bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Platelet Lysates as a Serum Substitute in Renal Epithelial Cell Culture
Authors: Gstraunthaler Gerhard , Rauch Caroline , Feifel Elisabeth
Published: June 2021

Cultured renal epithelia form monolayers of differentiated, polarized cells. On permeable supports, an apical and a basolateral compartment are separated by the cultured epithelium to study epithelial transport in vitro. However, culture conditions and culture media have a significant impact. In this respect, the quality of fetal bovine serum (FBS) seemed to be of major importance. The high lot‐to‐lot variablity of FBS was found to substantially influence the differentiation of epithelial cultures with regard to the generation of a transepithelial electrical resistance (TEER). Thus, batch‐testing of FBS was required. We recently reported on the use of human platelet lysates (PL) as a replacement for FBS. The growth promoting capacity of PL was tested on renal epithelial cell lines, for which differentiation end points are well established. PL support growth, proliferation and differentiation of LLC‐PK1, HK‐ 2 and MDCK cells. In addition, TEER was monitored in filter‐grown LLC‐PK1 and in MDCK II and MDCK I epithelia. Low‐resistance epithelia generated a TEER of 150–250 Ω•cm2 in PL‐media, as seen with FBS, and high‐resistance MDCK I retained their TEER of 8,000 Ω•cm2. PL are a valuable, animal‐derived component‐free substitute for FBS in renal epithelial cell culture with a proven high batch‐to batch uniformity.
Characterization of phosphorylation of NCC at serine 124
Authors: Rosenbaek Lena Lindtoft , Rasmussen Mette Assentoft , MacAulay Nanna , Fenton Robert Andrew
Published: June 2021

LC‐MS/MS‐based phosphoproteomic profiling identified phosphorylation of NCC at S124 (Feric M et al. Am J Physiol Cell 300: C755–770, 2011). We previously showed pS124‐NCC to be predominantly associated with the apical plasma membrane of DCT cells, where its abundance was regulated by AVP and ANGII. Semi‐quantitative 36Cl uptake studies of various NCC mutants in X. Laevis oocytes showed decreased activity of NCC when replacing S124 with Ala. SPAK or OSR1 protein kinases were unable to phosphorylate synthetic peptides corresponding to the region surrounding S124. Novel tetracycline inducible MDCKII cell lines expressing WT‐NCC and various mutants were generated and used for characterization of the S124 phosphorylation site. WT‐cells displayed thiazide‐sensitive 36Cl uptake, whereas a NCC‐S124A mutant demonstrated decreased activity. WT cells showed robust phosphorylation of NCC at T53 and T58 and membrane targeting following hypotonic low chloride stimulation. Treatment of WT cells with PMA decreased total NCC in the plasma membrane, but the ratio of pNCC to total NCC at the membrane increased, suggesting that non‐phosphorylated NCC is preferentially internalized. Funded by the Lundbeck Foundation.
GENISTEIN DIET: EFFECTS ON JEJUNUM FROM THE R117H CYSTIC FIBROSIS MICE
Authors: Polito Sarah , Drumm Mitch L. , Al-Nakkash Layla
Published: June 2021

We have previously shown wild‐type (Wt) C57BL/6J mice fed a genistein‐containing diet (600 mg/kg food, 600G) for 1‐month, have significantly elevated basal levels of jejunal chloride (Cl) secretion (measured as transepithelial short circuit current, Isc) compared to mice fed a genistein‐free diet (0G); a gender‐specific response to 600G, i.e. females produced a significant increase in Cl secretion, whereas no response was observed in males. We fed male and female cystic fibrosis R117H mutant mice, 600G and 0G diets for 1‐month. Basal Isc was unchanged in 600G male mice (40.4 ± 15.2 μA/cm2, n = 7) compared to 0G controls (34.9 ± 7.7 μA/cm2, n = 9). Basal Isc was significantly increased in 600G female mice (39.1 ± 7.4 μA/cm2, n = 3) compared to 0G controls (24.8 ± 3.0 μA/cm2, n = 3). Comparable steady‐state increases in Isc in response to forskolin (10 μm, bilateral) were observed in 600G and 0G male and female mice. There was no effect of the inhibitor of the Na+/K+/2Cl− co‐transporter, bumetanide (100 μm, basolateral), on the forskolin‐stimulated Isc in either 600G or 0G fed male and female mice. Addition of acetazolamide (100 μm, bilateral) to block the HCO3− secretory component, produced an increase in the percentage inhibition in 600G fed males (11.9 ± 3.0 %, n = 7) and 600G fed females (13.7 ± 1.9 %, n = 3), compared to their respective controls. In the male R117H mice there were no changes in jejunal morphology associated with the diet groups (villi length, number of goblet cells per villi, crypt depth and number of goblet cells per crypt). These data suggest that, in male R117H mice, a 4‐week diet treatment with 600G does not provide any benefits towards the overall magnitude of basal or forskolin‐mediated secretion, but in female R117H mice there is improvement in basal Isc with the 600G diet. Sarah Polito was supported by the Midwestern University Summer DO Fellowship Program. Layla Al‐Nakkash was supported by the Soy Health Research Fund.
Pre‐pregnancy BMI Impacts Bone Loss across Pregnancy in Adolescents
Authors: Young Bridget E , McNanley Thomas , Cooper Beth , Witter Frank , Harris Z. Leah , O'Brien Kimberly
Published: June 2021

