated substrates bind the proteasome more avidly. These factors can transduce signals to modulate proteasome activity positively or negatively. For example, UBP6 is induced when ubiquitin levels fall, while HUL5 is not. In addition to modifying ubiquitin chains, both factors regulate degradation noncatalytically. Hul5's noncatalytic effect is apparently to suppress Ubp6 deubiquitinating activity. Also, of the 910 residues in Hul5, only a short 13‐residue segment at the N‐terminus is required for the noncatalytic effect. The deubiquitinating activity of Usp14 (the mammalian ortholog of Ubp6) can be suppressed by small‐molecule inhibitors identified by high‐throughput screening. These compounds allow one to enhance the degradation rates of many proteins, including toxic proteins such as mutant forms of tau that have been implicated in neurodegeneration.
Green tea prevents aberrant crypt foci (ACF) in mice fed the total Western diet (TWD) but not the AIN93G diet
Authors: Hintze Korry Joseph , Ward Robert , Benninghoff Abby , Lefevre Michael
Published: June 2021

The new TWD emulates average American intakes (at the NHANES 50th percentile) for micro and macronutrients based on nutrient density. Mice were fed either TWD or AIN93G basal diets supplemented with or without 0.2% green tea extract in the drinking water. Green tea extract reduced bodyfat percentage in both the TWD and AIN93G groups (P < 0.05) and decreased fasting glucose levels in mice fed TWD (P = 0.02) but not AIN93G. Mice fed TWD without green tea had more ACF and a higher total crypt cell count compared to cohorts fed the AIN93G diet (P < 0.001). Interestingly, green tea extract decreased ACF and total crypt cells in the TWD group (P < 0.001) but not in mice fed the AIN93G basal diet. Cecal bacterial DNA from each treatment was pyrosequenced and a metagenomic analysis of the microbiome will be presented. These results suggest that the Western dietary pattern promotes carcinogenesis and that supplementation with chemopreventive bioactives, such as green tea, may be beneficial to populations consuming a poor diet. Funded by the Utah Ag Experiment Station.
Anti‐obesity and Anti‐inflammatory Effect of Acai Polyphenols in 3T3‐L1 Adipocytes
Authors: Martino Hercia , Noratto Giuliana , Dias Manoela , Talcott Stephen , Mertens‐Talcott Susanne
Published: June 2021

Many health benefits have been attributed to polyphenolics from acai, however, proclaimed anti‐obesity activities have not yet been investigated in much depth. The aim of this study was to determine the anti‐obesity and anti‐inflammatory effects of acai polyphenols (AP) in 3T3‐L1 mouse adipocytes. Cells were differentiated in the presence of AP at 2.5, 5 and 10 μg gallic acid equivalents (GAE)/mL. The activities of AP against lipid synthesis and inflammation were evaluated using RT‐PCR, Western blotting, and multiplex bead‐based immunoassay. Results show that AP reduced the accumulation of intracellular lipids in a dose‐dependent manner and suppressed mRNA and protein levels of PPAR‐γ2, a master regulator of fat synthesis and inflammation to 71% of the control. This was accompanied by downregulation of PPAR‐γ2 upstream and downstream genes (C/ebp α, C/ebp β, Klf5, Srebp1c), PPAR‐γ2‐target adipogenic and pro‐inflammatory adipokine genes. AP also protected against TNFα‐induced ROS and inflammatory nuclear factor kappa B (NFκ‐B), and the NFκ‐B‐target genes (interleukins (IL)‐6, IL‐1β, tumor necrosis factor (TNF‐α), interferon (IFN)‐γ and monocyte chemotactic protein‐1 (MCP‐1)). These results suggest that AP might be effective in the prevention of obesity and protect against obesity‐induced inflammation.
Eicosapentaenoic acid prevents high fat diet‐induced metabolic disorders: Genomic and metabolomic analyses of underlying mechanisms
Authors: LeMieux Monique , Kalupahana Nishan , Xin Wenting , Thanamool Catthareeya , Voorhees Victor , Siriwardhana Nalin , Claycombe Kate , Moustaid‐Moussa Naima
Published: June 2021

