bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling the inhibition of human MARK2 kinase by H. pylori CagA virulence factor
Authors: Farley Grace , Issroff Jemma , Kaplan Russell , Altaras Deborah , Cherston Caroline , Drucker Julian , Durkin Matthew , Edelstein Sophia , Feng Liang‐Liang , Jacobson Connor , Klabin Maya , Lovett Charlie , Shapiro Tessa , Susman Lily , Hackett Jennifer , Stebbins C. Erec
Published: June 2021

A partnership between students and Dr. Erec Stebbins enabled the Dalton School SMART team (Students Modeling a Research Topic) to model the interaction between the Helicobacter pylori CagA protein and the human PAR1b/MARK2 kinase.

Infection of the human stomach with H. pylori bacteria carrying the CagA virulence factor causes gastric ulcers and cancer. H. pylori inject CagA into host cells using a type IV secretion system. CagA interacts with several host proteins to contribute to gastric disease, including the MARK2 kinase, which helps maintain gastric cell polarity. A 14 amino acid portion of CagA mimics host proteins by binding in the MARK2 substrate‐binding site. Thus, CagA inhibits MARK2 in the same manner that the human protein kinase inhibitor, PKI, inhibits the protein kinase PKA by blocking the substrate‐binding site.

The inhibition of MARK2 kinase by CagA leads to loss of polarity in cells of the gastric epithelium, which is thought to lead to development of ulcers. Our model shows the interaction of the MARK2 kinase with the CagA peptide that binds in the substrate‐binding site. Supported by grants from the HHMI Pre‐College Program and the Camille and Henry Dreyfus Foundation.
Recognition of bacterial promoter ‐10 region by σ subunit of RNA polymerase
Authors: Jalal Erica J. , Lee Irvin , Lee Daniel , Chung Adam , Daci Genta , Li Hovey , Ung Bryan , Copp Richard P. , Manchanda Gita , Feklistov Andrey
Published: June 2021

RNA polymerase (RNAP), a bacterial cell regulation target, binds promoters, then opens DNA near the transcription start site. The crucial step in DNA promoter melting is −10 element (consensus sequence T−12A−11T−10A−9A−8T−7) recognition by the RNAP sigma (σ) subunit. X‐ray crystallography reveals details of ‐10 element recognition by σ. Single stranded (ss) DNA with the TATAAT sequence is draped across a σ conserved surface, with a 90° turn between the −11 and −10. Specific interactions occur with the most conserved bases: T‐12, A‐11, and T‐7, especially A‐11 and T‐7, which are flipped out of ss DNA and buried in σ pockets. A hydrophobic pocket tightly fits A‐11 between σ R246 and Y253. A‐11 hydrogen bonds with σ K241, F242 and E243. T‐7 fits in a large hydrophilic σ pocket with three ordered waters. T‐7 hydrogen bonds with L209 and N206 and has water‐mediated interactions with several σ residues. Less important central bases, ‐10T, ‐9A and ‐8A, point away from σ and make few or no base‐specific contacts with the protein. The structure reveals insights into initiation of transcription bubble formation and suggests interesting ideas for rational antimicrobial design. The Leonia High School SMART (Students Modeling a Research Topic) Team partnered with a researcher to create a physical model of σ interacting with the DNA ‐10 element using 3D printing technology. Supported by a grant from HHMI Pre‐College Program.
Structural Modeling of the PTF1‐J and PTF1‐L Heterotrimeric Transcription Factor Complexes
Authors: Stein Kelly , Kohrs Alix , Schuller Lauren , Marshall Andrea , MacDonald Raymond , Coats Ward
Published: June 2021

Students in Biomedical Research Studies at Hillcrest High School in Dallas, Texas participate in an ongoing research program at the University of Texas Southwestern Medical Center to study the structure and function of a mammalian transcription factor complex. Ptf1a is a sequence specific DNA‐binding transcription factor that is crucial to the development of the embryonic pancreas. Its functional form is in a trimeric complex composed of a common E‐box binding protein (E12/47, HEB, or TCF12), Ptf1a and either Rbpj or Rbpjl. The Rbpj form of the complex (PTF1‐J) is required for the early stage of pancreatic development. Subsequently, the Rbpjl‐form (PTF1‐L) is required for the formation of mature acinar cells. PTF1‐L is involved in an auto‐regulatory loop for the maintenance of transcription of both Ptf1a and Rbpjl genes. We have generated structure models for the PTF1‐J and PTF1‐L heterotrimeric transcription factor complexes bound to their consensus DNA binding sequence. The models provide the orientation of the Ptf1a/E12 heterodimer relative to the Rbpj and Rbpjl proteins and identify the protein surface areas at the heterotrimeric interface.
Toll‐Like Receptor 4 (TLR4) Myeloid Differentiation Factor‐2 (MD‐2) Complex
Authors: Tetzlaff Sarah , Larson Mary , Toll Molly , Garb Jodie , Tourdot Ben , Newman Debra K.
Published: June 2021

Sepsis, the tenth leading cause of death in the United States, is a whole‐body inflammatory response to infection. Sepsis leads to septic shock, a condition with a 30–40% mortality rate caused by multiple organ failure and development of hypotension. Lack of understanding of the pathophysiology of sepsis limits successful treatment options. Gram‐negative bacteria are a major cause of sepsis. The outer membrane of Gram‐negative bacteria contains lipopolysaccharide (LPS). LPS is recognized by a receptor complex expressed by certain immune cells that includes the transmembrane glycoprotein, Toll‐like receptor 4 (TLR4), and myeloid differentiation factor‐2 (MD‐2). Over‐stimulation of immune cells by LPS through TLR4/MD‐2 results in sepsis. TLR4‐mediated activation of immune cells is also responsible for allergic contact dermatitis due to nickel, a common and less fatal condition than sepsis. The crystal structure of TLR4‐MD‐2‐LPS has elucidated residues involved in LPS binding to TLR4/MD‐2 and in TLR4 dimerization, which are essential events involved in immune cell activation and induction of sepsis. A better understanding of interactions between TLR4, LPS and MD‐2 will help create better drugs to disrupt the interactions. The Laconia SMART (Students Modeling A Research Topic) Team used 3‐D printing technology to model the TLR4 dimer in collaboration with MSOE. Supported by a grant from the NIH‐NCRR‐SEPA.
Concerted Action of Aldehyde Dehydrogenases in Depot‐specific Fat Formation
Authors: Yasmeen Rumana , Reichert Barbara , Ziouzenkova Ouliana
Published: June 2021

