bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and Mechanism of the human Transcription Initiation Machinery
Authors: Taatjes Dylan , Bernecky Carrie , Poss Zachary , Ebmeier Christopher
Published: June 2021

The human Mediator complex (1.2 MDa, 26 subunits) is a general factor that helps regulate expression of perhaps all protein‐coding genes. Using cryo‐EM, we defined the structural organization of the 1.9 MDa Mediator‐pol II‐TFIIF assembly, in which Mediator was bound to the activation domain of VP16. The pol II orientation within the cryo‐EM map is entirely consistent with existing biophysical studies that determined the location of other PIC factors relative to pol II itself, allowing a structure‐based model for the entire 3.5 MDa human PIC to be proposed. Whereas TFIIF was shown to be important for stably orienting pol II within this assembly, the potential role of the activator (VP16) was not assessed. Remarkably, subsequent cryo‐EM studies of human Mediator–pol II–TFIIF complexes in which Mediator was not bound to an activator have revealed that pol II does not adopt a stable orientation in the absence of an activator. These data demonstrate that activator‐Mediator binding can control pol II orientation at the Mediator‐pol II interface and suggest that activators function in part by regulating PIC structure at the promoter. A major regulator of the Mediator–pol II interaction is the 600 kDa CDK8 module, which consists of the proteins CDK8, Cyclin C, MED12, and MED13. The human CDK8 module regulates Mediator structure and function and prevents Mediator– pol II binding. Emerging data now suggest additional, expanded roles for the human CDK8 module in regulating gene expression, which will be discussed in the context of the structural model of the human PIC.
Mapping out forces that act on transmembrane helices during membrane insertion
Authors: von Heijne Gunnar
Published: June 2021

Membrane proteins destined for insertion into the inner membrane of E. coli are synthesized by ribosomes bound to the SecYEG translocon. During co‐translational membrane integration, transmembrane α‐helical segments in the nascent chain exit the translocon through a lateral gate that opens towards the surrounding membrane, but the mechanism of gate opening and lateral exit is not well understood. In particular, little is known about how a transmembrane helix behaves when inside the translocon. Current work in our lab has shown that a force proportional to the free energy of membrane insertion is exerted on a transmembrane helix when it transits through the translocon channel, providing a direct insight into the dynamics of membrane integration.
Progress in development of dietary supplement (DS) composition and label databases for research
Authors: Dwyer Johanna T , Bailey Regan , Saldanha Leila , Holden Joanne , Andrews Karen , Betz Joseph , Gahche Jaime , Hardy Constance , Milner John , Roseland Janet
Published: June 2021

Lack of publicly available DS composition databases can hinder estimates of total nutrient intakes & studies of DS & on health and disease risk This research is needed for estimating nutrient needs, informing policy decisions, and developing dietary guidance messages for consumers. Federal collaborators are currently working to produce publicly available DS databases. The Dietary Supplement Ingredient Database (DSID‐2) provides estimates for 20 nutrients based on chemical analyses from national samples of adult & child multivitamin‐minerals (MVM), and provides online calculators for researchers to compare estimated analytically derived data with product label values. OTC prenatal MVM & omega‐3 fatty acid products will be added to DSID in 2013. Another research gap is the lack of a label database; the NHANES database contains label derived nutrient information from DS consumed by survey respondents. And work is underway to create a label database of most DS sold in the US in collaboration with the National Library of Medicine. A controlled vocabulary (LanguaL) has been developed to link these databases. Advances in these databases to address research needs will be described. Funding: ODS/NIH;NLM/NIH; Nutrient Data Laboratory, USDA; and NCHS/CDC.
Validating sugar‐sweetened beverage intake and adiposity among African‐American and White adults in a doubly labeled water study
Authors: Emond Jennifer A. , Patterson Ruth E , Jardack Patricia M , Arab Lenore
Published: June 2021

Evidence is mixed regarding sugar‐sweetened beverage (SSB) intake and adiposity among adults. To look at the effect of reporting bias, we compared SSB intake and overweight/obese status (BMI ≥25 kg/m2) among adults in a dietary assessment and doubly labeled water study (n=250). Four web‐based, 24‐hour recalls assessed dietary intake. SSB intake was categorized as none, 1–99 kcals/day, or >99 kcals/day; 99 kcals/day was median SSB intake among consumers. Logistic regression models, adjusted for total caloric intake, age, race, education and diet quality, compared SSB intake to overweight/obese status. We repeated analyses in a subset of “true reporters”: those with self‐reported total caloric intake within 25% of total energy expenditure per doubly labeled water (n=108). One‐half of participants were overweight/obese; more overweight/obese participants drank SSB than normal weight participants (69% vs. 47%; p<0.001). Intake of other beverages did not differ by adiposity. Compared to no intake, SSB intake up to the median doubled the risk of being overweight/obese (OR: 2.1, 95% CI: 1.0–4.3; p=0.046), and SSB intake over the median more than doubled the risk (OR: 2.6, 95% CI: 1.2–6.0; p=0.018). When limited to true reporters, SSB intake significantly increased the risk of being overweight/obese by nearly 4 fold. Results support that SSB consumption increases the risk of being overweight/obese among adults. This funding for this project was provided by the National Institutes of Health grant R01CA105048, as well as training grant T32 GM084896.

