bluuebunny/crossref_metadata_2025 huggingface dataset:
A High Flavonoid Diet Does Not Improve Markers of Insulin Resistance and Subclinical Inflammation in Overweight and Obese Men and Women
Authors: Lefevre Michael , Ward Robert , Bergeson Janet , Hergert Nancie , Hergert John , Aguilar Sheryl
Published: June 2021

Populations consuming healthy diets high in flavonoids have lower levels of subclinical inflammation, better insulin sensitivity and reduced risk for both cardiovascular disease and type 2 diabetes. We hypothesized that providing a diet high in flavonoids would reduce insulin resistance and subclinical inflammation relative to a low flavonoid diet. To test this hypothesis, 29 overweight/obese (BMI; 25.2–34.5 kg/m2) men (N=16) and women (N=13) were provided two well‐controlled diets that were identical in macronutrient content (Protein, 17% en; Fat, 30% en; Carbohydrate, 53% en), but differed markedly in flavonoid content (Low Flavonoid Diet, 10 mg/1000 Kcals; High Flavonoid Diet, 340 mg/1000 Kcals). All meals for both diets were prepared and fed for 6 weeks each in a randomized cross‐over design with endpoints determined in duplicate during the last week of each diet period. All meals were well tolerated by the participants and overall compliance as assessed by questionnaires and direct observations was excellent. Relative to the Low Flavonoid Diet (LFD), the High Flavonoid Diet (HFD) did not significantly affect serum chemistry (blood urea nitrogen, creatinine, estimated glomerular filtration rate, sodium, potassium, chloride, CO2, calcium, total protein, albumin, globulin, A/G ratio, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase). Surprisingly, the HFD had no significant effects on blood pressure and plasma lipids (triglycerides, LDL‐C, HDL‐C) and tended to increase (all non‐significant) markers of inflammation (C‐reactive protein, soluble TNFα receptor‐1, soluble TNFα receptor‐2, interleukin‐6). Contrary to expectations, markers of insulin resistance (fasting insulin, glucose, HOMA‐IR) were modestly, but significantly higher on the High Flavonoid Diet relative to the Low Flavonoid Diet. Under the conditions of this study, diets high in flavonoids failed to improve plasma lipids, insulin sensitivity or subclinical inflammation. Our results are at odds with the reported beneficial effects of high flavonoid diets on CVD risk factors, insulin sensitivity and systemic inflammation observed in both preclinical and epidemiological studies.

Support or Funding Information

This work was supported by USDA/NIFA grant 2014‐67017‐21749.
Dietary Glycomacropeptide Increases Fat‐Free Mass, Bone Mineral Content and Fatty Acid Oxidation Compared With Casein In Female Mice
Authors: Sawin Emily Ann , Stroup Bridget Marie , Murali Sangita G , Ney Denise Marie
Published: June 2021

Background

Glycomacropeptide (GMP) is a 64 amino acid (AA) glycophosphopeptide released from κ‐casein during cheese making that promotes satiety, reduces body fat, increases bone mass and infers prebiotic and anti‐inflammatory effects.

Objective

To determine if GMP, when fed as the primary protein source, modulates feed efficiency, weight gain and body composition and increases fat oxidation compared with casein.

Methods

Weanling female C57Bl6 mice were fed high and low fat diets (HF 60% en; LF 13% en) with casein or GMP for 28 wks with assessment of body weight and food intake. Body composition was assessed by DXA. Fatty acid oxidation was measured in liver, muscle, and fat tissues using 14C‐palmitate. Plasma concentrations of amino acids, hormones and cytokines were determined.

Results

Despite similar food intake, mice fed the HF GMP diet had significantly lower final body weight than mice fed the HF casein diet. Fat free mass was increased in mice fed the LF GMP diet relative to the LF casein diet with similar final body weight but greater food intake. Regardless of dietary fat content, GMP significantly increased bone mineral content (BMC) and fatty acid oxidation in subcutaneous fat compared to casein.

Conclusions

GMP reduces feed efficiency, modulates body composition to increase fat free mass and BMC, and increases fat oxidation compared to casein in female mice. GMP is a bioactive food ingredient with application to obesity and osteopenia in females.

Support or Funding Information

Supported by the Wisconsin Alumni Research Foundation, NIH T32 DK 007665, and USDA Hatch WIS01790.
Relationship between concentrations of lutein and StARD3 among pediatric and geriatric human brain tissue
Authors: Johnson Elizabeth J , Tanprasertsuk Jirayu , Li Binxing , Bernstein Paul S , Vishwanathan Rohini , Johnson Mary Ann , Poon Leonard W
Published: June 2021

Lutein, a dietary carotenoid, selectively accumulates in human retina and brain. While many epidemiological studies show evidence of a relationship between lutein status and cognitive health, lutein's selective uptake in human brain tissue and its potential function in early neural development and cognitive health have been poorly evaluated at a molecular level. The objective of this study was to evaluate the cross‐sectional relationship between concentrations of brain lutein and StARD3 (identified as its binding protein in retinal tissue) among three age groups: infants (1–4 months, n=10), older adults (55–86 years, n=8), and centenarians (98–105 years, n=10). Brain lutein concentrations were analyzed by high‐performance liquid chromatography and StARD3 levels were analyzed by Western Blot analysis. The strong relationship in infant brains (r=0.75, P <0.001) suggests that lutein has a role in neural development. The relationship remained significant but weaker in older adults (r=0.51, P <0.05) and insignificant in centenarians (r=0.08, P >0.05), eight of whom had dementia. Although the sample size for each group was small, these results suggest an age‐related decrease or abnormality of StARD3 activity in human brain. Given that StARD3 is also involved in cholesterol transportation, a process that is aberrant in neurodegenerative diseases, the potential protective function of lutein against these diseases remains to be explored.