Unraveling mechanisms regulating fat distribution in visceral and subcutaneous fat depots is necessary to improve treatments of a number of chronic diseases. Retinoic acid, a transcriptionally active metabolite of vitamin A, plays a key role in differentiation processes. It is produced by the aldehyde dehydrogenase‐1 family of enzymes (aldh1a1,a2,a3). Our objective was to investigate whether differences in RA production by Aldh1, in subcutaneous versus visceral fat, diverge fat formation in these depots.

We demonstrated that Aldh1a1 is the major enzyme regulating RA generation in adipocytes in vitro and in vivo. Deficiency in Aldh1a1 inhibits endogenous RA production and suppresses PPARg expression, a key event in 3T3‐L1 adipogenesis, that we rescued by overexpression of Aldh1a1,a3 or both. We identified that visceral fat expressed less Aldh1a2 and a3 than subcutaneous fat in C57/BL6 female mice on regular chow, although Aldh1a1 was predominantly expressed in these depots. Therefore, in Aldh1a1−/− mice, visceral fat essentially lacked all Aldh1 expression, which resulted in 70% decreased expression of PPARg and FABP4 in visceral fat of Aldh1a1−/− vs. WT mice. Consequently, Aldh1a1−/− mice specifically lost visceral fat.

Our data suggest that Aldh1 enzymes play a key role in adipogenesis and their concerted action may contribute to differences in fat formation in different depots.
Influence of Nutrient Timing Following Resistance Exercise on Sleep RQ and Glucose in Young Men with a Family History of Type 2 Diabetes
Authors: Stevens Michelle , Gutierrez Jean , Gribok Andrei , Rumpler William , Tadros Maryann , DiPietro Loretta
Published: June 2021

Objective

To examine the impact of post‐resistance exercise supplement timing on sleep glucose and respiratory quotient (RQ; indicator of metabolic flexibility and lipid metabolism).

Methods

Young male participants (n=6) underwent two 48‐h stays in a whole‐room calorimeter. The first 24‐h served as a control day, and on the second day participants completed a 40‐min novel resistance exercise protocol and consumed 600g of 1% chocolate milk either immediately after (IPEN) or 3‐h after completing resistance exercise (3h‐PEN). RQ was measured continuously throughout the 48‐h period and glucose concentrations were determined by a continuous glucose monitoring system.

Results

Sleep RQ following the exercise day, as compared to the control day, was significantly lower following IPEN (0.71±0.04 vs. 0.75±0.02; p= 0.04), but was not different in 3h‐PEN (0.72±0.03 vs. 0.72±0.03 p= 0.71). Glucose during the same sleep period following the exercise day, as compared to the control day, was not significantly different following IPEN (99±15 vs. 98±9 mg·dL−1; p= 0.77) or 3h‐PEN (92±10 vs. 92±12mg·dL−1; p= 0.96).

Conclusions

Preliminary results indicate IPEN may have promoted greater fat metabolism during sleep, as compared to 3h‐PEN, but blood glucose concentration was not affected by nutrient timing. This study was funded by the GWU Department of Exercise Science.
Congenital malformations in rats exposed to tap water
Authors: Ladd Nicholas K , Melin Vanessa K , Ferguson Elizabeth , Hrubec Terry C
Published: June 2021

Previous research has demonstrated increased rates of neural tube defects, cleft palates and rib malformation in CD‐1 mice exposed to tap water from a variety of municipalities. Exposure to tap water resulted in neural tube defect rates of 10–20%. Rates of cleft palates and rib malformations were 5% and 70% respectively. This research investigated whether analogous findings could be demonstrated in a second animal model, specifically Sprague Dawley and Wistar rats. Female rats were maintained on tap water for 12 weeks prior to breeding and throughout gestation. Resulting embryos were evaluated on day 18 of gestation for fetal weight, placenta weight, and neural tube, palate, facial, ocular, tail, limb and digital malformations. The fetal skeleton was then assessed using tissue clearing and staining with Alcian Blue – Alizarin Red. Both strains of tap water exposed rat embryos demonstrated no significant gross or skeletal malformations at gestational day 18. Lumbar ribs were observed in 8.3% of tap water exposed Sprague Dawley fetuses; however, this is similar to background rates for this strain. These results suggest that rats may be more resistant than mice to the possible tertogen present in tap water. This work was funded by the VA‐MD College of Veterinary Medicine and NIH‐NIEHS R21ES016886.
Novel use of confocal microscopy in conjunction with EdU technology to determine in vivo cellular proliferation in the piglet intestine
Authors: Reznikov Elizabeth Alexandra , Comstock Sarah S. , Sivaguru Mayandi , Donovan Sharon M.
Published: June 2021