Grant Funding Source: Training grant T32 GM084896
Coffee consumption and risk of chronic disease in the European Prospective Investigation into Cancer and Nutrition (EPIC)‐Germany study
Authors: Floegel Anna , Pischon Tobias , Bergmann Manuela M , Teucher Birgit , Kaaks Rudolf , Boeing Heiner
Published: June 2021

Early studies suggested that coffee consumption may increase risk of chronic disease. We investigated prospectively the association between coffee consumption and risk of chronic disease (type 2 diabetes, cardiovascular disease and cancer) in 42659 participants of the European Prospective Investigation into Cancer and Nutrition (EPIC)‐Germany study. Coffee consumption was assessed by self‐administered food frequency questionnaire at baseline and incident cases of chronic disease were identified and medically verified during follow‐up. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated using multivariate Cox proportional hazard regression. During 8.9 years of follow‐up, we observed 1432 cases of type 2 diabetes, 704 cases of cardiovascular disease, and 1801 cancer cases as first qualifying event. Coffee consumption was not associated with the overall risk of chronic disease (≥4 cups/d compared to <1 cup/d, HR: 0.94; 95% CI: 0.84–1.05). We observed an inverse association between coffee consumption and type 2 diabetes risk (≥4 cups/d compared to <1 cup/d: HR: 0.77, 95% CI: 0.63–0.94) and no association with risk of cardiovascular disease and cancer (≥4 cups/d compared to <1 cup/d: HR: 1.10, 95% CI: 0.84–1.44; and HR: 0.97; 95% CI: 0.83–1.14, respectively). Our data indicate that coffee consumption does not increase risk of chronic disease, but may be linked to lower risk of type 2 diabetes.

Grant Funding Source : Federal Ministry of Science, Germany (grant No. 01 EA 9401), the European Union (grant No. SOC 95 201408 05F02), and the German Cancer Aid (grant No. 70‐2201‐Bo2)
Salmonella Typhimurium induces HXA3 secretion by decreasing cellular levels and activity of the oxido‐reductase enzymes GPX 1 and PHGPX
Authors: AGBOR Terence , McCormick Beth
Published: June 2021

Salmonella Typhimurium (S. Typhimurium)‐induced transepithelial migration of neutrophils rapidly follows attachment of the bacteria to the epithelial apical membrane. We have previously reported that hepoxilin A3 (HXA3) mediates the final step in this process. HXA3 is a derivative of arachidonic acid formed from the enzymatic action of the 12‐lipoxygenase (12‐LOX) pathway. However, the molecular mechanisms regulating the activity/expression of 12‐LOX during S. Typhimurium‐induced intestinal inflammation remains unknown. Thus, we sought to decipher the mechanisms regulating 12‐LOX and increased HXA3 secretion during S. Typhimurium infection. We found that the protein expressions of Glutathione peroxidase (GPX) 1 and PHGPX (GPX 4), known to have an inhibitory effect on 12‐LOX activity, are significantly decreased during S. Typhimurium infection as well as an ~20% decrease in total cellular GPX activity. We also show that inhibition of total cellular GPX activity by iodoacatetate treatment, which increases 12‐LOX activity, resulted in a significant increase in neutrophil transmigration during S. Typhimurium infection. These results suggest that S. Typhimurium induces apical secretion of HXA3 by decreasing the expression of cellular GPX, which in turn leads to an increase in 12‐LOX activity. We are currently performing RNAi studies to determine which GPX isoform is responsible for these functions.
Synergistic Inhibition of Lung Cancer Cell Growth by Nobiletin and Atorvastatin in Combination
Authors: Charoensinphon Noppawat , Zheng Jinkai , Qiu Peiju , Ngauv Perline , Xiao Hang
Published: June 2021

Combination of different bioactive agents may produce enhanced inhibitory effects on cancer cells due to their synergistic interactions. We studied combination of nobiletin (NBT, a citrus flavonoid) and atorvastatin (ATST, a cholesterol‐lowering drug) in 3 human lung cancer cells. NBT/ATST co‐treatments at low doses produced much stronger inhibitory effects on all three cancer cells in comparison to those produced by NBT or ATST alone at higher doses. Isobologram analysis confirmed NBT/ATST combinations produced strong synergistic inhibition on lung cancer cells. Flow cytometry analysis showed NBT/ATST co‐treatments for 24 hrs induced G0/G1 cell cycle arrest, while the prolonged co‐treatments for 48 hrs caused extensive apoptosis. Immunoblotting showed NBT/ATST co‐treatments activated caspase‐3 and cleavage of PARP, increased levels of p21 (Cip1/Waf1) and p27 (Kip1), while decreased expression levels of Cdk‐2, Cdk‐4, Cdk‐6, Cyclin D1, hyper‐p‐Rb, and Mcl‐1. Expression levels of both EGFR and p‐EGFR (Tyr1068) in plasma membrane were decreased by NBT/ATST co‐treatment. qRT‐PCR assay showed that NBT/ATST co‐treatments profoundly modulated oncoginic miRNA (e.g., mir‐7) profile in lung cancer cells. Overall, our results demonstrated strong synergy between NBT and ATST in inhibiting human lung cancer cell growth, which warrants future investigation on the in vivo efficacy of NBT/ATST combination.

Grant Funding Source: NIH: CA139174, AICR: 10A044
Nutrient biomarker patterns and rates of cognitive decline in dementia‐free elders
Authors: Bowman Gene L. , Quinn Joseph F. , Kaye Jeffrey A. , Shannon Jackilen
Published: June 2021

Background

We previously identified three distinct nutrient biomarker patterns associated with both psychometric and neuroimaging indices in a cross‐sectional analysis. The objective of this study was to examine the relationship between the nutrient biomarker patterns and cognitive decline over 2 years.

Methods

Thirty biological markers of diet were assayed in plasma drawn from 104 dementia‐free participants of the Oregon Brain Aging Study. Principal component analysis constructed 8 distinct nutrient biomarker patterns. A linear regression model was used to examine the change in the Clinical Dementia Rating ‐ sum of box score over two years explained by the nutrient biomarker patterns.

Results

Mean age was 87±10, 62% are female, and 10% are carrying the ApoEe4 allele. Two distinct nutrient biomarker patterns were associated with rates of cognitive decline: a vitamin pattern high in vitamins B, C, E and D (p=0.001) and a high trans fat pattern (p<0.001) associated with less and more decline over two years, respectively. These findings were independent of age, gender, education years, ApoEe4 carrier status and vascular risk factors.

