bluuebunny/crossref_metadata_2025 huggingface dataset:
Higher intakes of low‐fat milk combined with 12 weeks of endurance training does not result in lower fat mass and higher lean mass.
Authors: Roberts Rebekah , Euiler Elizabeth , Ward Wendy , LeBlanc Paul , Tarnopolsky Mark , Roy Brian
Published: June 2021

Previous studies showed that a low‐fat milk intervention combined with resistance training resulted in lower fat mass and higher lean mass in males and females. The purpose of this study was to determine the effects of combining increased low‐fat milk intake with endurance exercise on fat and lean mass. 32 healthy young males were randomized and blindly assigned into one two groups 1) MILK (3 additional servings of low‐fat milk, 750 mL) or 2) CHO (maltodextrin beverage, isocaloric to MILK). All participants completed a 12‐week endurance‐training program. The exercise program consisted of cycling 1 hour per day at ~60% VO2 peak, 5 days per week for 12 weeks. Whole body fat and lean mass were determined using dual‐energy X‐ray absorptiometry (DXA) that was performed before and after training. 23 participants completed the study. Changes in body mass (MILK: 0.39±0.61 kg; CHO: 0.30±0.77 kg), fat mass (MILK: −420.7±522.1 g; CHO: 99.6±794.8 g) and lean mass (MILK: 831.2±509.8 g; CHO: 624.9±434.6 g) were similar between the 2 groups. Findings to date suggest that combining a higher intake of low‐fat milk with 12 weeks of endurance training does not improve changes in body composition. Further analyses are ongoing.

Funding provided by The Dairy Farmers of Canada.
Impact of prolonged mechanical ventilation on diaphragmatic protein synthesis
Authors: Wiggs Michael P , Hudson Matthew B , Smuder Ashley J , Nelson W Bradley , Shimkus Kevin L , Fluckey James D , Powers Scott K
Published: June 2021

Mechanical ventilation (MV) is a life‐saving intervention in patients suffering from respiratory failure. Unfortunately, prolonged MV promotes the rapid development of diaphragmatic atrophy due to increased proteolysis and decreased protein synthesis. These experiments investigated the influence of two different modes of MV on rat diaphragm protein synthesis. Specifically, we examined the mixed protein synthesis rate of diaphragm muscle proteins during controlled MV (CMV) which causes complete diaphragmatic inactivity and pressure support MV (PSV) which permits partial activity of the diaphragm. Our results reveal that compared to spontaneously breathing animals, diaphragm protein synthesis rates are not depressed during the first 6‐hours of PSV but are significantly decreased in animals exposed to CMV. However, the rates of diaphragm protein synthesis are depressed at similar levels in both CMV and PSV animals during 6–12 hours of MV. We conclude that while PSV can initially delay the MV‐induced decreases in diaphragm protein synthesis, this protective effect is rapidly lost after the first 6 hours of MV. NIH RO1 HL087839 (SKP)
Mutant Ataxin‐3 inhibits 3’ phosphatase activity of human polynucleotide kinase 3′‐phosphatase (PNKP)
Authors: Saha Saikat , Chatterjee Arpita , Mandal Santi M , Gao Rui , Hegde Muralidhar L , Hegde Pavana M , Ashizawa Tetsuo , Sarkar Partha S , Hazra Tapas K
Published: June 2021

Ataxin‐3 (ATXN3), a polyglutamine repeat‐containing protein is mutated to abnormally expanded poly Q tracts in Spinocerebellar Ataxia type 3 SCA3 / Machado‐Joseph Disease (MJD). This disease is one of the most common dominantly inherited Ataxia worldwide; the molecular defect is CAG repeat expansion (from 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is poorly understood. Here, we show that, expanded poly Q form of human Ataxin‐3 (Q72), stably associates and inhibits 3’[prime]‐phosphatase activity of PNKP one of the major DNA end processing enzyme in mammalian cell. Activities of other single strand break repair (SSBR) proteins are not affected. A conditionally expressing pathological ATXN3 cells also showed reduced 3’[prime]‐phophatase activity of PNKP and subsequent accumulation of DNA strand breaks in nuclear genomes. Moreover, LA‐QPCR analysis of human MJD patients’ brain samples showed significant accumulation of DNA strand breaks that supported the cause. In summary, selective inhibition of 3’[prime]‐phosphatase activity of PNKP may be the signature mechanism contributing to accumulated DNA damage and pathophysiology of SCA3/MJD.
MutS and DNA dynamics during mismatch recognition and repair.
Authors: Sharma Anushi , Hingorani Manju
Published: June 2021

