bluuebunny/crossref_metadata_2025 huggingface dataset:
Epinephrine Does Not Influence Baroreflex Sensitivity During Lower Body Negative Pressure to Physiological Tolerance
Authors: Scruggs Zachariah M. , Petersen‐Jones Humphrey G. , Convertino Victor A. , Joyner Michael J. , Curry Timothy B. , Baker Sarah E.
Published: June 2021

Background

The baroreflex plays an integral role in blood pressure maintenance by altering heart rate and sympathetic activity in response to changes in arterial pressure. Baroreflex sensitivity (BRS) has been shown to decrease linearly during progressive exposure to central hypovolemia as induced by lower body negative pressure (LBNP), and can be used as an index of decompensation status during hemorrhage. Circulating epinephrine is significantly elevated during high stress situations such as hemorrhage; however, it is unclear whether epinephrine influences BRS during progressive exposure to central hypovolemia. Based on previous work, we hypothesized epinephrine would attenuate any reduction in spontaneous cardiac baroreflex sensitivity during LBNP.

Methods

Ten young (35±3 yrs), healthy (BMI 24±1 kg/m2) men and women (6M/4F) underwent LBNP protocols on 2 separate days consisting of a 10‐min pre‐infusion baseline, 15‐min infusion period (randomized to saline or epinephrine, 0.1 μg/kg/min), 10‐min post‐infusion baseline, and 5 minutes of LBNP at −15, −30, −45, −60, −70, and −80 mmHg until the onset of presyncope (range= −30 to −80 mmHg). Presyncope was defined as a rapid decrease in systolic blood pressure greater than 15 mmHg, systolic blood pressure less than 80 mmHg, bradycardia, or voluntary termination due to presyncopal symptoms. Heart rate (HR; electrocardiogram) and blood pressure (BP; radial arterial catheter) were recorded continuously. Spontaneous cardiac BRS was determined using the sequence method (WinCPRS). Data are reported from both 10‐min baselines and from 1 minute at 20, 40, 60, 80, and 100 percent of each individual's total LBNP exposure time.

Results

We observed no significant differences in total exposure time (1475±150 vs. 1415±79s; p=0.69) or cumulative stress index (820±135 vs. 699±69 min*mmHg; p=0.37) between saline and epinephrine visits, respectively. Baroreflex sensitivity did not change during post‐infusion baselines (ΔDown‐Down =−2±1 vs. −2±2 ms/mmHg; saline vs. epinephrine, respectively; p=0.09). Baroreflex sensitivity was significantly reduced with LBNP (p<0.01 main effect of LBNP). There was no difference between the change in baroreflex sensitivity during LBNP with saline or epinephrine infusion (ΔDown‐Down =−14±4 vs. −8±4 ms/mmHg, p=0.17).

Conclusions

Consistent with previous work, spontaneous cardiac baroreflex sensitivity fell during LBNP. Contrary to our hypothesis, epinephrine infusion did not attenuate the fall in spontaneous cardiac baroreflex sensitivity observed during simulated blood loss. Because epinephrine did not alter the effect of LBNP on BRS, these data suggest that BRS may accurately reflect decompensation status during traumatic hemorrhage independent of elevated circulating epinephrine.

Support or Funding Information

US Army Medical Research and Materiel Command Contract W81XWH‐13‐2‐0038
Neurovascular Complexity Index: A Potential Quantitative Measure for Assessment of Traumatic Brain Injury
Authors: Howard Jeffrey T , Janak Jud C , Bukhman Vladislav , Robertson Claudia , Frolov Iurii , Nawn Corinne D , Schiller Alicia M , Convertino Victor A
Published: June 2021

Multimodal monitoring of brain physiology following a traumatic brain injury (TBI) shows promise as a strategy to improve management and outcomes of TBI patients. Valid and reliable measures of different aspects of brain physiology following a TBI could prove critical to accurately capturing these changes. Using a case‐control study design, we evaluated a new proprietary algorithm of cerebral blood flow variability (CBFV) called the neurovascular complexity index (NCI). Baseline NCI data from 169 healthy control participants were compared to 51 TBI patients. Unadjusted and age‐sex adjusted logistic regression models were used to assess the association between NCI and TBI. TBI patients exhibited significantly greater variability in NCI scores than healthy controls (F=131.88; p<0.0001). Compared to the continuous measure of NCI, the threshold of ± 1.96 standard deviations from the mean for the healthy control group measure (5.888.10) had superior ability to identify TBI patients based on the Receiver Operator Characteristic Area Under the Curve (0.84 vs. 0.61; p=0.0007), and greater sensitivity (74.5 vs. 51.0; p=0.0013). Individuals with NCI scores outside the 5.88–8.10 range had a 29‐fold increase in the odds of TBI in multivariate logistic regression models adjusted for age, sex and end tidal CO2 (OR=29.44; 95% CI 5.98–144.87; p<0.0001). Advancement in multimodal monitoring of TBI patients is important in reducing the potential risk of secondary injury. This study reports results for a new non‐invasive quantifiable assessment of TBI based on a measure of CBFV showing potential for continuous monitoring and early identification of brain‐injured patients to better inform individualized management.