Conclusion

A nutrient biomarker pattern high in specific vitamins and low in trans‐fat may offer a prudent profile for promoting cognitive health in older populations. Funding: NIH/NCCAM AT004777 (GLB), NIH/NIA AG08017 (JAK), NIH/NCRR UL1 RR024140

Grant Funding Source : NIH AT004777
Early Obesity Prevention: A Randomized Trial of a Practice‐Based Intervention in 0–24 Months Infants
Authors: Schroeder Natalia , Rushovich Berenice , Bartlett Edward , Gittelsohn Joel , Caballero Benjamin
Published: June 2021

A pediatric office‐based intervention was implemented following a randomized, controlled design, aimed at improving child feeding practices, growth and ultimately reducing risk for overweight and obesity later in life. Educational materials on feeding and physical activity called Growing Leaps and Bounds was delivered by the health care provider during each well‐baby clinic visit. A total of 232 infants at 4 clinics (2 urban, 2 suburban), followed for 24 months completed the study. The intervention (I) group offered significantly less soda (p=0.006), sweetened tea (p=0.01), punch (p=0.02) or cow's milk (p=0.001) to infants compared to the control (C) group. I group delayed introduction of drink/food other than breast milk (p<0.05). Furthermore, parents in I group had higher perceived parental monitoring (p=0.05) and restriction (p=0.01) on infant feeding. While I group exhibited at baseline more adverse socioeconomic indicators than the C group, growth trajectory or body size indices did not significantly differ between the groups at the end of the follow‐up period. Education provided by health care providers may help modify parental behaviors related to child feeding and increase parental sense of responsibility toward child eating behaviors.

Funded by the Dannon Institute
The Feeding Infants and Toddlers Study (FITS) 2008: Dramatic changes in the amount and quality of vegetables in the diet occur after the first year of life
Authors: Deming Denise M , Reidy Kathleen C , Briefel Ronette R , Fox Mary Kay , Condon Elizabeth
Published: June 2021

Population trends in vegetable consumption patterns were assessed among U.S. children from FITS 2008. Dietary intake interviews (24‐hr recalls) were conducted by telephone with parents or primary caregivers of infants 0–11 months (n=887), toddlers 12–23 months (n=925), and preschoolers 24–47 months (n=1,461). Based on per capita intakes, the amount of any vegetable consumed (AVC) dropped from 84.0±7.5 g/day among older infants (6–11 months) to 64.2±3.8 g/day among toddlers and 63.6±3.9 g/day among preschoolers, a downward shift that occurred as baby food vegetables fell out of the diet. A concurrent shift in the types of vegetables in the diet occurred with white potatoes (WP) replacing orange vegetables (OV) as the dominant vegetable after infancy. OV dropped from 39% of the amount of AVC among older infants to 19% and 14% among toddlers and preschoolers, respectively, while WP rose from 12% of the amount of AVC among older infants to 29% among toddlers and preschoolers. By the age of 2, about half the amount of WP consumed were French fries (9.4±1.5 g/day), and French fries (22.9±3.1 kcal/day) contributed more than two thirds of the average total caloric intake of WP (32.2±3.2 kcal/day). FITS data indicate that dietary patterns begin early in life and that parents need to focus on offering a variety of colorful, nutrient‐rich vegetables, especially during the transition to table food. Funded by Nestlé Infant Nutrition.
Intestinal Effects of Alanyl‐Glutamine Dipeptide‐Supplemented Diet in a Mouse Model of DSS‐Induced Colitis
Authors: Benton Shana , Hao Li , Merlin Didier , Jones Dean P. , Ziegler Thomas R.
Published: June 2021

Enteral and parenteral glutamine administration has cytoprotective effects in animal models of gut injury. We investigated intestinal effects of oral diet supplemented with alanyl‐glutamine dipeptide (AG) in dextran sulfate sodium (DSS)‐induced colitis in mice.

Methods

C57BL/6 mice were given 3.5% DSS in water and concomitantly pair‐fed iso‐N oral diets without AG (DSS) or with AG supplementation (AG‐DSS). Control mice did not receive DSS or AG. Mucosal injury scores, myeloperoxidase (MPO) activity, TNF‐α and IL‐6 mRNA (qPCR) and GSH/GSSG redox status were determined in jejunum, ileum and colon after 7 days; clinical status was assessed daily.

Results

Food intake was similar in all groups. DSS‐treated mice had weight loss, bloody diarrhea, and increased small bowel and colonic MPO activity vs controls regardless of diet (P <0.05). The AG‐DSS group exhibited greater histologic injury in both ileum and colon vs the DSS and control groups (P<0.05). Colonic TNF‐α mRNA was increased in the DSS group vs the AG‐DSS and control groups (P<0.05). IL‐6 mRNA and GSH/GSSG redox in jejunum, ileum and colon was similar in all 3 groups.