Mismatch repair (MMR) is essential for correcting base‐pairing errors in DNA. MutS protein recognizes mispaired bases in DNA and signals excision and resynthesis of the mismatched strand. The DNA binding and ATPase activities of MutS are under active investigation‐especially the transient events involved in mismatch recognition and initiation of repair. We utilize transient kinetic methods coupled with fluorescence spectroscopy to determine the mechanisms of action of Thermus aquaticus MutS. Our results indicate a two‐step DNA binding mechanism for MutS, wherein an initial rapid encounter establishes a weak binding equilibrium (KD1 = 3 μM), followed by slow formation of a mismatch‐specific MutS‐DNA complex with the DNA in bent conformation (kconf ~ 20 s−1and KD2 = 5 nM). ATP binding to MutS‐DNA complex (kON = 0.3 μM−1s−1) is followed by two slow steps involving conformational changes in MutS and the mismatch site in DNA (~ 2 s−1) followed by MutS release from the mismatch (~ 0.5 s−1). With detailed kinetic analysis, we are developing a comprehensive view of MutS actions on DNA, and gaining novel mechanistic insights into the mechanism of initiation of MMR.
Arctigenin improves glucose metabolism in line of mighty mice
Authors: Bawany Mohammad Husain , Savage Kathleen
Published: June 2021

Many transforming growth factor β (TGFβ) protein family members signal through the activin receptor IIB in order to impact skeletal muscle. Mice expressing a muscle specific dominant negative activin receptor IIB (dnActRIIB) have muscle hypertrophy, increased lean mass, decreased fat mass, improved glucose metabolism on standard and high‐fat diets, and resistance to diet‐induced obesity. It has recently been discovered that AMPK activation can lead to endurance training like adaptations. The current study examined the ability of Arctigenin, an herbal extract known to activate adenosine monophosphate kinase (AMPK), to change glucose metabolism in dnActRIIB mice. Wildtype and dnActRIIB mice were treated with Arctigenin or saline 5 days per week for 6 weeks. Mice were fasted overnight and a standard intraperitoneal glucose tolerance test was conducted. Treatment with Arctigenin did not alter glucose disposal in wildtype mice. However, Arctigenin treatment improved glucose disposal in dnActRIIB mice. Arctigenin treatment did not alter body weight or muscle weights, and ongoing studies are being performed to determine if Arctigenin stimulates muscle plasticity.
The influence of O‐GlcNAc in the motility of alveolar epithelial cancer cells
Authors: Dias Wagner B. , Donadio Joana L. , Medina Ana C. B. , Todeschini Adriane R.
Published: June 2021

It's already well known that cancer cells increase glucose and glutamine uptake. The Hexosamine Biosynthetic Pathway (HBP) is highly dependent on both molecules and recent showed that cancer cells upregulate the HBP, including increased O‐GlcNAc levels, that we confirmed by immunohistochemistry. Two enzymes are responsible for the addition and removal of O‐GlcNAc (OGT and OGA, respectively). The transition of epithelial cells to mesenchymal cells (EMT) is crucial in metastasis of carcinoma cells by losing cellular polarity and increasing the cellular motility. In this work we used two approaches (Wound Assay and Colloidal Gold Assay) to investigate the role of O‐GlcNAc in cellular motility. Here, we showed that increase of O‐GlcNAc levels through OGA silencing or inhibition, increase the cell motility in alveolar epithelial cancer cells. Additionally, the reduction of O‐GlcNAc levels by OGT silencing showed a significant decrease the cell motility. In addition we showed increased levels of OGT and OGA during EMT induced by TGF‐β. These results suggest a role of O‐GlcNAcylation in the process of cell motility and may have implications in tumorigenesis. FAPERJ, CNPq
Effects of Captan, a fungicide, on mammalian slow‐twitch muscle
Authors: Darwich Walaa , Ramadan Wiam , Khachfe Hassan , Akhdar Hanane , Rammal Wassim , Koubaissi Marwan , Sabra Lina , Bach Veronique , Joumaa Wissam H
Published: June 2021

The population exposure to pesticides is dramatically increasing in the last few decades because of the widespread uncontrolled use of pesticides especially in the developing countries. In our study, we examine the effect of 6 mg/kg of Captan, a fungicide, mixed with diet, on body weight, muscle mass, proteins content and contractile response of slow‐twitch skeletal muscles (soleus). As compared with controls placebo animals, 2 weeks of treatment induces a significant increasing in body weight (15%) and muscle mass (14%) which could be due in part to the increasing in soluble and Myofibrillar proteins content. Moreover, our results show that Captan treatment induces a decreasing by 15% in the expression of MHC I isoforms (Control: 88.7 ± 3.2 ; Captan: 75.3 ± 2.3) and an increasing in the expression of MHCII isoforms (Control: MHC IIa: 1.09 ±0.61; MHC IIb: 10.34±2.33; MHC IIx/d: 1.28±1.02; Captan: MHC IIa: 9.79±1.61; MHC IIb: 17.91±1.40; MHC IIx/d: 12.28±1.72; p<0.05; n= 10). Thus, this study shows that treatment by Captan mixed with diet can induce an effect in myofibrillar proteins of slow‐ twitch mammalian skeletal muscles by shifting in the expression of slow MHC type into fast MHC type.
Heat shock protein 70 overexpression attenuates botulinum neurotoxin type A induced skeletal muscle atrophy in rats
Authors: Houston Fraser Edwards , Hain Brain Allen , Dodd Stephen
Published: June 2021