Previously our lab demonstrated eicosapenaenoic acid (EPA)'s ability to prevent high‐fat (HF) diet‐induced obesity by decreasing insulin resistance, glucose intolerance and inflammation. In the current study, we used genomic and metabolomic approaches to further investigate the molecular basis for these effects. Gene (microarrays and RT‐PCR) and protein expression studies (immunoassays) revealed changes in levels of genes/proteins associated with cell proliferation, apoptosis, adipocyte differentiation and glucose metabolism. Specifically, expression of lipogenic/adipogenic markers (Fasn, Srebf1, Cebpa, Ncoa2, Wnt signaling) were decreased by EPA. Metabolomic analyses confirmed enrichment of adipose tissue in n‐3 polyunsaturated fatty acids (PUFAs) and a decrease in n‐6 PUFAs in both visceral and subcutaneous fat. These studies also indicated increased oxidation of stored fatty acid and increased fatty acid oxidation related‐metabolites such as acetylcarnitine. Together the genomic and metabolomic findings were in‐line with other metabolic and physiologic outcomes including decreased adiposity and fat cell size and increased lipid oxidation. In conclusion, our studies demonstrate that EPA ameliorates glucose homeostasis at least in part by reducing adipose tissue inflammation and lipid accumulation and increasing fatty acid oxidation.

Grant Funding Source: UT AgResearch and College of Human Sciences / Texas Tech University
Severe sepsis cytomix elicits inflammation in cerebrovascular endothelial cells and polymorphonuclear (PMN) leukocytes in vitro.
Authors: Blom Christopher , Deller Brittany , Fraser Douglas D , Martin Claudio , Young Bryan , Cepinskas Gediminas
Published: June 2021

The mechanisms of severe sepsis induced activation/dysfunction of the cerebrovascular endothelium are poorly understood. Our findings indicate that severe sepsis in humans results in up‐regulation of 8 (p<0.01; out of 41 measured) inflammation‐relevant analytes in the blood plasma. We employed Severe sepsis Cytomix (SS‐CM) consisting of 8 cytokines/chemokines (at the levels detected in Severe sepsis‐plasma) to assess activation/dysfunction of human‐derived cerebrovascular endothelial cells (hCMEC/D3; provided by Dr. P.O. Couraud, INSERM) and neutrophilic leukocytes (PMN). Various inflammation‐relevant endpoints were assessed. The obtained results indicate that stimulation of hCMEC/D3 with SS‐CM failed to induce ROS production, activation of NF‐κB, and increase in hCMEC/D3 permeability; however ICAM‐1 gene expression was up‐regulated in hCMEC/D3. The latter was accompanied by increased PMN adhesion to hCMEC/D3 under conditions of “flow” (0.7 dyn/cm2 shear stress). The most potent increase in PMN adhesion, however, occurred when both PMN and hCMEC/D3 were stimulated with SS‐CM. PMN adhesions was prevented by interfering (neutralizing MAb) with β‐2‐integrin function. Taken together our findings indicate that increased adhesive interaction between circulating PMN and cerebrovascular endothelium may contribute to BBB dysfunction in severe sepsis via β‐2‐integrin. HSFO NA‐6914, PSI 11–01)
Adaptation to chronic alcohol intake alters STAT3 genome‐wide binding dynamics during liver regeneration
Authors: PATRA BISWANATH , Kuttippurathu Lakshmi , Vadigepalli Rajanikanth , HOEK JAN B
Published: June 2021

The objective of this study is to characterize effects of adaptation to chronic alcohol intake on STAT3 mediated transcriptional regulatory networks during the priming phase of liver regeneration. Rats were fed liquid diet containing 36% of total calories derived from ethanol for 5 weeks, and control animals were pair‐fed an isocaloric liquid diet. A 70% partial hepatectomy (PHx) was performed. Genome‐wide STAT3 binding targets were detected by ABI SOLiD sequencing of Chromatin Immunoprecipitated (ChIP) samples. Our results indicate that (1) STAT3 targets 114 promoters in control samples, and 794 promoters in chronic ethanol at the baseline adapted state; (2) PHx induced a significant increase in STAT3 binding at 6h, in chronic ethanol samples (771 genes) versus controls (246 genes). STAT3 target genes specific to the chronic ethanol group participate in key processes such as protein complex assembly, cell signaling, RNA metabolic process, and programmed cell death etc. Based on identified STAT3 motifs in target promoters, we found combinatorial interaction between multiple transcription factors. We conclude that chronic ethanol effects on liver regeneration are mediated by altered STAT3 binding at specific pathway genes. Research Support: NIH R01 AA018873, K05 AA017261, and T32 AA007463.
The non‐neuronal cholinergic system: sources of vascular ACh
Authors: Davis Harrison , Smith Kirsty , Bagher Pooneh , Dora Kim , Garland Christopher
Published: June 2021