Conclusion

Dietary supplementation with AG increased histologic gut injury and decreased colonic TNF‐α in this model of colitis. Supported by NIH grants K24 RR023356 and T32 DK007298.
Understanding the patterns and trends of potassium intake and sodium/potassium ratio in China, 1991–2009
Authors: Du Shufa , Neiman Andrea , Wang Huijun , Zhang Bing , Popkin Barry M
Published: June 2021

While recent studies reported an association between the sodium/potassium ratio (Na/K) and mortality from cardiovascular diseases and all‐cause mortality, little is known about patterns and trends of potassium intake and Na/K. Data was used from the China Health and Nutrition Survey, an ongoing open cohort study initiated in 1989 and followed up through 2009 of 16,869 adults aged 20–60 y. Detailed dietary data was collected in three consecutive 24–h records with weighing schemes. Multinomial logit model was used to determine trends and patterns of Na/K. Potassium intake was 1.9 g/day in 2009, an increase from 1.5 g/day in 1991. Na/K was 2.8 in 2009, a decrease from 4.8 in 1991. <0.3% of those surveyed met adequate intake (AI) of potassium as recommended by Institute of Medicine, while >60% met the AI as recommended by China. 6% had Na/K reached the level recommended by WHO. Na/K was the highest in central China, where the increased prevalence of hypertension was more rapid as compared to the north and south. The population of central China, low income, and low educational attainment were least likely to consume Na/K at recommended level. Unlike current thinking, adults in central China have the highest Na/K intake and greater increase in hypertension. Replacing sodium with potassium in salt to control and prevent hypertension in China should be considered among other public health and clinical prevention options.
Relationship between maternal diet, plasma lipids and human milk cholesterol and fatty acids
Authors: Woo Jessica Graus , Summer Suzanne S , Morrow Ardythe L , Heubi James E
Published: June 2021

Human milk cholesterol and fatty acids (FA) are important for infant development. Few studies have examined the relationship between maternal diet or plasma lipids on human milk composition. From 120 longitudinally‐studied breastfeeding mother‐infant pairs, mothers with the 15 highest (HC) and 15 lowest (LC) dietary cholesterol were analyzed. Maternal diet (4–13 weeks postpartum) was assessed, and plasma and milk were collected at 4 weeks postpartum. Demographics and anthropometrics were similar between LC and HC, but HC had higher intake of kcal and macronutrients, except carbohydrate. Plasma and milk total cholesterol did not differ by group. LC had lower milk palmitic acid (median 19.1 %wt/wt vs. 21.1, p=0.03), higher milk linoleic acid (20.5 vs 17.9, p=0.003) and marginally higher plasma linoleic acid (42.8 vs 40.4, p=0.06). In HC, milk oleic and stearic acids were correlated with kcal (Spearman rho=0.45 and 0.43, respectively). In LC, plasma and milk FAs were significantly correlated with each other. These data suggest that milk FA, but not total cholesterol, may be partially influenced by maternal diet and plasma FA. This work was funded by the CCHMC CTRC and Mead Johnson Nutrition, Inc.
Disparities in childcare and breastfeeding rates among US children of single mothers
Authors: kim juhee
Published: June 2021

Objectives

This study is to evaluate the characteristics and disparities in childcare and breastfeeding rates among children of single mothers compared to those of two parents using a nationally representative US children.

Methods

The study sample consisted of children aged 0 to 5 years enrolled in the National Study of Children's Health 2007 (children of single mothers ( n=3,803, 16.6%) and children of two‐parents (n=22,510, 83.4%). The primary caregiver of the study child responded the following outcome measures: 1) childcare type, 2) employment problems due to childcare, 3) last‐min change of childcare arrangement, 4) breastfeeding initiation, 5) exclusive breastfeeding for 6 months, 6) any breastfeeding for 6 months, and 7) started complementary foods before 6 months.

Results

The profiles of race/ethnicity, birth weight, education, poverty, employment and nativity of mothers in children of single mothers may present higher risk for low breastfeeding rates. There were disparities in childcare use and problems among children of single mothers. Exclusive breastfeeding and early initiation of complementary foods were not significantly different by family structure; however, breastfeeding initiation (OR= 0.78: 95% CI=0.64–0.97) and any breastfeeding for 6 months (OR=0.76: 95% CI=0.61–0.95) were much lower among children of single mothers compared to those of two parents.

Conclusion

This study documented disparities in childcare use and problems as well as breastfeeding rates among children of single mothers, raising a need for public health interventions targeting single mothers.

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government, (NRF‐2011‐330‐B00190).
B‐Lymphocyte Stimulator in neuroendocrine tumors: correlation with disease behaviour
Authors: Fabris Martina , Tonutti Elio , Pizzolitto Stefano , Isola Miriam , Curcio Francesco , Grimaldi Franco
Published: June 2021

Aim of the study

To test B‐Lymphocyte Stimulator (BLyS) as a new serological marker in the follow‐up of patients with neuroendocrine tumors (NET).

Methods

Eighty‐one consecutive NET patients and 56 controls were enrolled in the study. Patients were classified in 2 subgroups: persistent but stable disease or remission (n = 45) vs patients with recurrent disease (progressive patients, n = 36). BLyS and Chromogranin A (CgA) serum levels were analyzed by ELISA.

Results

BLyS levels were more elevated in NET patients than in controls (1153±529 pg/ml vs 655±158 pg/ml; p <0.0001) and correlated with tumor differentiation (1058±398 pg/ml in gastroenteric G1‐typical lungs vs 1325±640 pg/ml in gastroenteric G2–atypical lungs; p=0.026). Stable/remission patients displayed lower BLyS than progressive patients (889±251 pg/ml versus 1461±623 pg/ml; p <0.0001). BLyS did not change in patients who remained stable after 6.6±2.8 months (from 864± 283 pg/ml to 809±235 pg/ml), while further increased in patients with disease progression (from 1575±810 pg/ml to 1887±1163 pg/ml; p=0.045). Whereas, CgA showed contradictory changes. Metastatic patients displayed higher BLyS than non metastatic (1391±724 pg/ml vs 1079±422 pg/ml; p=0.022).

Conclusion

Elevated BLyS serum levels characterize more aggressive NET patients. BLyS appears as a new potential prognostic marker in the follow‐up.
PEZH2 and H3K27me3 are associated with Mallory‐Denk body forming liver cell phenotype and HCC
Authors: French Barbara A. , Bardag-Gorce Fawzia , Oliva Joan , Li Jun , Zhong Jin , French Samuel W.
Published: June 2021

Background

Mallory‐Denk body (MDB) forming hepatocytes in chronic liver disease (CLD) and hepatocellular carcinoma (HCC) share phenotypic characteristics including altered expression of pEZH2 and H3K27me3.