The method most commonly used to label newly divided cells is a thymidine analogue, 5‐bromo‐2′‐deoxyuridine (BrdU). BrdU detection requires extensive DNA denaturation. However, an alternative thymidine analogue, 5‐ethynyl‐2′‐deoxyuridine (EdU) can be used. EdU that has been incorporated into newly formed DNA is tagged by a covalently bound fluorescent azide molecule allowing for highly specific recognition without DNA disruption. We have optimized EdU technology for in vivo use in pigs using confocal microscopy as a novel approach to quantify proliferation in ex vivo stained sections. Piglets were intraperitoneally injected with 10mM EdU at 1 ml per kg body weight 2 hours prior to euthanasia on day 7 or 14 postpartum. Formalin‐fixed jejunum and ileum sections were stained using the Click‐iTTM EdU Alexa Fluor 555 Imaging Kit (Invitrogen). DAPI was used to detect total nuclei. Samples were imaged using Confocal and Nanozoomer microscopy. AxioVision was used to quantify the area of EdU per unit area of DAPI. We have shown the use of confocal microscopy provides better specificity and depth discrimination than Nanozoomer microscopy. This is necessary to accurately detect small changes in proliferation from thick tissue specimens. These findings will be used in future studies to detect changes in intestinal proliferation caused by feeding of bioactive compounds to neonatal piglets.
Effects of a High‐Carbohydrate Diet on the Proinflammatory Interleukin Levels of Older Adults
Authors: Maddy LaDonna , Gordon Scott E , Tharrington India H , Macesich Jennifer L , Harper Bradley M , Heidal Kimberly , Pofahl Walter E , Wall‐Bassett Elizabeth D
Published: June 2021

Immunosenescence involves changes on the cellular and molecular levels in the immune system, and is associated with aging. Higher incidence of autoimmune disorders and other inflammatory conditions, as well as increased rates of infectious disease, cancer, and mortality have been linked to these conditions. Proinflammatory interleukins (IL‐6) have been shown to increase while anti‐inflammatory interleukins (IL‐4, IL‐10) decrease with age. Energy restriction has been associated with altered cytokine production; thus, the purpose of this study is to examine the effects of a prescribed diet on immunosenescence in older adults.

IL‐6 concentrations were analyzed from venous samples of 10 older adults (46–85 yrs) and compared to 9 younger adults (18–45 yrs). Five older adults consumed a high‐carbohydrate diet for three days. All other subjects consumed a normal diet. Older adults who consumed less than the Acceptable Macronutrient Distribution Range for fat (20–35% of total calories) had lower IL‐6 levels comparable to younger subjects. Contrary to our hypothesis, a high‐carbohydrate diet did not lower IL‐6 levels however fat intake may contribute to a reversal of immunosenescence. Further investigation is warranted to investigate anti‐inflammatory factors contributing to changes in immunosenescence.

This research was supported by the East Carolina University Division of Research and Graduate Studies.
Characterization of Gene Targets Regulated by Lmx1b during Limb Dorsalization
Authors: Feenstra Jennifer M , Pira Charmaine U , Kanaya Kohei , Hoffman Sarah E , Eppey Rick J , Oberg Kerby C
Published: June 2021

Lmx1b is a homeodomain transcription factor that regulates dorsal identity during limb development and Lmx1b knockout (KO) mice develop nearly symmetrical ventral‐ventral limbs with hypoplastic scapulae. Currently, downstream targets of Lmx1b within dorsal mesoderm that impart the limbs' unique dorsal asymmetry are unknown.

To identify genes targeted by Lmx1b, we compared gene arrays from Lmx1b KO and wild type (WT) mouse limbs during limb outgrowth and patterning, i.e., 11.5, 12.5, and 13.5 days post coitum (dpc). Real‐time PCR confirmed microarray results and whole mount in situ hybridization was used to localize and verify differential dorsal‐ventral expression.

Microarray analysis identified 23 targets differentially expressed in all three arrays. Real‐time PCR confirmed 17 up‐regulated and 2 down‐regulated targets. Whole mount in situ hybridization of WT 12.5 dpc embryos demonstrated a dorsal‐ventral asymmetric pattern for the validated targets, further categorized as skeletal, soft tissue or other. Skeletal targets, including Emx2, Matrilin1 and Matrilin4, demonstrated a loss of scapular expression in the Lmx1b KO mice. Soft tissue targets which demonstrated dorsal mesodermal staining in WT were drastically reduced in KO limbs. This study provides the most comprehensive characterization of skeletal and soft tissue genes regulated by Lmx1b during limb development to date. Our data also suggest targets which Lmx1b may augment to ensure proper scapular development. Further studies are warranted to determine the mechanism of Lmx1b target regulation during limb dorsalization. NIH HD39421
Comparative effectiveness of two popular weight loss programs in women IV: quality of life
Authors: Jagim A , Mardock M , Lockard B , Simbo S , Byrd M , Kresta JY , Baetge C , Jung Y , Koozehchian m , Khanna D , Oliver JM , Kyul H , Greenwood M , Rasmussen C , Kreider R
Published: June 2021

42 sedentary women (33±7 yrs, 164±7 cm; 93±19 kg; 46±7% body fat, 35±6 kg/m2) were randomized to participate in the Curves (C) or Weight Watchers (W) programs for 16‐wks. Participants in the C program followed a 1,200 kcal/d diet for 1‐wk and 1,500 kcal/d diet for 3 wks (30:45 CHO:PRO). Subjects then ingested 2,000 kcals/d for 2‐wks (45:30) and repeated this diet while participating in the Curves with Zumba program 3‐d‐wk. Remaining subjects followed the W point‐based diet program. Data were obtained at 0, 4, 10, & 16 wks and analyzed by MANOVA. Both programs increased ratings of physical functioning (16.5±34%, p=0.002), vitality (19.9±30%, p=0.002), bodily pain (15.7±33%, p=0.03), and general health (16.7±40%, p=0.001) while tending to increase role physical (14.7±46%, p=0.11) and role emotion (−6.5±4%, p=0.08). Significant interactions were seen in physical function (C 89.7±11, 91.8±9, 93.8±7, 94.1±10; W 75.2±87.6, 87.6±14, 88.6±14, 88.1±16, qp =0.05) and vitality (C 53.5±14, 61.8±13, 62.1±13, 60.0±12; W 48.1±12, 55.2±10, 54.0±15, 57.9±16, qp =0.05) with participants in the C group experiencing significantly higher values. Results indicate that participation in the C and W programs generally improved markers of quality of life and that subjects participating in the C group may experience greater improvement in vitality than those in the W group.
Is FFM the best factor to predict the resting energy expenditure (REE) in children with obesity or leanness?
Authors: Kim Myung‐Hee , Kim Eun‐Kyung
Published: June 2021