Support or Funding Information

US Army MRMC Combat Casualty Care Research Program
GLP‐1 Mediates the Intrinsic Gut‐Liver Metabolic Signaling in Anti‐VLDL Overproduction and Insulin Resistance in Vagotomized Mice
Authors: Khound Rituraj , Su Qiaozhu
Published: June 2021

Perturbations in hepatic lipid and very low density lipoprotein (VLDL) metabolism are involved in the pathogenesis of obesity and hepatic insulin resistance. In this study, our objective was to investigate the intrinsic peripheral gut‐liver vagal nerve circuit in the regulation of hepatic VLDL secretion and insulin sensitivity in sub‐diaphragmatic vagotomized mice. Specifically, by disrupting both hepatic and gastric divisions of the vagal nerve and using fast protein liquid chromatography (FPLC) to subclassify the lipoprotein particles, we discovered that vagotomy significantly decreased triglyceride (TG) contents in VLDL particles compared to that in the control mice with sham‐surgery. Vagotomy further prevented overproduction of VLDL‐TG and VLDL‐apoB induced by an acute fat load. Espousing both sham control and vagotomized mice to a long term high fat diet (HFD) for 22 weeks, we found that vagotomy protected mice from HFD induced hyperlipidemia, obesity, hepatic steatosis and insulin resistance. In an attempt to delineate the molecular mechanism for the anti‐hyperlipidemia and anti‐insulin resistant effect of vagotomy, we investigated the gut hormones and discovered that plasma glucagon‐like peptide‐1(GLP‐1) was significantly higher in the vagotomised mice compared to sham controls, which was associated with significant reduction on mRNA and protein expression of hepatic stearoyl CoA desaturase‐1 (SCD‐1), an enzyme that catalyzes the synthesis of oleyl CoA from stearolyl CoA. In vitro, pre‐treating a rat hepatoma cell line, McA‐7777 cells, with GLP‐1 inhibited SCD‐1 and Fasn mRNA expression which resulted in significant reduction on hepatic de novo lipid synthesis and VLDL secretion. Vagotomy further sensitized insulin sensitivity by upregulating expression of insulin receptors and enhanced activation of downstream signaling molecule, AKT. In conclusion, we demonstrated that increased secretion of GLP‐1 in vagotomized mice mediates the intrinsic gut‐liver metabolic signaling in preventing VLDL overproduction and insulin resistance induced by HFD. GLP‐1 inhibits SCD‐1 activity which in turn inhibits hepatic de novo lipogenesis and VLDL biogenesis. These novel findings, for the first time, provide mechanistic rationale for the application of vagotomy in clinical treatment in patients with obesity and diabetes.

Support or Funding Information

Hatch Funds from USDA/NIFA to Q. Su
Absorption and Excretion of Vitamin K Varies by Age and Triglycerides: A Metabolic Study in Older and Younger Adults Using Deuterium‐Labeled Collard Greens
Authors: Ellis Jessica Lea , Fu Xueyan , Al Rajabi Ala , Naumova Elena N , Grusak Michael A , Saltzman Edward , Booth Sarah L
Published: June 2021

Vitamin K is an essential fat‐soluble nutrient, with its most established role as a cofactor in coagulation. Emerging roles in soft tissue calcification have been proposed. However, further progress in understanding absorption and metabolism is needed, including knowledge of the factors influencing the physiologic response to vitamin K. Phylloquinone (PK) is the primary form of vitamin K in the diet and circulation. Large intra‐individual variances in circulating PK exist, and little is known about the dietary and non‐dietary factors that influence PK metabolism.

The purpose of this study was to compare absorption efficiency and body retention of PK in older and younger adults following dietary vitamin K depletion and repletion.

Forty‐two older (55–80 y; n=21) and younger (18–40 y; n=21) adults were maintained on sequential 28‐day vitamin K depletion (~10 ug PK/day) and vitamin K repletion (~500 ug PK/day) diets. At the end of each phase, subjects were fed deuterated collard greens (2H‐PK) and monitored over 72 hours. Liquid chromatography‐mass spectrometry (LC‐MS) was used to measure labeled and unlabeled PK in plasma and triglyceride‐rich lipoprotein (TRL) fractions, and labeled and unlabeled menadione (a PK metabolite) from 24‐hr urine collections.

Absorption efficiency of 2H‐PK did not differ between PK depletion and repletion, but was significantly higher in older adults than in younger adults (p=0.02). 2H‐PK in TRL fractions was correlated with the total 2H‐PK in plasma (r=0.95, p<<0.001), corroborating previous data that PK is primarily transported in circulation associated with triglycerides. Age (p=0.015) and plasma triglycerides (p<0.001) were significant linear predictors of 2H‐PK area under the curve (AUC). Both age and plasma triglycerides retained significance in the multivariate model, and together explained 31.6% of the variance in absorbed plasma 2H‐PK during the depletion period, and 29.9% in the repletion period. Analysis of urinary 2H‐menadione showed no differences in body retention of vitamin K by age, sex, or depletion or repletion.

In conclusion, age and circulating triglycerides are determinants of vitamin K absorption efficiency and together explained about 30% of the variance observed in absorbed PK. Body retention of vitamin K did not vary by age, sex, or dietary intake. There is a need to elucidate non‐traditional factors, such as genetics, that may influence absorption and metabolism of vitamin K.