Support or Funding Information

This work was supported by : USDA agreement 58‐1950‐4‐003, NIH 1PO1‐AG17553, Abbott Nutrition, DSM Nutritionals, NIH EY‐11600 (PSB), NIH EY‐14800 (PSB) and an unrestricted grant to the Moran Eye Center of the University of Utah from Research to Prevent Blindness
Differential Expression of Genes Involved in Inflammatory Immune Response and Protein Ubiquitination in the Prefrontal Cortex of Rhesus Macaque with High and Low Lutein Content
Authors: Mohn Emily S , Erdman John W. , Neuringer Martha , Kuchan Matthew J , Johnson Elizabeth J
Published: June 2021

Lutein, a carotenoid with antioxidant and anti‐inflammatory functions, accumulates in the primate brain. Studies have shown that brain levels of lutein are more consistently related to better cognitive test scores in humans than other antioxidants found in higher concentrations in the brain, such as vitamin E. Supplementation with lutein can improve verbal fluency, memory scores, and rates of learning in older adults. However, the mechanism by which lutein functions in cognition is unclear. The aim of this study was to determine the relationship between lutein concentration and gene expression profiles in rhesus macaque brain. Brain tissue was dissected from monkeys fed a chow diet containing lutein (~16 μmol/kg diet). Next generation RNA sequencing was used to determine differentially expressed genes in the prefrontal cortex of monkeys (11–13y) with high lutein content (mean=34.5 pg/mg wet weight, n=3) versus those with low lutein content (mean=11.8 pg/mg wet weight, n=3). Lutein concentrations were determined in these brain samples using HPLC. A total of 415 genes were differentially expressed between the two groups. Ingenuity Pathway Analysis (IPA) revealed the up‐regulation of pro‐inflammatory immune response pathways, such as chemokine signaling, antigen presentation, leukocyte extravasation, and neuropathic pain signaling, in brains with low lutein content. A total of 16 genes were upregulated in this pro‐inflammatory network, with specific genes of interest including beta‐2‐microglobulin and p38 MAPK (1.5 fold change for each), which are both associated with age‐related cognitive impairment and neurodegeneration. Additionally, up‐regulation of the protein ubiquitination pathway (9 genes), a hallmark of Alzheimer's disease, was observed in brains with low lutein content. These results indicate that lutein may play a role in suppressing neuroinflammation at the transcription level, and provide the first steps towards understanding the mechanisms underlying the beneficial effect of lutein on cognition.

Support or Funding Information

This work was supported by grants from Abbott Nutrition through the Center for Nutrition, Learning, and Memory at the University of Illinois, USDA (58‐1950‐4‐003), and NIH grant P51OD011092 to the Oregon National Primate Research Center
High‐fat diet exacerbates bone loss in mice implanted with low‐dose slow‐release lipopolysaccharide pellets
Authors: Cao Jay J , Shen Chwan‐Li
Published: June 2021

Obesity is associated with chronic up‐regulation of inflammatory cytokines which stimulate osteoclast activity and bone resorption. Osteopenia or low bone mass is observed in a variety of physiological conditions with chronic inflammation including aging and post‐menopause with estrogen deficiency. This study investigated whether obesity induced by a high‐fat diet alters bone loss in an animal model of chronic inflammation with low‐dose, slow‐release of lipopolysaccharide (LPS). Forty‐eight 6‐wk‐old female C57BL/6 mice were randomly assigned to four treatment groups (n=12/group) in a 2×2 factorial design: control (placebo) or LPS treatment and fed either a normal‐fat (NF, 10% energy as fat) or a high‐fat (HF, 45% energy as fat) diet ad libitum for 3 mon. LPS (E‐coli serotype 0127:B8) was incorporated into time‐release pellets designed to provide a consistent dose (1.3μg/day) for 3 months The LPS and placebo (containing just biodegradable carrier binder) pellets were implanted subcutaneously in the dorsal region of the neck. Mice implanted with LPS had similar body weight, fat mass and lean mass to those in the placebo (NF + HF) groups. LPS treatment increased serum concentration of tartrate‐resistant acid phosphatase (TRAP, a bone resorption marker), expression of proinflammatory cytokines (TNF‐α and IL‐6) in abdominal adipose tissue, and bone marrow osteoclast differentiation, while decreasing tibial and vertebral trabecular bone volume, trabecular thickness, connectivity density, and bone mineral density (P < 0.05). Mice fed the HF diet had higher body weight (P < 0.05) mainly due to increased fat mass (P < 0.05) than animals fed the NF diet. Obesity induced by the HF diet increased serum TRAP concentration, bone marrow osteoclast differentiation, and expression of TNF‐α and IL‐6 in adipose tissue (P < 0.05). Compared to the NF groups, mice in the HF groups had decreased bone volume, connectivity density, trabecular number while had increased trabecular separation, and structure model index (P < 0.05). Mice implanted with LPS pellets and fed the high‐fat diet had the lowest bone mineral density compared to all other groups. These results show that obesity exacerbates bone loss in mice with chronic inflammation induced by low‐dose slow‐release LPS administration.

Support or Funding Information

the USDA Agricultural Research Service program “Bone Metabolism in Obesity,” Current Research Information System no. 5450‐51000‐046‐00D
Leucine ameliorates endotoxin‐induced alterations in protein‐protein interactions within mTORC1 complex in neonatal piglets
Authors: Hernandez‐Garcia Adriana D , Suryawan Agus , Nguyen Hanh , Columbus Daniel A , Manjarin Rodrigo , Orellana Renan A , Davis Teresa A
Published: June 2021

