bluuebunny/crossref_metadata_2025 huggingface dataset:
Attitudes of nutrition education teachers and dietitians on implementation of school breakfast program in Korea
Authors: Choi Young-Sun , Oh Hyang
Published: June 2021

There has been increasing concern on skipping breakfast among children in Korea; one of strategies for this issue is school breakfast program. This study was aimed at investigating attitudes of nutrition education teachers and dietitians of schools in Gyeongbuk area on implementation of school breakfast program and action plans. The survey was carried out in April and May, 2010, and 194 questionnaires were used for statistical analysis. Seventy three percent objected to school breakfast program, and only 27% favored the program. The subjects who have more working experience and higher age objected more to the program. Reasons for favor were ‘good for children's growth’, ‘no more skipping breakfast’, ‘balanced diets’, ‘better concentration on learning’, and etc. Reasons for objection were ‘eating with family preferable’, ‘heavy burden of school lunch service’, ‘food service environment unsuitable for breakfast’, ‘recruiting difficulty of cooking staffs’, and etc. Direct operating system by the school, meal cost with 2,000~2,500 won a day, preparation time of 2~3 hours, serving meals 50 minutes before classes start, and convenient and simple style menu were favored for action plans. Therefore, school principal, teachers, nutrition education teachers, and parents should become consorted together to find out the most tailored program for each school situation.
Are the dietary reference intake (DRI) values for estimated energy requirements (EER) accurate for girls in life stage group 9–13 years?
Authors: Bandini Linda G , Lividini Keith , Must Aviva
Published: June 2021

We measured resting metabolic rate (RMR) by indirect calorimetry and daily total energy expenditure (TEE) by doubly labeled water (DLW) in 154 non‐obese pre‐menarcheal girls ages 9–12y. We compared EER based on the DRI equations to TEE measured by DLW. The level of physical activity (required to estimate EER) was based on the activity factor, TEE/RMR. 58% of subjects were pre‐pubertal (Tanner 1) and 71% were white. Mean (SD) age was 10.3(0.85) y, body fat was 23.1(5.2) %, TEE was 1962(277) kcal/d and EER was 1861(234) kcal/d. TEE and EER were highly correlated (Pearson r=0.79; p<0.0001). In unadjusted linear regression analysis, EER significantly predicted TEE (R2 =0.62; beta (SE) =0.93(0.06); p<0.0001). The association remained significant with adjustment for pubertal status and physical activity level (R2 = 0.67; beta (SE) = 1.08(0.09), p< 0.0001). To assess the accuracy of the DRI prediction equation we compared the individual‐level EER to the value of TEE from DLW. The mean (SD) difference between EER and TEE was −101(171) kcal/d and the average %difference was −4.6(8.5). 73% of participants had EER values within 10% of TEE. We conclude that for preadolescent girls the DRI equations predict well on a group basis. Use of these equations for individuals, however, may result in underestimation of energy requirements for a substantial proportion of girls. Supported by NIH grants MO1‐RR‐00088, DK‐HD50537 and 5P30 DK46200.
Differential expression of insulin receptor isoforms A and B in highly proliferative stem and tumor cells vs. differentiated intestinal epithelial cells
Authors: Bortvedt Sarah F , Mah Amanda T , Van Landeghem Laurianne , Santoro M. Agostina , Magness Scott , Lund P. Kay
Published: June 2021

Little is known about expression and roles of insulin receptor (IR) in the intestinal epithelium despite emerging links between obesity, hyperinsulinemia, and intestinal dysfunction or cancer risk. IR exists as an IR‐B isoform that mediates insulin's metabolic actions and an IR‐A isoform implicated in fetal growth and recently, cancer. This study used Sox9‐EGFP reporter mice, ApcMin/+ mice, and human colorectal cancer cell lines (CRC) to test the hypothesis that IR‐A and IR‐B have distinct expression profiles in highly proliferative vs differentiated normal intestinal epithelial cells (IEC) or CRC.

Results

IR‐A predominates in highly proliferative intestinal epithelial stem cells and progenitors, while IR‐B predominates in differentiated lineages. In ApcMin/+ mice, the IR‐B:IR‐A ratio is dramatically decreased in tumor vs non‐tumor tissue in small intestine (0.65 ± 0.02 vs 1.3 ± 0.03) and colon (0.63 ± 0.1 vs 1.27 ± 0.9). The IR‐B:IR‐A ratio is higher in CRC with the ability to differentiate vs undifferentiated, aggressively growing CRC. The ratio of IR‐B:IR‐A is significantly increased in differentiated vs undifferentiated Caco2 cells (2.2 ± 0.2 vs 1.5 ± 0.1). We conclude that relative expression levels of IR‐B:IR‐A may be critical determinants of normal IEC or CRC differentiation. Grants: F31AG040943 (SFB) and NIH DK040247‐19 (PKL).
Adult stem cells in therapy: a practical approach
Authors: Curcio Francesco , Fontanini Alessandra , Cattaruzzi Giacomo , Vitrani Francesco , Moretti Massimo , Campolo Marina , Soldati Alfredo , Ambesi-Impiombato Francesco Saverio
Published: June 2021