Support or Funding Information

Study supported by the USDA ARS under Cooperative Agreement 58‐1950‐7‐707 and the NIH (5R01DK69341‐4).
1α,25‐dihydroxyvitamin D3 Inhibits Adipocyte Mediated Metastatic Capability of Breast Cancer Cells
Authors: Yum Chae Hyun , Wilmanski Tomasz M , Sheeley Madeline , Teegarden Dorothy
Published: June 2021

Excess adiposity is thought to be associated with approximately 20% of all cancers, including breast cancer. However the mechanism underlying this relationship remains controversial. Adipocytes secrete a wide array of bioactive molecules which act as key mediators in several obesity‐associated diseases. Further, the active form of vitamin D, 1α,25dihydroxyvitamin D (1,25(OH)2D) regulates multiple aspects of adipocyte biology, including adipogenesis and adipocyte apoptosis, as well as modulating the release of factors associated with cancer promotion. The purpose of these studies was to investigate 1,25(OH)2D‐mediated regulation of differentiated adipocytes to determine the impact on breast cancer progression. Differentiated 3T3‐L1 adipocytes were treated with 1,25(OH)2D (10 nM) for 2 days, followed by harvesting the adipocytes and collection of adipocyte conditioned media (ACM). ACM from adipocytes treated with 1,25(OH)2D decreased cell proliferation in metastatic MDA‐MB‐231 and MCF10CA1a cells compared to vehicle treated ACM, (42% and 37% of vehicle ACM, respectively), as measured by an MTT assay. In addition, ACM from adipocytes treated with 1,25(OH)2D inhibited cell migration and wound healing in MDA‐MB‐231 cells (37% and 30% respectively relative to vehicle ACM). In order to explore the mechanism underlying these effects on breast cancer metastatic capability, the mRNA expression of IGF‐1, IL‐6 and leptin was measured in adipocytes treated with either vehicle or 1,25(OH)2D. Treatment of adipocytes with 1,25(OH)2D decreased mRNA expression of IGF‐1, IL‐6 and leptin by 23%, 85%, and 39%, respectively. Further studies aim at demonstrating the mechanism underlying the impact of 1,25(OH)2D on adipocytes to inhibit their capability of promoting breast cancer metastasis. In summary, these results suggest that 1,25(OH)2D alters adipocyte secretions to inhibit breast cancer metastasis.

Support or Funding Information

This work was supported in part by the Higher Education Challenge Grant from the National Institute of Food and Agriculture, United States Department of Agriculture (#2013‐70003‐20922).
Processing Human Milk to Increase Nutrient Density for Preterm Infants
Authors: Ulus Hande Z. , Watkins Ruth H. , Allen Jonathan C
Published: June 2021

Breast milk is the optimal food for infants. It is tailor‐made for each species and each birth. Preterm birth can cause problems with initiation of lactation especially if it is the first pregnancy of the mother, and infants may have inadequate suckling reflexes. In this situation direct intragastric feeding can use donor breast milk from a milk bank, infant formula, or a combination. Concerns regarding the choice of feed are catch‐up growth and health effects. Formula might cause necrotizing enterocolitis and negative long term health effects but has faster catch‐up growth. Preterm infants have better tolerance for human milk, but lower caloric density of term mothers' milk or donor milk might not meet preterm infant growth needs. Preterm infants have higher needs for calories and protein and have a very limited stomach capacity.

Aims of this study were to concentrate donor breast milk to have a higher caloric density and protein, but at the same time avoiding side effects of higher lactose concentration and hyperosmolarity by precipitating lactose at low temperature.

Donor breast milk was obtained from WakeMed Mothers' Milk Bank. For preliminary results 10 human milk samples of 50 mL were concentrated by evaporation (Rotavap Buchi R‐300) to different concentration levels, held at 0°C overnight, followed by refrigerated centrifugation for lactose removal. Measurement of lactose was performed with enzymatic analysis via Roche Lactose/D‐Galactose kit on skim milk supernate and remixed skim milk.

Preliminary data found that low temperature removal of lactose from unprocessed human milk was minimal. Therefore milk was concentrated to varying extents prior to freezing. Volume reductions were 63%, 48%, 40%, 30% and 28%. Macronutrients were concentrated in the milks during the evaporation procedure in proportion to the amount of water removed, so caloric density was higher. Volume reduction (%) vs. Lactose concentration (g/L) yielded a linear relationship (Lactose =1.25 * volume‐reduction +3.50, r2=.77, p<0.05). Lactose was not consistently precipitated at the lower levels of volume reduction.

Water can be effectively removed from human milk by low pressure evaporation at temperatures that are unlikely to denature bioactive substances. Regression analysis found that every percent of concentration increased 1.2 g/L of lactose in milk, but the effectiveness of low temperature lactose precipitation was not statistically significant. Similar concentration should be true for protein and other bioactive factors. Additional research is needed to devise techniques to further reduce the osmotic activity in the donor milk concentrate generated by the increased lactose concentration.

Support or Funding Information

USDA HatchProject NC02435; Fulbright Foundation Scholarship to HZU.
Evo‐Devo, epigenetics, niche construction and the Extended Evolutionary Synthesis: crucial implications for anatomical and medical sciences in the 21st century
Authors: Diogo Rui
Published: June 2021

It is a puzzling paradox that eco‐morphological mismatches occur so frequently in an evolutionary process that often leads to macroevolutionary trends and in which organisms are said to be ‘designed’ for the habitats they inhabit. Here I present a new framework ‐ Organic Nonoptimal Constrained Evolution (ONCE) ‐ to address this paradox, and to explain why organisms look as they look: organisms themselves, and in particular their behavior, are the major active players of evolution. That is, within this framework, internal factors can both decrease and increase plasticity/hidden variation and therefore, together with epigenetic factors influenced by the external environment, can allows organisms to shift their behavior, for instance as a response to environmental changes. Importantly, due to behavioral persistence related to behavioral/ecological inheritance, organisms as diverse as bacteria, plants and animals help to construct their own niches, thus being crucial to direct evolution. Darwinian natural (external) selection then comes into play as a secondary ‐ but still crucial ‐ player. That is, due to organismal behavioral persistence, the random mutations/epigenetic factors that happen to be advantageous within the niches constructed by the organisms will be selected, further directing evolution and increasing the match between behavior, phenotype, and environment. This process can extend for long periods of time, leading to macroevolutionary trends and further increasing this match, potentially resulting in successful phenotypic overspecialization. However, behavioral persistence, loss of plasticity due to natural selection, genetic drift, overspecialization, and internal constraints can often make it difficult for the organisms to respond behaviorally and/or anatomically to new environmental changes, resulting in potential mismatches between behavior, ecology and form, and eventually in extinction. This new framework therefore bridges the gap between ideas that have been long considered to be opposing views of biology since Aristotle, including internalism and externalism, Cuvier's vs. Geoffroy's take on form and function, Darwinism and Lamarkism, the ideas of Baldwin, Waddington and Goldschmidt, and current Evo‐Devo ideas such as a physicalist framework, niche construction and epigenetic plasticity. This talk therefore covers ‐ and aims to link ‐ areas as diverse as evolutionary developmental biology, paleontology, evolutionary biology, comparative anatomy, ecomorphology, functional anatomy, ecology, ethology, biological anthropology, evolutionary medicine and history and philosophy of biology.