Heat shock protein 70 (Hsp70) is a ubiquitous, cytoprotective protein that is up‐regulated in response to cellular stress. Botulinum neurotoxin type A (BoNT/A) administration causes a chemical denervation which leads to skeletal muscle atrophy in rats. Previous work in skeletal muscle has shown that Hsp70 inhibits both NF‐κB and FOXO activation in response to certain stimuli. During periods of atrophy, Hsp70 levels are decreased, while NF‐κB and FOXO activity are increased and required for atrophy in some models. Therefore, the purpose of the current study was to determine if electrotransfer of Hsp70 into the soleus muscle of rats, prior to BoNT/A administration, is sufficient to inhibit atrophy and NF‐κB/FOXO activation. BoNT/A administration induced ~70% atrophy of the soleus. BoNT/A + Hsp70 administration elicited only ~50% atrophy. Hsp70 prevented the BoNT/A‐induced increase in total ubiquitin conjugated proteins. Furthermore, BoNT/A‐induced NF‐κB transactivation was abolished by Hsp70 and levels of FOXO3a‐phosphorylation were maintained. In conclusion, while Hsp70 decreases total ubiquitin conjugated proteins and attenuates a portion of the observed skeletal muscle atrophy, these data suggest that pathways other than NF‐κB/FOXO may predominate in control of protein degradation. Supported by Ipsen Pharmaceuticals.
Effect of EUK‐134 on Insulin Signaling and Morphology Alterations by 7 Days of Hindlimb Unloading in the Rat Soleus
Authors: Lawler John M , Lee Yang , Hord Jeff M , Botchlett Rachel E , Fluckey James D
Published: June 2021

Removal of mechanical loading causes rapid and profound atrophy of skeletal muscle due to an increase in contractile protein degradation coupled with a decrease in protein synthesis. Emerging data suggest that oxidative stress may contribute to unloading‐induced translocation of nNOS from the sarcolemma, FoxO3a activation, and fiber atrophy. Insulin resistance can altered in unloaded skeletal muscle and could contribute to atrophy; however, the mechanisms are unknown. We hypothesized that oxidative stress may contribute to insulin signaling and morphological changes in the rat soleus with 7 days of unloading. F344 rats were divided (n=9/gr) into ambulatory control (CON), hindlimb unloaded (HU), and HU + EUK‐134 (HUE) groups. The superoxide dismutase/catalase mimetic EUK‐134 was injected daily (3 mg/kg/d) beginning 24 hours prior to HU. HU significantly decreased muscle weight/body weight ratio (18%) and fiber cross‐sectional area (−32%) in the soleus, effects ameliorated by EUK‐134. GLUT4 co‐localization with the membrane protein β‐sarcoglycan significantly increased with HU; while HUE muscles were not different than CON. Akt phosphorylation (Ser473) was elevated by HU, with a lesser effect in HUE. Our data suggest a complex relationship between HU‐related alterations in insulin signaling and oxidative stress that may be independent of protective effects against atrophy. Supported by NASA (NNX13AE45G), NIH (AR054084).
Acute high‐intensity exercise increases the nuclear abundance of PGC‐1α in equine skeletal muscle
Authors: Mukai Kazutaka , Kitaoka Yu , Ohmura Hajime , Takahashi Toshiyuki , Matsui Akira , Aida Hiroko
Published: June 2021

Peroxisome proliferator‐activated receptor gamma coactivator (PGC) ‐1α is a transcriptional coactivator that plays a key role in coordinating mitochondrial biogenesis. It was recently shown in rodent and human skeletal muscles that acute exercise causes a shift in the subcellular localization of PGC‐1α from the cytosol to the nucleus, allowing PGC‐1α to coactivate transcription factors and increase mitochondrial gene expression, but there are no equine data. Our purpose was to examine PGC‐1α protein content in cytosolic and nuclear fractions of equine skeletal muscle following an acute bout of high‐intensity exercise. Ten Thoroughbred horses ran at 115% of maximal oxygen consumption until exhaustion and needle biopsy samples were obtained from the gluteus medius muscle before and immediately after exercise, and after 4 and 24 h of recovery. In response to acute exercise, nuclear PGC‐1α protein significantly increased after 24 h of recovery (p<0.05), but cytosolic PGC‐1α protein was unchanged compared with rest. These results suggest that acute exercise causes translocation of cytosolic PGC‐1α protein to the nucleus to activate mitochondrial biogenesis in horses.
Excitatory effects of neurokinin 2 and bombesin receptor peptide agonists vivo in urinary tract of rats with voiding dysfunctions
Authors: Kullmann Florenta Aura , Zheng Jihong , Wells Grace , McKenna David , Burgard Ed , Thor Karl
Published: June 2021

Bladder function was examined in anesthetized and awake adult female Sprague Dawley rats. Voiding dysfunction models included STZ‐induced diabetic (streptozotocin; STZ; 65mg/kg i.p., 9–20 weeks post treatment), polyuria (5% sucrose‐fed; >;3 weeks post treatment) and spinal cord injured (SCI; T8‐T9 cut; acute or 3 days – 6 weeks post SCI) rats. Telemetry transmitters were implanted into polyuria group to measure bladder pressure in freely moving rats. [Lys5,MeLeu9,Nle10]‐NKA(4–10) (LMN‐NKA), a neurokinin 2 receptor (NK2R) agonist, gastrin‐releasing peptide (GRP), a bombesin receptor 2 (BBR2) agonist, and neuromedin B (NMB), a BBR1 agonist, were delivered via jugular vein catheters.