Pregnant teens may be at increased risk for pregnancy‐induced bone loss. To assess the impact of dietary Ca and vitamin D status on bone loss, 171 teens (13–18 yrs) were followed from 22.3±5.9 weeks of gestation through delivery. Bone quality was assessed up to three times across pregnancy using calcaneus heel ultrasound (US) measures. Vitamin D, calcitropic hormones, and osteoprotegerin (OPG) were assessed at mid‐gestation and delivery. Speed of sound (SOS) at the heel decreased as pregnancy progressed (p<0.001, after controlling for multiple measures). SOS decreased by 8.6±17.0 m/s (over an 11.7±5.6 week interval), which is equivalent to a 0.28±0.54 decrease in T‐score. The change in SOS/week was positively associated with pre‐pregnancy weight (p=0.012) and BMI (p=0.007), such that heavier teens experienced less bone loss. Bone loss did not differ by race, age, gynecological age, or diet (caloric, protein, or Ca intake). The decrease in SOS across pregnancy was not influenced by mid‐gestation or delivery 25(OH)D, calcitriol, or PTH concentrations. Teens who did not lose bone SOS exhibited a trend for higher OPG at delivery (p=0.089). Maternal bone loss was not associated with infant weight, ponderal index, or gestational age at birth. These findings suggest that teens are at risk for pregnancy‐induced bone loss and BMI pre‐conception plays a role in adolescents’ skeletal response to pregnancy.

USDA: 2005‐35200
Choline dehydrogenase polymorphism rs12676 is a functional variation associated with changes in human sperm cell function
Authors: Johnson Amy Rose , Lao Sai , Wang Tongwen , Galanko Joseph , Zeisel Steven
Published: June 2021

Approximately 15% of couples are affected by infertility and up to half of these cases arise from male factor infertility. Unidentified genetic aberrations such as chromosomal deletions, translocations and single nucleotide polymorphisms (SNPs) may be the underlying cause of many cases of idiopathic male infertility. Choline dehydrogenase (CHDH), a mitochondrial enzyme, catalyzes the conversion of choline to betaine, a methyl group donor and organic osmolyte. Deletion of the Chdh gene in mice results in decreased male fertility due to diminished sperm motility; sperm from Chdh−/− males have decreased ATP concentrations likely stemming from abnormal sperm mitochondrial morphology and function in these cells. SNPs have been identified in the human CHDH gene that may result in altered CHDH enzymatic activity. rs12676 (G233T), a non‐synonymous SNP located in the CHDH coding region, is associated with increased susceptibility to dietary choline deficiency and risk of breast cancer. We now report evidence that this SNP is also associated with altered sperm motility patterns and dysmorphic mitochondrial structure in sperm. Sperm produced by men who are GT or TT for rs12676 have 40% and 73% lower ATP concentrations, respectively, in their sperm. rs12676 is associated with decreased CHDH protein in human hepatocytes. We hypothesize that nutritional intervention involving dietary betaine supplementation may improve the function of sperm produced by men with the rs12676 SNP. This research was funded by NIH grants DK055865 and DK056350.

Grant Funding Source: National Institutes of Health
Regulatory T cells: a mechanism for impaired immunity to influenza infection in obese mice?
Authors: Milner J Justin , Paich Heather A , Sheridan Patricia A , Beck Melinda A
Published: June 2021

Obese individuals are at greater risk of morbidity and mortality when infected with pandemic influenza. The underlying mechanism for the increased risk is unknown, although our work in a vaccinated obese population suggests an impaired immune response. Using a mouse model to study the immune response to influenza infection under conditions of diet‐induced obesity, we previously found reduced NK cell activity, impaired dendritric cell function, and failure of CD8+ T cells to respond to virus. What could cause this seemingly global immune failure? One possibility is alterations in the highly suppressive T cell subset, regulatory T cells (Tregs). CD25+Foxp3+ Tregs regulate nearly all aspects of the immune response, making them a plausible candidate for the impaired immunity in obese mice and likely obese humans. Diet‐induced obese and lean mice were infected with a mild dose of influenza A/PR/8/34. Obese mice had a greater number of Tregs in the lungs during the peak of the adaptive immune response. At an increased dose, obese mice had a greater percentage and number of Tregs in mediastinal lymph nodes. Further, Tregs isolated from obese mice were found to be less suppressive and more proliferative than those of lean mice. This data suggests that alterations in Treg distribution and function may be a novel, modifiable mechanism contributing to the impaired immune response and worse outcome to infection in the obese.

Grant Funding Source : NORC DK56350
Teaching Beyond the Classroom
Authors: Sherman Lloyd R.
Published: June 2021

Over a hundred years ago, John Dewey complained that education was static in subject matter, authoritarian in its methods, mainly passive and receptive … and did not go beyond the provision of a fixed and rigid environment. Dewey echoes our current struggle to bring meaning to anatomical education beyond knowledge of anatomy itself. Knowledge of anatomy takes on meaning through a pedagogy of practical experiences. An amalgam ‐‐ content, pedagogy, arrangement of students, and depth of experiences designed ‐‐ makes an Anatomy Curriculum. Activities configured within the cultural milieu are the dynamic agents that give meaning to what anatomy students learn. Methods that arrange individuals into small groups have the greatest potential for engagement; and foster learning, self‐expression, creative problem‐solving, collaborative learning, “professional” behaviors, creative use of resources, mutual responsibility and coordination. Students teaching and learning from each other increases feedback per individual ‐‐ compared to large‐group instruction. Anatomical content formulated in terms of activities ‐‐ configured within a pedagogy that facilitates interactive expression, inquiry, and problem‐solving ‐‐ maximizes individual feedback, the underlying mechanism of learning, and prepares students for success in their occupation, and for life itself.
Early life growth trajectories and future risk for overweight
Authors: Jones-Smith Jessica C , Laraia Barbara , Neufeld Lynnette M , Fernald Lia C.H.
Published: June 2021

Using a newly developed statistical technique, our objective was to test whether key aspects of growth trajectories between 0–24 months of age, such as size, velocity, and timing of peak velocity, were independently associated with overweight in late childhood. We used data from a birth cohort in Mexico (n=586) and the SuperImposition by Translation and Rotation (SITAR) method to estimate infant/childhood (0–24 months) growth trajectories for BMI, height, and weight. The SITAR method uses a nonlinear random‐effects model to estimate the average growth curve and each individual's deviation from this average curve on three dimensions—size, velocity, and timing of peak velocity. In separate models for each of the anthropometric measures (BMI, height, and weight), logistic regression estimated the association between overweight in late childhood and size, velocity, and timing of the BMI/height/weight trajectory. In crude models, relative BMI/height/weight (size) and BMI/height/weight velocity during 0–24 months were each associated with increased odds of overweight. In the mutually‐adjusted models, only relative BMI/height/weight (size), but not velocity, remained statistically significant. We found no evidence that the association between velocity and overweight varied by size. These results are not consistent with the hypothesis that growth velocity in infancy programs future risk for overweight.