The localisation of muscarinic receptors in vascular endothelial cells and the consequences of activation of these receptors are extensively documented. However, it is not known if endogenous acetylcholine is available to activate receptors, and if so where it originates. As acetylcholine (ACh), its receptors and synthesising enzymes are widely expressed in extra‐neuronal cells and tissues (the non‐neuronal cholinergic system) including blood cells, we investigated a potential role for these cells as a source of endogenous ACh to stimulate the endothelium. Isolated rat mesenteric arteries were cut open and fixed en‐face. Endothelial cells were loaded with a calcium dye to measure levels of intracellular calcium using confocal microscopy. Endothelial cells exhibited transient but pronounced calcium increases when exposed to rat whole blood. The frequency of endothelial cell calcium events, average fluorescence and the number of active cells increased significantly above the spontaneous basal activity. ATP was shown not to be a component of this response, however, a trend in sensitivity to block with atropine (1μM) was observed. Taken together these data suggest that ACh may originate in sufficient quantities from blood cells to stimulate endothelial cell calcium increases. As such this may represent a significant auto or paracrine influence on vascular tone.

Work funded by the British Heart Foundation.
Identifying key residues important for CGRP binding to its receptor
Authors: Kuteyi Gabriel , Barwell James , Poyner David
Published: June 2021

The calcitonin gene‐related peptide (CGRP) is a 37 amino acid neuropeptide. Its receptor is a heterodimeric complex of calcitonin receptor‐like receptor (CLR) – a family B G‐protein coupled receptor – and a single‐pass transmembrane protein, receptor activity modifying protein 1 (RAMP1). Here, we identify residues, within the N‐terminal extracellular domain (ECD) of CLR, potentially involved in ligand binding.

Certain residues presumed to be possible sites of contact for the CGRP were picked from the CLR/RAMP1 ECD crystal structure (PDB 3N7S). Residues were mutated to alanine (A) by site‐directed mutagenesis (QuikChange™, Stratagene). Mutants were transiently co‐transfected with HA‐tagged CLR and RAMP1into COS7 cells using PEI. After 48 hr, cells were treated with 10pM – 100nM CGRP for 15 min. cAMP production was assessed by a radio‐receptor assay. Concentration‐response curves were analysed with Graphpad Prism 4.00 to give pEC50 values. Cell surface expression was determined by enzyme‐linked immunosorbent assay (ELISA) (Barwell et al., 2011).

All mutants (Table 1) showed reduced potency, though to varying degrees. W69A and D70A showed significant reduction in cell surface expression

These findings suggest that these residues are important for the interaction of CGRP with its receptor. W69A and D70A, part of the WDG motif of family B GPCRs, are thought to rather play a role in receptor stability (Kumar et al., 2012). The data is consistent with CGRP binding in a groove between CLR and RAMP1.

This project was supported by Aston School of Life and Health Sciences
The role of the kappa‐opioid receptor on age‐specific behaviors induced from co‐administration of alcohol and nicotine
Authors: Larraga Armando , Belluzzi James D. , Leslie Frances M.
Published: June 2021

The two most concurrently used recreational drugs are EtOH and tobacco. Adolescence is the age of onset for use of both these drugs. We have previously found concurrent self‐administration of EtOH and nicotine (EtOH+Nic) is reinforcing in adolescent, but induces kappa‐opioid receptor (KOR)‐mediated aversion in adult rats. We also found acute administration of EtOH+Nic is anxiolytic in adolescents, but not adults. This study focused on the mechanism of action of EtOH+Nic anxiolysis in adolescents, and tested if the KOR has a role in the lack of effect in adults. We hypothesize that KOR activation prevents EtOH+Nic‐induced anxiolysis in adults. To test this, we pretreated male adolescent, aged postnatal day (P)29, and adult (P87) Sprague Dawley rats with the irreversible KOR antagonist, nor‐BNI (10 mg/kg, i.p.). Three days later, we acutely administered saline, or Nic (30 μg/kg) and EtOH (4 mg/kg), then monitored behavior in a novel open field for 30 minutes. Brains were immediately collected after the open field behavior experiment and processed for in situ hybridization for c‐fos mRNA. C‐fos is an immediate early gene commonly used as a neuronal activity marker. Anatomical analysis comparing c‐fos activation induced by acute EtOH+Nic will help understand the mechanism of action by which EtOH+Nic causes unique behaviors in adolescent rats, and how KOR is involved in EtOH+Nic aversion in adult rats.
Modulation of sirtuins during acute inflammatory pain: the role of ROS
Authors: Muscoli Carolina , Lauro Filomena , Ilari Sara , Pucci Bruna , Dagostino Concetta , Gliozzi Micaela , Tafani Marco , Palma Ernesto , Ventrice Domenica , Russo Matteo , Fini Massimo , Mollace Vincenzo , Salvemini Daniela
Published: June 2021