The purpose of this study was to compare REE between obese and nonobese children. Subjects were 24 obese (12 boys, 12 girls) and 45 normal weight (16 boys, 29 girls) Korean students aged 7–12y. REE was measured by indirect calorimetry(TrueOne 2400). REE were significantly higher in obese groups(boys 1448±211 kcal/day, girls 1291±132 kcal/day) than in nonobese groups(boys 1157±84 kcal/day, girls 1122±108 kcal/day). But, REE/weight of obese students(boys 27±2 kcal/day, girls 29±3 kcal/day) were significantly lower than that of nonobese students(boys 36±4 kcal/day, girls 31±3 kcal/day). And, there was no significant difference in REE/fat‐free mass(FFM) between obese and nonobese groups. FFM was also positively correlated with REE(r=0.84, p<0.001). These data explain that FFM is one of the most important factors which influence on REE. Therefore, to prevent the reduction of REE during weight loss, it is necessary to maintain or increase the percentage of FFM.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology(2010‐0004472).
Antioxidant Activity of Angelicae gigas Nakai Leaves
Authors: PARK SUNG‐JIN , JUNG JI‐HOON , KIM JONG‐DAI
Published: June 2021

Angelicae gigas Nakai is used in traditional medicine. The leaves have been consumed as a vegetable in Korea. In this study, we examined the antioxidant capacity of the extracts from Angelicae gigas Nakai leaves in vitro using hydroxyl radical scavenging assay, reducing power activity, metal chelating assay, DPPH free radical scavenging assay, phenolic acids. The effect of the ethanol extract was stronger than that of the methanol and water extracts in all of the assays. Moreover, the total phenolic contents in the extracts were measured by HPLC. Our data suggest that Angelicae gigas Nakai leaves possess significant antioxidant properties. Its strong antioxidant activity suggests that it has great potential in the food industry as functional food ingredient.
Polycystic ovary Syndrome (PCOS) and its nutritional antecedents in a multiethnic population
Authors: Dabrell Rachel , Nichols Selby
Published: June 2021

Polycystic ovary Syndrome (PCOS) is a complex disorder with many nutritional implications. In this case‐control study we compared anthropometry and nutrition behaviours among women clinically diagnosed with PCOS with an age‐matched group of women without the condition. Forty‐three (43) women with PCOS and 30 non‐PCOS participated in the study. Participants were interviewed on socio‐demographic charateristics, symptoms and dietary behaviours. Weight, height and Waist circumference were measured using standard procedures. Our results showed that compared to without PCOS those PCOS were more likely to have BMIs greater than 25(17% vs. 51%, P<0.01) and waist‐to‐height ratios> 0.5(70% vs. 47%). PCOS was positively associated with higher intakes of white rice (P<0.05) and processed meats (p=0.06), coffee (P<0.05), peas and peas (P= 0.07) carrots and pumpkin (P<0.05) and inversely associated with foods high in refined sugars (P<0.010) and citrus fruits (P<0.05). These results suggest that women with PCOS tended to have greater adiposity than their non‐PCOS counterparts. They also avoided foods that are known to have a high glycaemic index
Human milk mucins inhibit salmonella typhimurium invasion of human epithelial cells
Authors: Liu Bo , Newburg David S.
Published: June 2021

Salmonella infection is more frequent among infants than the general population, but the incidence is lower in breastfed babies. This study was to test whether human milk per se inhibited salmonella invasion of human intestinal epithelial cells in vitro, and to identify the milk components likely to be responsible for any inhibition. Salmonella typhimurium SL1344 invasion of FHs 74 Int and CaCo‐2 cells were the models of human intestinal epithelium infection. Internalization of FITC‐labeled salmonella into intestinal cells was measured by FACS analysis to quantify infection. Pooled human milk and its fractions were tested for their ability to inhibit infection, including two human milk mucins, mucin 1 and mucin 4. At 150 ng/mL, approximating the amount in milk, human milk mucin 1 and mucin 4 inhibited salmonella invasion of both cell types. They also bound Salmonella typhimurium SL1344 in a dose‐dependent manner. Thus, mucins may prove useful as a basis for the development of novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by salmonella, and perhaps other pathogens. Supported by NIH HD013021 and AI075563.
Use of ethnogenetic layering to identify and quantify geographically localized sources of chronic stress and their impact on health among African Americans
Authors: Jackson Fatimah LC , Hardin Jesse
Published: June 2021

Economically and socially disadvantaged African Americans have faced a number of important sources of chronic physiological and psychological stressors that have contributed to an increased vulnerability to poor health. Although there appears to be a direct relationship between economic and social disadvantage and health disparities, the sources and precise impact of such stressors often remain inadequately quantified. The strain of discrimination is associated with unequal exposures to stress and unequal access to ameliorative interventions throughout the lifespan, across geographical space, and over multiple generations. Applying the allostatic stress process model to GIS‐facilitated ethnogenetic layering, we present data that suggests that stress and its sequelae can be predicted, identified, and quantified for the purposes of addressing health disparities. Protracted adversity responses are correlated with increased vulnerabilities to infection, injury, and disease and increases in the associated selective coefficients for exposed groups. We report on the development of computer‐assisted models that quantify the intensity and duration of particular stressors and detail their roles in health disparities.
IIn vitro study of green tea, tangerine pulp and prickly pear cactus extracts on platelet aggregation, RBC Na‐Leak and TBARS production
Authors: Kim Eun Hee , Kim Ji Mi , Hwang Hye Jin , Kang Min Sook , Chae In Sook
Published: June 2021