Method

Livers from DDC primed mice were induced to form MDBs by refeeding DDC with or without SAMe for 7 days after 1 month DDC withdrawal. These mice were used to test the role of pEZH2 and H3K27me3 using immunohistochemistry (IHC), Western blot and qRTPCR.

Results

Livers from mice refed DDC showed MDB forming hepatocytes and decreased staining for H3K27me3 in the nuclei compared to neighboring normal hepatocytes. The MDBs double stained for ubiquitin (Ub) and pEZH2 showed colocalization of Ub and pEZH2 in MDBs. Double stains of human livers with AH and HCC, showed colocalization of pEZH2 and Ub in the MDBs formed. Stains for H3K27 showed decreased staining of nuclei in liver cells forming MDBs. qRTPCR of EZH2 showed that DDC refeeding with SAMe reduced EZH2 levels significantly. Western blots of pEZH2 levels showed that pEZH2 was largely concentrated in the MDB band at the top of the gel. This correlated with the IHC results.

Conclusion

Changes in H3K27me3 and pEZH2 observed in MDB forming cells in AH, HCC and DDC fed mice indicated that MDBs are a phenotypic change related to loss of gene silencing, dedifferentiation and liver cell proliferation. Supported by NIH PA50‐011999.
Dietary supplementation with aromatic amino acids improves net protein synthesis in children with severe acute malnutrition during hospitalization
Authors: Hsu Jean W , Badaloo Asha , Taylor-Bryan Carolyn , Reid Marvin , Forrester Terrence , Jahoor Farook
Published: June 2021

The stress of infection in children with severe acute malnutrition (SAM) imposes a metabolic demand for increased synthesis of acute phase proteins which have a higher content of aromatic amino acids (AAA), phenylalanine (Phe), tyrosine and tryptophan. We investigated whether supplementation with an AAA cocktail (330 mg·kg−1·d−1
) compared with isonitrogenous amounts of alanine (ALA) would improve measures of protein kinetics in 22 (11 with AAA, 11 ALA) children with SAM (age 4–31 mo) during treatment for SAM. Protein kinetics were assessed by measuring Phe kinetics in the fed state using a primed‐continuous infusion of 13C‐Phe. Supplementation started after the baseline experiment at the end of maintenance period (Phase 1) and continued to recovery. The 2nd and 3rd experiments were done at mid‐catch up growth when the children had replaced 50% of their weight deficit (Phase 2) and at recovery (Phase 3).

Phase 1
Phase 2
Phase 3

AAA
ALA
AAA
ALA
AAA
ALA

Phe Kinetics
μmol·kg−1·h−1

 Total Intake
18±1
18±0.4
81±2 a
42±2
74±3a
35±2

 Flux
48±4
53±5
60±5
47±6
81±15
72±13

 NOD
7.5±2
9.8±1
32±5a
12±1
35±10a
23±3

 Balance
12±2
10±2
49±5a
31±2
37±8a
11±3

Mean±SEM; NOD = Non‐oxidative disposal

significantly different from ALA group in same phase, P < 0.05

We conclude that dietary supplementation with AAA improves net protein synthesis in children with SAM.

Supported by NIH Grant # 5R01DK75018
Protein requirement of elderly women determined using the indicator amino acid oxidation technique
Authors: Tang Minghua , McCabe George P , Elango Rajavel , Pencharz Paul B , Ball Ronald O , Campbell Wayne W
Published: June 2021

Objective

This study was designed to determine the protein requirement of elderly women age 80 y and older using the indicator amino acid oxidation technique.

Design

Eight Caucasian women aged 80–87 y (83 ± 3 y and BMI 27 ± 4 kg/m2, mean ± SD) completed a 3‐day protocol 7 times. Each woman consumed an adaptation diet for 2 days and on day 3 consumed a crystalline amino acid mixture with one of 7 protein intakes (0.1, 0.3, 0.6, 0.9, 1.2, 1.5 or 1.8 g·kg‐1·day‐1) tested randomly. Group‐based protein requirement was assessed using a biphase linear regression model of protein intake and L‐[1–13C] phenylalanine oxidation. The break point, at which there was no further decline in the 13C rate of appearance in breath, represented the mean protein requirement.

Results

The mean protein requirement was 1.06 g·kg‐1·day‐1 (standard error of the group mean estimate: 0.095). This requirement is 61 % higher than the current Estimated Average Requirement (EAR) for adults of 0.66 g·kg‐1·day‐1 based on nitrogen balance.

Conclusion

This study is the first to assess the dietary protein requirement of women in their 80s and the first to use the indicator amino acid oxidation technique with elderly participants. These findings bring into question the accuracy and adequacy of the current nitrogen balance based EAR for elderly women.

Support: NIH RR025761; Bilsland Fellowship from Purdue Univ.