Organs and tissues for transplantation are dramatically too few to fulfill patients’ needs. Especially if not only life saving procedures are considered but also procedures that deal with quality of life. Tissue engineering represents a possible solution. To construct a viable graft substitute, cells, scaffolds, biomolecules, safe culturing devices and procedures are all needed. The scaffold 3D architecture as well as the medium fluid dynamic during the in vitro culture are also very relevant since it is crucial to exclude the presence of necrotic cells in the tissue substitute as much as possible to avoid scarring in the site of implantation. We designed a scaffold made of polycaprolactone and CaPO4 with a 3D structure optimized by computational fluid dynamic and built an automated bioreactor. Bone marrow stem cells have been cultured in the bioreactor on such scaffold, characterized and tested in vivo in different animal models for the ability to produce bone substitutes. Results show the absence of acute toxicity, the ability to form bone and receive optimal vascularization subcutaneously in SCID mice and the ability to repair critical lesions in rabbits and sheep. All animal experiments have been conducted according to Italian Laws, in conformance with the FASEB Statement of Principles for the use of animals in research and education and in ISO certified facilities.
Recruitment of the Vascular Endothelium into Neurovascular Coupling
Authors: Longden Thomas , Nelson Mark
Published: June 2021

In neurovascular coupling (NVC), neuronal activity in the brain leads to elevated local blood flow to satisfy the increased metabolic demands of the active tissue. The vascular endothelium has a major role in the modulation of blood flow, and here we explore the hypothesis that the endothelium has an important role in NVC.

Brain slices were prepared from male C57 Bl/6 mice. Intraparenchymal arterioles were pre‐constricted with U46619 (125 nM). Neurons were depolarized with electrical field stimulation (EFS) to induce NVC and arteriolar dilation. In some experiments, the endothelium was loaded with di‐8‐ANEPPS (20 μM).

EFS‐evoked arteriolar dilation was accompanied by endothelial hyperpolarization, as indicated by a 1.4% increase in the fluorescence ratio of di‐8‐ANEPPS (n=7). Blockade of I prostanoid receptors with 1 μM R01138452 had no effect on NVC (n=4). Blockade of neuronal nitric oxide synthase (NOS) with 7‐NI (100 μM) reduced the magnitude of vasodilation by 63% (n=7) with no effect on vessel tone (n=5). Blockade of both neuronal and endothelial NOS with L‐NNA (100 μM) caused a tonic constriction (17%, n=5) and reduced vasodilation by 89% (n=7). In the absence of U46619, L‐NNA constricted arterioles by 28% and reduced vasodilation by 90% (n=5).

These data are consistent with the concept that the vascular endothelium is actively engaged during neurovascular coupling. Supported by NIH award P01 HL095489.
Breast milk represses UDP‐glucuronosyltransferase (UGT) 1A1 expression in the gastrointestinal tract, increasing the risk for severe hyperbilirubinemia and brain damage
Authors: Fujiwara Ryoichi , Chen Shujuan , Tukey Robert H.
Published: June 2021

Infants develop physiological jaundice, which can be severe and cause irreversible brain damage, a physical property called kernicterus. Accumulating evidence indicates that breast‐fed infants have a higher risk for kernicterus than formula‐fed infants. However, the underlying mechanism linking severe hyperbilirubinemia to breast feeding has not yet been elucidated. Here we describe a new mechanism that places breast‐fed neonates at risk for developing kernicterus using recently developed humanized UDP‐glucuronosyltransferase 1 (hUGT1) mice. Breast‐fed hUGT1 mice develop hyperbilirubinemia. In contrast, formula‐fed mice have significantly lower levels of serum bilirubin. Compared to breast‐fed neonates, formula‐fed mice have dramatically higher levels of UGT1A1, the sole bilirubinconjugating enzyme, in the gastrointestinal tract. None of the formula fed mice develop severe hyperbilirubinemia or kernicterus. Furthermore, the oral treatment of neonatal hUGT1 mice with agents that activate NF‐kB results in induction of intestinal UGT1A1. We are proposing that breast milk suppresses UGT1A1 expression in the GI by stimulating inhibition of the NF‐kB pathway. These findings indicate that intestinal control of UGT1A1 in neonatal children is sufficient in preventing developmentally induced hyperbilirubinemia and brain toxicity. (Supported by grants GM086713 and ES010337)
Effect of an extract of Withania somnifera on Human Breast Cancer Cells
Authors: Khazal Kamel , Samuel Temesgen , Hill Donald L. , Ge Xin , Grubbs Clinton
Published: June 2021

The main goal of this study was to examine the effects of a root extract of Withania somnifera (WS), a plant in the nightshade family, on breast cancer cells in vitro and in vivo. For evaluation of WS in intact animals, 16 athymic nude mice were subcutaneously xenografted with MCF‐7 or MDA‐MB‐231 cells and, after development of tumors, divided into control and treated groups. The treated mice were dosed daily for 8 weeks by oral gavage with WS (300 mg/kg). WS reduced cells viability and induced cell cycle arrest and apoptosis in a concentration‐dependent manner. After treatment with WS (50 μg/ml or 100 μg/ml), there were 71% and 90% reductions in viability of MCF‐7 and 75% and 88% reductions in viability of MDA‐MB‐231 cells, respectively. Treatment of MCF‐7 cells with WS (25 μg/ml) for 24 hr increased the percentage of cells in the G1 phase of the cell cycle by 7.5% as compared to the vehicle‐treated cells, whereas treatment of the cells with the same dose for 48 hr increased the percentage of G1 cells by 17.4%. Treatment of both types of cells with a higher concentration (50 μg/ml) for 24 hr induced a predominantly G2/M phase arrest and increased apoptosis (sub‐G1). WS also caused concentration‐and time dependent stimulation of vimentin degradation. In nude mice, WS caused 64% and 60% reductions in size of the xenografted MCF‐7 and MDA‐MB‐231 cells, respectively.
Effects of protocatechuic acid on endothelium‐dependent vascular function and superoxide level in hypertensive rats
Authors: Lin Yu-Mei , Yang Ai-Lun , Su Chia-Ting
Published: June 2021