In urethrane anesthetized STZ and control rats, LMN‐NKA (0.1–100μg/kg), GRP (0.1–300μg/kg) and NMB (0.1–300μg/kg) dose dependently increased bladder contraction (BC, up to 4x) and micturition frequency. In STZ rats, LMN‐NKA reduced residual volume by half. In acute anesthetized SCI rats, LMN‐NKA (>;10μg/kg) induced urine release (voiding efficiency ~40%). In awake polyuria rats, LMN‐NKA and GRP (1–100μg/kg) induced BC that was often associated with voiding. In awake SCI rats, LMN‐NKA (100μg/kg) and GRP (100, 300μg/kg) triggered voiding in 25 – 80% of rats.

Activation of NK2R and BBRs enhanced or triggered voiding in naïve rats and in several models of voiding dysfunction including STZ and SCI rats.
Role of hydrogen sulfide (H2S) on the ventilatory responses to hypercapnia
Authors: da Silva Glauber S F , Kwiatkoski Marcelo , Giusti Humberto , Glass Mogens L , Branco Luiz G S
Published: June 2021

Hydrogen sulfide (H2S), an endogenous gasotransmitter, is predominantly produced by the enzyme cystathionine‐β‐synthase (CBS) within the central nervous system. There is no evidence showing the role of H2S in the ventilatory responses to hypercapnia. The objective of this study was to verify a possible role of H2S on the ventilatory responses to hypercapnia. Adult male Wistar rats were submitted to the stereotaxic surgery to implant a guide cannula into the fourth ventricle (4V). Pulmonary ventilation (VE, plethismography) and body temperature were measured before and during a 30 min hypercapnic challenge (7% CO2). AOA (CBS enzyme inhibitor) and Na2S (H2S donor) were injected into the 4V during room air and hypercapnia. aCSF was used as vehicle and injected for the control group. Either AOA or Na2S did not affect VE during room air. Ventilatory response to CO2 was attenuated after the injection of AOA, whereas Na2S elicited an enhanced response. In addition, CBS‐H2S pathway seems to be activated during hypercapnia, since H2S levels were found higher, at least in the Nucleus of Tract Solitary (NTS). These results are consistent with the notion that CBS‐H2S pathway is involved in ventilatory response to CO2.
Neurochemical assessment of identified phrenic motor neurons via flow cytometry
Authors: Dougherty Brendan J. , Smith Stephanie M. , Mitchell Gordon S. , Watters Jyoti J.
Published: June 2021

Flow cytometry (FCM) utilizes high powered, laser‐based tools to perform multi‐parametric analyses on cell populations. Although FCM is routinely utilized in immunology and cancer biology fields, it is rarely used in the study of neurobiology. Here, we show a novel use of FCM to identify and isolate phrenic motor neurons, a small, but critical pool of respiratory motor neurons innervating the diaphragm. Rat phrenic motor neurons were retrogradely labeled via bilateral intrapleural injections of fluorescently‐conjugated cholera toxin fragment B (CtB‐AlexaFluor 647). After 72 hours, rats were perfused and the cervical spinal cords dissected, dissociated, fixed in a modified zinc‐based fixative, and stained with DAPI to discriminate intact cells from debris. Using FCM, a clearly defined group of intact cells were identified as CtB‐AlexaFluor 647 positive, corresponding closely with available estimates of phrenic motor neuron numbers in rats. These cells were confirmed to be neurons by co‐staining with the neuronal marker NeuN. Our results demonstrate the feasibility of using FCM to identify, characterize, and sort phrenic motor neurons. Coupling FCM with neurophysiology provides an innovative approach to the study of phrenic motor neuron biology, while lending new insights into mechanisms of spinal respiratory neuroplasticity in models of traumatic or neurodegenerative injury.
Expression of sphingosine‐1‐phosphate receptor subtypes in rat lung microvessels
Authors: Kandasamy Kathirvel , Bezavada Lavanya , Parthasarathi Kaushik
Published: June 2021