We compared the radical scavenging activity of the extracts of green tea, prickly pear cactus, tangerine pulp and vitamin E and their invitro effects of antioxidant on erythrocyte Na leak, platelet aggregation and TBARS production using SD rats. Platelet aggregation incubated with green tea extract was significantly decreased in the maximum and initial slope compared with other extracts or vitamin E (p<0.05). Na leak in AAPH treated erythrocyte was significantly lower when incubated with cactus extract compared with other extracts or vitamin E. Hemolysis of AAPH treated RBC was significantly lower when incubated with vitamin E compared with control and the extracts (p<0.05). TBARS production in AAPH treated platelet rich plasma (PRP) was significantly increased compared to the untreated PRP (p< 0.01), and vitamin E significantly decreased TBARS production in the AAPH treated PRP (p<0.01). Vtamin E significantly decreased TBARS production in the AAPH treated human LDL compared with all other groups (p<0.01). Present study showed that green tea in vitro system has lowering effect of platelet aggregation and vitamin E has a protective effect on membrane preventing lipid peroxidation and membrane damage.
Role of NOX2 in Insulin resistance
Authors: Jamali Amina EL
Published: June 2021

Insulin resistance due in part to impaired glucose uptake in skeletal muscle is consistently associated with oxidative stress. Skeletal muscle cells express NADPH oxidase type 2 (NOX2), a potent generator of reactive oxygen species (ROS). Our central hypothesis is that NOX2 is a key factor in the development of insulin resistance. In vivo, our preliminary data show that insulin resistance induced by a high‐fat diet (HFD) is associated with increased ROS production, NOX2 expression, and leukocyte infiltration in skeletal muscle. Furthermore, superoxide production in skeletal muscle, fasting blood glucose and insulin levels, and the insulin resistance index were all reduced in HFD‐fed NOX2‐null vs. wild‐type mice. These results suggest that NOX2 is implicated in the development of insulin resistance. A full understanding of NOX2 regulation is likely to help for the prevention or treatment of type 2 diabetes patients.
Genetic variation in antioxidant enzymes and rate of decline in lung function
Authors: Tang Wenbo , Bentley Amy , Kritchevsky Stephen , Cassano Patricia
Published: June 2021

The oxidant/antioxidant balance in the lung is hypothesized to play an important role in chronic obstructive pulmonary disease (COPD). Deficits in antioxidant defenses due to variation in genes encoding antioxidant enzymes may cause accelerated lung function decline and increase disease risk. This study explored the association between genetic variation in a network of antioxidant enzymes and rate of decline in lung function phenotypes, including forced expiratory volume in the first second (FEV1) and the ratio of FEV1 to forced vital capacity (FEV1/FVC). 287 SNPs in 56 genes (MAF ≥ 1%) were analyzed using mixed effects models in 1,487 European American participants in the Health, Aging, and Body Composition study. The 7 top hits (nominal p<0.02, range 0.007 to 0.017) for FEV1 were in 4 genes: mGST3, GSTA4, GPX1, and GPX2. The rs6904771 locus in GSTA4 was associated with a 12 ml/yr steeper rate of decline in FEV1 per T allele. Markers in two genes (GCLC, GSTO2) were associated (nominal p<0.02) with the ratio of FEV1/FVC. mGST3, GPX1, and GPX2 have not been previously identified in human studies of lung outcomes, but agree with prior animal studies, and all 6 genes have known functional relevance to lung outcomes. In summary, genetic variants in antioxidant enzymes are associated with rate of decline in lung function. These pathways should be further investigated for possible antioxidant approaches to prevent and/or treat COPD. The project was supported by RC1 AG035835, R01 HL74104, R01 HL071022, genotyping services were provided by the Johns Hopkins University under N01‐HV‐48195 from the NHLBI, and NIA contracts N01‐AG‐6‐2101, N01‐AG‐2103, and N01‐AG‐6‐2106.
Human cadaveric tissue is suitable for histological, immunohistochemical and ultrastructural analysis
Authors: Jensen Cynthia Graham , Slim M. Atif Mohd , Wang Tom A , Al‐Ali Saad , McGlashan Susan R
Published: June 2021

Objective

This study examined the use of cadaveric material for morphological and pathological studies using histology, immunohistochemistry and TEM.

Methods

Paraffin‐embedded sections of liver, myocardium, kidney, testis and trachea were prepared from cadavers embalmed using the Dodge arterial injection method. Sections were either stained with H&E or incubated with antibodies against α‐smooth muscle actin (α‐SMA), acetylated α‐tubulin or connexin 43 and detected using either DAB or fluorescence. In addition, some samples were processed for TEM.

Results

All tissues showed excellent histological morphology comparable to freshly‐fixed tissue; ultrastructural samples yielded moderate resolution. DAB‐positive labelling was observed for α‐SMA and α‐tubulin in all tissues. For immunofluorescence, all tissues were autofluorescent in the yellow‐green spectrum. However, we found that red fluorescent secondary antibodies positively labelled tubulin in tracheal cilia, sperm flagella and kidney primary cilia, as well as connexin 43 in myocardial intercalated disks and α‐SMA in all blood vessels.

Conclusion

Although it is often believed that there is poor microstructural preservation during embalming, we have shown that cadaveric tissues are an excellent source of ethically‐approved human tissue for histological and immunohistochemical studies in biomedical research. Funded by U of Auckland.
The Role of Estrogen in Gender Differences in Adipocyte Biology
Authors: Stubbins Renee Elaine , Holcomb Valerie , Hong Jina , Nunez Nomeli P
Published: June 2021

Background

It has been established that males and females differ in their adipose tissue deposition; however, a full understanding of the adipocyte biology involved is needed. The objective of our study is to explore the role estrogen plays in gender differences in obesity, specifically in adipocyte biology.