Support or Funding Information

Support from NSF (1516557) and American Association of Anatomists Innovations Program
A Race‐Specific Interaction Between Vitamin K Status and Statin Use During Warfarin Therapy Initiation
Authors: Kelly Jennifer M , Harshman Stephanie G , Brensinger Colleen M , Barger Kathryn , Kimmel Stephen E , Booth Sarah L
Published: June 2021

Vitamin K (VK) is required for the post‐translational modification of several clotting factors. Warfarin is a vitamin K antagonist and anticoagulant. The most common dietary and circulating form of VK is phylloquinone (PK). PK is lipid soluble, carried by triglyceride‐rich lipoproteins, and shares a metabolic pathway with cholesterol. Thus, there is biological plausibility for an interaction between serum PK and lipid‐lowering medications (statins) in warfarin therapy.

The objective of this study was to examine if serum PK and/or statin treatment was associated with anticoagulation control in African Americans and Caucasians initiating warfarin therapy. Warfarin sensitivity differs between African Americans and Caucasians, so analyses were race‐specific.

Cox proportional hazards and linear regression models were used in a cross‐sectional analysis of the 2009–2013 International Normalized Ratio Adherence and Genetics II cohort of African Americans (n=331; 40% female) and Caucasians (n=140; 36% female) initiating warfarin therapy. Primary exposures included serum PK (categorized as quartiles 1: PK<0.6, 2: 0.6≤PK≤1.0, 3: 1.01.7nmol/L) and statin use. Outcomes included number of days to maintenance dose and percent time in the therapeutic range (PTTR). Covariates included age, sex, smoking status, serum triglycerides, BMI, and clinic site.

A significant interaction between serum PK quartile and statin use with respect to days to maintenance was detected in Caucasians (p=0.04), but not African Americans (p=0.81). In Caucasians taking statins (n=60), the rate to reach maintenance was 88% (HR=0.12, 95%CI 0.04–0.39) and 81% (HR=0.19, 5%CI 0.06–0.53) slower for those in the second and third serum PK quartiles, respectively, compared to those in the lowest quartile. The rate to reach maintenance did not differ between the highest and lowest serum PK quartiles (HR=0.84, 95% CI 0.26–2.70) or between any quartiles among Caucasians not taking statins (n=80, p=0.67). In African Americans, serum PK was not associated with the rate to reach maintenance whether they used statins (n=140, p=0.98) or not (n=191, p=0.23). Serum PK and statin use were not associated significantly with PTTR in either race.

Statin use appears to slow the rate to establish maintenance warfarin dose among Caucasians with moderate serum PK. This apparent drug‐drug‐nutrient interaction suggests PK status and lipid‐lowering medication may influence anticoagulation for Caucasians during warfarin therapy initiation. The role of vitamin K with statin use in anticoagulation therapy in different races requires further examination.

Support or Funding Information

Supported by: USDA, Agricultural Research Service under Cooperative Agreements No. 58‐1950‐7‐707 and NIH‐NHLBI R01HL06617606A1.
Dietary cranberry alleviated colonic inflammation and altered gut microbiota in mice
Authors: Cai Xiaokun , Gu Min , Song Mingyue , Li Zhengze , Li Fang , Goulette Timothy , You Xiaomeng , Sela David A. , Xiao Hang
Published: June 2021

Increased consumption of fruits such as cranberry may decrease the risk of several chronic diseases such as inflammatory bowel disease (IBD). However, the exact mechanism of actions of dietary cranberry is unknown. In this study, we determined the effects of whole cranberry powder on IBD as well as the structure of gut microbiota in a mouse model of IBD induced by dextran sodium sulfate (DSS). Our results showed that dietary cranberry treatment significantly decreased the severity of IBD in DSS‐treated mice, evidenced by that cranberry treatment significantly increased colon length, and decreased disease activity and histologic score of IBD in DSS‐treated mice compared to the positive control group (p < 0.05). Moreover, the colonic levels of pro‐inflammatory cytokines (IL‐1β, IL‐6 and TNF‐α) were significantly reduced by cranberry treatment (p<0.05). Analysis of the relative abundance of fecal microbiota in phylum and genus levels reveled that DSS treatment significantly altered the microbial structure of fecal microbiota in mice, and dietary cranberry treatment partially reversed these alterations. Our results also demonstrated that DSS treatment significantly increased the relative abundance of Akkermansia and Sutterella, and decreased the abundance of Bifidobacterium and Lactobacillus in the mouse feces, which was associated with the development of IBD. Importantly, dietary cranberry treatment inhibited Akkermansia and Sutterella, and promoted Bifidobacterium and Lactobacillus in DSS‐treated mice. Overall, our results for the first time suggested that modification of gut microbiota by dietary whole cranberry might contribute to its inhibitory effects against the development of IBD.