Free radicals plays a crucial role in the enhanced pain sensitivity experienced during inflammatory diseases. ROS accumulation modifies the activity of sirtuins (SIRT 1–7), proteins that deacetylate histones and non‐histone proteins playing an important role in regulation of inflammation and neurodegenerative diseases. SIRT1 either directly or indirectly influences the redox property of the cell and it is also regulated by oxidative stress. SIRT3 plays a role in the maintenance of basal levels of reactive oxygen species; it stimulates MnSOD and catalase production by FOXO activation as SIRT1 and deacetylates two critical lysine residues on MnSOD itself to promote its antioxidative activity. We show that removal of free radicals by antioxidants is able to block the thermal hyperalgia in the carrageenan‐induced inflammation model. This effect was associated with inhibition of edema, lipid peroxidation, nitrotyrosine formation and PARP activation in the paw exudates and to nitrotyrosine formation in the spinal cord. We report that SIRT1 and SIRT3 are decreased in the spinal cord of carrageenan treated rats and that removal of free radicals by antioxidant enhanced their expression. These findings demonstrate for the first time that sirtuins activation by antioxidants is beneficial during oxidative stress induced hyperalgesia and inflammation.

This work has been supported by PON a3–00359 and GR‐2010–2318370
Effects of Ethanol and Oxidized Metabolites of Linoleic Acid on Caco‐2 cell Model of Intestinal Epithelial Barrier
Authors: Liu Huilin , Joshi‐Barve Swati , Barve Shirish , McClain Craig , Ramsden Christopher , Kirpich Irina
Published: June 2021

Introduction

The intestinal epithelial barrier plays a critical role in host defense against luminal pathogens. Ethanol (EtOH) and dietary components may alter barrier integrity resulting in increased intestinal permeability, endotoxemia and multi‐organ pathology. The aim of the present study was to evaluate the effects of EtOH, linoleic acid (LA, a major unsaturated fatty acid in the Western diet), and its bioactive oxidized metabolites (OXLAMs) on Caco‐2 cells, an in vitro model of intestinal epithelial barrier.

Materials and Methods

Caco‐2 cell monolayers were exposed to LA, OXLAMs (9‐and 13‐hydroxyoctadecadienoic acids, HODEs) and/or EtOH. Transepithelial resistance (TEER), expression of intestinal tight junction (TJ) proteins, and pro‐inflammatory response were evaluated.

Results

Decreased TEER in response to EtOH, LA, and multiple HODE isoforms was observed after 24 hours of exposure. Combined effects of EtOH and 13(S)HODE or +13HODE on TEER were greater compared to EtOH and 9(S)HODE or +9HODE. The combination of EtOH and 13(S)HODE or +13HODE resulted in greater down‐regulation of TJ proteins and increased expression of TNF‐α, IL‐1β, and IL‐6 mRNA compared to EtOH alone.

Conclusion

Combined exposure of EtOH and OXLAMs exacerbates disruption of intestinal barrier integrity and pro‐inflammatory response in Caco‐2 cells.
The identification of novel modulators for the SIRT‐6 protein
Authors: Moaddel Ruin , Singh Nagendra , Ravichandran Sarangan , Norton Darrell D. , Fugmann Sebastian
Published: June 2021

Objective

SIRT6 has been proposed as a potential therapeutic target for metabolic disorders and the prevention of age‐associated diseases. The aim of this study was to develop a screening method for the identification of novel modulators of SIRT6 from a natural plant extract.

Methods

SIRT6 was immobilized onto the surface of magnetic beads (SIRT6‐MB), and the enzymatic activity was measured. Subsequently, SIRT6 was immobilized onto the surface of an open tubular capillary and characterized by frontal chromatography.