Grant Funding Source : This work was supported by pilot funds from the Berkeley Population Center NICHD R21 (HD056581).
Overweight duration and diabetes risk among Filipino women
Authors: Feranil Alan B , Lee Nanette R , Adair Linda S
Published: June 2021

Few studies have examined the influence of the duration, rather than the degree, of overweight on diabetes (DM) risk, especially among Asians. We examined the association of OW (OW=BMI>=25kg/m2) duration with having impaired fasting glucose or type 2 diabetes (IFG/DM = fasting glucose >110mg/dL) and insulin resistance (HOMA‐IR), independent of current BMI, using data from 1,871 women (ages 35–68 y) from the Cebu Longitudinal Health and Nutrition Survey in 2005. Mean HOMAIR was 2.7 and about 9% had IFG/DM. Overweight duration was categorized as >=14 y (19%), 11–13 y (6%), 7–10 y (5%), <7 y (12%), had a history of OW but not currently OW (10%) and never OW (48%). Logistic regression analysis adjusted for current BMI, age and other confounders showed that the odds of having IFG/DM increased with OW duration. Compared to never OW women, odds (95% CI) of having IFG/DM was highest among women with longest OW duration: 5.1 (2.4,10.6) for OW>=14 y, and 3.0 (1.3, 6.97) for OW 11–13 y. We observed a similar pattern for log HOMA‐IR where linear regression coefficients (95% CI) for OW>=14 y and OW 11–13 y were 0.23 (0.10, 0.36) and 0.21 (0.06, 0.36), respectively. With DM as an increasing major cause of mortality and morbidity in low‐ and middle‐income countries like the Philippines, these results underscore the need to pursue programs that prevent chronic OW. R01TW008288
Dermal Carotenoids as Measured by Resonance Raman Spectroscopy as a Biomarker of Response to a Fruit/Vegetable Intervention Study
Authors: Jahns Lisa , Whigham Leah , Johnson LuAnn , Mayne Susan , Cartmel Brenda , Ermakov Igor , Gellermann Werner
Published: June 2021

Dermal carotenoid status may have utility as a biomarker for vegetable and fruit consumption. Resonance Raman spectroscopy (RRS) is a valid, non‐invasive method to assess dermal carotenoids as a biomarker of usual vegetable and fruit intake, but has not been evaluated in response to a whole‐diet intervention. Thirteen outpatient subjects who reported a mean intake of 16 mg total carotenoids/day followed a depletion diet (0.76 mg/d) for 6 weeks, and then consumed a controlled repletion diet based upon the Dietary Guidelines for Americans that contained an average of 62 mg carotenoid/d for 8 weeks. RRS measures of the palm were taken at least 2x week during depletion and 5x week during the repletion phase. To model depletion and repletion, the Glimmix procedure in SAS was used and included a linear term for the depletion phase, linear and quadratic terms for the repletion phase, and subject as a random effect. Subjects’ RRS scores decreased by 35 ±12.5% (p<0.001) during depletion and increased by 380 ±180.0% (p<0.001) during repletion. Depletion and repletion slopes did not differ between subjects (p>0.55), despite there being a broad range of baseline values (49% CV). No subject achieved a maximal value during the 8‐week repletion period. RRS is a non‐invasive method that shows great promise as an objective biomarker of response to changes in consumption of carotenoid‐rich foods by individuals.
The National Cancer Institute's automated self‐administered 24‐hour dietary recall (ASA24)
Authors: Subar Amy F , Kirkpatrick Sharon I , Mittl Beth , Zimmerman Thea P , Thompson Frances E , Bingley Christopher , Willis Gordon , McNutt Suzanne , Potischman Nancy
Published: June 2021

Extensive evidence indicates 24‐hour recalls (24HR) provide high‐quality dietary data; multiple administrations can be used to model usual intakes. Traditional 24HR, however, are expensive and impractical for large‐scale research as they rely on trained interviewers. To address this, we developed ASA24, a web‐based tool that enables multiple, automated, self‐administered 24HR. It consists of a Respondent website to collect recall data and a Researcher website to manage study logistics and obtain analyses. The Respondent website is modeled on USDA's interviewer‐administered Automated Multiple Pass Method. Unique to the Respondent site are multi‐level food probes to obtain details on food types and amounts. A dynamic user interface, including a talking, animated guide, facilitates finding foods and beverages, jogs respondents’ memories, and assists in estimating portion sizes from multiple images. Resulting data files include food code, nutrient, and food group variables for each day and food reported. Optional modules collect data on location of meals, who one ate with, TV/computer use during meals, and supplement intake. ASA24 will be compared to interviewer‐administered 24HR in a large sample of adults and validated in a small feeding study. Converting interviewer‐administered 24HR to a self‐administered tool makes the collection of high‐quality dietary data feasible and economical. Funding: NIH.
CX3CR1 regulates intestinal macrophage homeostasis, bacterial translocation and colitogenic TH17 responses in mice
Authors: Medina-Contreras Oscar , Geem Duke , Laur Oskar , Williams Ifor R , Lira Sergio A , Nusrat Asma , Parkos Charles A , Denning Timothy L
Published: June 2021