Support or Funding Information

This study was partially supported by fund from USDA and Cranberry Institute.
Oral administration of curcumin and salsalate attenuates high fat diet‐induced up‐regulation of pro‐inflammatory colonic cytokines and suppresses Akt/NFκB signaling in azoxymethane‐treated mice
Authors: Wu Xian , Pfalzer Anna C , Koh Gar Yee , Crott Jimmy W , Tang Sanyuan , Thomas Michael J , Meydani Mohsen , Mason Joel B
Published: June 2021

Background

Obesity, a robust risk factor for colorectal cancer (CRC), is known to elevate the concentrations of pro‐inflammatory cytokines in the murine colon. Also, signaling through the Akt pathway, which is known to be activated by pro‐inflammatory cytokines, is thought to play a role in colorectal carcinogenesis, in part by enhancing NFκB signaling. Further, we have previously demonstrated that high fat diets (HFD) and excess adiposity each alter the expression of the regulators of colonic Akt signaling in a pattern suggestive of Akt activation.

Objective

We sought to determine whether the combination of a pharmacologic agent and a dietary component that each possesses anti‐inflammatory properties might suppress obesity‐induced elevations in colonic cytokines and Akt/NFκB signaling, with the ultimate intent of mitigating the cancer‐promoting effect of obesity.

Methods

Curcumin (CUR), a natural polyphenol in turmeric, and salsalate (SAL) a non‐steroidal anti‐inflammatory drug (NSAID) that lacks the gastrointestinal toxicity of other NSAIDs, were tested alone and in combination in the azoxymethane (AOM) model of colonic carcinogenesis in 110 A/J mice, randomized to five groups including a control group receiving a low‐fat diet. AOM (7 mg/kg body weight, 4 weekly injections) in combination with a HFD that contained 60% kcals as fat was used induce a pro‐tumorigenic milieu in the colons of mice.

Results

The results showed that HFD‐mice had 30.4% greater fat mass and a lower lean mass than did low‐fat diet (10% kcal fat, LFD, p<0.005)‐mice. In the colonic mucosa of the obese mice addition of CUR and SAL (0.2% and 0.15% w/w, respectively) to the diet significantly diminished concentrations of IL‐1β and IL‐6 (p<0.015). Moreover, CUR/SAL co‐treatments also profoundly suppressed phosphorylation of Akt (p<0.044) and concurrently suppressed NF‐κB p65 signaling (p<0.042).

Conclusions

Our results demonstrate, for the first time, that co‐administration of CUR and SAL in very low, well‐tolerated, concentrations attenuates inflammatory cytokine levels, Akt activation, and NFkB signaling in the colons of AOM/HFD mice, providing a scientific rationale for using this combination as a chemopreventive regimen to mitigate the risk of obesity‐associated colon cancer.

Support or Funding Information

Tufts Collaborates Grant; United States Department of Agriculture, Cooperative Agreements #58‐1950‐0‐014 and #58‐1950‐4‐003
Quantitative Real Time PCR (qRT‐PCR) Evaluation of Pig Mitochondrial DNA Damage
Authors: Barr Johnny L , Thomas Theo W , Gibson Ruby S , Dubick Michael A , Bowman Phillip D
Published: June 2021

Mitochondrial DNA (MtDNA) damage has generated much interest in recent years as a potential biomarker after ischemia/reperfusion and traumatic injury and in critical illness. MtDNA is particularly susceptible to damage due to is close proximity to the source of generation of reactive oxygen species during metabolism and its relatively low ability to repair DNA damage compared to nuclear repair mechanisms. Reduction in MtDNA damage appears to be a good indicator of a successful intervention. QRT‐PCR has been shown to be the most sensitive and accurate method to evaluate mtDNA damage requiring only nanogram amounts of DNA. While validated primer pairs for mtDNA damage for human and rodent mtDNA damage are available, comparable sets for pig have not previously been described, yet many studies in trauma and critical illness have utilized swine models. To select primer pairs for pigs, we aligned the pig mitochondrial DNA sequence to the pig genome in the UCSC Genome Browser and primers for regions not exhibiting overlap with the nuclear genome were selected with NCBI Primer3. Using the bleomycin injury model, porcine pulmonary artery endothelial cells obtained from Cell Biologics and cultivated in MCDB 131 medium were treated for 1 hr with bleomycin (10 μg/ml). Short and long sequences were tested with pig total DNA and demonstrated that one set detected specific induction of DNA lesions. The assay was able to detect 11 lesions per 10,000 base pairs. The short primer pairs also accurately predicted mitochondrial copy number DNA as corroborated for mtDNA by published results for the pig ND4 gene. We are currently employing this assay to detect mtDNA damage in a swine model of hemorrhagic shock.