Methods

To determine the role of estrogen in gender differences in the susceptibility to obesity we used C57BL/6J mice (15/group): 1) males 2) nonovariectomized females 3) ovariectomized females and 4) ovariectomized females supplemented with estrogen, which were randomized to the following diets: 30% calorie‐restricted, low‐fat or high‐fat diet. We measured weight gain, percent body fat, abdominal adipose tissue, and adipocyte size. Additionally, we assessed adipocyte biology by using QRTPCR to detect mRNA levels of key adipocyte biology genes.

Results

Male mice were more susceptible to diet induced obesity compared to female mice. Removal of the ovaries caused the female mice to mimic the males' susceptibility to obesity, and supplementation of estrogen to the ovariectomized females restored their protection to obesity. In the high‐fat diet groups, males and ovariectomized females gained more abdominal adipose tissue due to increased adipocyte size. Moreover, males and ovariectomized females had significantly higher peroxisome proliferator‐activated receptor‐gamma and leptin mRNA levels. Male mice also had significantly higher lipin1 and Ccaat‐enhancer‐binding protein‐beta mRNA levels compared to intact females and ovariectomized females supplemented with estrogen.

Conclusion

Estrogen protects female mice from obesity by modulating adipocyte biology thru key adipogenic genes.

Grant Support: This work was supported by American Cancer Society grant.
Estrogen receptor‐alpha genotype affects exercise‐related bone mass in young healthy women
Authors: Kondo Hiroyo , Fujino Hidemi , Murakami Shinichiro , Fujita Naoto , Nagatomo Fumiko , Ishihara Akihiko
Published: June 2021

Osteoporosis is a systemic skeletal disease characterized by low bone mass and/or fragile bone. The estrogen receptor alpha (ESR1) is a good genetic candidate for a prime regulator of bone metabolism. The purpose of this study was to assess the interactive effects of exercise and/or calcium intake and ESR1 polymorphism on bone mass.

Eighty healthy young women (20–23 yr) were recruited in this study. Bone mass and body composition were measured. Food frequency and exercise‐related questionnaires were used to estimate calcium intake and exercise intensity. The ESR1 polymorphisms at intron 1(rs2234693 and rs9340799) were genotyped using the TaqMan‐based SNP method.

Exercise and milk‐intake were significantly associated with bone mass in the whole subjects. The bone mass in non‐exercised participants with CC homozygote was lower than that in TT homozygote. The bone mass in high‐intensity exercised participants with CC homozygote was higher than that in low‐intensity. The bone mass in high‐intensity exercised participants carrying both CT and AG heterozygotes was significantly higher than that in low‐intensity.

These results suggest that exercise attenuates are the increase of peak bone mass in young women carrying both C allele and AG heterozygote in estrogen receptor alpha.

This study was supported by Grants for Research and Education from Nagoya Women fs University.
Effect of procyanidin fractions from defatted grape seed meal on adipogenesis in 3T3‐L1 cells
Authors: Kim Younghwa , Seo Seulgi , Choi Youngmin , Park Yooheon , Park Yeonhwa , Lee Junsoo
Published: June 2021

Grape seed has been known to be abundant in various bioactive compounds. Known beneficial effects of bioactive compounds from grape seed are anticancer, anti‐inflammation, and the prevention of heart disease. Procyanidins are considered as representative bioactive compounds from grape seed as well as various fruits and plants including cocoa, apple and tea. Grape seed procyanidins can be fractionated as monomeric, oligomeric, and polymeric fraction by their structures and chemical characteristics and each fraction have different biological activities. We obtained various procyanidin fractions containing crude polyphenols, oligomeric procyanidin, and polymeric procyanidin from defatted grape seeds meal and tested the effect of each fraction on adipogenesis in 3T3‐L1 cells. 3T3‐L1 pre‐adipocytes were differentiated by the addition of insulin (1μg/ml), dexamethasone (1μM), IBMX (10mM), and 10% FBS in DMEM medium. Cells were treated with or without CPE (crude polyphenol fraction), FCO (catechin and oligomeric procyanidin mixed fraction), and FPP (polymeric procyanidin fraction), respectively. Lipid accumulation was measured by Oil‐red O staining and intracellular triacylglyceride contents were analyzed. Level of mRNA and protein expression of the lipid metabolism related transcription factors were measured by RT‐PCR and Western Blot analysis, respectively. FCO and FPP have more significant anti‐adipogenic effects than CPE and these results may provide the preliminary data for the utilization of agricultural byproducts as functional food substances.
Signaling molecules differentiate single versus dual ligation of complement receptor 3
Authors: Johnson Courtney M. , Byrd Angel , Reichner Jonathan
Published: June 2021

Complement Receptor 3 (CR3), an integrin found on neutrophils, regulates firm cell adhesion to the extracellular matrix and serves as a pattern recognition receptor for the fungal Pathogen Associated Molecular Pattern (PAMP) β‐glucan. Coincident ligation of CR3 with fibronectin and β‐glucan is possible due to spatially distinct ligand binding domains. Current work demonstrates that dual ligation of CR3 with fibronectin at its I‐domain and β‐glucan at its lectin‐like site causes homotypic aggregation of primed human neutrophils. The downstream signaling pathways resulting from differential CR3 ligation are unknown. Using phosphoproteomics, we have identified several proteins that are significantly phosphorylated when ligated with fibronectin and β‐Glucan but not either ligand alone. Phosphoinositide 3 Kinase (PI3K), Protein Kinase Cδ (PKCδ), Glycogen Synthase Kinase 3β (GSK3β), and ERK show increased phosphorylation and each may play a role in the homotypic aggregation of neutrophils via CR3. Western Blotting has validated our initial phosphoproteomic findings for each protein. Use of inhibitors obviated homotypic aggregation of neutrophils for all aforementioned proteins except for GSK3β. Thus PI3K, PKCδ, and ERK are all components of the signaling pathway of dually ligated CR3 that are also relevant to homotypic aggregation.
The knock‐down of Caveolin‐1 inhibits STAT3 signaling in human metastatic lung cancer in vitro
Authors: Gasperi‐Campani Anna , Pancotti Fabia , Maggiolini Marcello , Roncuzzi Laura
Published: June 2021