Oxidative stress has been recognized as an important factor for the pathogenesis of hypertension. Recently, protocatechuic acid (PCA; 3,4‐dihydroxybenzoic acid), a simple phenolic acid, has been found to be a strong antioxidant. In this study, we investigated the effects of PCA on endothelium‐dependent vascular function and superoxide level in spontaneously hypertensive rats (SHR). The SHR were randomly divided into SHR with PCA supplement (SHR+PCA) and without supplement (SHR) groups. Wistar‐Kyoto (WKY) rats were used as the normotensive control group. The SHR+PCA group was supplemented with PCA adjusted from their daily water consumption for 8 weeks. Rat thoracic aortas were isolated for the measurement of endothelium‐dependent and endothelium‐independent vasorelaxant responses in the organ baths. The serum levels of superoxide dismutase (SOD) and thiobarbituric acid reactive substance (TBARS) were also evaluated. We found that the endothelium‐dependent vasorelaxant responses to acetylcholine were significantly (p<0.05) decreased in the SHR compared with the WKY. The PCA supplement significantly (p<0.05) ameliorated the endothelium‐dependent vasorelaxation. However, there was no significant difference in the endothelium‐independent vasorelaxation among three groups. Moreover, the PCA supplement tended to increase the SOD levels, but not TBARS level. We suggested that the PCA supplement could improve the endothelium‐dependent vascular dysfunction induced by hypertension.
Influence of obesity on insulin‐mediated dilation in the human microcirculation
Authors: Borbouse Lena , Gutterman David D.
Published: June 2021

Obesity is known to reduce both insulin sensitivity to glucose and endothelial‐derived NO‐dependent vasorelaxation to insulin, but little is known about the effect of obesity on insulin‐induced dilation mediated by endothelial‐derived hyperpolarizing factors (EDHFs). We examined the influence of obesity on insulin‐mediated dilation in human adipose arterioles. Vasodilation in microvessels from human adipose tissue were examined in response to physiological concentrations of insulin (0.3 – 100 ng/ml) using videomicroscopy. Endothelial denudation or treatment with high K+ (45 mM) abolished dilation to insulin in obese subjects, indicating an EDHF mechanism. Treatment with PEG‐catalase reduced insulin dilation in arterioles from lean (BMI 22.5 ± 1.1; m ± SE) and obese (BMI 31.0 ± 1.0) subjects by 47% and 45% respectively. In the presence of L‐NAME and indomethacin (INDO), PEG‐catalase abolished the remaining dilation to insulin in arterioles of lean subjects; but had no further effect in obese subjects. The mono‐oxygenase inhibitor MS‐PPOH had no effect on insulin dilation in obese subjects. We conclude that H2O2 contributes significantly to EDHF dilation to insulin in lean subjects, but obesity induces a mechanistic switch to a non‐H2O2, non‐epoxyeicosatrienoic acid EDHF. (Supported by NHLBI grants HL‐094971 and HL‐080704 (DDG) and AHA Midwest Postdoctoral Fellowship Award).
Angiotensin II decreases the levels of PKA‐mediated NHE3 phosphorylation in renal proximal tubule
Authors: Rossetto Silmara Larissa , Queiroz-Leite Gabriella Duarte , Crajoinas Renato Oliveira , Omae Samantha V , Malnic Gerhard , Girardi Adriana C
Published: June 2021

NHE3 transport function is inhibited by PKA. Binding of angiotensin II (ANG II) to AT1 receptor has been reported to activate Gi‐protein signaling pathway, resulting in the inhibition of adenylyl cyclase, and consequently, in decreased PKA activation. We therefore tested the hypothesis that decrement of PKA‐mediated NHE3 phosphorylation is one of the mechanisms by which ANG II acutely stimulates NHE3 activity in renal proximal tubule. To this end, OKP cells were treated with 100 μM Dopamine (DOP) for 30 min in the presence or absence of 10−10 M ANG II for 5, 15, and 30 min; or only with ANG II for 30 min. Extrusion of H+ from OKP cells was measured. DOP significantly inhibited NHE3 activity (0.2013 ± 0.009 vs. 0.3036 ± 0.0019 pH units/min in control). Simultaneous incubation of DOP with ANG II for 15 (0.2347 ± 0.044 pH units/min) and 30 min (0.3295 ± 0.032 pH units/min) completely blocked this inhibitory effect. As expected, ANG II stimulated NHE3 activity. DOP increased the levels of NHE3 phosphorylated at the PKA consensus phosphorylation sites [serines 552 and 605] by 64 ± 8% and 288 ± 37% respectively vs. control. ANG II treatment for 15 and 30 min prevented this increase. PKA activity was also measured in these groups of cells. We found that PKA activity was significantly increased in cells treated with DOP for 30 min and in cells treated with DOP plus ANG II for 5 min. Longer incubation times with ANG II (15 and 30 min) in the presence of DOP restored PKA activity near to control levels. Losartan blocked all the effects of ANG II here reported. Collectively, these data suggest that inhibition of PKA activity leading to lower levels of endogenous phosphorylation of NHE3 is one of the mechanisms by which ANG II stimulates NHE3 activity in the proximal tubule.