Support or Funding Information

Funded by US Army Medical Research Materiel Command
Phylogenetic Analysis of RuBIsCO: An Active Learning Strategy for Teaching Plant Evolution
Authors: Van Stry Melanie
Published: June 2021

Lane College is a small historically black college located in Jackson, TN. The majority of the students are underserved African Americans from areas of high poverty. General Biology at Lane College is a foundational survey course required for Biology majors and Physical Education majors. Topics covered in the second semester of the course include natural selection, evolution, biodiversity, and ecology. This course is especially challenging for students given the large amount of information that they are typically required to memorize. We are currently implementing a peer‐led team learning strategy to enable students to actively engage in learning core concepts outlined in Vision and Change. The learning goal for the exercise presented here is to explore the evolutionary relatedness of different plant species. The learning objectives for this exercise are for students to understand plant taxonomy and to analyze the DNA evidence for the origin of the chloroplast through a phylogenetic comparison of the ribulose‐1,5 bisphosphate carboxylase/oxidase large subunit (rbcl) gene and to understand plant taxonomy. Prior to coming to class, the students are given a reading assignment for the Land Plants chapter in their textbook. During the first class meeting, at the beginning of the exercise, the students are divided into teams of four. Each team is assigned a set of four unknown DNA sequences from closely related plant or algal species, and each student chooses a sequence. Each student then uses the standard Basic Alignment Search Tool (BLAST) to identify the gene and their plant species for their sequence. The students then answer specific questions about their plant or algal species regarding taxonomic classification. Next, each student creates a sequence alignment of the entire class set of sequences and constructs a phylogenetic tree using CLUSTAL Omega. They then compare their phylogenetic tree with the one from their textbook and discuss the similarities and differences between the trees. At the next class meeting, each group presents a short summary of the characteristics for their assigned plant clade.

Support or Funding Information

National Science Foundation: HRD 1623340
Experiences of Undergraduate Students Identifying Proteins of Unknown Function As Part of a Teaching Laboratory in a Biochemistry Course
Authors: Daubner Susan Colette , Beckman John , Gastelum Jazmin Beltran , Mallet Sarah , Vogt Ellysa
Published: June 2021

In the laboratory section of Biochemistry II at St. Mary's University, we are performing a series of experiments involving authentic scientific inquiry. The experiments' goal is to identify the functions of a handful of unknown proteins. The curriculum relies heavily on both in silico and in vitro biochemistry techniques. Faculty and students pick the proteins by looking through the PDB list of crystallized proteins that have no known function. Since these proteins have been crystallized, they have already been cloned and purified; we know we are likely to be able to isolate suitable quantities of the proteins for work. We ascertain ahead of time that their coding vectors are available to us from sources such as DNASU or AddGene. We analyze the sequences of potential proteins using internet programs such as Pfam, Dali, BLAST, and HHPred. Good alignment with characterized proteins allows us to make hypotheses for the function of the unknown ones. We use the PyMol plug‐in ProMol to look for arrangements of active site residues common to certain categories of enzymes. For example, we used the triad of serine, histidine, and aspartate from chymotrypsin to identify possible hydrolases. We use the three‐dimensional alignment function of the program Chimera to see whether our unknown proteins resemble the known proteins that are thought to be similar. We overexpress the unknown proteins in E. coli, and purify the enzymes taking advantage of their engineered tags. The spring 2016 class focused on and was successful in broadly identifying four unknown hydrolases. Of these, three were lipases and one was a nuclease. We are extending that success by purifying and assaying additional hydrolases in spring 2017. After every lab in the pilot semester students reported on their experiences, and they presented posters at a University research symposium. The student authors of this poster will either have performed the pilot semester (SM) or be involved in the second round of the curriculum (JB, JB, and EV). They will report on their experiences, their experiments, their findings and whether this innovative teaching lab affected their attitudes toward the inquiry process.

Support or Funding Information

This work is funded by NSF DUE‐1502720.
Architecture of the Nucleosome Remodeling and Deacetylase (NuRD) Complex
Authors: Low Jason K.K. , Silva Ana P.G. , Sharifitabar Medhi , Torrado Mario , Schmidberger Jason , Webb Sarah R. , Saathoff Hinnerk , Parker Benjamin L. , Paudel Bishnu , van Oijen Antoine , Landsberg Michael J , Shepherd Nicholas E. , Mackay Joel P.
Published: June 2021

The Nucleosome Remodeling and Deacetylase (NuRD) complex is a ~1‐MDa 10‐subunit transcriptional co‐regulator that plays essential roles in hematopoietic development, stem cell renewal and DNA repair, and is emerging as a central player in diseases ranging from cancer to thalassemia.

Despite the importance of this complex, little is known about its structure and biochemical activity. Hence, knowledge of NuRD structure and function will provide broad insight into mechanisms of eukaryotic gene regulation.

To address this deficit, we are using a barrage of biochemical and biophysical approaches. We recently showed that the NuRD complex can exist as a separate stable entity (without CHD4) dubbed the NuDe (Nucleosome Deacetylase) complex — disrupting the architectural paradigm that CHD4 is an integrated core component of the complex (1). In addition, a combination of mass spectrometry and single‐particle electron microscopy has allowed us to piece together the architecture of NuRD through its subcomplexes at (quite) low resolution (2). Last but not least, we are now utilising single‐molecule fluorescence to observe the remodeling activity of individual NuRD complexes in real time. This could reveal the biochemical mechanism by which this complex remodels chromatin during gene transcription and DNA repair.

Support or Funding Information

Australian Research Council (DE120102857) Australian National Health and Medical Research Council (APP1012161 and 571099)
Assessing nutrition knowledge using Healthy Plate photo method among youth participating in WeCook
Authors: Chai Weiwen , Walther Ashley , Dunker Tara , Franzen‐Castle Lisa , Krehbiel Michelle
Published: June 2021

Objective

Mounting evidence suggests that poor dietary habits are linked to a lack of cooking at home and insufficient nutrition knowledge such as making a healthy plate. This study assessed the changes in nutrition knowledge among youth participating in WeCook.