Sepsis decreases protein synthesis in skeletalmuscle in part by limiting the phosphorylation of translation initiationregulatory factors, eukaryotic initiation factor (eIF) 4E‐binding protein (4EBP1)and S6 kinase (S6K1), that are downstream of the mammalian target of rapamycincomplex 1 (mTORC1). Previous work inmature rats suggests that decreased raptor binding activity to eIF3b mayunderline the sepsis‐induced reduction in protein synthesis in skeletal muscle. Raptor‐eIF3b interaction is crucial for thestimulation of protein translation and its alteration during endotoxemia inmature animals appears to block the anabolic response to the branched‐chainamino acid, leucine (Leu). We have shown in healthy neonatal pigs that Leu supplementation increases muscle protein synthesis by enhancing the activation of mTORC1 and its downstream targets, 4EBP1 and S6K1. To determine if supplemental Leu can stimulate raptor‐eIF3b binding and muscle protein synthesis in theneonate during endotoxemia, overnight fasted neonatal pigs (n=6–8/group) were infused for 8 h with LPS or saline while plasma amino acids, glucose, and insulin were maintained at fasting levels during pancreatic‐substrate clamps. Leu (400 μmol·kg−1·hr−1) or saline was infused during the last h. Phosphorylation of 4EBP1 and S6K1, eIF4E‐eIF4G complex formation, raptor‐eIF3b binding, protein synthesis rates, ribosomal translational capacity (Cs) and translational efficiency (KRNA) were determined in longissimus dorsi (LD), gastrocnemius (Gast) and soleus muscles. LPS decreased protein synthesis and KRNA in LD, Gast, and soleus muscles (P<0.05) while Cs did not change compared to controls. Leu infusion in the LPS group increased 4EBP1 and S6K1 phosphorylation, eIF4E‐eIF4G complex formation, KRNA, and the binding of raptor to eIF3b (by 2 to 3 fold) and protein synthesis (by over 80%) in LD, Gast, and soleus muscles compared to LPS infusion alone (P<0.05). Compared to saline, Leu infusion increased protein synthesis, KRNA, raptor‐eIF3b interaction, 4EBP1 and S6K1 phosphorylation and eIF4E·eIF4G complex formation in all muscles (P<0.05). Raptor‐eIF3b binding showed a linear correlation with 4EBP1 and S6K1 phosphorylation, eIF4E‐eIF4G formation, and protein synthesis in LD, Gast, and soleus muscles that was independent of treatment(P<0.01). These findings suggest that parenteral Leu supplementation attenuates the reduction in muscle protein synthesis induced by endotoxemia by stimulating the interaction of raptor with eIF3 within the mTORC1 complex in the neonate.

Support or Funding Information

Supported by NIH AR444474, USDA/ARS 6250‐51000‐055, NIH HD072891, and USDA NIFA 2013‐67015‐20438
Intracerebroventricular Oxytocin Effects on mRNA Abundance Differ in Adipose Tissue of Chicks from Low and High Body Weight‐Selected Lines
Authors: Koskinen Anna , McConn Betty , Wang Guoqing , Siegel Paul B , Cline Mark A , Gilbert Elizabeth R
Published: June 2021

Neuropeptide oxytocin (OXT), an important anorexigenic factor in the central nervous system, has been shown to have effects on adipose tissue lipid metabolism in mammals. The purpose of this study was to determine the effect of intracerebroventricular (ICV) administration of OXT on subcutaneous adipose tissue mRNA abundance in chicks from lines selected for low (LWS) or high (HWS) juvenile body weight. These lines differ in adiposity. At 5 days post‐hatch, LWS and HWS chicks were fasted for 3 hours, injected ICV with 0 (vehicle), 0.039, 0.312, or 2.5 nmol OXT (n=10 per group).Their food intake was monitored, and subcutaneous adipose tissue was collected at 3 hours post‐injection. Total RNA was isolated, reverse transcribed and real time PCR performed to measure mRNA abundance of the following adipose metabolism‐associated factors: acyl‐CoA dehydrogenase, long chain, 1‐acylglycerol‐3‐phosphate O‐acyltransferase 9, CCAAT/enhancer‐binding protein alpha, CCAAT/enhancer‐binding protein beta, fatty acid binding protein 4 (FABP4), lipoprotein lipase (LPL), monoglyceride lipase (MGLL), OXT, peroxisome proliferator‐activated receptor gamma, and sterol regulatory element‐binding transcription factor 1. The relative quantity values were analyzed by ANOVA in JMP Pro 11 (SAS Inst.). The model included the main effects of genetic line, OXT treatment, and the interaction between them. Significant interactions prompted ANOVAs within each line, and means were separated by Tukey's test. Abundance of FABP4 (P = 0.02), MGLL (P = 0.0002), and OXT (P = 0.001) mRNA was greater in LWS than HWS chicks. There were no main effects of OXT treatment, however there were two‐way interactions of genetic line and OXT treatment on LPL (P = 0.003), MGLL (P = 0.02), and OXT (P = 0.02) mRNA. ANOVAs conducted within each line revealed an effect of treatment on LPL (P = 0.004), MGLL (P = 0.02), and OXT (P = 0.04) in LWS, but not HWS (P > 0.05 for all three genes). In LWS, LPL mRNA was greater in the fat of chicks treated with 0.312 nmol OXT than in those treated with 0.039 or 2.5 nmol (P < 0.05), whereas expression of MGLL was greater in chicks treated with 0.312 nmol OXT than in those injected with vehicle or 2.5 nmol OXT (P < 0.05). Oxytocin mRNA was greater in the fat of LWS injected with 0.312 nmol OXT than in those injected with 2.5 nmol OXT (P < 0.05). Results show that central administration of oxytocin to young chicks may have effects on adipose tissue physiology and that they are genetic line‐dependent.

Support or Funding Information

Funding for this work was provided in part, by the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture
Redox Regulation of the M1/M2 Shift in Microglia: Programming the Deleterious Phenotype
Authors: Block Michelle L.
Published: June 2021

Microglia, the resident innate immune cells in the brain, are implicated in the progressive nature of diverse neurodegenerative diseases associated with aging, including Parkinson's disease. Our recent findings point to the loss of NF‐κB p50 function due to reactive oxygen species as a key mechanism driving microglial failure to resolve the M1 (pro‐inflammatory) response and impaired M2 (alternative) activation, where loss of NF‐κB p50 function leads to a CNS‐specific vulnerability to chronic inflammation. Moreover, our current work indicates that aging synergistically augments the brain M1 activation response in NF‐κB p50−/− mice, implicating a role for loss of NF‐κB p50 function in age‐associated microglial priming (heightened sensitivity to pro‐inflammatory stimuli) and dopaminergic neurotoxicity. Together, these findings implicate NF‐κB p50 as pivotal redox signaling mechanism regulating the M1/M2 activation balance in microglia, where loss of function tips this balance to favor a M1 polarized and neurotoxic phenotype. These findings support that loss of NF‐κB p50 function and aging may interact to excessively augment microglial priming and point to a novel pro‐inflammatory signaling mechanism independent the NF‐κB p50/p65 transcription factor in this process.