The two most common forms of inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, affect approximately 1 million people in the United States. Uncontrolled APC reactivity toward commensal bacteria is implicated in the pathogenesis of the disease. A number of distinct APC populations exist in the intestinal lamina propria (LP), but their relative contributions to inflammation remain unclear. Here, we demonstrate important roles for the chemokine receptor CX3CR1 in maintaining LP macrophage populations, preventing translocation of commensal bacteria to mesenteric lymph nodes (mLNs), and limiting colitogenic Th17 responses. CX3CR1 was expressed in resident LP macrophages but not DCs. LP macrophages were decreased in two strains of CX3CR1‐knockout mice and in mice deficient in CX3CL1. All these knockout strains displayed markedly increased translocation of commensal bacteria to mLNs. Additionally, the severity of DSS‐induced colitis was dramatically enhanced in the knockout mice as compared with controls. Disease severity could be limited by either administration of neutralizing IL‐17A antibodies or transfer of CX3CR1‐sufficient macrophages. Our data thus suggest key roles for the CX3CR1/CX3CL1 axis in the intestinal mucosa; further clarification of CX3CR1 function will likely direct efforts toward therapeutic intervention for mucosal inflammatory disorders.
Conduction abnormalities in metabolically stressed CD36 deficient mouse
Authors: Sulkin Matthew S , Pietka Terri A , Abumrad Nada A , Efimov Igor R
Published: June 2021

CD36 facilitates myocardial uptake of fatty acids (FA), and has recently been shown to be involved in the regulation of intracellular Ca2+ dynamics in both non‐electrically excitable cells and cardiomyocytes. The heart derives the majority of its energy from FA oxidation and during fasting as glucose utilization diminishes, the heart has an even greater reliance on FA. We investigated whether CD36 dysfunction would accelerate metabolic changes during fasting (18 hrs) that would lead to electrophysiological abnormalities. Conscious non‐anesthetized wild type (WT) (n=5) and CD36 knock out (CD36 KO) (n=5) mice were placed in a restraining device to obtain a baseline ECG before and after fasting. Following fasting, hearts were excised, Langendorff perfused, and subjected to several pacing protocols while being optically mapped. Before fasting, RR, PR, and QTc values were not significant (NS) (p>.05) between groups. After fasting, WT mice displayed a NS increase in RR (75.7 ± 1.0 to 85.4 ± 2.9 ms) and PR (32.8 ± 0.9 to 36.2 ±0.9); whereas, CD36 KO mice showed a significant increase in RR (78.2 ± 1.2 to 120 ± 7.5 ms) and PR (35.9 ±0.9 to 58.7 ± 3.8). CD36 KO mice exhibited a significant increase in the effective refractory period of the atrioventricular (AV) node, ventricular action potential duration, cycle length with which Wenchebach block was observed, and a NS increase in AV‐delay.

This work was supported by NIH Grants P01‐HL057278, R01 DK33301.
Growth of Triple Negative Human Breast Cancer Cells is Regulated by the OGF‐OGFr Axis
Authors: Zagon Ian S , Porterfield Nancy K , McLaughlin Patricia J
Published: June 2021

Breast cancer is the most common cancer, and the leading cause of cancer death, in females worldwide. Triple negative breast cancer (TNBC) accounts for 10–20% of all breast cancers. There are no targeted therapies for TNBC. The opioid growth factor (OGF) and its receptor (OGFr) is a tonically active inhibitory pathway that regulates normal and neoplastic cell proliferation, and we hypothesize that the OGF‐OGFr axis is a determinant of the progression of TNBC. Both OGF and OGFr were detected in TNBC human cell line MDA‐MB‐231 by immunohistochemistry. Receptor binding assays revealed specific and saturable binding (Bmax = 8.5 ± 1.8 fmol/mg protein; Kd = 4.1 ± 1.1 nM). Exogenous OGF depressed growth by 19 to 28% beginning at 24 hr and persisting to 120 hr. Growth inhibition was dose‐dependent (10−4M to 10−10 M), reversible, and receptor‐mediated, and suppression of cell proliferation was dependent on RNA and protein synthesis. Other endogenous and synthetic opioids, including those targeting μ, δ, and κ opioid receptors, did not alter growth at a concentration of 10−6 M. Cells with a knockdown of OGFr using siRNA did not respond to OGF. A complete blockade of the OGF‐OGFr axis with the opioid antagonist naltrexone increased cell number, indicating that this peptide‐receptor complex is critical in maintaining homeostasis of cell proliferation. These data show that the OGF‐OGFr system is present and functioning as a native biological regulator of human breast cancer, and will be important in designing treatment strategies for this group of deadly neoplasias. Supported by the Paul K. and Anna E. Shockey Family Foundation.
Inhibitory effect of adiponectin on breast cancer cell growth: evidences of the crucial role of ERα expression
Authors: Mauro Loredana , Pellegrino Michele , De Amicis Francesca , Giordano Cinzia , Andò Sebastiano
Published: June 2021

Adiponectin (adipo) has emerged as a possible link between obesity and breast cancer. Here, we demonstrated that adipo inhibited proliferation in ER‐negative cells, while stimulated growth in MCF‐7 cells. Adipo transactivated ERα in MCF‐7 cells and upregulated mRNA and protein content of the estrogen target gene cyclin D1 (CD1) involved in cell cycle progression. In contrast, in MDA‐MB‐231 cells adipo induced a downregulation of CD1 expression. The latter findings fitted with the opposite effects of adipo on CD1 promoter elicited in the two different cell lines. Mutagenesis studies and ChIP assays revealed that the Sp1 motif, present on CD1 promoter, was important for the modulatory role of adipo on CD1 promoter activity. In the presence of ERα, ChIP analysis revealed an increase in Sp1/ERα complex, concomitant with an enhanced Pol II and pCAF recruitment, addressing the involvement of an activator complex that mediated the adipo‐induced transcriptional activation of CD1. In contrast, in the absence of ERα, adipo exposure increased the recruitment of corepressors SMRT and NCoR and the association of HDAC‐1, that correlated with a significant decrease in Pol II on Sp1 site. Thus, we propose that the possible mechanism through which adipo may modulate CD1 expression affecting breast cancer growth is tightly related to the estrogen receptor dependency.