Sphingosine‐1‐Phosphate (S1P) plays a major role in lung vascular pathophysiology. While S1P receptor subtypes that mediate the signaling in lung vessels are being defined, the expression of the receptors in these vessels remains unclear. Toward this, isolated blood‐perfused rat lung were pump‐perfused at 14 ml/min with autologous blood. The pulmonary artery, left atrial, and airway pressures were maintained at 10, 3, and 5 cmH2O, respectively. Through a microcatheter inserted via the left atrial cannula, we cleared blood cells from a small lung region and determined expression of S1P receptor 1–4 in microvessels by indirect in situ immunofluorescence. In two lungs for each receptor, we captured images of surface microvessels using a confocal microscope and quantified fluorescence intensity along the wall of the vessels. S1P4 immunofluorescence in venules and capillaries was 76.7±3.7 (n=11) and 27.7±1.8 (n=36), respectively. Immunofluorescence of S1P1, S1P2, and S1P3 in venules was 33.9±3.4 (n=15), 27.0±5.1 (n=10), and 31±2.9 (n=9), respectively, and in capillaries was 15.4±0.7 (n=50),11.8±0.8 (n=50) and 11.4±1 (n=43), respectively. These data revealed that S1P4 immunofluorescence was higher compared to other subtypes (p< 0.001). Thus, we show for the first time that in lung venules and capillaries (1) S1P receptors 1–4 are expressed, and (2) S1P4 may be the predominant receptor subtype. NIH HL75503
Induction of Apoptosis by ER Stress in Lung Alveolar Epithelial Cells (AECs) Is Blocked by Angiotensin 1–7 Through Mas
Authors: Uhal Bruce , Nguyen Hang , Dang MyTrang , Gopallawi Indiwari , Dang Vinh , Ono Shinji , Morimoto Konosuke
Published: June 2021

STUDY OBJECTIVE

Our prior work showed that apoptosis of AECs is regulated by autocrine generation of angiotensin (ANG) II and the counterregulatory ANG1–7. Mutations in surfactant protein C (SP‐C) induce endoplasmic reticulum (ER) stress and apoptosis. This study tested the hypothesis that ER stress‐induced apoptosis of AECs might also be regulated by autocrine ANGII/ANG1–7.

METHODS

A549 cells or primary cultures of human AECs were challenged with MG132 or the SP‐C BRICHOS domain mutant G100S. The ANGII‐generating and –degrading enzymes cathepsin D and ACE‐2 were measured by enzyme assay and western blot. Apoptosis was measured by mitochondrial function, JNK and caspase activation and nuclear fragmentation.

RESULTS

ER stress activated cathepsin D and decreased ACE‐2. Both changes were inhibited by TAPI‐2, a blocker of ADAM17/TACE. Apoptosis of A549 or primary human AECs induced by MG132 or the SP‐C BRICHOS mutant G100S was significantly inhibited by the nonselective ANG receptor blocker saralasin and was completely abrogated by ANG1–7 (both p<0.01). Inhibition by ANG1–7 was blocked by the specific mas antagonist A779.

CONCLUSIONS

These data show that ER stress‐induced apoptosis is mediated by the autocrine ANGII/ANG1–7 system in human AECs and demonstrate effective blockade of SP‐C mutation‐induced apoptosis by angiotensin1–7 through a mas‐dependent mechanism.

Support: PHS HL‐45136
Tet1/3 mediated epigenetic regulations in male germ cell development
Authors: Luk Alfred CS , Ng Shuk Hang , Tao Jiajie , Chan Wai‐Yee , Lee Tin‐Lap
Published: June 2021

Previous epigenetic studies focused mainly on 5‐methylcytosine (5mC), emerging evidence suggests that 5‐hydroxymethylated cytosine (5hmc) could be another functional epigenetic modification in eukaryotic genome as demonstrated during differentiation of neurons and embryonic stem cells. However, its roles in male germ cell development remain unexplored.

Our preliminary data demonstrated decreasing expression of ten‐eleven translocation (Tet) protein family after serial passages of C18–4 cells (mouse type A spermatogonia), suggesting potential correlation on epigenetics and aging. Moreover, we showed a higher expression level of Tet1/3 in stem‐cell like C18–4 cells than in somatic TM4 (mouse Sertoli) cell. Our dot‐blot results also demonstrated a higher genomic 5hmC content in C18–4 than in TM4 cells, implying possible stemness‐related regulation of 5hmC. Taken together, these data revealed a potential regulation of 5hmC/Tet in male germ cell development, which furthered our knowledge on male reproductive health and infertility control.
Role of sphingosine‐1‐phosphate receptor‐2 gene in neural cell migration as revealed by in utero electroporation of S1P2 into telencephalic neuroepithelial cells
Authors: Oh Sung‐Min , Kim Nam‐Ho , Huh Sung‐Oh
Published: June 2021

Sphingosine‐1‐phosphate, S1P, is known to be involved in various biological processes including cell migration. The cellular effect of S1P, mediated through its cognate G protein‐coupled receptors, are well studied but the transcriptional regulation of S1P2 during brain development has not been elucidated in detail yet. Here, we report the identification of the S1P2 promoter in neocortical neuroblast cells and a functional characterization of S1P2 during mouse brain development. By measuring the expression level of the S1P2 gene in embryonic mouse brain by reverse transcriptase polymerase chain reaction (RT‐PCR) analysis, we found low but clearly detectable expression of S1P2 at embryonic days 14.5, 16.5, and 18.5 in whole embryo brain. By promoter analysis experiment, we identified that transcriptional regulatory region of S1P2 gene promoter is located in 5’‐flanking proximal region of S1P2 gene. Introduction of multiple copies of S1P2 gene into the neuroepithelial cells in the ventricular zone at embryonic days 13.5 by in utero electroporation revealed perturbation of radial migration of ventricular zone cells, suggesting a role S1P2 in embryonic neural cell migration. To test whether S1P2 is able to induce neurite retraction, we analyzed morphological changes of primary cultured telencephalic neuroblast cells transfected with S1P2. Together, we demonstrate that S1P2 plays important role in radial migration of neuroepithelial cells in the ventricular zones.
Decreased functionality of A164S variant of apolipoprotein A‐I in cardiovascular disease (1002.1)
Authors: Petrlova Jitka , Dalla‐Riva Jonathan , Lagerstedt Jens
Published: June 2021