Supported by FAPESP
The use of human embryonic kidney (HEK 293) cells to enhance characterization of the LMX1B pathway
Authors: Stump Robert Patrick , Feenstra Jennifer M , Castillo Michael A , Soriano Salvador , Oberg Kerby C
Published: June 2021

LMX1B is a homeodomain transcription factor critical to kidney development and glomerular filtration. In humans, LMX1B haploinsufficiency causes a condition known as Nail Patella syndrome (NPS). Individuals with NPS typically have under developed nails, absent patellae, and impaired kidney function. In Lmx1b knockout mice, the absence of Lmx1b function disrupts glomerular filtration and abates urine production. The molecules targeted by LMX1B that regulate glomerular development and function, however, are poorly characterized. Immortalized human embryonic kidney (HEK 293) cells provide a potential tool for high‐throughput identification of LMX1B targets and an in vitro system for dissecting the functional role of targets. Thus, we characterized the known LMX1B pathway in HEK 293 cells and evaluated sites of LMX1B binding. We demonstrate the expression of LMX1B and its renal targets, COLLAGEN 4 ALPHA 4 and PODICIN in HEK 293 cells by RT‐PCR. Protein expression of LMX1B was verified by western blot analysis. In addition, using Chromatin immunoprecipitation we determined that LMX1B binds to regulatory sequences associated with both target genes. Further characterization of the LMX1B pathway in HEK 293 cells is underway and will likely enhance the identification of relevant downstream targets and the molecular cascade that regulates glomerular development and function.

Grant Funding Source: (NICHD) HD 39421
Changes in the activin A‐myostatin‐follistatin system within bone and muscle of aging mice
Authors: Hamrick Mark , Bowser Matthew , Fulzele Sadanand , Ahsan Sahir , Arounleut Phonepasong , Isales Carlos M
Published: June 2021

The activin A‐myostatin‐follistatin system is thought to play an important role in the regulation of muscle and bone mass, but changes in the relative levels of these factors in muscle and bone with age are not well understood. We investigated age‐related changes in the activin A‐myostatin‐follistatin system using femoral bone marrow aspirates from young (12 months) and aged (24 months) mice, and soleus and extensor digitorum longus (EDL) muscles from these same animals. Bone marrow supernatant samples were analyzed using ELISA, and muscle samples were analyzed using real‐time PCR and ELISA. Results indicate that myostatin levels were significantly (+75%) increased in mouse bone marrow with age, whereas levels of activin A and follistatin did not differ significantly between young and aged mice. Myostatin and activin A levels were not significantly altered with age in either soleus or EDL, however follistatin levels decreased by ~30% in mouse EDL with age. These findings suggest that age‐associated loss of muscle mass in the predominantly fast‐twitch EDL muscle may be due in part to declining levels of follistatin. Funding for this research was provided by the Congressionally Directed Medical Research Programs (USAMRAA).
Proliferation, osteogenic and chrondrogenic differentiation of equine‐derived bone marrow mesenchymal stem cells on bacterial cellulose scaffolds
Authors: Favi Pelagie M. , Stephens Christopher , Neilsen Nancy , Hammonds Ryan , Benson Roberto , Dhar Madhu
Published: June 2021

The purpose of this study was to evaluate the cell proliferation and viability, osteocyte and chondrocyte differentiation of equine‐derived bone marrow mesenchymal stem cells (Eq‐MSCs) when seeded onto biocompatible bacterial cellulose (BC) scaffolds. BC was synthesized using the bacterium Gluconacetobacter sucrofermentans. Previously isolated, expanded and characterized Eq‐MSCs were seeded on the BC scaffolds and fluorescence microscopy confirmed adherence and expansion. Confocal imaging confirmed the cells positively stained for CD90 antibody, a common surface marker for mesenchymal stem cells. MTS assay demonstrated increasing proliferation and viability with time (days 2, 7 and14). Cell‐scaffold constructs were cultured for 14 days under osteogenic and chondrogenic conditions. Osteocytes were positive for alizarin red and chondrogenesis was confirmed by alcian blue staining of the chondrocytes. In summary, biocompatible BC scaffolds support the proliferation and viability, osteogenic and chondrogenic differentiation of Eq‐MSCs cultured onto its surface in‐vitro, allowing for future potential use for tissue engineering therapies. This research is sponsored by the Physicians Medical and Education Research Foundation Grant, the Cardiothoracic Surgery Gift Fund, and the National Institute of Health Training Grant.

Grant Funding Source: Physicians Medical and Education Research Foundation Grant, the Cardiothoracic Surgery Gift Fund, and the National Institute of Health Training Grant
Biological Timekeeping in Obesity and Diabetes
Authors: Bass Joseph T.
Published: June 2021

Circadian systems are programmed by a transcription‐translation feedback loop that generates daily cycles of energy storage and utilization in synchrony with the 24 hr rotation of the Earth. In mammals, master pacemaker neurons located within the suprachiasmatic nucleus are entrained to light, and generate 24 hr oscillations in extra‐SCN neurons and most peripheral tissues, including those involved in glucose and lipid metabolism. During wakefulness and feeding, the molecular clock promotes glucose‐stimulated insulin secretion within b‐cells of the Islets of Langerhans. Clock ablation limited to pancreas results in hypoinsulinemic diabetes, indicating a primary function of peripheral clocks in glucose homeostasis. In contrast, ablation of the liver clock impairs oxidative metabolism, indicating that peripheral tissue clocks exert opposing effects in response to feeding and fasting. At the molecular level, clock disruption causes reduced NAD+ biosynthesis and leads to alterations in function of NAD+‐dependent ribosylases and de‐acetylases involved in oxidative metabolism. Circadian systems thus contribute to long‐term energy and glucose homeostasis across alternating behavioral and nutrient states.
Tumor‐Derived IDO Affects the Lifespan in a Mouse Model of Glioblastoma
Authors: Wainwright Derek Alan , Lesniak Maciej S.
Published: June 2021