The research has been supported by AIRC MFAG 6180.
Diesel particulate matter (DPM) induces receptor for advanced glycation end‐products (RAGE) expression by pulmonary macrophages
Authors: Curtis Cameron S. , Earley Tyler D. , Reynolds Paul R.
Published: June 2021

RAGE is a cell‐surface receptor implicated in mechanisms of sustained pulmonary inflammation. In the present study, we tested the hypothesis that diesel particulate matter (DPM) up‐regulates RAGE in pulmonary macrophages (PMs) and influences infammation. Using real time RT‐PCR, we demonstrate that RAGE was up‐regulated in immortalized mouse macrophages (RAW) exposed to DPM for four hours. Because DPM increased RAGE expression, we exposed primary AMs isolated from RAGE null and wild type Balb/C mice to DPM. Immunohistochemistry using Mac‐3, a macrophage‐specific antibody, ensured a pure cell population. Utilization of a reporter vector containing NF‐kB response elements upstream of the luciferase gene revealed that NF‐κB‐mediated inflammation in cells exposed to DPM is mediated in part by RAGE. Lastly, a PCR based cDNA amplification approach revealed that TNF‐α, IL‐4, 5, 12, and 13 were all up‐regulated following DPM exposure which may occur in part through RAGE signaling. Collectively, these data offer insights into potential mechanisms whereby RAGE influences macrophage‐mediated inflammation exacerbated by DPM exposure. Further research may demonstrate RAGE signaling as a potential target in the successful treatment of inflammatory lung diseases. This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).
Evidence for proliferation of airway‐delivered donor type II cells in lungs of recipient mice following intratracheal bleomycin
Authors: Wang Ping M. , Martin William J
Published: June 2021

As a model of cell‐based therapy for acute lung injury, we used airway delivery of donor alveolar type II (AT II) epithelial cell into recipient mice after intratracheal instillation of bleomycin (3.33 U/kg). We harvested and purified donor AT II cells from wild type or GFP healthy male adult murine lungs and airway delivered 10^6 cells into female adult recipient mice at day 4 post‐bleomycin. Lungs were isolated and sectioned at day 6 and 18–21 following post bleomycin. We monitored the donor cells pre‐labeled with DiI or with GFP expression in the recipient lungs and identified AT II cells by immunostaining with surfactant protein C (SPC) using microscopy. We found GFP expression in lung tissue to become more diffuse from day 2 to 14 after airway delivery of GFP donor AT II cells. There was co‐expression of ki67, a marker for proliferation, and Dil or GFP labeled AT II cells identified by SPC, indicating the ability of donor AT II cells to proliferate in recipient lungs. Furthermore, we found evidence of adherens junctions (stained with E‐cadherin) between donor AT II cells and recipient alveolar cells labeled with SPC. These results suggest that donor AT II cells following airway delivery proliferate and incorporate within alveolar structures in bleomycin‐injured lungs providing evidence for these cells to act as repair cells during acute lung injury.
LXR agonists protect against saturated fatty acid‐induced cytotoxicity in liver cells
Authors: Wang Dong , Wei Yuren , Pagliassotti Michael
Published: June 2021

Free fatty acid‐induced toxicity is a characteristic feature of obesity, type 2 diabetes and non‐alcoholic fatty liver disease. Long chain saturated fatty acids, including palmitate and stearate, are particularly cytotoxic. Diversion of saturated fatty acids into the triglyceride pool reduces saturated fatty acid‐induced cytotoxicity in CHO cells and β‐cells. LXR agonists and downregulation of Phosphatase and Tensin Homologue Deleted on Chromosome 10 (PTEN) promote triglyceride storage in the liver. In the present study we examined the role of LXR agonists and PTEN in saturated fatty acid‐induced cytotoxicity in HepG2 and H4IIE liver cells. The presence of the LXR agonists, TO901317 or GW3965, increased liver cell triglyceride stores and reduced palmitate‐ (250 μM) and stearate‐ (250 μM) mediated ER stress and apoptosis in both cell types. Palmitate and stearate increased the expression of PTEN, whereas the unsaturated fatty acids, oleate and linoleate (250 μM), reduced PTEN expression in both cell types. SiRNA‐mediated knockdown of PTEN did not increase liver cell triglyceride stores nor did it reduce palmitate‐ or stearate‐induced ER stress or apoptosis. These results suggest that LXR‐mediated triglyceride storage protects against saturated fatty acid‐induced cytotoxicity in these liver cell models.
Ethanol and hydrogen peroxide modify the expression of EGFR‐Tyr845 in hepatic cells WRL68
Authors: Rodríguez-Fragoso Lourdes , Ayala Elizabeth Alvarez , Esparza Jorge Reyes
Published: June 2021