Apolipoprotein A‐I (apoA‐I) is the main protein involved in the formation of high‐density lipoprotein (HDL). HDL is the principal mediator of the reverse cholesterol transfer (RCT) pathway and provides cardio‐protection. Currently, we are interested in analyzing the structural and functional properties of the A164S variant of apoA‐I (1:500 in the general population), which is the first known mutation of apoA‐I that leads to an increased risk of ischaemic heart disease (IHD), myocardial infarction and mortality without low HDL cholesterol levels. Despite the fact that epidemiologically IHD is associated with low plasma levels of HDL, the A164S mutation is linked to normal plasma levels of lipids, HDL and apoA‐I, suggesting impaired functionality of this variant. Our biophysical analysis (e.g., Circular Dichroism Spectroscopy, Electron Microscopy) of secondary structure, stability and pro‐amyloidogenic property of the lipid free A164S apoA‐I variant suggest similarity in structural properties between wild type apoA‐I and the A164S apoA‐I variant. However, the lipid‐binding ability experiments suggest functional deficiency of the A164S variant. Specifically, the A164S apoA‐I variant exhibits lower binding affinity to lipids but forms similar sized HDL particles to those produced by WT. Also, cholesterol efflux from macrophages was employed to study functionality of the A164S apoA‐I variant.
Role of endothelial sialic acid expression on xenogenic neutrophil adhesion (1003.4)
Authors: French Beth , Harris Donald , Benipal Prabh , Pierson Robin , Azimzadeh Agnes
Published: June 2021

Objective: Transplantation of organs across species, xenotransplantation, is limited by the sequestration of platelets and neutrophils in injured organs; we hypothesized that sialic acids on porcine endothelial cells affects adhesion of human neutrophils.

Methods: An adhesion assay was developed with freshly isolated human neutrophils and porcine aortic endothelial cells (pAECs). Neutrophils labeled with 0.5 uM of Calcein were quantified using a Spectramax fluorescent reader. Neutrophil adhesion was expressed as arbitrary fluorescence units. 25 mU of Neuraminidase (Clostridium Perfringens) was added to remove sialic acids from pAECs 30 minutes before neutrophil addition. Sialic acid expression was assessed by flow cytometry using lectins (Elderberry lectin, (SNA), Peanut agglutinin (PNA) and Maackia amurensis hemoagglutinin (MAC)). Some pAECs wells were incubated with human TNF (25ng/ml) for 3 hrs at 37 °C before addition of neuraminidase and neutrophils.

Results: Human neutrophil adhesion was detectable on resting porcine endothelial cells (52 +/‐15) and was increased by TNF activation (72 +/‐ 4.7). Neuraminidase treatment increased neutrophil adhesion on resting pAECs to a level similar to TNF activation (71 +/‐ 8.8).

Conclusion: Removal of sialic acid with neuraminidase increased the adhesion of human neutrophils to swine endothelial cells. Thus, targeting levels of sialic acid expression may be a novel therapy to limit neutrophil adhesion during xenotransplantation. Future studies will examine the role of endothelial sialic acid on platelet adhesion as well as the role of neutrophils and platelet sialic acid expression.

Grant Funding Source: Supported by: NIH 5T32HL007698‐19 & U19AI090959; United Therapeutics unrestricted gift
Investigating the role of protein O‐fucosyltransferase 1 in Toll‐like receptor signaling (1004.6)
Authors: Mann Jessica , Li Ning , Laky Karen , Fraser Iain , Nita‐Lazar Aleksandra
Published: June 2021

Protein O‐fucosyltransferase 1 (Pofut1) adds fucose to the serine or threonine residues in the consensus sequence C2‐X(4,5)‐[S/T]‐C3 of EGF domains, where C2 and C3 are the second and third conserved cysteines. The most widely studied Pofut1 substrate is Notch, and the modification is essential in many developmental events involving Notch signaling, such as the development of the nervous system, myelopoiesis and lymphopoiesis, and later T‐cell and B‐cell development. It has not been, however, previously implicated in innate immune signaling.

We have identified protein O‐fucosyltransferase 1 (Pofut1) as a significant hit in a genome‐wide RNAi screen of Toll‐like receptor (TLR) signaling in mouse RAW264.7 cells, where Pofut1 knockdown led to a reduction in LPS‐induced TNF expression.

To further investigate the role of Pofut1 in the TLR signaling, we are using both Pofut1 siRNA transfected RAW264.7 cells and bone marrow derived macrophages (BMDM) from Pofut1 conditional knockout mice. We have studied the TLR4 pathway upon stimulation with lipopolysaccharide, combining cytokine expression assays, protein phosphorylation and protein expression profiling using Western blotting and mass spectrometry, and protein localization experiments.