Support or Funding Information

NIEHS/NIH ONES Award R01ES016951; DOD GW120068
Response of Skeletal Muscle Arterioles In Vivo to Modulation of Endothelial Glycocalyx (EG) by Resuscitation Fluids after Hemorrhagic Shock (HS)
Authors: Torres Luciana , Salgado Christi , Sondeen Jill , Dubick Michael , Filho Ivo Torres
Published: June 2021

EG shedding may be associated with deleterious endothelial function, leading to edema, inflammation, poor vasomotor control and tissue hypoxia. Previous studies reported that fluid shear stress may stimulate glycocalyxsynthesis and modulate arteriolar diameter. Administration of resuscitation fluids in severely hemorrhaged patients may result in hemodilution, edema and impairment of microvascular perfusion. Therefore this study aims to elucidate the effects of such fluids on arteriolar glycocalyx and the overall hemodynamic response after HS followed by hypotensive resuscitation. Anesthetized rats (n=30) subjected to HS for 1h (25mL/kg blood loss) were resuscitated with fresh frozen plasma (FFP), fresh whole blood (FWB), 5% Albumin (ALB), lactated Ringer's (LR) or normal saline (NS). The volume infused was 15 mL/kg (FFP, FWB and ALB) or 45 mL/kg (LRor NS). In addition of systemic parameters, EG thickness, red cell velocity, blood flow, wall shear rate (WSR) and wall shear stress (WSS), as well as permeability were studied on over 30 arterioles using intravital microscopy. Post‐resuscitation arterial pressure was 20% lower than baseline in alltreated‐rats. Also post‐resuscitation hematocrit decreased equally to 24±1 % in all groups, except in FWB group (33±1 %). Arteriolar blood flow was significantly lower on rats treated with LR and NS compared to FFP, FWB and ALB treatments. After resuscitation, arteriolar WSR returned to baseline levels in all groups, however WSS was significantly lower in LR and NS compared to FFP, FWB and ALB. Arteriolar EG thickness was lower in LR and NS, whereas it recovered in FFP, FWB and ALB, compared to baseline (p<0.05). A linear correlation between changes in arteriolar diameter (relative to baseline) and changes in EG thickness (R=0.56, p<0.05) as well as changes in WSS (R=0.60, p<0.05)were observed. To conclude, as previously shown in venules, shedding/recovery of the EG occurs also within arterioles of the rat cremaster network. In addition, fluid resuscitation after HS may improve arteriolar EG recovery, endothelium function and microhemodynamics, possibly due to higher viscosity fluids that modulates shear stress and its effect on the endothelium, without volume overload.

Support or Funding Information

Supported by US Army Medical Research & Materiel Command
Maternal High Fat Diet Induces the Expression of an Inhibitory Glycinergic Current in Rat Dorsal Motor Nucleus of the Vagus Neurons Regulating Gastric Functions
Authors: McMenamin Caitlin A , Browning Kirsteen N
Published: June 2021

The perinatal period is critically important in the development of autonomic neurocircuits and maternal overnutrition during this period has been shown to predispose offspring to obesity and associated comorbidities. Previous studies suggest there is an increased inhibition of gastric projecting dorsal motor nucleus of the vagus nerve (DMV) neurons in rats fed a high fat diet (HFD) during the perinatal period, but the underlying neural mechanisms and the effects on gastric motility have yet to be determined. This aim of this study was to assess the effects of perinatal HFD on (i) inhibitory synaptic inputs to gastric‐projecting DMV neurons and (ii) gastric tone and motility.

Whole cell patch clamp recordings were made from gastric projecting DMV neurons in thin brainstem slices from Sprague‐Dawley rats (4–6 weeks of age) fed either a control or HFD (14 or 60%kcal from fat, respectively) from embryonic day 13. Gastric motility and tone was measured via miniature strain gauges attached to the antrum of anesthetized rats.

In the present study, perinatal HFD rats were not significantly heavier than age‐matched control rats (6 weeks; 232±17g vs 209±5g). The glycine receptor antagonist, strychnine (1μM), induced a larger inward current in perinatal HFD vs control DMV neurons (23±2pA vs 3±1pA, n=8, P<0.05), suggesting that glycine is a tonically released neurotransmitter in perinatal HFD, but not control, rats. Strychnine also increased the rise time (5.0±0.3ms vs 3.5±0.2ms), decay time (9.5±0.8ms vs 5.3±0.6ms), and half width (8.8±0.9ms vs 5.0±0.5ms) of miniature inhibitory postsynaptic currents (mIPSCs) in perinatal HFD (n=5, P<0.05 vs baseline for each parameter) but had no effect in control neurons rise time (3.45±0.2ms vs 3.7±0.2ms), decay time (4.5±0.3ms vs 4.9±0.5ms), or half‐width (4.3±0.3ms vs 4.8±0.5ms) of miniature postsynaptic currents (n=5, P>0.05 for each). Microinjection of strychnine (300pmoles/60nl) into the DMV increased gastric tone and motility in the antrum in perinatal HFD rats (82–101mg, 20–25% of baseline, respectively, n=2) but not in control rats (11–21mg, 2–10% of baseline, n=2, P<0.05 vs perinatal HFD for each), suggesting the tonic glycinergic inputs onto DMV neurons decreased vagal efferent outflow and reduced basal gastric motility and tone.