Caveolin‐1 (Cav‐1) is an essential structural constituent of caveolae, implicated in mitogenic signaling, oncogenesis, neurodegenerative diseases and senescence. A complex role is exerted by Cav‐1 in human malignancies, which seems to be dependent on histotype, stage and grade of each neoplastic disease. As regards lung cancer, emerging evidence indicates a close relation between Cav‐1 and the development of it. In the present study we investigated the role of Cav‐1 in metastatic lung cancer proliferation. Cell lines were from metastatic lesions of lung adenocarcinoma (RAL) and of small cell lung carcinoma (SCLC‐R1), in which Cav‐1 protein was expressed at high levels. Results show that siRNA‐mediated down‐regulation of Cav‐1 caused stable arrest of cell cycle in both cell lines. A marked reduction of cyclin D and of CDK2‐CDK4 expressions were evident in the cells transfected with Cav‐1 siRNA and consequently of phosphoRb on ser795 and ser780. A significant decrease of the expression of phosphorylated Akt and its down‐stream effectors phosphorylated ERK and STAT3 was also evident. This is the first report of a block of metastatic lung proliferation obtained by Cav‐1 silencing, in the same time suggesting a new cell cycle inhibiting pathway, mediated by Cav‐1 knock‐down, in lung cancer and providing new insights into the molecular mechanism underlying the pro‐survival and tumor‐promoting functions of Cav‐1

Grants from MIUR (RFO, PRIN), Pallotti's Legacy for Cancer Research, University of Bologna, Italy
Ubiquitination and Lipid Rafts in Erythropoietin Receptor Internalization and Signaling
Authors: Dao Chinh T.
Published: June 2021

Signaling through the erythropoietin receptor (EpoR) is important in the proliferation and differentiation of red blood cells and their precursors. Many of the molecular details of this signaling pathway have been described. In these experiments, we endeavored to more fully map the determinants of EpoR signaling by addressing the importance of membrane cholesterol and the possible participation of membrane lipid rafts. The results of our experiments show that depletion of cholesterol from the surface of Ba/F3 cells stably expressing the receptor results in decreased STAT5 activation. Tagged EpoR can be detected within the lighter fractions of a density gradient cellular fractionation, suggesting that the receptor may co‐localize with membrane lipid rafts. Interestingly, cells expressing a modified EpoR lacking intracellular domain lysine residues were seemingly unaffected by the pharmacologic agent methyl‐β‐cyclodextrin. The significance and mechanism of this are unknown, but point to the possibility that receptor ubiquitination may be involved in receptor localization and/or migration to lipid rafts.
Development stages of anaplastic liver cancer in the experiment at rats
Authors: Manekenova K. , Omarov T. , Peradze M. , Suleymanova A. , Sakipova A.
Published: June 2021

Development stages of anaplastic liver cancer were investigated in the experiment at rats. Carcinogen (DMBA) diluted in the solution of medical bile, used as cocancirogen, injected intrahepatically. Histologic discomplexation signs of stringer structures in the liver lobules were revealed after 30 days from the beginning of experiment, mostly, subcapsular zones, hyperplasia of hepatocytes with enlargement nuclear‐cytoplazmatic correlation. Formation trabecular and tubular structures from large hepatocytes with large hyperchomic nuclears and signs of hepatocytes' dysplasia of different expressed degree was marked on 60 day of the experiment. Singular massy hepatocytes proliferation with signs of cells' atipism was revealed after 90 days from the beginning of experiment. Trabecular and tubular structures from hepatic cells in the condition of severe dysplasia are remained among massy foci. Morphologic picture of anaplastic cancer is observed after 120 days in the liver of rats.

Research results confirm hepatocellular origin of anaplastic liver cancer.Development stages of anaplastic liver cancer of rats are following: progressing dysplasia of hepatocytes; formation of atypical trabecular and tubular structures from large hepatocytes, discomplexation of tubular and trabecular structures with hepatocytes' anaplasia.
ATM is a Novel Regulator of Nucleolar Transcription
Authors: Smith Scott Carl , Hetman Michal
Published: June 2021

ATM (Ataxia‐telangiectasia mutated protein kinase) mediates DNA double‐stranded break (DSB) signaling. ATM deficiency results in the autosomal recessive disorder known as ataxia‐telangiectasia (A‐T). The cause of the neurodegeneration associated with this disorder remains unknown. Here we report the unexpected finding that, in neurons, ATM is a regulator of RNA‐Polymerase‐1 (Pol‐1) ‐mediated transcription of nucleolar rRNA genes (rDNA). Low concentrations of the ATM‐activating DSB inducer, etoposide, stimulated rDNA transcription in both cultured rat cortical neurons and cerebral cortices of postnatal day seven (P7) rats. The etoposide‐mediated activation of rDNA transcription was reduced by ATM inhibition. Importantly, in the absence of etoposide, the specific ATM inhibitor, KU55933, reduced basal levels of nucleolar transcription and attenuated its induction in response to enhanced neuronal activity. Finally, ATM is robustly present in neuronal nucleoli and several critical regulators of Pol‐1 display potential ATM phosphorylation sites. Our results suggests that ATM is a major positive regulator of nucleolar transcription. Hence, defective ribosomal biogenesis may contribute to the A‐T‐associated neurodegeneration.
Characterization of FGF receptors expression on human neutrophils and their contribution to chemotaxis
Authors: Haddad Lydia E. , Khzam Lara Bou , Hajjar Fadi , Merhi Yahye , Sirois Martin G
Published: June 2021