Grant Funding Source: This research was supported by the Intramural Research program of NIAID, NIH.
The commensal glycoantigen PSA mitigates inflammation through a novel mechanism of T cell‐T cell interaction (1004.7)
Authors: Jones Mark , Johnson Jenny , Cobb Brian
Published: June 2021

Asthma and airway inflammatory disease has become the leading cause for ER visits in the US, a four‐decade trend linked to the increasing sterility of western lifestyles and a proposed lack of early microbial antigen exposure. Here, we demonstrate that gastrointestinal contact with the Bacteroides fragilis glycoantigen polysaccharide A (PSA) educates the peripheral immune system to limit the degree of inflammation. Effective immune responses upon re‐exposure to an antigen is typically mediated through antigen‐experienced memory T cells, whereas regulatory T cells are known to play key roles in suppressing immune responses, generally through the secretion of immunosuppressive cytokines like IL‐10. Here, we show that gut exposure to PSA increases a population of antigen experienced CD4+CD45Rblo T cells which synergistically amplify IL‐10 production in peripheral tissues upon inflammatory stimulus. We propose a novel pathway of T cell‐crosstalk in which PSA treatment generates commensal glycoantigen experienced T cells in the gut environment that are capable of dampening the magnitude of antigen‐specific memory responses in the periphery. These findings provide a mechanistic framework for the hygiene hypothesis where communication between the microbiota in the gut and the T cell compartment links commensal products to non‐specific peripheral immune quiescence resulting in reduced susceptibility to inflammation.

Grant Funding Source: Supported by: NIH DP2 (1‐DP2‐OD‐004225), NIH R01 (5‐R01‐GM082916), AAF (EEA 10‐0187), T32 (AI089474)
The long non‐coding RNA HAS2‐AS1 enhances the transcription of hyaluronan synthase 2 (1005.1)
Authors: Vigetti Davide , Deleonibus Sara , Viola Manuela , Karousou Evgenia , D'Angelo Maria Luisa , De Luca Giancarlo , Passi Alberto
Published: June 2021

As the accumulation of the glycosaminoglycans hyaluronan (HA) into arteries wall supports cardiovascular pathologies and protein modification by attachment of beta‐N‐acetylglucosamine (GlcNAcylation) is involved in diabetic angiopahties, we studied the involvement of O‐GlcNAcylation in the regulation of HA synthesis. Here we report that the long non coding RNA HAS2‐AS1 enhances the transcription of hyaluronan synthase 2 (HAS2), the main HA synthetic enzyme in humans.

We found that O‐GlcNAcylation increased both HAS2 mRNA as well as HAS2‐AS1 RNA, the natural antisense transcript of HAS2 ,which is complementary to exon 1 of HAS2. Such HAS2 mRNA increase could be reduced by the abrogation of HAS2‐AS1.

Further, we found that the HAS2‐AS1 promoter was activated by O‐GlcNAcylation via NF‐kB, whereas HAS2 promoter did not respond to such glycosylation. The presence of HAS2 exon 1 to its promoter increased transcriptional activity after O‐GlcNAcylation.

As it is known that long non coding RNAs modulate epigenetic modifications, we studied the chromatin conformation by nuclease accessibility assays and found that HAS2‐AS1 was critical to maintain the chromatin around HAS2 promoter in a “open” conformation.

Our results indicate that HAS2 transcription is finely regulated not only simply by binding transcription factors to the promoter, but also by modulating chromatin accessibility through long non coding RNA.
The improvement of neuronal function in normal aging by melatonin is mediated by altering mitochondria bioenergetics (1009.3)
Authors: Idowu Abimbola , Olatunji‐Bello Ibiyemi , Bai Yidong
Published: June 2021

The changes in mitochondria bioenergetics play a critical role in neuronal function with age. Mitochondria from synaptosomes in the cerebral cortex of young (6 months), middle‐age (13 months) and old (26months) mice were isolated and transferred to mitochondria DNA‐less LL/2‐m21 cell line (rho‐zero) to generate cybrids. Mitochondria bioenergetics studies of the cybrids show that there was significant reduction with age in mitochondria membrane potential (MMP), P<0.05, in the young, middle‐aged and old synaptosomal mitochondria bearing cybrids respectively. Adenosine triphosphate (ATP) levels were also significantly lower in the old mitochondria bearing cybrids compared with middle age and young cybrids (P<0.05). Melatonin pre‐treatment at a concentration of 1mM in glucose replaced with galactose medium containing 1mM melatonin after 6 hours significantly improved cell viability and restored MMP in the old mitochondria bearing cybrids , S‐O‐48 (P<0.05). Furthermore, melatonin treatment in young mitochondria bearing cybrids, S‐Y‐24 lowered MMP (P<0.05) and increased MMP in middle aged mitochondrial bearing cybrid S‐M‐29 (P<0.05) in galactose medium. In the young cybrids, there was no significant difference in ATP levels between the melatonin treated cybrids and cybrids without treatment (P>0.05). The young mitochondria bearing cybrids were able to produce ATP optimally in the galactose medium which shows they have functional mitochondria. ATP was significantly increased in the middle‐aged mitochondria cybrid, S‐M‐29, and old mitochondria bearing cybrids, S‐O‐48, with melatonin treatment. These results establish a strong relationship between melatonin and mitochondria bioenergetics. It also gives support to other evidences that melatonin may be a potential therapeutic mitochondria target in aging.
Chemopreventive effects of resveratrol derivatives on breast cancer cells (1011.5)
Authors: Campos Nathali , Costa Danielly , Silva Jerson
Published: June 2021