The studies suggest that perinatal overnutrition induces the expression of a novel tonic glycinergic input onto gastric projecting DMV neurons, increasing the inhibitory drive to the stomach. Such alterations in brainstem neurocircuit development as a consequence of maternal over‐nutrition may induce gastric dysmotility and dysregulated gastointestinal reflexes, hence influencing offspring to develop obesity.

Support or Funding Information

Funded by NIH 078364 and NSF IOS1148978
An update on arsenic in rice, impact on dietary arsenic intakes and U.S. Department of Agriculture efforts at mitigation
Authors: Finley John W
Published: June 2021

Dietary arsenic from rice came to the attention of the public in 2012 following the release of analyses by Consumer Reports and Dr. Ozshowing levels of concern. Subsequently the U.S. Food and Drug Administration (USFDA) conducted their own analyses as well as a risk analysis for consumption of arsenic‐containing rice and rice foods (unreleased). Potentially high inorganic arsenic (iAs) levels previously have been reported in drinking water and apple juice. To understand the impact that rice consumption has on arsenic intake relative to other sources, an analysis was conducted using NHANES consumption data and USFDA data from analyses of arsenic in rice. These data show that water intake and arsenic levels in water are the primary determinants of iAs intake; however rice can be a significant source, especially among consumers of brown rice in areas where arsenic levels in wate rare low. The U.S. Department of Agriculture is committed to maintaining a safe and nutritious food supply and has put efforts into mitigating these problems. Agricultural Research Service projects include development of varieties of rice that take up less arsenic, agronomic techniques such as water management that reduce arsenic accumulation and methods of analysis that are quicker and cheaper and allow identification of arsenic content of harvested rice before mixing at grain elevators.

Support or Funding Information

This work was conducted by the U.S> Department of Agriculture, Agricultural Research Service
Does Attenuated Skin Blood Flow Lower Sweat Rate and Thereby the Critical Environmental Limit for Heat Balance?
Authors: Jay Ollie , Cramer Matthew N , Gagnon Daniel , Crandall Craig G
Published: June 2021

Attenuated skin blood flow (SkBF) is often assumed to critically impair core temperature (Tc) regulation. Recent evidence has shown that a profound pharmacologically‐induced attenuation of SkBF leads to a reduction in local sweat rate. Whether attenuated SkBF to a level more commonly observed in aged or diseased populations leads to decrements in sweat rate, and consequently, a reduction in heat loss potential is presently unknown. Therefore, the purpose of the present study was to test the hypothesis that attenuated SkBF would lead to lower sweat rate and heat loss potential, defined as the critical ambient vapour pressure (Pcrit) at which core temperature (Tc) inflected, while maintenance of a high SkBF would preserve sweat rate and heat loss potential. Seven healthy males (28±4 y) completed the protocol, which consisted of non‐encapsulated passive heat exposure (41°C), while ambient vapour pressure increased by 0.17 kPa (~2% RH) from 2.57 to a maximum of 5.95 kPa every 6 min until an upward inflection in Tc occurred. Three separate treatments elicited changes in plasma volume (ΔPV) with the goal of achieving three distinct levels of SkBF: control (CON), diuretic‐induced iso‐osmotic dehydration to lower SkBF (DEH), and continuous saline infusion to maintain SkBF (SAL). Tc, mean skin temperature (Tsk), heart rate (HR), mean laser‐Doppler flux (forearm, thigh; LDFmean), mean local sweat rate (LSRmean), and metabolic rate were measured. In DEH, a −14.2 ± 5.7% ΔPV resulted in a ~20% lower LDFmean (DEH: 139 ± 23, CON: 176 ± 22, SAL: 186 ± 22 PU; P = 0.034). However, LSRmean and whole‐body sweat losses were unaffected by treatment (P > 0.482). Pcrit for Tc was similar between treatments (CON: 5.05 ± 0.30, DEH: 4.93 ± 0.16, SAL: 5.12 ± 0.10 kPa; P = 0.166). Further, no differences were observed in the Tsk‐Ta gradient, metabolic rate, and changes in Tc (P > 0.197). In conclusion, a physiological reduction in SkBF consistent with those reported in the elderly and diseased populations alters neither local and whole‐body sweating nor heat loss potential during non‐encapsulated passive heat stress.

Support or Funding Information

Funded by National Institutes of Health GM068865 and Department of Defense W81XWH‐12‐1‐0152 (CGC), and a Natural Sciences and Engineering Research Council Discovery Grant #386143‐2010 (OJ).
Van‐Gogh‐Like 2, Frizzled, and Knypek Control Distinct Aspects of Polarized Cellular Migration During Neural Convergence
Authors: Singh Karndeep , Brown Sharlene , Werner Jonathan , Vital Eudorah , Sanchez Stephanie , McFarland Rebecca , Brewster Rachel
Published: June 2021

The planar cell polarity (PCP) pathway plays a significant role in facilitating neural convergence (NC) – the narrowing of the neural plate before the formation of the neural tube. Evidence from the literature and our laboratory suggests that NC in zebrafish requires elongation and midline‐directed polarized migration of neural plate cells. Failure of NC or delayed stages of neural tube morphogenesis can result in severe neural tube defects (NTDs) which have been observed in all vertebrates studied. Although perturbation of the PCP pathway is associated with NTDs in model organisms and humans, the underlying neural cell behaviors remain elusive.