Methods

Twenty‐four 4th and 5th grade students from two Title I elementary schools in Nebraska were recruited to participate in WeCook, an on‐going, semester‐based, 12‐week after‐school prevention program focusing on youth cooking and physical activity. Surveys were administered at each site before and after the program. Youth's nutrition knowledge was assessed using a novel Healthy Plate photo method (based on MyPlate guidelines) and previously validated instrument. Youth were asked to build a healthy plate using a blank MyPlate template and food models pre‐ and post‐intervention, with healthy plate photos taken by the researchers. Healthy plate photos were assessed using a scoring system based on the five food groups present on healthy plate; one point was assigned to each of the correct food groups present with a maximum total score of five. The differences in pre‐ and post‐intervention scores of healthy plate photos and related nutrition knowledge were assessed using paired t‐test and chi‐square test.

Results

The average score of correct food groups on healthy plate photos was significantly increased after WeCook (Pre: 3.8±0.8, Post: 4.4±0.8; P=0.003). Fifty‐six percent of participants received the maximum score (5 points) after the program whereas only 17% scored five pre‐ program. Additionally, there were non‐statistically significant increases in knowledge on daily fruit and vegetable consumption and the importance of breakfast after the program.

Conclusion

Our preliminary results suggest WeCook appears effective to improve nutrition knowledge among 4th and 5th grade participants in the study.

Support or Funding Information

This material is based upon the work of CYFAR University of Nebraska‐Lincoln Sustainable Community Project, a project supported by the National Institute of Food and Agriculture, United States (U.S.) Department of Agriculture, through a cooperative agreement with the University of Nebraska‐Lincoln under award number 2014‐41520‐22207.
Identification of Glycoproteins on the Surface of Bovine Milk Exosomes and Intestinal Cells that Facilitate Exosome Uptake in Human Colon Carcinoma Caco‐2 Cells
Authors: Sukreet Sonal , Zhang Hanyuan , Adamec Jiri , Cui Juan , Zempleni Janos
Published: June 2021

Background

Exosomes are small, cargo‐containing vesicles that are secreted by donor cells to elicit changes in gene expression and metabolism in recipient cells. In previous studies, we provided evidence that, in addition to endogenous synthesis, exosomes may also be obtained from dietary sources such as bovine milk, and intestinal transport is facilitated by endocytosis.

Hypothesis

Glycoproteins on the surface of bovine milk exosomes and human intestinal cells play essential roles in exosome transport.

Methods

Exosomes were isolated from bovine milk by differential centrifugation and authenticated as recommended by International Society for Extracellular Vesicles (Fig. 1). Surface proteins or glycan modifications in exosomes or Caco‐2 cells were removed using proteases (Glu‐C, trypsin, Arg‐C, Asp‐N, or proteinase K) or glycosidases [N‐glycosidase F (PNGase F), galactosidase, O‐glycosidase, neuraminidase, N‐acetyl glucosaminidase, or a combination of all]. Controls were incubated with solvent. Exosomes were labeled with FM4–64, and unlabeled fluorophore was removed. Surface peptides released after the treatment were identified using LC/MS‐MS, Mascot and Sequest databases. Glycoproteins on the external surface were identified using a series of tools based on neuronal networks including NetNglyc, NetOglyc, NetCglyc 1.0 and GlycoEP. Transport kinetics were modelled using the Michaelis Menten equation. One‐way ANOVA and Bonferroni's multiple comparison were used for statistical analyses.

Results

When exosomes or cells were treated with individual proteases (Fig. 2) or glycosidases (Fig. 3), exosome uptake decreased up to 80% when compared to controls even at prolonged incubation times. Treatment with galactosidase, PNGase F and O‐glycosidase had a stronger effect than other glycosidases. When exosomes or cells were treated with the glycosidase mixture, no uptake was detected or uptake was decreased by 80%, respectively, in comparison to controls (Fig. 4). 149 total proteins were identified in bovine exosome, including 4 (N), 2 (O) and 2 (C) glycan binding sites; 298 proteins were identified in Caco‐2 cells, including 46 membrane proteins that included 29 (N), 33 (O) and 6 (C) glycan binding sites.

Conclusions

Glycoproteins on the surface of bovine milk exosomes and intestinal cells facilitate the uptake of exosomes in human intestinal cells. The β‐galactoside, Core 1 & Core 3‐O‐linked disaccharide and N‐acetylglucosamine modifications appear to be of greater importance than other glycan features present on complex glycoproteins.

Future Directions

The transport of bovine exosomes will be studied in conditional glycan knockout mice.

Support or Funding Information

NIFA 2015‐67017‐23181, NIFA 2016‐67001‐25301/NIH DK107264, NIH 1P20GM104320, the Gerber Foundation, and USDA Hatch Act and W3002.
Association of Added Sugars Intake with Physiologic Parameters in Adults: NHANES 2001–2012
Authors: O'Neil Carol E , Nicklas Theresa A , Fulgoni Victor L
Published: June 2021

The association of added sugars intake with liver enzymes, cardiovascular risk factors, and markers of red cell counts was determined using NHANES 2001–2012 data from adults (19+ years; n=26,402). Dietary intake was determined using 24‐hour dietary recall interviews using an Automated Multiple‐Pass Method. The usual intake of added sugars as a percent of energy was estimated using the Markov Chain Monte Carlo (MCMC) ratio method as implemented in v1.0 of the SAS programs published by the National Cancer Institute (NCI). Balanced repeated replication (BRR) was used for variance estimation. Subjects were separated into six groups: 0<5, 5 to <10, 10 to<15, 15 to <20, 20 to <25, and ≥25% of energy as added sugars. Group trends and linear trends for were determined using linear regression (p<0.01) for: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma‐glutamyl transferase, lactate dehydrogenase, blood pressure, high‐ and low‐density lipoprotein cholesterol, triglycerides, glucose, c‐reactive protein, waist circumference, ferritin, serum and red blood cell folate, hemoglobin, and hematocrit. None of the liver enzyme levels showed any significant association with added sugars intake. Mean plasma glucose levels (mg/dL) showed an inverse group (beta = −0.71; p=0.0003) and linear trend (beta = −0.13); p=0.0002) association with increasing percent energy of added sugars. No other cardiovascular risk factors, including metabolic syndrome, showed any association with added sugars intake. Only hemoglobin (g/dL) (beta = −0.03, p=0.0007; beta = 0.005, p=0.0013) and hematocrit (%) (beta = −0.09, p=0.0001; beta = −0.02, p=0.0002) showed a group or linear trend, respectively. No other statistical associations were found. These results suggest that the association of usual added sugars intake, as determined by sophisticated models, up to >25% of energy, had limited effect on physiologic parameters in adults.