Grant Funding Source : NIH RR025761; Bilsland Fellowship from Purdue Univ
Maternal vitamin D supplementation to optimize the vitamin D status of breastfed infants
Authors: Oberhelman Sara Schwind , Meekins Michael , Fischer Phillip , Lee Bernard , Gardner Brian , Singh Ravinder , Cha Stephen , Pettifor John , Thacher Thomas
Published: June 2021

Vitamin D supplementation of breastfed infants is recommended because breast milk levels are low. We sought to determine if maternal supplementation would increase breast milk levels to achieve sufficiency in their infants. Forty mothers with exclusively breastfed infants were randomized to receive oral cholecalciferol (vitamin D3) 5000 IU/d for 28 days or 150,000 IU once. Maternal serum, breast milk and urine were collected on days 0, 1, 3, 7, 14 and 28 and infant serum on days 0 and 28. In mothers receiving daily vitamin D, serum and breast milk cholecalciferol attained steady levels of 18 and 8 ng/ml, respectively, from day 3 through 28. In mothers given the single dose, serum and breast milk cholecalciferol peaked at 160 and 40 ng/ml, respectively at day 1, before rapidly declining. Breast milk and maternal serum cholecalciferol were related (r=0.87). Infant serum 25OHD (mean±SD) increased from 17±13 to 39±6 ng/ml in the single dose group and from 16±12 to 39±12 ng/ml in the daily dose group (P= 0.88). All infants achieved sufficient 25OHD serum levels (≥20ng/mL). This study suggests that maternal supplementation can achieve sufficient infant serum levels of 25OHD via breastfeeding. A large single dose of vitamin D was equivalent to daily dosing for optimizing infant vitamin D status. This research was funded by the National Center for Research Resources and Mayo Clinic.

Grant Funding Source: NCRR and Mayo Clinic
Visualizing homologous recombination and illustrating DNA repair pathway interaction in vivo via a bioengineered fluorescent reporter system
Authors: Sukup Jackson Michelle R. , Kiraly Orsolya , Rowaland Elizabeth , Schulte Kelly , Niedernhofer Laura J , Engelward Bevin P.
Published: June 2021

Homologous recombination (HR) is a DNA repair process critical for maintaining genomic integrity. HR is generally beneficial, but over‐ or under‐utilization of HR can lead to can lead to deleterious rearrangements and cancer. To study HR, our laboratory previously developed the Fluorescent Yellow Direct Repeat (FYDR) mouse in which HR yields a fluorescent signal.

We have utilized this mouse to study the interplay between HR and non‐homologous end‐joining (NHEJ) by knocking out the Ku86 protein. Both pathways are known to repair double strand breaks (DSB); preventing NHEJ in Ku86−/− mice resulted in an increase in HR. The newly proposed alternative end‐joining pathway was explored by knocking down the repair protein Ercc1. The Ercc1−/Δ mice showed an increase in HR in the pancreas, confirming in vivo an alternative DSB repair pathway and implicating ERCC1 in this pathway.

While an excellent tool, the FYDR mouse is limited to study of HR in the pancreas and skin. We created the RADR (Recombination at a Direct Repeat) mouse, an improved model that has enabled our study of HR in a wide range of tissues including gut and liver. We can now study DNA damage and HR in multiple tissues in response to a treatment or exposure.

Grant Funding Source : NIEHS Grant T32‐ES07020, NSF GRF, NIH Grant# P01‐CA026731‐31, R33‐CA112151‐01A2
Gene therapy as a cure for color blindness
Authors: Neitz Maureen , Neitz Jay , Mancuso Katheirne , Kuchenbecker James , Connor Thomas B
Published: June 2021

Red‐green color blindness most often results from the absence of the long‐(L) or middle‐(M) wavelength sensitive visual photopigments. It is the most common single locus genetic disorder. Recent advances in the development of gene therapy vectors have offered the promise of gene therapy cures for inherited vision disorders; however, classical visual deprivation experiments show that for at least some features of the visual system, there is a “critical period” in development in which visual input is required for proper circuitry to form. Here, we performed subretinal injections of a gene therapy vector designed to introduce a third cone type in the retina of dichromatic (color blind) squirrel monkeys. The vector was adeno‐associated virus carrying the gene for a human L cone opsin under control of an L/M cone specific enhancer and promoter. The effects of the gene therapy treatment were monitored using color vision behavioral testing and electroretinography. Results indicate that trichromacy can arise from a single addition of a third cone class and it does not require an early developmental process. This provides a positive outlook for the potential of gene therapy to cure adult vision disorders and as a tool for exploring neural plasticity of the visual system.
Novel regulation of PDGFRα activation in Glioblastoma
Authors: Phillips Joanna J. , Huillard Emmanuelle , Robinson Aaron , Rosen Steven D. , Rowitch David H. , Werb Zena
Published: June 2021

Glioblastoma (GBM) is characterized by abnormal activation of receptor tyrosine kinase (RTK) signaling pathways. We demonstrate that the extracellular sulfatase, SULF2, an enzyme that regulates multiple heparan sulfate proteoglycan‐dependent RTK signaling pathways, is a novel regulator of RTK‐signaling in GBM. In primary human GBM SULF2 protein is expressed in 29/57 (50%) tumors and using data from The Cancer Genome Atlas SULF2 transcript is increased in 197/424 (46%) tumors (http://cancergenome.nih.gov). To determine the functional role of SULF2 in GBM we knocked down SULF2 in GBM cell lines and generated murine malignant astrocytomas from Sulf2−/− tumorigenic neurospheres. These data demonstrated a striking SULF2‐dependence in activity of PDGFRα, a major signaling pathway in GBM. Ablation of SULF2 resulted in decreased PDGFRα phosphorylation and decreased downstream MAPK signaling activity. In addition, in vivo tumor growth was decreased. Interestingly, the proneural GBM subtype, characterized by aberrations in PDGFRα, demonstrated the strongest SULF2 expression. SULF2 may help identify a subset of GBMs that may be more dependent on exogenous growth factor‐mediated signaling.

This work was supported by funds from the National Institutes of Health (K08 NS063456 to J.J.P.) and the Preuss Foundation.
The angiogenesis suppressor gene AKAP12 is under the epigenetic control of HDAC7 in endothelial cells
Authors: CASTRONOVO Vincent , MATHEUS Nicolas , DUMONT Bruno , PEIXOTO Paul , HENNEQUIERE Vincent , DEROANNE Christophe , COLIGE Alain , DE PAUW Edwin , BELLAHCENE Akeila , MOTTET Denis , TURTOI Andrei
Published: June 2021

Previous studies have shown that inhibition of HDAC7 blocks angiogenesis both in vitro and in vivo, however the underlying molecular mechanisms are not fully understood. The goal of the present study was to further the understanding of HDAC7 epigenetic control of angiogenesis in human endothelial cells.