Glioblastoma multiforme (GBM) is a malignant brain tumor with an average survival time of one year. Our laboratory previously demonstrated that a subset of T cells, known as Tregs, infiltrate both human‐ and mouse models of‐GBM. Importantly, depleting Tregs leads to increased survival in a mouse model of GBM. Indoleamine 2,3 dioxygenase (IDO) has emerged as a molecule that controls Treg recruitment and/or activation and has recently been shown to be expressed in human GBM. Based on this association, we hypothesized that IDO is involved in GBM progression. Thus, this work set out to establish: 1) if IDO expression affected survival and, 2) where IDO is required to affect survival, in a mouse model of GBM. To determine if and where IDO is required to affect lifespan, we used wild‐type (WT) and IDO‐deficient (IDO−/−) mice, intracranial injections of the GBM‐like cell line, GL261, histological quantification of tumor size, immunofluorescence for IDO expression, small hairpin RNA knockdown for IDO, as well as mouse survival analysis. Here, we demonstrate that both WT and IDO−/− mice intracranially‐injected with GL261 cells and knocked‐down for IDO (GL261Kd) live significantly longer than WT and IDO−/− mice intracranially‐injected GL261 cells transduced with a scrambled control shRNA (GL261Vc) (p < 0.001). Histology confirms that the GL261Kd GBM grows significantly slower, when compared to the GL261Vc GBM (p < 0.001). This is co‐incident with a significant decrease in immunofluorescence for IDO expression in the GL261Kd‐, when compared to the GL261Vc‐GBM (p < 0.001). By using a pre‐clinical mouse model of GBM whereby IDO is deficient or knocked‐down in different anatomical compartments, this work demonstrates that IDO is an important molecule, required and sufficient for promoting brain tumor growth, supporting the future investigation of pharmacological inhibitors of IDO in patients with GBM.

Grant Funding Source: 5R01CA138587‐03; 1F32NS073366‐ 01A1
Switch in amyloid clearance mechanisms leads to vascular fragility in cerebral amyloid angiopathy
Authors: Zabel Matthew K , Schrag Matthew , Crofton Andrew , Tung Spencer , Vinters Harry V , Kirsch Wolff M
Published: June 2021

Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) are two very common pathologies associated with amyloid accumulation and inflammatory processes, which are not well understood. The objective of this study was to evaluate human post mortem brains of cases with pure AD, those with AD and either concomitant mild, moderate or severe CAA and age matched controls (all n=4) for differing expression of microglia‐associated Aβ clearance receptors and complement activation. We used brain parenchymal homogenate and an enriched microvessel fraction from the occipital lobe for western blot analysis to measure the levels of C3b and C6. Immunoprecipitation was used to pull down microglial CD11b to look for binding complex levels with C3b and Aβ. Confocal microscopy was used to visualize these interactions and light microscopy to visualize late complement deposition. C3b was significantly increased in all CAA severities compared to AD and controls. Co‐IP showed significantly increased CD11b/C3b complexes with Aβ. Additionally, C6 was significantly increased on isolated CAA blood vessels compared to AD and control vessels. These findings suggest an Aβ clearance mechanism via microglial CD11b that delivers Aβ and complement factors to the blood vessels in CAA, which leads to Aβ deposition and vascular fragility due to the lysing effects of late complement.

Grant Funding Source : NIH grant AG20948, NSF‐MRI 0619774 and by Loma Linda University School of Medicine.
Functional changes in the brain caused by neonatal influenza infection
Authors: Kim Minsoo , Nahm Sang-Soep
Published: June 2021

Influenza infection in the brain is not a common manifestation but various clinical cases with influenza associated neurological disorders have been reported. This study was undertaken to assess long‐term functional changes in the brain caused by neonatal influenza infection. Balb/c mice at postnatal day 5 were infected intraperitoneally with influenza virus (A/NWS/33) at a dose of LD50 (105 TCID50/ml). The brain metabolism of the survived mice was monitored with positron emission tomography at 10 weeks post infection (PI). Changes of fluorodeoxyglucose (FDG) uptake in the brain were further analyzed by statistical parametric mapping (SPM). Consecutive behavioral tests including open field test, water maze test and rotarod test were also performed from 10 weeks PI. The SPM analysis revealed regional differences in FDG uptake in the infected mice. The affected brain regions were located around hippocampus, amygdala and thalamus. Influenza infected mice showed significant increase in the latency and distance during swimming in water maze test. Moreover, influenza infected mice showed decreased running time in accelerating rotarod test. However, no difference was observed in open field test. Our results suggest neonatal influenza infection could elicit learning impairment in the brain. Supported by Basic Science Research Program through the National Research Foundation of Korea (2011‐0005442)
Valproic acid attenuates neuronal apoptosis in APP/PS1 transgenic Alzheimer′s disease mice model
Authors: He Guiqiong , Zhao Lei
Published: June 2021

One of the hallmarks of Alzheimer's disease (AD) is the accumulation of senile plaques(SP) composed of extracellular aggregates of amyloid ¦Â peptides (A¦Â). It is proved that A¦Â triggers apoptotic cell death via the activation of caspase‐dependent and ‐independent cell death effectors, respectively. Valproic acid (VPA) is a widely used mood stabilizer and antiepileptic drug. Our previous study showed that VPA treatment significantly reduced SP formation and improved memory deficits in AD model mice. The present study intended to explore the protective effect of VPA on neuronal apoptosis in transgenic AD model mice and the possible mechanisms involved. Immunohistochemical staining showed that VPA reduced the number of swollen neurons and promoted neurite outgrowth in AD mice. MTT assay and Tunel staining showed that VPA increased cell viability and reduced the number of apoptotic cells. Western blot revealed that VPA downregulated the expression of Caspase‐ 3, Caspase‐9 and Caspase‐12, reduced cytochrome C level. The expression of Bcl‐2 was increased by VPA treatment. Flow cytometry revealed that VPA decreased intracellular level of Ca2+ and elevated mitochondrial membrane potential. Our results indicate that VPA protected AD mice model via inhibition of both mitochondrial and endoplasmic reticulum pathway of apoptosis.