Support or Funding Information

USDA Hatch Project LAB 94209 and USDA/ARS through specific cooperative agreement 58‐6250‐6‐003 was received. Partial support was also received from the Corn Refiners Association.
Neonatal diet composition modulates ileum mitochondrial function in a neonatal pig model
Authors: Carvalho Eugenia , Moreira Ana R , Bowlin Anne K , Saraf Manish K , Adams Sean H , Borsheim Elisabet , Yeruva Laxmi
Published: June 2021

Background

The composition of postnatal diet (i.e., breastmilk vs. formula) has a strong influence on a variety of physiological outcomes in infants, but the impact on bioenergetics and mitochondrial function remains an open question. In a published study (1), early ingestion of dairy‐based infant formula vs. human breast milk differently modulated liver mitochondrial function and gut microbiota in rodents. However, the effects on bioenergetics in other tissues, including the gut, and the persistent effect after weaning remain unknown. In the current study we used a piglet model to determine the effects of these two milk types on bioenergetics phenotypes in the ileum.

Methods

Neonatal piglets were fed isocaloric diets of either human breast milk (n=9) or a dairy‐based infant formula (n=9) from 48 hours post‐delivery until day 21, followed by an ad libitum solid diet until day 51. Subsequently, ileum biopsies were collected for evaluation of permeabilized cell mitochondrial function by high resolution respirometry, following a substrate and inhibitor protocol. In addition, gene expression for a key regulator of mitogenesis and mitochondrial function, peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) was assessed.

Results

ATP‐linked respiration tended to be increased in the ileum of piglets fed a dairy‐based infant formula compared to those fed with human breast milk, even at 4 weeks of post‐weaning neonatal diet (10.4 +/− 2.2 vs. 5.8 +/− 0.9 pmol·s−1·mg−1; p=0.07). Cytochrome c oxidase (COX) activity was significantly elevated in the formula compared to the human breast milk (23.3 +/− 4.8 vs. 9.9 +/− 1.8 pmol·s−1·mg−1; p=0.02). Thus, the substrate control ratio for succinate relative to COX was significantly lower in the dairy fed group (0.07 +/− 0.02 vs. 0.14 +/− 0.02; p=0.03). Interestingly, PGC‐1α gene expression tended to be elevated (2.3 fold, p=0.06) in ileum of formula‐fed piglets compared to those fed human breast milk.

Conclusion

We conclude that different milk diets may have a sustained impact on ileum bioenergetics in neonatal piglets, even a month after discontinuation of the milk diets. Future efforts are needed to understand the implications of these novel findings on infant tissue function and whole‐body macronutrient metabolism.

Support or Funding Information

Funded by USDA ARS Project 6026 51000 010 05S. FEDER and FEDER funds through the Operational Programme Competitiveness Factors ‐ COMPETE and UID/NEU/04539/2013.
Cytotoxicity of ethanol extracts from glanded and glandless cottonseed in cultured mouse RAW macrophages
Authors: Cao Heping , Sethumadhavan Kandan
Published: June 2021

Cotton plant provides two economically important products, fiber as the major product and cotton seed as the minor product. However, cotton plant produces more seed in terms of quantity than fiber with a seed/fiber ratio of 1.5 – 1.7. Cottonseed typically accounts for 15 – 25% of the crop value. Due to presence of toxic gossypol in the commonly cultivated cotton (glanded cotton), the uses of cottonseed is limited to feeding cows and not for human and other animal consumption. To explore the potential use of glandless cottonseed which has only trace amount of gossypol, we investigated the cytotoxicity of extracts from glandless cottonseed along with those from glanded cottonseed and compared directly with gossypol and lipopolysaccharide (LPS) using cultured mouse macrophages. Cottonseeds were separated into coat and kernel fractions. Both fractions were homogenized in 0.1 N acetic acid, defatted with chloroform and hexane and extracted with ethanol. The ethanol extracts were rotoevaporated to remove ethanol and the dried extracts were reconstituted in DMSO before being added to cultured mouse RAW264.7 macrophages using gossypol and LPS as controls. Cell cytotoxicity was determined with MTT method. Dosage study showed that no cytotoxicity effect was observed in cells treated for 2–24 h with extracts (up to 1 mg/ml) from glandless cottonseed coat or kernels, nor with glanded cottonseed coat. However, mitochondrial activity measured by the MTT method was decreased by 40% with glanded cottonseed kernel extract after cells were treated with 0.1–1 mg/ml for 24 h. LPS (up to 1 ug/ml) did not show toxic activity but gossypol (≥5 ug/ml, 24h) almost completely inhibited cell viability. These results suggest that glandless cottonseed extractis harmless towards the cultured macrophages but glanded cottonseed extract from its kernel, like gossypol, is harmful to the immunological cells.

Support or Funding Information

Supported by USDA‐ARS Quality and Utilization of Agricultural Products Research Program 306 through CRIS 6054‐41000‐103‐00D.