Results

The identification of quercetin, vitexin as inhibitors from Trigonella foenum‐graecum was carried out. Structurally related flavonoids were then tested for their activity on SIRT6. In addition to obtaining their binding activity, they were also ranked based on their ability to displace quercetin. Further, preliminary results with ligand fishing experiments using the SIRT6‐MB suggest the identification of additional compounds from fenugreek seed extract with activity at the SIRT6‐protein.

Conclusion

The SIRT6‐MBs were successfully used for the identification of an active ligand from a botanical matrix, which sets the basis for identification of SIRT6 modulators from plant extracts.
Construction of a novel system for developing inhibitors of influenza virus neuraminidase by yeast cell surface engineering
Authors: Shigemori Tomohiro , Nagayama Mitsuru , Yamada Junki , Miura Natsuko , Yongkiettakul Suganya , Kuroda Kouichi , Katsuragi Tohoru , Ueda Mitsuyoshi
Published: June 2021

Neuraminidase (NA) is a glycoprotein of the influenza virus membrane and is involved in releasing newly made virus from infected cells, and thus it is the main target of the current influenza chemotherapy. Novel inhibitors of NA which is easily mutated are urgently required to be developed because of the recent emergence of oseltamivir‐resistant strains and highly pathogenic avian influenza viruses. Yeast cell surface engineering is one of the useful technologies for screening binding proteins with specific affinity to target molecules. In this study, we attempted to construct the efficient system for developing inhibitors of NA using this technology. The head domain of wild‐type or oseltamivir‐resistant (H275Y) H5N1 NA was displayed on yeast cell surface and their activities were evaluated. These results suggested that the displayed head domain of NAs retained the innate characteristics of NAs and was useful for evaluating inhibition activity[1].
Molecular topology as novel strategy for Alzheimer's disease drug discovery
Authors: Pasinetti Giulio Maria
Published: June 2021

Molecular topology (MT) uses and expands upon the principles governing molecular connectivity theory of numerically characterizing the molecular structure of, in the present case, active anti‐Alzheimer's disease (AD) drugs/agents using topological descriptors to build models. Topological characterization has been shown to embody sufficient molecular information to provide a strong correlation to therapeutic efficacy. In the present study, we used MT to include multiple bioactive properties to identify multifunctional single agent compounds, in this case, the dual functions of Aβ –lowering and anti‐oligomerization. Using this technology, we identified and designed novel compounds in chemical classes unrelated to current anti‐AD agents that exert dual Aβ lowering and anti‐Aβ oligomerization activities in animal models of AD. AD is a multifaceted disease with different pathological features. Our study for the first time demonstrated that MT can provide a novel strategy in designing molecules that target multiple pathological mechanisms of AD.
Design of complement inhibitors that block IgG‐induced C3 activation through its properdin binding site
Authors: Petitclerc Eric , Mercier Virginie , Drouin Mathieu , Simard Carl , Aubin Eric
Published: June 2021

Complement dysregulation appears important in the etiology of many autoimmune and inflammatory diseases. IVIG, the C3 inhibitor compstatin and the humanized anti‐C5 antibody Eculizumab modulate complement through direct inhibition of protein activation. In an effort to decipher the mechanisms by which protein‐protein interactions modulate the activation of the complement protein C3 through the classical and alternate cascade, we investigated whether small peptides could interfere with the specific interaction between C3 and properdin. First, we verified that a 34 amino acid peptide, derived from C3, inhibits its interaction with properdin but cannot displace either C3 or properdin from aggregated IgG. Interestingly, a 9‐mer peptide that span within the original 34 amino acids is able to replicate the effect of the 34‐mer toward C3‐properdin interaction. Of utmost interest, further experiments demonstrate that this 9‐mer is also able to inhibit the binding of C3 to IgG. On a molar basis, the active peptide is as powerful as IVIG and an optimized cyclic peptide that inhibits the activation of C3, compstatin. Further analyses demonstrate that the active 9‐mer peptide is able to inhibit complement activation in sensitized red blood cells (SRBC) hemolysis assays. Altogether, these results showed that inhibitory effect of IVIG on in vitro complement activation can be mimicked, at least in part, by a 9‐mer peptide. A quantitative structure‐activity relationship (QSAR) was performed using the minimal active 9‐mer peptide. Linearity and the C‐terminus amino acids were shown to be critical for its activity, and further length reduction of the peptide strongly reduces its activity.