This work was supported by National Natural Science Foundation of China (30970986).
Mobilization of endothelial progeitor cells in patents with acute ischemic stroke
Authors: Zheng Min , Wang Jian
Published: June 2021

Objectives

To observe the mobilization of Endothelial Progenitor cell (EPCs) following acute ischemic stroke (AIS) and the possible mechanism involved.

Methods

Level of circulating EPCs(CD34+/KDR+) of patients with AIS were examined by flow cytometry at day1, 7, 14 and 21 after acute stroke. Infarct volume was checked by MRI. Stromal derived factor‐1(SDF‐1) content was determined by ELISA. The change rate of EPCs was defined as (EPC14d‐EPC1d)/EPC1d.

Results

1. The number of circulating EPCs began to increase at day 7 after AIS, reached peak level at day 14 and decreased at day 21. EPCs change rate was significantly correlated with infarct volume (r = 0.692; P<0.01). 2. The content of SDF‐1 significantly increased at day 7 after AIS, reached peak level at day 14 and decreased at day 21. SDF‐1 content at day 14 was significantly correlated with infarct volume (r = 0.774; P<0.01). 3. EPCs change rate in patients with AIS was significantly correlated with SDF‐1 content at day 14(r = 0.682; P<0.01).

Conclusion

The increased circulating EPCs may result from the mobilization of EPCs in response to stroke stress. The mechanism of EPCs mobilization may be associated with an increased expression of SDF‐1.

This work was supported by National Scientific Foundation Committee of China ( 31100985)
MRI and DTI characterization of spinal cord severe contusion injury in the rat
Authors: Lim Wootaek , Baligand Celine , Keener Jonathon , Ye Fan , Vohra Ravneet S , Ruhella Arjun , Bose Prodip K , Walter Glenn A , Thompson Floyd J , Vandenborne Krista H
Published: June 2021

Spinal cord injury (SCI) induces loss of structural integrity of spinal cord tissue, resulting in locomotor deficit and muscle wasting. Thus, it is important to accurately assess the severity of injury. In this study, we characterized the spinal cord changes using diffusion tensor imaging (DTI) and 3D magnetic resonance imaging (MRI) following severe contusion injury. Sixteen‐week old female Sprague‐Dawley rats were given severe contusion injury at the T8 level using a NYU impactor (10g, 50 mm weight drop) (SCI, n=5). 3 animals were used for control (CTR). Ex vivo 3D MRI and 2D DTI were performed 3 weeks post‐injury in a Bruker 750MHz magnet. Imaging parameters were: field of view of 4.5 × 4.5 mm2; matrix size of 128 × 128; in‐plane resolution of 35.16 × 35.16 μm2. Images were acquired over 1 cm of the length of the cord, including the epicenter. All procedures were approved by the University of Florida institutional animal care and use committee. In CTR, spinal cord cross‐sectional was 4.38 ± 0.21 mm2 on average. This value was decreased to 1.58 ± 0.09 mm2 at the epicenter in SCI 3 weeks post‐injury. Fractional anisotropy (FA) for CTR was 0.76 ± 0.01 in white matter and 0.51 ± 0.05 in gray matter with a mean FA of 0.72 ± 0.02. In SCI, mean FA was decreased to 0.40 ± 0.09 at the epicenter. These findings demonstrate that the injured cords show lower FA value with significant atrophy following severe contusion injury.

Grant Funding Source : NIH Grant P01 HD059751
Z‐Bisdehydrodoisynolic Acid (Z‐BDDA), a putative estrogenic seco‐steroid, induces profound lordosis behavior in ovariectomized female rats
Authors: Viscomi Natalie S , Clough Rich W , Arbogast Lydia , Banz William J , Hou Yuqing , Meyers Cal
Published: June 2021

Z‐Bisdehydrodoisynolic Acid (Z‐BDDA) has been described as highly estrogenic, and a selective estrogen receptor modulator, based exclusively on uterotrophic activity in female rats. Exploring possible neuroprotective effects of Z‐BDDA, we thought it important to determine whether Z‐BDDA has estrogenic effects in the brain. Accordingly, female Sprague‐Dawley rats were ovariectomized (ovx) under isofluorane anesthesia and assigned to various steroid treatment protocols intended to induce sexual receptivity. Z‐BDDA (300ug/rat or 30ug/rat) or Estradiol benzoate (10 ug/rat) was administered (sc) two weeks following ovx and some rats were also given Progesterone (1mg/rat) 48 hours later. Lordosis behavior was assessed by placement of female rats in an arena containing a sexually mature male rat. Mount attempts by the male and lordotic responses in the females were recorded. Results showed that female rats treated with either Z‐BDDA or Estrogen (followed or not by Progesterone) exhibited profound receptivity assessed by display of lordosis in response to male mount attempts. Thus, this study further establishes the estrogenicity of Z‐BDDA and demonstrates a positive feedback effect on the central nervous system that appears to parallel the peripheral (uterotrophic) effects of the compound. Supported by the SIU School of Medicine Center for Integrated Research in Cognitive and Neural Sciences.