Our aim was to study the effect of the ethanol and hydrogen peroxide on EGFR expression and their phosphorylation at Tyr845 in the hepatic cells (WRL68). For the study, we used the WRL68 cells cultured in MEM supplemented with 10% fetal bovine serum, 2mol/L glutamine, 1% non‐essential amino acid. Cells were kept at 37°C, under an atmosphere of 5% CO2. The cells were treated with ethanol at concentrations of 5, 12, 25, 50, 100, 200, 400 and 800 mg/dL, and hydrogen peroxide at concentrations of 0.34, 3.4, 34, 340, 3400 ug/dL. The EGFR expression was analyzed by WB and the phosphorylation was detected by IP and WB. Ethanol and hydrogen peroxide did not modify EGFR expression at 24 h to exposure. However ethanol decreased the EGFR expression (20–50%) before to 24h to exposure and phosphorylation at Tyr845 (40%). While hydrogen peroxide induced changes in EGFR expression (10–50% less) and phosphorylation at Tyr845 was inhibited (50%). In conclusion, ethanol and hydrogen peroxide diminish EGFR expression and phosphorylation at Tyr845 in hepatic cells WRL‐68.
β‐Catenin suppression induces CYP2b10 expression by activating CAR activity
Authors: Yang Jing , Misse Amalea , Monga Satdarshan P
Published: June 2021

Constitutive androstane receptor (CAR) is a nuclear orphan receptor. The translocation of CAR from cytoplasm to nucleus triggers the expression of its downstream targets, cytochrome P450 (CYP) enzymes, which play a critical role in the metabolism of drugs and xenobiotics. A potent synthetic inducer, 1,4‐bis‐ [2‐(3,5, ‐dichloropyridyloxy)] benzene (TCPOBOP), induces the activity of CAR. Once stimulated, CAR increases the expression of CYP genes. Activation of CAR also induces non‐compensatory liver growth, and it has been well established that wnt/β‐catenin signaling pathway is a key determinant in liver growth and regeneration, however, the interplay between β‐catenin and CAR signaling is not yet clear. In the current study, we investigated the interaction between β‐catenin and CAR. CAR reporter assay showed that knocking down β‐catenin by siRNA increased CAR transcriptional activity, and this induction effect was more potent than treatment with TCPOBOP. Further, we found that in liver tissue of liver specific β‐catenin knockout mice, CYP2b10, a downstream target of CAR, was up‐regulated in both mRNA and protein level. We conclude that CAR is a regulator of β‐catenin activity, while knocking down β‐catenin serves as a negative feedback to CAR, which further induces the expression of its downstream target, CYP2b10. Funded by 1R01DK62277 and 1R01CA124414 to SPM
Cell proliferation in liver in response to iron overload is dependent on β‐catenin in male mice
Authors: Monga Satdarshan , Misse Amalea , Ramos Emelio , Ganz Tomas
Published: June 2021

Iron overload as observed in diseases such as hemochromatosis is associated with development of hepatocellular cancer (HCC). Since β‐catenin signaling is known to be a key molecular mechanism in HCC, we interrogated the impact of chronic iron‐containing diet on mice with normal or absent β‐catenin in the hepatocytes. Male and female β‐catenin conditional knockouts (KO) or littermate controls (Con) were fed high iron diet for 3 months. Male and female Con and female KO tolerated the regimen, while male KO showed signs of morbidity. At time of harvest, the liver weight to body weight ratio of KO males was 50% of controls. Serum biochemistry showed only a mild elevation of serum bilirubin in KO. Histological analysis showed increased apoptotic hepatocytes and increase in CD45 cells in KO. There was no increase in fibrosis. KO male mice show 1.6‐fold more liver iron (p=0.012), lower serum iron and similar hepcidin and Id1. There was about 64‐fold increase in Saa‐1 (p=0.05) coinciding with inflammation. All controls & KO females showed an increase in cell proliferation and number of cyclin‐D1‐positive hepatocytes, while KO males showed absent cell proliferation and cyclin‐D1. Thus, reactive proliferation in mice in response to excess iron may be a reparative response and is dependent on β‐catenin‐cyclin‐D1 axes. Eventually, however this response may be a mechanism of neoplastic transformation in males.
The nucleolinus: a disappearing, forgotten, and (maybe) misnamed organelle
Authors: Alliegro Mark Collin
Published: June 2021

Many of the cell components we study today were discovered more than a century ago. Some have been renamed due to a newer understanding of their physiology or composition, and the old terminology abandoned. It is unusual, however, to find a structure that has not been renamed but simply forgotten. This appears to be the case for the nucleolinus (NLi), discovered at least 150 years ago and studied by Agassiz, Haekel, Montgomery, and others until it virtually disappeared from the literature in the early 1970s. The NLi is an RNA‐rich compartment, closely apposed to or embedded within the nucleolus. It has been identified in invertebrate oocytes, mammalian and amphibian epithelial cells, neurons, and several transformed cell lines. The NLi was thought to have a role in cell division, but with so little knowledge of its composition and no molecular markers available for its study, it has been difficult to address this hypothesis. Recently, a method has been developed to isolate NLi from oocytes of the surf clam, Spisula solidissima. This has allowed for biochemical analysis and the generation of specific probes. The dynamics and macromolecular composition of the NLi reveal a unique cellular compartment with functional links to the nucleolus on one hand, and the cell division apparatus on the other. Although the NLi may be a forgotten organelle, these new observations suggest it deserves renewed attention.
Regeneration of the Drosophila epidermal barrier after wounding
Authors: McGinnis William , Juarez Michelle
Published: June 2021