Fibroblast Growth Factors (FGFs) can induce inflammatory mediators release by endothelial cells (ECs) and adhesion molecules expression at their surface, thereby favoring neutrophil recruitment and transvascular migration. Neither the expression nor the biological activities that could be mediated by FGFRs have been investigated on human neutrophils. We observed that circulating human neutrophils from healthy individuals expressed varying levels of FGFRs in their cytosol and at their surface. FGFR‐2 was identified as the sole cell‐surface receptor, with FGFR‐1 and ‐4 localizing in the cytosol and FGFR‐3 being undetectable. Using the murine matrigel plug assay, we observed an inflammatory response in FGF‐2‐containing matrigel plugs (inflammatory score ‐ IS: 2.2/3) as compared to control plugs (IS: 0.2/3). In vitro, FGF‐2 stimulation produced a maximal increase of neutrophil migration at 10−9 M, and by 2.3‐fold as compared to control. Treatment with blocking anti‐FGFR‐2 antibodies abrogated the effect of FGF‐2, while the blockade of FGFR‐1 and FGFR‐4 reduced neutrophil transmigration by 28.9 ± 20.6% and 14.6 ± 13.6%, respectively. In summary, our study is the first one to report FGFRs expression on human neutrophils, with FGF‐2 promoting neutrophil chemotaxis through FGFR‐2 activation. This work was supported by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Quebec.
A Novel Epithelial Proallergic Cytokine IL‐33 Serves As A Biomarker For Ocular Allergic Inflammation
Authors: zhang lili , zhao Guiqiu , De Paiva Cintia S. , Pflugfelder Stephen C. , Li De‐Quan
Published: June 2021

Purpose

To explore a novel role of interleukin (IL)‐33 in Th2‐dominant allergic inflammation on ocular surface.

Methods

IL‐33 and its signaling molecules were evaluated in patients with atopic conjunctivitis and in experimental allergic conjunctivitis (EAC) mice induced by short ragweed (SRW). Human corneal epithelial cells (HCECs) were cultured for IL‐33 regulation.

Results

IL‐33 and ST2 were highly detected in conjunctival impression cytology samples from atopic conjunctivitis patients. In the SRW‐induced EAC mice, IL‐33 and ST2 transcripts and immunoreactivity significantly increased in the corneal and conjunctival epithelia with largely infiltrated CD11c+/OX40L+ dendritic cells. Th2 cell infiltration was evidenced by increased transcripts and immunoreactivity of CD4, IL‐4, IL‐5 and IL‐13 in the conjunctiva, with increased expression of OX40, STAT6 and GATA3. In culture model, IL‐33 was dramatically stimulated in HCECs exposed to microbial ligands through toll‐like receptor (TLR) and NF‐kB pathways.

Conclusions

Our findings demonstrate an important role of mucosal epithelial cells in initiating Th2‐dominant ocular allergic inflammation by IL‐33‐ST2 signaling pathways.
Dietary flaxseed oil decreases interleukin‐1 and alpha‐smooth muscle actin in a rat bleomycin‐induced fibrosis model
Authors: Choi Seojin , Lin Dingbo , Ransom Brian , Molteni Agostino , Herndon Betty , Wang Weiqun , Baybutt Richard
Published: June 2021

Dietary fish oil, containing long chain ω‐3 fatty acids, has been previously found to protect against monocrotaline‐induced pulmonary fibrosis. This study is investigated the effects of ω‐3 fatty acids‐enriched flaxseed oil on bleomycin‐induced pulmonary fibrosis. Forty‐two Sprague‐Dawley rats were assigned into seven groups with various doses of flaxseed oil at 0, 2.5, 5, 7.5, 10, 12.5, and 15% (w/w) in the diet for one month prior to treating with bleomycin at 8 U/ kg oropharyngeally. Two weeks after bleomycin treatment, the rats were sacrificed and three proteins including TGF‐β, IL‐1, and α‐Smooth Muscle Actin (α‐SMA), commonly associated with fibrotic inflammation in the lung, were examined by Western blot. The amount of IL‐1 and α‐SMA decreased significantly as the amount of ω‐3 fatty acids increased, whereas TGF‐β did not change significantly. In addition, fatty acid profiles from the liver and lung tissues were analyzed by lipidomics, resulting an increased ratio of ω‐3:ω‐6 fatty acids in both tissues as the amount of flaxseed oil increased. These results indicated that the ω‐3 fatty acids in flaxseed oil inhibited the formation of pulmonary fibrosis in a dose‐dependent manner via anti‐inflammatory mechanisms, which appears associated with the modulated fatty acid composition in the tissues.
Using dose‐equivalence theory to quantify endothelial function
Authors: Lamarre Neil , Tallarida Ronald
Published: June 2021

Currently, evaluation of endothelial function in vitro typically examines a dose‐response to acetylcholine following preconstriction with phenylephrine. However, endothelial modulation of vasoconstrictors, such as angiotensin II, is well known. Our novel methodology derives a dose‐response relationship for this endothelium‐dependent component of action (EDC), which can be fit to a standard four‐parameter dose response equation. Our method is advantageous in that it can provide quantitative information about the EDC of overt vasoconstrictors, and thus does not require the confounding presence of another agonist. In addition, further testing with L‐NAME and IP receptor antagonists can provide information about the agonist‐specific mechanism of the EDC. This may prove useful, since the standard method (acetylcholine vasodilatory response) is primarily nitric oxide (NO)‐dependent in rat aorta, and thus is limited to measuring changes in eNOS function or NO bioavailability. In addition, our methodology allows for statistical evaluation of changes in endothelial function in response to disease and/or treatments, with respect to individual agonists. Four endogenous mediators of vessel tone are examined: angiotensin II, endothelin‐1, norepinephrine, and urotensin II. Research supported by NIDA T32 DA07237