We have identified a potent complement inhibitor with potential therapeutic applications in inflammatory and autoimmune diseases.
Evidence of centrally‐induced cholinergic vasodilatation in skeletal muscle during voluntary one‐legged cycling and motor imagery
Authors: Ishii Kei , Matsukawa Kanji , Liang Nan , Endo Kana , Idesako Mitsuhiro , Kataoka Tsuyoshi , Ueno Kazumi , Hamada Hironobu
Published: June 2021

Centrally‐induced vasodilatation occurs in non‐contracting vastus lateralis muscle (VL) at the onset of voluntary one‐legged cycling (Ishii et al. J Appl Physiol, 2012). To test whether the vasodilatation is mediated by a cholinergic mechanism in humans, seven subjects were asked to perform voluntary one‐legged cycling and motor imagery of the one‐legged cycling before and after intravenous injection of atropine sulfate. The concentration of oxygenated‐hemoglobin (Oxy‐Hb) in the bilateral VLs was measured as an index of muscle blood flow with near‐infrared spectroscopy. The Oxy‐Hb increased not only the non‐contracting VL but also the contracting VL at the onset of voluntary one‐legged cycling. Atropine blunted the increases in the Oxy‐Hb of both non‐contracting and contracting VL. Furthermore, motor imagery of the one‐legged cycling induced increases in the Oxy‐Hb of bilateral VLs, which were decreased by atropine. These results suggest that centrally‐induced cholinergic vasodilatation occurs in both non‐contracting and contracting skeletal muscle during voluntary exercise and motor imagery.
Depressive Symptoms, Inflammation and Microvascular Dysfunction: Presence of a Gender Disparity
Authors: Stanley Shyla C , Butcher Joshua T , Frisbee Stephanie J , D'Audiffret Alexandre C , Frisbee Jefferson C
Published: June 2021

Patients suffering from depressive symptoms have blunted vascular responses to dilator stimuli, with increasing evidence suggesting that this may be due to an inflammation/ROS‐induced loss in dilator metabolite bioavailability. We used the established unpredictable chronic mild stress (UCMS) model to simulate human clinical depression in mice. Endothelium‐dependent dilator responses in both conduit arteries (methacholine) and arterioles (acetylcholine) were attenuated by UCMS; more severely in males vs. females (responses to sodium nitroprusside were intact). Treatment with TEMPOL improved dilation in vessels from both genders. NOS inhibition had minimal impact on dilator responses in vessels of UCMS males, while reducing responses in females. Inhibition of COX blunted reactivity in UCMS males while exhibiting a muted effect in UCMS females. Impaired agonist‐induced vascular production of NO and PGI2 in UCMS males versus females paralleled mechanical responses to NOS inhibition, but less well with COX inhibition (suggesting constrictor metabolites via COX may also contribute). The circulating levels of inflammatory markers MCP‐1 and TNF‐α were similar in both genders, despite the differences in dilator reactivity. These results suggest a greater susceptibility of males to UCMS‐induced production of ROS and inflammatory markers while protective mechanisms may blunt these effects in females.
Blunted hypertensive response to Ang II infusion in IFN‐g knockout mice: molecular mechanisms
Authors: Kamat Nikhil V , Fletcher Nicholas K.H. , Thabet Saleem , Harrison David G. , McDonough Alicia A
Published: June 2021

The hypertensive response to AngII infusion is blunted in CD8−/− and IFNg −/− mice.

AIM

Determine the molecular basis for the blunted response by evaluating renal Na transporter and SPAK abundance and phosphorylation in IFNg KO vs WT mice using quantitative immunoblots. At baseline, blood pressure (BP) in IFNg KO mice was similar to WT, yet abundance of NKCC2p, NCCp and SPAKp were elevated, evidence for Na transporter activation in the distal nephron. AngII infusion into WT mice raised BP 38 mmHg and increased abundance of NKCC2p, NCCp, SPAK and SPAKp and cleaved form of α ENaC. AngII infusion into IFNg KO raised BP 15 mmHg yet did not change NKCC2p, NCCp, SPAK or SPAKp; however, AngII infusion increased cleaved form of αENaC.