An epidermal wound provides signals that initiate a variety of localized responses that in favorable circumstances results in regeneration and repair of the wound. The Drosophila melanogaster embryonic epidermis provides an excellent system to discover new genes that regulate wound‐healing processes. Using fluorescent epidermal “wound” reporters that are locally activated around wound sites, we have screened almost 5,000 Drosophila mutants for functions required to activate or delimit wound‐induced transcriptional responses to a local zone of epidermal cells. Among the seven new genes required to delimit the spread of wound responses are Flotillin‐2 and Src‐42A. These two genes are also sufficient, when overexpressed at high levels, to inhibit wound‐induced transcription in epidermal cells. One new gene required to activate epidermal wound reporters encodes Dual oxidase, an enzyme that produces hydrogen peroxide. We also find that four biochemical treatments (a serine protease, a Src kinase inhibitor, methyl B‐cyclodextrin, and hydrogen peroxide) are sufficient to globally activate epidermal wound regeneration genes in Drosophila embryos. Our results define new genetic functions, and the interactions among them, that regulate the local transcriptional response to puncture wounds.
Growth Factor Signaling Pathways in Lung Development and Cancer
Authors: Yin Yongjun , Hill Ashley , Ornitz David M
Published: June 2021

Lung mesenchyme is a critical determinant of the shape and size of the lung, the extent and patterning of epithelial branching, the formation of the pulmonary vasculature, and the formation of mesenchymal components of the adult lung. Fibroblast Growth Factor 9 (FGF9) is expressed in developing lung epithelium and mesothelium and has an essential primary role in regulating mesenchymal growth and differentiation through signaling to mesenchymal FGF receptors (FGFRs) 1 and 2.

Pleuropulmonary Blastoma (PPB) is a pediatric lung cancer syndrome with onset in late gestation and early childhood. PPB is a unique lung cancer in that its cellular origins are derived from lung mesenchyme. Germline loss‐of‐function mutations in Dicer1, an essential gene for the production of microRNAs, predisposes to PPB in humans. In this talk I will discus mechanisms by which FGF9 regulates mesenchymal growth and differentiation, evidence that Fgf9 may be regulated by microRNAs during lung development, and evidence that FGF9 may be an important factor for the initiation PPB.

This work is funded by a grant from the March of Dimes foundation.
Integrating MALDI Imaging Mass Spectrometry and Shotgun Proteomics for a Systems Biology Understanding of Congenital Heart Valve Disease
Authors: Angel Peggi M , Mettler Brett M , Bichell David P , Baldwin H. Scott , Caprioli Richard M.
Published: June 2021

Our objective is to define protein regulation and interaction involved in cellular microenvironments of congenital aortic valve stenosis (CAVS) towards therapies delaying valve deterioration. We are using matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) to identify pathology related protein distributions within CAVS leaflets. Protein interaction networks (PINs) are constructed around MALDI IMS protein nodes from proteomic data produced using multidimensional protein identification technology (MudPIT). We illustrate the technique with a case study of bicuspid CAVS. MALDI IMS showed 39/44 novel proteins following patterns of extracellular matrix disarray. MudPIT identified 2238 proteins with main pathways of focal adhesion, leukocyte transendothelial migration, and complement system, indicating a primary component of immune response. Spatially defined PINs showed thymosin beta 4 involved in cytoskeletal organization within areas of glycosaminoglycans. Thymosin beta 10 overlapping with S100A6 expression in areas of elastin was implicated in regulation of adhesion and structural development. These studies may be applied to other areas of cardiovascular biology.
Dynamic Imaging of VEGF Relative to the ECM and its Effects on Endocardial Cell Behavior During Cardiovascular Morphogenesis
Authors: Rongish Brenda J , Aleksandrova Anastasiia , Lansford Rusty , Little Charles D , Czirok Andras
Published: June 2021

Vascular endothelial growth factor (VEGF) interacts with extracellular matrix (ECM) constituents; setting up morphogenetic gradients and regulating ligand presentation to cells. We are investigating the behavior of ECM‐interacting VEGF165 and soluble VEGF121 during endocardial assembly in quail. We use fluorescent anti‐VEGF antibodies and VEGF receptor bodies to visualize VEGF. ECM constituents are visualized by microinjection of fluorescent antibodies against fibronectin and fibrillin‐2. All experiments are performed in Tie1::H2B‐YFP quail embryos (nuclear labeling of endocardial cells). Endocardial cell, ECM, and VEGF tissue components are recorded using time lapse imaging, with their relative movements analyzed using tracing and image analysis techniques. During formation of the endocardium, ECM components move in a coordinated manner with endocardial precursors (tissue motion). However, neither VEGF over‐activation (hypervascularization) or inactivation (blocking of endocardial differentiation) halts the tissue movement that results in formation (albeit abnormal) of a midline heart tube. Our results further the current understanding of the coordinated behavior of VEGF and ECM constituents of embryonic tissues and their collective role in cardiovascular morphogenesis. HL085694 (BJR); HL087136 (AC); Mathers Foundation (BJR, AC, CDL); AHA predoctoral award (AA).
Mitochondrial stress and metabolic dysfunction in skeletal muscle
Authors: Muoio Deborah
Published: June 2021

Application of targeted metabolic profiling tools has led to our finding that both diet‐induced and genetic forms of insulin resistance are accompanied by high rates of “incomplete” fat oxidation, perturbations in carnitine metabolism and acylcarnitine production, and impaired switching from fatty acid to carbohydrate substrate during the fasted to fed transition. These metabolic perturbations and the accompanying glucose intolerance are mitigated by genetic, nutritional or pharmacological maneuvers that alleviate mitochondrial carbon load. The role of mitochondrial stress as a potential cause of insulin resistance will be discussed.
The unfolded protein response in health and disease
Authors: Walter Peter
Published: June 2021