Grant Funding Source : None
Extracellular phosphorylation is a novel target for regenerative medicine against spinal cord injury
Authors: Takei Yoshinori , Suehiro Kenji , Shimizu Kazuharu , Tsujimoto Gozoh
Published: June 2021

The mammalian adult central nervous system (CNS) can be hardly regenerated after traumatic damage, and therapeutic methods for promoting CNS regeneration are strongly desired. Nogo‐A is a membrane protein present in CNS myelin. Models of spinal cord injury (SCI) in rats and macaque monkeys demonstrate that treatment with neutralising antibodies against Nogo‐A results in the formation of new functional connections in the spinal cord. A Phase I clinical trial applying anti‐Nogo‐A antibody to subjects with acute SCI has been successfully conducted. We reported that phosphorylation of extracellular domains of Nogo‐A receptors inhibits ligand binding, enabling neurite outgrowth even in the presence of Nogo‐A, in vitro (Takei, Science signaling, 2009). Here, we examined the effects of the extracellular kinase treatment on the recovery from SCI with rat model. Marked behavioural improvement was observed in the lesioned animals after the extracellular kinase treatment. These results provide a launching point for developing methods to stimulate neuronal regeneration in the adult mammalian CNS. Thus, our results provide evidence that artificial modification of extracellular domains of membrane receptors can be novel targets for drug discovery.
Regulation of Alpha‐Myosin Heavy Chain in cardiac remodeling associated with pregnancy
Authors: Sizemore Johnna McKelvey , Dixon Emily Nicole , Baute Audrey Joy , Waikel Rebekah L
Published: June 2021

During pregnancy, an increase in blood volume occurs to compensate for fetal development, resulting in adaptive cardiac hypertrophy, which typically remodels back to pre‐pregnancy size shortly after the mother gives birth. In contrast, pathologic cardiac hypertrophy may begin as a compensatory process, but transitions to a maladaptive state and can lead to heart failure. During pathologic cardiac hypertrophy, levels of MHC are altered: beta‐myosin heavy chain (β‐MHC) is increased and alpha‐myosin heavy chain (α‐MHC) is decreased. Activation of α‐MHC in animal models of heart failure has been shown to preserve cardiac function and prevent of heart failure. Interestingly, we have shown that α‐MHC is upregulated in the heart during pregnancy in a rat model, potentially contributing to preserved heart function and adaptive remodeling typically associated with pregnancy. We propose that by studying the mechanisms of α‐MHC regulation that aid in adaptive cardiac remodeling associated with pregnancy, we will gain a better understanding of mechanisms to promote adaptive remodeling in pathologic situations. To characterize the regulation of MHC and MHC regulatory factors that occur during pregnancy induced cardiac remodeling, we subjected rats to timed matings and collected morphological and biochemical data from not pregnant, 19 days pregnant, and 24 hours postpartum. Our rat model for pregnancy induced hypertrophy demonstrated that heart size and weight is increased at 19 days pregnant, and begins to return to normal 24 hours postpartum. Real‐time PCR analysis revealed unique expression patterns of MHC genes and regulatory factors in the hearts of non‐pregnant, late pregnant, and postpartum rats.
Role of the Ccr4‐Not Complex in Histone Acetylation
Authors: Mehrotra Swati , Vancura Ales
Published: June 2021

The Ccr4‐Not complex is a global regulator of gene expression that affects expression both positively and negatively. The complex, conserved from yeast to human, is composed of at least nine subunits. The Ccr4 subunit functions in the control of mRNA deadenylation and transcriptional initiation/elongation. The NOT proteins are involved in transcriptional repression by restricting access of TFIID to promoters. The Ccr4‐Not complex also plays a key role in sensing nutrients and stress. Our earlier results implicated Ccr4‐Not complex in regulation of global untargeted histone acetylation. This notion is also supported by the synthetic negative genetic interactions between the ccr4Δ and not4Δ mutations and mutations in SAGA and NuA4, the main histone acetyltransferases in yeast. The histone hypoacetylation phenotype of ccr4Δ and not4Δ mutants can be partially suppressed by attenuated expression of acetyl‐CoA carboxylase ACC1. This result suggests that ccr4Δ and not4Δ mutants have altered regulation of intermediary metabolism, reduced level of nucleocytosolic acetyl‐CoA, and consequently display histone hypoacetylation. Together, our data highlight the connection between intermediary metabolism and histone acetylation and indicate that the nucleocytosolic level of acetyl‐CoA is an important determinant of histone acetylation.

This work was funded by NIH.
Changes in genes methylation of the TLR pathway in livers of mice fed DDC
Authors: Oliva Joan , French Samuel W.
Published: June 2021

Background

Mallory‐Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. Proteins of the TLR pathway were shown to be involved in the formation of MDBs, in mice fed DDC. TLR genes are upregulated and SAMe supplementation prevents this up regulation and prevented the formation of MDBs.

Method

DNA of livers from control mice, from mice fed DDC 10 weeks, refed 1 week with DDC and with DDC+SAMe were extracted and used to study the methylation pattern of genes involves in the TLR pathway. A PCR array was used to analyze it.

Results

Using PCR array for the mouse TLR pathway (24 genes) were found whose the expression of IL12A was regulated by the methylation of its gene. DDC treatment reduced the methylation of the IL12A gene expression, with a similar level when DDC was refed. However, in the presence of SAMe, the intermediate level of methylation of IL12A was up regulated and the methylation of the promoter decreased compared to DDC refeeding or DDC 10 weeks.

Conclusion

IL12a is known to induce the production of IFNg by NK and LT. We showed in previous publication that IFNg is one of the major cytokines involved in the induction of MDB formation. The low expression of IL12A associated with the intermediate methylation of its promoter could explain one step in the mechanism which leads to the formation of MDBs. Supported by NIAAA PA50‐011999.
CTGF/CCN2 induction by TGF‐1 β requires Ets‐1 in osteoblasts
Authors: Arnott John , Geisinger Max , Astaiza Randy , Butler Tiffany , Popoff Steven , Planey Sonia
Published: June 2021