WT+AngII vs. WT ‐ baseline
IFNg KO vs. WT ‐ baseline
IFNg KO+AngII vs. IFNg KO

NKCC2
0.79 ± 0.03*
1.01 ± 0.09
1.46 ± 0.18

NKCC2p
4.17 ± 0.60*
6.12 ± 1.17*
1.25 ± 0.31

NCC
0.88 ± 0.03
1.28 ± 0.10
1.22 ± 0.09

NCCpS71
8.96 ± 2.53*
8.33 ± 2.55*
1.66 ± 0.40

α‐ENaC (90 kDa)
1.42 ± 0.12*
1.09 ± 0.06
1.54 ± 0.15*

α‐ENaC (20 kDa)
2.41 ± 0.12*
1.28 ± 0.1*
1.64 ± 0.25*

SPAK
1.41 ± 0.12*
1.28 ± 0.11
1.18 ± 0.10

SPAKp
1.71 ± 0.24*
1.81 ± 0.10*
0.96 ± 0.13

In summary, we postulate that IFNg KO mice have a sodium‐losing phenotype that provokes a pronounced activation of NKCC and NCC phosphorylation mediated by SPAK activation, and that the response to AngII infusion is blunted because the AngII targets are already activated. NIH RO1 DK 083785
Renal Mitochondria Predominantly Express [des‐Ang I]‐Angiotensinogen and Renin
Authors: Guan Meijian , Nautiyal Manisha , Hakuda Debroah , de Lima Rariane , Rose James , Pirro Nancy T , Chappell Mark C
Published: June 2021

Increasing evidence supports an intracellular‐based renin‐angiotensin system (RAS) expressed in a variety of cell types and tissues. Functional AT1, AT2 and AT7 receptors are evident on nuclei and mitochondria (Mito) of the kidney. The presence of intracellular Ang receptors may well portend for the local synthesis of Ang II and Ang‐(1–7) ligands within subcellular organelle. The present study assessed the expression of angiotensinogen (Aogen) in the Mito from renal cortex and proximal tubule cells using 2 antibodies directed to the Ang I and N terminal domains of rat Aogen. Mitos were isolated by differential centrifugation and a discontinuous Percoll gradient from the renal cortex of both rat and sheep, as well as from the rat NRK52E proximal tubule cell line. Immunoblots revealed a prominent 60 kDa band in the purified Mito fraction using the Aogen antibody specific for the N domain. However, the antibody directed to the Ang I sequence of Aogen revealed faint detection of protein bands in the Mito fraction from rat and sheep kidney cortex, as well as the NRK epithelial cells. Since the Mito apparently lack the predominant form of intact Aogen, the fractions were further probed for immunoreactive renin and a 55 kDa band was strongly evident for the enzyme. We conclude that the des‐Ang I form of Aogen is predominantly expressed in renal Mito which may reflect active processing of the precursor protein by renin.
Regulation of plasma aldosterone levels and renal sodium handling by the circadian clock protein Per1
Authors: Richards Jacob , Cheng Kit‐Yan , All Sean , Skopis George , Jeffers Lauren , Lynch Jeanette , Wingo Charles S. , Gumz Michelle L.
Published: June 2021

Regulation of sodium balance by the kidney is an important determinant of long term blood pressure (BP) control. We have previously shown that the circadian clock protein Per1 regulates expression of the alpha subunit of the renal epithelial sodium channel (αENaC). Together with our recent finding that Per1 knockout (KO) mice exhibit dramatically lower BP than wild type (WT) mice, these data led to the hypothesis that the BP phenotype involves a renal Na handling mechanism. Consistent with our hypothesis, metabolic balance studies showed that Per1 KO mice excreted more urinary Na compared to WT mice. We measured plasma aldosterone (Aldo) levels in WT and Per1 KO mice at noon and midnight. WT mice experienced the expected circadian surge in Aldo. Importantly, the circadian Aldo surge did not occur and Aldo levels were significantly lower in Per1 KO mice. Thus we examined the expression of the circadian target gene Hsd3b6, an adrenal glomerulosa cell‐specific enzyme in the Aldo synthesis pathway. Whereas Hsd3b6 was expressed in a circadian manner in WT mice, that pattern was lost in Per1 KO mice. Hsd3b6 expression was significantly less in Per1 KO mice, providing one possible explanation for the lower plasma Aldo levels observed in Per1 KO mice. Taken together, these data support the hypothesis that the BP phenotype observed in mice lacking Per1 may involve a primary adrenal defect in addition to a renal defect in Na conservation.