In order to investigate the cellular effects of the PCP pathway, we used Knypek (Knyfr6), Van gogh‐like 2 (Vangl2vu67), and Frizzled 7a−/7b− (Fzd7ae3−; Fzd7bhu3495), three zebrafish lines carrying null mutations. We confirmed the published result that mutations in the PCP pathway delayed NC. We analyzed the width of the neural plate in 4–5 somite stage embryos to confirm NC by performing in situ hybridization using probes against the hindbrain and neural crest mRNAs Krox20 and Dlx3, respectively. Next, our comparative cellular analysis revealed how cell elongation, membrane dynamics, and trajectory are affected in homozygotes. We show that wild type (WT) neural plate cells elongate and medially restrict membrane protrusions, thus narrowing the neural tube. Our preliminary data show that cells in all mutants failed to elongate initially, specifically Vangl2 and Fzd7a/b mutant cells extend randomized protrusions while Kny mutant cells show temporally restricted protrusive activity. Furthermore, cell behavioral analysis revealed that neural plate cells in PCP mutant embryos are unable to polarize or migrate toward the midline effectively. Live cell behaviors were assessed by confocal fluorescence microscopy of embryos neural convergence extension movements.

Evidence from the literature revealed that Kny is a Fzd co‐receptor thought to present Fzd with various Wnt ligands in the PCP pathway. Differences between Kny and Fzd7a/b cellular phenotypes could suggest a ligand‐independent aspect of PCP signaling during NC. While current literature understands that PCP genes regulate cell polarity and migration, the mechanisms of these genes in neural tissue and how they contribute to NTDs is poorly understood. By studying cell behaviors in the neural plate, our laboratory aims to further reveal how the PCP pathway promotes NC and identify additional genes affecting NTDs.

Support or Funding Information

U.S. DOD W81XWH‐15‐RTR‐IDA NIH R01‐GM085290 NIH R03‐3HD076615 NSF DBI‐0722569 Meyerhoff Graduate Fellowship U.S. Department of Education GAANN Fellowship NIH/NIGMS MARC U*STAR T3408663 National Research Service Award to UMBC
Depletion of Caveolin‐1 in Lung Vasculature and Increase in Caveolin‐1 Positive Circulating Extracellular Vesicles as Early Biomarkers of Endothelial Injury
Authors: Oliveira Suellen Darc Santos , Castellon Maricela , Machado Roberto F , Elliott Michael H , Bonini Marcelo G , Minshall Richard D
Published: June 2021

Rationale

Endothelial cell activation and dysfunction are hallmark features of Acute Lung Injury and Acute Respiratory Distress Syndrome (ALI/ARDS). Caveolin‐1 (Cav‐1) is highly expressed in pulmonary microvascular endothelial cells (PMVECs) and plays a key role in maintaining vascular homeostasis. The aim of this study was to determine whether the lung inflammatory response to endotoxin promotes PMVEC Cav‐1 depletion and appearance of Cav‐1 positive circulating extracellular vesicles (Cav‐1+ EVs) and whether this contributes to ALI.

Methods

ALI was induced in C57BL6 WT mice (male; 2.5–3 months old) by nebulized Escherichia coli lipopolysaccharide (LPS; 10 mg over 1 hr daily for 1 or 4 days). Lung pathology, Cav‐1 and eNOS expression, Cav‐1 ubiquitination, and Cav‐1+ EVs were quantified in total lung or platelet‐free plasma by immunoshistochemistry and western blot, respectively. Broncho‐alveolar lavage fluid (BALF) obtained from control and LPS‐treated mice was used to measure inflammatory cell, cytokine, and albumin levels.

Results

Alveolar accumulation of plasma albumin, immune cells, and pro‐inflammatory and regulatory cytokines (TNF‐α, IL‐1β, IL‐6, TGF‐β) was signficantly elevated in LPS‐treated WT mice. Intermittent airway LPS exposure for 96 hrs also increased Cav‐1 phosphorylation and ubiquitination (Ub) and decreased total lung Cav‐1 expression by 48 ± 4.16 % (n=3–4 mice; P<0.05) when compared to untreated WT mice. Depletion of EC Cav‐1 was accompanied by a decrease in eNOS dimerization indicative of endothelial dysfunction. In addition, circulating Cav‐1+ EVs increased by 38% after 24 hrs of LPS exposure (0.61 ± 0.05 to 0.98 ± 0.16 a.u.) in control and LPS‐treated mice, respectively (n= 8–10 mice; P<0.05) and decreased below basal level after 96 hrs indicating PMVEC Cav‐1 depletion may be due to both Cav‐1 ubiquitination and proteosomal degradation as well as release Cav‐1+ EVs into the circulation. PMVEC Cav‐1 depletion in response to endotoxin exposure was associated with endothelial dysfunction and vascular injury suggesting EC Cav‐1 depletion may play a significant role in the onset of ALI. Similar lung pathology was observed in EC‐specific Cav‐1−/− mice.

Conclusion

Endothelial cell Cav‐1 depletion and Cav‐1+ EVs may be an early biomarker of pulmonary vascular injury that may predict pulmonary vascular inflammatory disease due to endothelial dysfunction.

Support or Funding Information

Financial support: CNPq Fellowship‐CsF/Brazil (SDO) and HL125356 and DOD W911NF‐15‐R‐002 (RDM, MGB).
Prolonged Heat Stress Altered Autophagy Signaling in Oxidative Skeletal Muscle
Authors: Selsby Joshua , Ganesan Shanthi , Brownstein Alexandra J , Gabler Nicolas K , Pearce Sarah C , Baumgard Lance H , Rhoads Robert P
Published: June 2021