We performed siRNA‐mediated gene expression silencing of HDAC7 in human umbilical vein endothelial cells (HUVECs) followed by proteomic analysis. A‐kinase anchor protein 12 (AKAP12) was the most reproducibly up‐regulated protein and its overexpression was responsible for the inhibition of migration and tube formation in HDAC7‐depleted HUVEC. Mechanistically, H3 histones associated with AKAP12 promoter are acetylated following the removal of HDAC7, leading to an increase in its mRNA and protein levels. AKAP12 is responsible for protein kinase C‐β‐mediated phosphorylation of signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 increasingly binds to the chromatin and is necessary for maintaining the elevated levels of AKAP12 following HDAC7 knockdown.

We demonstrated that AKAP12 is an angiogenesis suppressor gene, an epigenetic target of HDAC7 and a negative regulator of HUVEC migration and tube‐like structures formation.

This work is supported by Walloon Region (NEO‐ANGIO), the University of Liège and the National Fund for Scientific Research, Belgium.
FRONTIERS IN BIOMEDICAL EDUCATION: INTEGRATIVE TEACHING/LEARNING OF PATHOPHYSIOLOGY AND CONCEPT OF ETIOPATHOGENETIC CLUSTERS
Authors: Kovac Zdenko
Published: June 2021

Pathophysiology education has been following many pathways, including concepts of general principles, nosology and organ‐related pathophysiology. During the studies adults construct the knowledge in self‐directed, autonomous and previous experience‐related manner. They try to apply new knowledge directly to a problem solving. Evaluation and active resynthesis of data seem to facilitate a gain and retain of knowledge. In postgenomic era, students of medicine are faced with various levels of cognitive certainty of knowledge (from a clear cause‐effect to a fuzzy‐logic relation), complexity, variability and endless quantities of data.

We developed educational method that contains four elements: 1. exposition of problem 2. repetition of relevant knowledge 3. algorhythmic resynthesis, and 4. feedback integration of the problem. Four reiterative steps are an efficient way to deal with complexities. The method is 2.3 times more efficient in comparison to classical teaching. It nurtures vertical, horizontal and longitudinal conceptualization of the problem. Etiopathogenetic pathways tend to form the clusters in which branching and integration take place. Therapeutical corrections of such cluster values lead to a clinical improvement. There are 91 principal clusters within the 30000 diseases. The concept of clusters helps to grasp a nonlinearity and complexity of pathobiology within the real study‐time framework.
Effects of heroin dependence on the expression of SP and NPY in the VTA and NAc regions of rat brain
Authors: Liang Wen-mei , Li Yi-xin
Published: June 2021

In previous studies from our laboratory, heroin use has been shown to cause abnormal expression of several neuroendo‐peptides in the nervous system and other organs. In the present study, the expression of substance P (SP) and neuropeptide Y (NPY) was examined in the ventral tegmental area (VTA) and nucleus accumbens (NAc) regions of rat brain after heroin use. Rats were chronically treated by subcutaneous injection of heroin for up to 38d. Immunohistochemical staining was performed to test the expression of SP and NPY in the VTA and NAc regions. The results revealed that the expression of SP and NPY was observed as brown yellow particle in the cytoplasm of neurons in the VTA and NAc regions. Furthermore, SP‐immunoreactive cells and NPY‐immunoreactive cells expressed less SP and NPY in the VTA and NAc regions from heroin dependence rats than that from saline control and normal control rats at different time points. Immunohistochemical analysis showed higher grey value was found in heroin dependence rats than that in controls (p<0.05). The findings highlight the secretion of SP and NPY is inhibited during heroin dependence and it may result in the inhibition of endogenous opioid peptides, suggesting that SP and NPY may be key molecules during drug addiction.
Comparative Neuronutritive Protection of Citrus sinensis & Vitamin C on Differential Gene Expression in Brain Tissues of Female Sprague Dawley Rats
Authors: Ogunleye Olorunwa Olufunmilayo , James Ayorinde Babatunde , Ebuehi Osaretin Albert , Iwalokun Bamidele , Saibu Gbemisola Morounke , Odesanmi Selina Omolola , Magbagbeola Olubunmi , Omilabu Sunday Aremu
Published: June 2021

Citrus sinensis (Orange), a popular fruit has been known to contain various antioxidant compounds in which vitamin C is one of them. These antioxidants scavenge free radicals that may cause neurodegenerative diseases. In this study, we investigated the comparative effects of orange juice and vitamin C on the differential gene expression of tumor necrosis factor receptor (Tnfr), p53, superoxide dismutase (Sod), and estrogen receptor genes in female rats.

Minerals and vitamin C composition was determined from extracted orange juice. 5ml/kg of the extracted orange juice and 20mg/kg Vit C were administered to the animals which were divided into control, Vit C and orange groups. On the 7th day, rat brain tissues were collected in liquid nitrogen and total RNA was extracted and cDNAs were made from 100ng normalized RNA samples. Gene expression was assayed using SyBR green relative quantification. Data was analyzed using Applied Biosystems SDS software and Graphpad Prism 5.

Our findings show that orange juice caused higher levels (46 Fold) of gene expression in antioxidant gene (Sod), anti‐tumour p53 gene (36 Fold) and estrogen receptor gene (3.5 Fold) which explains the promising neuro‐protective effect of orange juice.