Virtually all proteins that eukaryotic cells display on their cell surface or secrete into the extracellular space are folded and assembled in the endoplasmic reticulum (ER). When the protein folding capacity of the ER is exceeded, misfolded proteins accumulate and trigger the unfolded protein response (UPR), an intracellular signaling pathway that reestablishes homeostasis by adjusting ER abundance according to need. The UPR transmits the signal that controls its transcriptional output form the ER lumen to the nucleus. Mechanistic studies have revealed unprecedented ways through which eukaryotic gene expression can be controlled. A key player is Ire1, a bifunctional ER‐resident transmembrane kinase/endoribonuclease, that senses misfolded proteins in the ER lumen and initiates a non‐conventional mRNA splicing reaction in the cytosol. Splicing results in the production of a transcription factor that activates UPR target genes. If proper balance between the load and the protein folding capacity in the ER cannot be established, metazoan cells undergo apoptosis. This role in making life/death decisions places the UPR as a potential player at the center of many diseases, including virus infections, protein diseases, diabetes, and cancer. Our current ideas regarding how Ire1 monitors conditions inside the ER lumen will be discussed.
Association of selenoprotein gene expression with pancreatic atrophy in broiler chicks
Authors: Li Dai-Lin , Huang Jia-Qiang , Zhao Hua , Xia Xin-Jie , Wang Kang- Ning , Lei Xin Gen
Published: June 2021

Pancreatic atrophy is one of the classical Se‐deficiency diseases in chicks, but the pathological mechanism remains unclear. Our objective was to replicate this disease to study novel roles and mechanisms of selenoproteins in its pathogenesis. Four groups of day‐old chicks (n=60/group) were fed a corn‐soy basal diet (BD, 14 μg Se/kg) or the BD + all‐rac‐α‐tocopheryl acetate at 50 mg/kg, Se (as sodium selenite) at 0.3 mg/kg, or both for 4 wk. Acinar pancreatic degeneration was seen in chicks fed the BD. Pancreatic length was decreased (P < 0.05) by both Se and vitamin E deficiencies, whereas pancreatic weight was decreased (P < 0.05) by only the Se deficiency. Among 15 selenoprotein genes assayed in pancreas, 11, 9, and 11 genes were affected (P < 0.05) by Se, vitamin E, and their interactions, respectively. Gene expression of 3 key transcriptional factors (Pdx1, Mafa, and Foxa2) for pancreatic development and differentiation was down‐regulated (P < 0.05) by Se or vitamin E deficiency. While pancreatic mRNA levels of a key cell‐proliferation factor (Tgfb2) were decreased (P < 0.05) by the Se deficiency, gene expression of 2 cell death‐related factors (Casp3 and p53) was affected (P < 0.05) by the interactions of the two nutrients. In conclusion, pancreatic atrophy was experimentally induced in chicks to show association of multiple selenoprotein gene expressions with the disease. (NSFC; Chang Jiang Scholars; NIH 53018).
In vivo magnetic resonance spectroscopy of lipid handling in steatotic rat liver
Authors: Janssens Sharon , Jonkers Richard AM , van Riel Natal AW , Nicolay Klaas , Prompers Jeanine J
Published: June 2021

Objective

Examine lipid handling in liver of rats fed with different high‐fat diets using 1H‐[13C] magnetic resonance spectroscopy (MRS) together with oral administration of 13C labeled lipids.

Methods

6 male Wistar rats (11 weeks old; 348 ± 8g) were divided into three diet groups: low‐fat (10% fat, CON), high‐fat lard (45% fat, HFL), and high‐fat palm oil (45%, HFP). After 10 weeks of diet, MRS experiments were performed at baseline, and 4 and 24 h after oral administration of 1.5 g [U‐13C] Algal lipid mixture per kg body weight.

Results

At 4 h after administration of the 13C labeled lipids, 13C enrichment of intracellular liver lipids was similarly increased in all three groups compared to baseline (CON: 0.031 ± 0.017 %; HFL: 0.045 ± 0.022 %; HFP: 0.033 ± 0.013 %), demonstrating that lipid uptake was not affected by the diet regimen. At 24 h, on the other hand, 13C enrichment of liver lipids decreased in CON, whereas in both high‐fat diet groups the 13C enrichment did not change compared to 4 h, indicating a lower turnover of the stored liver lipids.

Conclusion

High‐fat diet feeding did not alter liver lipid uptake in rats, but resulted in a decreased turnover of the lipids stored in the liver. This research was funded by the Netherlands Consortium for Systems Biology (NCSB) which is part of the Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research.
Identification of genetic loci controlling intestinal calcium (Ca) absorption using BXD recombinant inbred (RI) mice fed high or low dietary Ca
Authors: Replogle Rebecca , Wang Libo , Zhang Min , Fleet James
Published: June 2021

Intestinal Ca absorption (Ca Abs) is affected by race and is similar between mothers and daughters suggesting genetic regulation of this trait. However, the impact of genetics on CaAbs and the adaptation of Ca Abs to inadequate dietary Ca intake is not known. We examined the role of genetics on Ca Abs efficiency in 51 lines of BXD RI mice. Mice were fed AIN93G diets with either 0.5% or 0.25% Ca from 4 to 12 wks of age (n=8/line/diet). Afterwards, Ca Abs was measured by Ca45 appearance in the blood 10 min after an oral gavage of 0.1 mM Ca. After correcting for covariates, considerable diversity was observed between the lines in Ca Abs on each diet. Narrow‐sense heritability for Ca Abs was 0.37 and 0.56 in the 0.5% and 0.25% Ca diets, respectively, demonstrating increased penetrance of genetic effects controlling Ca Abs with low Ca diets. Linkage mapping was conducted using both traditional and Bayesian methods. Quantitative trait loci (QTL) affecting Ca Abs were identified that were diet‐specific (0.25%: on Chr 5, 8 (distal), 9, 12, 17; 0.5% on Chr 8 (proximal), 10) and shared (Chr 4, 7). No known Ca regulatory genes were found around these QTL demonstrating the potential of this approach for discovery of new genes controlling Ca Abs. This study provides a foundation for determining genetic regulators that may underlie individual differences in Ca Abs and ultimately dietary Ca requirements. Supported by NIEHS award ES019103.

Grant Funding Source : NIEHS