Prolonged heat stress represents a continuing threat to human health and agricultural production economics. Despite this broad, negative impact little is known about underlying pathological mechanisms, which has limited the development of etiological interventions and left clinicians and producers with only cooling and rehydration strategies. The purpose of this investigation was to determine the extent to which prolonged heat stress altered autophagic signaling in oxidative skeletal muscle. We hypothesized that heat stress would induce autophagy independent of the volitional caloric restriction that accompanies heat stress. To test this hypothesis pigs were treated as follows: thermoneutral (TN; 20°C), heat stress (HS; 35°C), or were house in thermoneutral conditions but pair‐fed to the heat stress group (PFTN) for seven days. Upon euthanasia the red portion of the semitendinosus muscle was collected for subsequent measures of relative protein abundance. We determined that markers of autophagy initiation, including phosphorylated adenosine monophosphate activated protein kinase (AMPK) and Unc51‐like kinase 1 (Ulk1), were increased approximately 3‐fold (p<0.05) in HS compared to TN and PFTN. Similarly, nucleation markers, PI3 kinase class III and Beclin‐1, were increased approximately 3‐fold (p<0.05) in HS compared to TN and PFTN. We also discovered that elongation markers autophagy related gene (Atg)12 and Atg16 were increased 2‐ and 4‐fold (p<0.05), respectively, in HS compared to TN and PFTN, while Atg5 was similar between groups. Markers of autophagosome formation including microtubule‐associated protein 1A/1B‐light chain (LC3) II and the LC3 II/I ratio were increased 5‐fold (p<05) in HS and PFTN compared to TN while LC3 I was similar between groups. Sequestosome 1 (SQSTM1/p62) is consumed during autophagosomal degradation by the lysosome and is thus an inverse correlate of autophagic degradation. We discovered that relative protein abundance of p62 was increased 5‐fold (p<0.05) in HS compared to TN and PFTN. In partial support of our hypothesis, these data indicate heat stress‐mediated induction of autophagic signaling is largely independent of reduced feed intake. Further, these data also suggest a HS‐mediated stalling of autophagic degradation despite prolonged reduced feed intake.

Support or Funding Information

This work supported by USDA grants 2014‐67015‐21627 and 2011‐67003‐30007.
Measurement of the Compensatory Reserve Predicts Racial Differences in Orthostatic Tolerance
Authors: Wenner Megan M. , Hinds Kumba , Howard Jeffrey T , Nawn Corrine D , Stachenfeld Nina S , Convertino Victor A
Published: June 2021

The Compensatory Reserve Index (CRI) has recently been established to accurately measure the body's integrated capacity to compensate for physiological conditions of reduced central blood volume, and predict hemodynamic decompensation. We previously demonstrated that African American (AA) women have a higher tolerance to orthostatic stress. Therefore, we tested the hypothesis that the CRI would identify racial differences in orthostatic tolerance prior to the onset of traditional signs and symptoms. We measured beat‐by‐beat blood pressure (BP) and heart rate (HR) in 23 AA (22 ± 1 years; 24 ± 1 kg/m2) and 31 Caucasian women (20 ± 1 years; 23 ± 1 kg/m2) during progressive lower body negative pressure (LBNP) to presyncope. Blood pressure waveforms were analyzed using a machine‐learning algorithm to derive the CRI at each LBNP stage; stroke volume (SV) was derived from the Finometer (Model flow). Resting mean arterial BP (AA, 78 ± 3 vs. Caucasian, 74 ± 2 mmHg) and HR (AA, 68 ± 2 vs. Caucasian, 65 ± 2 bpm) were similar between groups. The CRI progressively decreased during LBNP in both groups, however the CRI in Caucasian women was 4% lower than AA women at −15 mmHg LBNP and decreased to 21% lower at −50 mmHg LBNP (P<0.05). The rate of decline in SV during LBNP was greater in Caucasian women (ANOVA P<0.05), and differences were noted as early as −20 mmHg. However changes in BP and HR during LBNP were not different between groups. At presyncope, mean arterial BP (AA, 66 ± 3 vs. white, 65 ± 4 mmHg) and HR (AA, 102 ± 4 vs. white, 97 ± 3 bpm) were similar between groups, as was the change in SV (AA, Δ −33 ± 2 vs. white, Δ −35 ± 3 mL). Finally, the time to presyncope was 1.6 minutes longer in AA women. These data support the notion that greater tolerance to the orthostatic stress induced by LBNP in AA women can be explained by their greater reserve to compensate for progressive central hypovolemia compared to Caucasian women. An important clinical translation is that the CRI provides a more sensitive and specific measure for early assessment of differences in orthostatic tolerance in comparison to traditional vital signs and symptoms or changes in hemodynamics.

Support or Funding Information

Research supported by funding from the Combat Casualty Care Research Program of the US Army Medical Research and Materiel Command and NIH HL071159.
Determination of the Bioactive Components in a Grape Seed Procyanidin Extract Responsible for Enhanced Farnesoid X Receptor Transactivation
Authors: Rodriguez Kelvin , Ricketts Marie‐Louise
Published: June 2021

Dietary procyanidins are beneficial for maintaining health. Grape seed procyanidin extract (GSPE) in particular has been shown to regulate cholesterol and triglyceride (TG) homeostasis in vivo (1–5). We have identified several underlying molecular mechanisms by which GSPE decreases serum TG levels, including farnesoid x receptor (FXR)‐dependent decreased hepatic triglyceride synthesis, inhibition of enterohepatic bile acid recirculation and increased hepatic TG catabolism (3–5). Furthermore, we identified GSPE as a histone deacetylase (HDAC) inhibitor, leading to enhanced peroxisome proliferator‐activated receptor alpha (PPARα) target‐gene transcription and increased fatty acid β‐oxidation (6). During our initial investigations we discovered that GSPE acts as a co‐agonist ligand for FXR (2). The present study was designed as a follow‐up to this initial observation and aimed to identify which component (or components) within GSPE are responsible for the observed FXR‐dependent co‐agonist activity. GSPE was fractionated by column chromatography to separate compounds based on molecular size (n=3). The degree of polymerization for each fraction was determined by high performance liquid chromatography. Using a cell‐based transient transfection assay, each fraction was tested in combination with the bile acid, chenodeoxycholic acid (CDCA), to assess its ability to increase FXR transactivation (n=3, analyzed in triplicate), and compared to the effect seen with GSPE. Five different fractions from each column consistently enhanced FXR transactivation and were subsequently analyzed by mass spectrometry (MS). The MS results show that the fractions that enhance FXR transactivation are enriched in dimers and dimer gallates. Ultimately, this study facilitates enhanced understanding regarding the observed GSPE‐mediated FXR‐dependent regulatory effects on metabolic homeostasis observed in vivo.