Grant Funding Source: AAA
The Functional Role of the Rad50 Zinc‐Hook Motif
Authors: Fisher Tasida Caron , Herdendorf Tim , Albrecht Dustin , Nelson Scott
Published: June 2021

The Rad50‐Mre11 complex (MR complex) is implicated in the initial processing of DNA double stranded breaks (DSBs). Mre11 is a nuclease and Rad50 is an ATPase belonging to the ATP Binding Cassette (ABC) protein superfamily. Rad50 forms a dimer at two interfaces: the pair of ATP binding sites that are formed in trans between two Rad50 subunits, and the zinc‐hook motif (CXXC) at the apex of an intramolecular coiled‐coil. T4 phage contains functionally equivalent orthologs of Rad50 and Mre11, gp46 and gp47 respectively. These orthologs are well expressed and easily purified from a bacterial system. The function of the zinc‐hook motif was investigated in this model system by mutating the conserved cysteines to serine (Rad502CS). The mutant was evaluated in vitro for DNA and Mre11 binding, ATP hydrolysis activity, and the activation of Mre11 nuclease activity. The results demonstrate that although Rad502CS binds Mre11 and DNA normally, the inability of the zinc‐hook to bind Zn2+ broadly affects both the ATP hydrolysis and nuclease activity of the complex. These data indicate a high degree of allosteric communication between the zinc‐hook and the active sites of both Rad50 and Mre11.
Hyperglycemia‐induced O‐GlcNAcylation of 4E‐BP1 enhances its expression through a reduced rate of degradation
Authors: Dennis Michael Don , Kimball Scot R , Jefferson Leonard S
Published: June 2021

4E‐BP1 binds the mRNA cap‐binding protein eIF4E and thereby represses cap‐dependent mRNA translation. Phosphorylation of 4E‐BP1 releases it from eIF4E, allowing 4E‐BP1 to be poly‐ubiquitinated and subsequently degraded. Previous studies from our laboratory have established that 4E‐BP1 is also modified by addition of N‐acetylglucosamine to Ser and/or Thr residues (O‐GlcNAcylation) in the liver of diabetic mice concomitant with increased association with eIF4E. In the present study, we examined the hypothesis that increased O‐GlcNAcylation of 4E‐BP1 leads to an increase in its expression through a reduced rate of degradation. In support of this hypothesis, in both diabetic mice and in cells in culture exposed to 25 mM glucose, 4E‐BP1 protein expression was increased relative to controls. However, there was no significant difference in the rate of 4E‐BP1 synthesis between cells exposed to 25 mM compared to 5 mM glucose. In contrast, the rate of 4E‐BP1 degradation was significantly prolonged in cells maintained in 25 mM compared to 5 mM glucose. Together these findings suggest that the increased 4E‐BP1 expression observed in diabetic mice and in cells incubated in 25 mM glucose is the result of impaired degradation of the protein. These findings provide insight into the pathogenesis of metabolic abnormalities associated with diabetes. (Supported by NIH grant 13499 (LSJ) and an NIH Postdoctoral Fellowship (MDD))
Measurement of Acetyl‐CoA turnover ( ≈citric acid cycle flux) in perfused rat hearts by isotope dilution
Authors: Li Qingling , Ibarra Rafael A. , Brunengraber Henri , Zhang Guo-fang
Published: June 2021

We developed an isotope dilution technique to assess acetyl‐CoA turnover (≈ citric acid flux) in perfused rat hearts. In hearts, almost all acetate is used in citrate synthesis. Heart is perfused with buffer containing a low concentration of [2H3‐13C2] acetate (0.03 mM) which forms M5 and M4 acetyl‐CoA. The M4 acetyl‐CoA (greater than the small M4 component of M5 acetate) results probably from reversible enolization of acetyl‐CoA with partial 2H loss. The buffer may also contain one labeled substrate which generates M2 acetyl‐CoA ([13C6] glucose or [1,2‐13C2] palmitate). Total acetyl‐CoA turnover is calculated from (i) the M5 acetate uptake, and (ii) the M5 acetyl‐CoA enrichment + the increase in M4 enrichment of acetyl‐CoA compared with the small M4 enrichment of [2H3‐13C2] acetate. The contribution of glucose or palmitate to acetyl‐CoA is calculated from the M2 enrichment of acetyl‐CoA. Loss of label between M5 acetate and citrate synthesis (acetylcarnitine, ketone bodies) is negligible. Rates of acetyl‐CoA turnover calculated by our method are similar to those calculated from O2 uptake. The method was applied to measurements of acetyl‐CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). Supported by NIDDK RoadMap and the Case Mouse Metabolic Phenotyping Center.
Alcohol effect on recombinant adenylyl cyclase
Authors: Qualls-Creekmore Emily , Gupta Ratna , Yoshimura Masami
Published: June 2021

Research suggests that the cyclic AMP (cAMP) signaling pathway is implicated in the development of alcoholism. Previous work in our laboratory has demonstrated that alcohol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; human type 7 AC (AC7) is most enhanced by ethanol as measured by cAMP accumulation assay in whole cells. We hypothesize that alcohol enhances the activity of the AC protein by directly interacting with it at a specific site(s) and that alcohol effects on AC can be studied using recombinant AC expressed in bacteria. We have shown that a bacteria‐generated AC7 recombinant consisting of the C1a and C2 domains retains alcohol responsiveness. To examine whether bacteria‐generated recombinant AC proteins maintain isoform specificity of alcohol effects, we expressed in bacteria, and purified recombinant AC proteins carrying the C1a or C2 domains of AC2, AC7, and AC9. Recombinant AC7 C1a and AC7 C2 include the ethanol responsive domains identified in our previous chimeric studies. AC2 and AC9 recombinant proteins were designed to include the corresponding regions. Activity of combined AC7 C1a and AC7 C2 was enhanced by ethanol. Chimeric preparations of AC7 C1a and AC2 C2 or AC9 C1a and AC7 C2 were less responsive to ethanol exposure. This suggests that the isoform specificity of the alcohol effect is maintained in recombinant AC. Supported by NIAAA grant AA13148.