Erythroblastosis virus E26 oncogene homologue 1 (Ets‐1) is the founding member of the Ets family of transcription factors that control cell proliferation, differentiation and ECM regulation. Ets‐1 participates in osteoblast differentiation; however, its mechanism of action remains largely undetermined. CTGF acts as an anabolic growth factor to regulate osteoblast differentiation and function. CTGF is induced by TGF‐β1 and mediates TGF‐β1 induced matrix production in osteoblasts, but the mechanisms that control CTGF induction by TGF‐β1 are not understood. This study investigates the role of Ets‐1 in CTGF induction by TGF‐β1 in osteoblasts. We demonstrate that: Ets‐1 expression is induced by TGF‐β1 treatment, that overexpression of Ets‐1 induces CTGF protein, and promoter activity similar to TGF‐β1 treatment alone and that Ets‐1 expression synergizes with TGF‐β1 to induce CTGF promoter activation and that Ets‐1 siRNA impaires CTGF expression. Bioinformatic analysis identified eight putative Ets‐1 binding motifs in the CTGF promoter. Using site‐directed mutagenesis, we found that mutation of EBE sites, especially in close proximity to the Smad binding element (SBE), had a more severe impact on CTGF expression suggesting that the Ets‐1 sites may cooperate with Smads to achieve CTGF expression following TGF‐β1 treatment in osteoblasts. This study was funded by NIAMS.
Towards predicting the behavior of E. coli LacI and FadR transcription factors to variations in their operator sequences
Authors: Maity Tuhin Subhra , Jha Ramesh , Strauss Charlie , Dunbar John , Kuske Cheryl
Published: June 2021

Transcription factors (TFs) typically activate or repress gene expression by binding to specific DNA sequences in the genome called operators. To deeply understand this process, it is necessary to catalog the behavior of transcription factors to variations of their operator sites. This offers the potential for recognition of alternative binding sites, and, synthetically, offers a prescription for tuning binding affinity. Ultimately, one hopes to acquire a predictive understanding of the sequence‐behavior relationship and use that knowledge for metabolic engineering.

In this study, we evaluated the effect of operator DNA sequence changes to the transcriptional repression properties of two E. coli TFs, LacI and FadR. We started with a known DNA operator sequence of the E. coli LacI TF and discovered the preferred FadR binding DNA sequence using a protein‐binding microarray experiment, and then randomly mutated a small subset of the nucleotides in their operator sites. We cataloged the effect of sequence change to the transcriptional repression/induction properties by the two TFs using an in vivo GFP reporter assay. For both the TFs, we observed a wide range of interesting and characteristically distinct repression and induction behaviors, and some of them exhibited a strong correlation with a specific nucleotide at a specific position of the operator DNAs. We also performed Rosetta modeling calculations to estimate relative TF‐binding energies of our operator variants from the existing 3D structures of the TF‐DNA complexes. Our results suggest that 3D structure calculations can infer the behavior of a useful subset of the sequence variations, but is not as yet sufficient to completely explain the behavior of all sequence variations.
Human Cosmc and T‐synthase Genes Are Transcriptionally Regulated by SP1/SP3 Transcription Factors
Authors: Ju Tongzhong , Mi Rongjuan , Wang Yingchun , Ding Xiaokun , Song Lina , van Die Irma , Chapman Arlene B. , Cummings Richard D.
Published: June 2021

Background

Cosmc is the specific molecular chaperone for core 1 β3‐galatosyltransferase (T‐synthase), which is the key enzyme for synthesizing the core 1 structure in mucin type O‐glycans. Interestingly, human Cosmc and T‐synthase are ubiquitously and coordinately expressed but vary by cell and tissue type. Genetic mutations in Cosmc have been identified in several human diseases. Recent studies have also shown that both Cosmc and T‐synthase are transcriptionally suppressed in the B cells from some patients with IgA nephropathy. Yet, how these two genes are regulated is unknown.

Study Objective

Identify and characterize the promoters for human Cosmc and T‐synthase.

Methods

Luciferase reporter assay and chromatin immunoprecipitation (ChIP) analysis.

Results

the proximal GC‐rich region (CpG island) at the 5′‐flank of human Cosmc (cCpG‐II) which contains two Specificity Protein 1 and 3 (SP1/3) binding sequences has the promoter activity. Similarly, the tCpG in human T‐synase which also contains two SP1/3 sites is the promoter for T‐synthase. Site direct mutagenesis demonstrated that both SP1/3 sequences for Cosmc and T‐synthase are essential to their promoter activity. Furthermore, ChIP analyses confirm that the putative SP1/3 sequence in the promoter associates with the SP1/3 for both Cosmc and T‐synthase.

Conclusion

Human Cosmc and T‐synthase are transcriptionally active at a basal level by SP1/SP3.
VMP1 is a novel inducible gene which activation is a switch for autophagy
Authors: Re Andrea Lo , Molejón Maria Ines , Ropolo Alejandro , Boggio Veronica , Vaccaro Maria Ines
Published: June 2021

Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents, which participates in cell homeostasis as well as in response to disease. Autophagy acts in a myriad of cellular events leading to survival or cell death. The aim of this work was to study the effector molecules that regulate the transcription of the inducible gene vmp1, which expression triggers autophagy. Using a combination of luciferase assays, expression analysis and shRNA strategies, we report the identification of the promoter region of the human vmp1 gene and the molecular pathways that lead to its activation. We show that vmp1 promoter responds to autophagy stimuli through mTOR‐dependent and mTOR‐independent pathways. Besides we present the essential sequence required for this activation containing the TATA Box and several consensus binding sites for transcription factors related to the cellular stress such as CREB, E2F1, c‐Jun and c‐Fos. We demonstrate that overexpression of any of this factors induces VMP1 expression. Moreover using shRNA technology, p62 degradation and LC3 cleavage or recruitment we show that these factors induce VMP1‐mediated autophagy. Finally we found that p300 acts and is required as a coactivator for VMP1 activation and autophagy. Our results define VMP1 as a target for cellular stress mediators, which activation is a switch for autophagy. AL:CONICET;MIV:UBA‐ CONICET‐ ANPCyT.