Support or Funding Information

USDA NIFA Hatch Project NEV0738; USDA NIFA Multi‐state Project W‐3122: Beneficial and adverse effects of natural chemicals on human health and food safety; USDA NIFA Hatch Project NEV0749; A grant from the National Institute of General Medical Sciences (P20GM103440) from the National Institutes of Health.
Knockout of the PHLDA1 gene in breast cancer cells reveals multiple roles for PHLDA1 in cancer phenotypes
Authors: Zimnicka Adriana M. , Sharma Tiffany , Regan Maureen , Merrill Brad J. , Frasor Jonna
Published: June 2021

PHLDA1 or pleckstrin‐homology‐like domain family A, member 1, also known as TDAG51, is a small, PH‐domain containing protein that acts as a potential modulator of apoptosis and cell proliferation in breast carcinomas. However, many contradictory findings have been reported and the mechanisms by which PHLDA1 affects cancer cells are not well defined. We have reported that in estrogen receptor (ER)‐positive breast tumors, high PHLDA1 mRNA expression was found to be associated with a high risk of distant metastasis, while in ER negative breast tumors, high PHLDA1 expression coincided with a more favorable outcome, suggesting that PHLDA1 has opposite effects depending on the ER status of a tumor. We have recently shown that PHLDA1 contributes to the survival and growth of breast cancer stem cells in ER positive mammospheres, where PHLDA1 mRNA and protein are regulated directly by a crosstalk between ER and the inflammatory nuclear factor κB (NFκB) pathway, and indirectly through inhibition of miR‐181a and b, which targets PHLDA1 for degradation. Here, using CRISPR/Cas9 mediated genome editing, we have permanently knocked out PHLDA1 gene from ER positive MCF‐7 and ER negative MDA‐MB‐231 breast cancer cell lines, and characterized their phenotype. While MDA‐MB‐231 cells are still under investigation, here we report that MCF‐7 cells missing the PHLDA1 gene showed decreased cell proliferation, migration, and invasion across a matrigel layer, consistent with a role for PHLDA1 in more aggressive ER+ breast cancers. In contrast, PHLDA1‐knock out cells were less susceptible to apoptosis than control cells, as shown by decreased activity of caspase‐3 and −7, and were rendered more resistant to doxorubicin and paclitaxel‐induced cytotoxicity, suggestive of a pro‐apoptotic role for PHLDA1. This phenotype was reversed by overexpressing PHLDA1 in the PHLDA1 (−/−) cells. We also compared PI3K activation and Akt phosphorylation in parental and knock out cells in order to determine the signaling pathway mediating the effects of PHLDA1. The PH domains, or phosphoinositide binding protein modules, are known to govern the function of two other PHLDA isoforms, PHLDA2 and PHLDA3, but no information on the role of PH domain in the function of PHLDA1 has been described. We created a PH domain deletion mutant (PHLDA1‐ΔPH), and using confocal microscopy assessed its subcellular localization, as well as Akt/PI3K signaling pathway activity, and compared it to the WT PHLDA1. Our study sheds new light on the function of PHLDA1 in breast cancer cells, suggesting a broad role as an anti‐proliferative/anti‐invasive but pro‐apoptotic protein. Moreover, we describe the signaling pathway involving PHLDA1, an understanding of which will potentially define novel therapeutic targets in treatment of breast carcinomas.

Support or Funding Information

This study was supported by the NIH fund 1 R01 CA200669 01 and DOD fund W81XWH‐16‐1‐0044 to JF
Insight into the Evolution of the Avian Vocal Organ, or Syrinx, from Enhanced‐Contrast X‐ray Computed Tomography and Fossil Data
Authors: Clarke Julia , Li Zhiheng , Riede Tobias , Goller Franz
Published: June 2021

From complex songs to simple honks, squawks, or caws, birds produce sounds using a unique vocal organ, the syrinx. Located close to the heart at the tracheobronchial junction, vocal folds or membranes attached to modified mineralized rings vibrate to produce sound. Not thought to commonly enter the fossil record, the few reported fossilized parts of the syrinx are geologically young (i.e., Pleistocene and Holocene). The only known older syrinx is an Eocene specimen, which was not described or figured. Data on the relationship between soft tissue structures and syringeal three‐dimensional geometry has also been limited. Our team of physiologists and paleontologists recently described the first remains of a fossil syrinx from the Mesozoic, which are preserved in three dimensions in a specimen from the Late Cretaceous of Antarctica. With both cranial and postcranial remains, the new specimen is the most complete to be recovered from a part of the radiation of living birds, Aves. Enhanced‐contrast x‐ray computed tomography (CT) of syrinx structure in twelve extant non‐passerine birds, as well as CT imaging of the Cretaceous and the Eocene syrinxes, informs both the reconstruction of ancestral states in birds and properties of the vocal organ in the extinct species. Fused rings in the Cretaceous fossil form a well‐mineralized pessulus, a derived neognath bird feature, proposed to anchor enlarged vocal folds, or labia. Left‐right bronchial asymmetry, only known in extant birds with two sound sources is similarly present.

The new data show the fossilization potential of the avian vocal organ and beg the question why these remains have not been found in other dinosaurs. The lack of other Mesozoic tracheal and bronchial ring remains, and the poorly‐mineralized condition in archosaurian taxa without a syrinx, may indicate a complex syrinx was a late arising feature in the evolution of birds, well after the origin of flight and respiratory innovations. While a role for complexity in social structure and mating system has been extensively investigated with reference to increases in brain size in other vertebrates including humans, in the early evolution of birds these hypotheses have not been explored. We propose that first more complex visual signals and then vocal communication and song may have played important roles in the evolution of the larger extant avian brain.

Support or Funding Information

Gordon and Betty Moore Foundation Grant GBMF4498, US National Science Foundation (OPP ANT‐1141820, OPP 0927341 and EAR 1355292 to J.C.); Argentine Agencia Nacional de Promoción Científica y Técnica (PICT 2010‐066; F.E.N).