Pterostilbene and piceatannol, two derivatives of resveratrol, are bioactive food compounds that mediates many cellular targets involved in cancer signaling pathways. The p53 tumor suppressor protein plays an essential role in preventing cancer development by inducing cell cycle arrest or apoptosis in response to cellular stress. This protein has been suggested to have a role in the anticancer properties of resveratrol and its structural analogues. Thus, the present study was aimed to check the cytotoxic and pro‐apoptotic effects of pterostilbene and piceatannol on human breast cancer cells. MTT reduction cell viability assays showed that resveratrol derivatives (0‐100 µM) promoted a cytotoxic effect on MCF‐7 and MDA‐MB‐231 breast cancer cells in a dose‐ and time‐dependent manner. The effects induced by pterostilbene were more pronounced than those triggered by piceatannol in both cell lines. Furthermore, cell treatment with 100 µM of pterostilbene for 24h increased phosphatidylserine exposure on cell surface, which is suggestive of apoptosis. This effect was partially prevented when cells were pretreated with pifithrin‐α, a specific p53 inhibitor. Taken together, our results indicate that pterostilbene can be suggested as a promising chemopreventive agent and the cytotoxicity promoted by this compound in tumor cells possibly requires p53 function.
Basonuclin 2 overexpression promotes keratinocyte proliferation and may contribute to basal cell carinoma tumorigenesis (1011.8)
Authors: Muller Bradley , Cornett Jonathan
Published: June 2021

Basal cell carcinoma (BCC) is the most common form of human skin cancer. Alterations in the sonic hedgehog‐patched signaling pathway are considered necessary events in BCC progression. To identify additional driver genes in BCC tumorigenesis, we carried out a piggyBac transposon mutagenesis screen in mice. The basonuclin 2 (bnc2) gene was identified as a common insertion site in multiple independent tumors. Further analysis revealed overexpression of bnc2 mRNA in these tumors when compared with normal skin. Bnc2 is a zinc finger protein of unknown function. To gain insight into its role in normal cells, we overexpressed bnc2 in human keratinocytes. Bnc2 overexpression increases proliferation of these cells and may prevent terminal differentiation. Taken together, these results suggest that bnc2 gene may be a novel oncogene involved in the progression of basal cell carcinomas.

Grant Funding Source: Supported by Lee University faculty research grant
Multiplex measurement of human kidney biomarkers in serum using the Magnetic Luminex® Performance Assay (1012.2)
Authors: Anderson Michael , Rynning Michael , Fuerstenberg Richard , Schmidt Jane
Published: June 2021

The kidneys play an important role in organismal homeostasis by regulating osmolality and blood pressure, aiding in the reabsorption of water and nutrients, excreting wastes, and secreting hormones. Renal function is also important in the metabolism and excretion of drugs; therefore, analyzing nephrotoxicity using renal biomarkers is an important experimental step during drug development. Historically, renal function has been evaluated by measuring serum creatinine and blood urea nitrogen levels. Recently, more sensitive kidney biomarkers have been identified including TIM‐1/KIM‐1/HAVCR, Lipocalin‐2/NGAL, Osteopontin, Cystatin C, Clusterin, CXCL10/IP‐10, RBP‐4, Fetuin A/AHSG, and TFF3. Serum samples from 10 healthy individuals, 10 acute kidney injury (AKI) individuals, and 10 individuals diagnosed with renal failure were evaluated using the Human Kidney Biomarker Magnetic Luminex Performance Assay. We observed statistically significant differences in the AKI group compared to the healthy group for Lipocalin‐2, Osteopontin, Fetuin A, and Cystatin C. We observed statistically significant differences in the renal failure group compared to the healthy group for TFF3, Lipocalin‐2, Clusterin, Fetuin A and Cystatin C. We also observed statistically significant differences in the AKI group compared to the renal failure group for the following biomarkers: TFF3, Fetuin A, and Cystatin C.

The Human Kidney Biomarker Magnetic Luminex Performance Assay from R&D Systems, Inc. is designed for simultaneous quantitative determination of biomarkers in serum, plasma or urine.
Profiling 200 biomarkers in cancer cell culture supernates and serum using Luminex® screening assays (1012.3)
Authors: David James , Anderson Michael , O'Brien Isabel , Schwartz Matthew , Schmidt Jane
Published: June 2021

Cancer progression involves metabolic pathways related to cell growth and proliferation, adhesion, angiogenesis, apoptosis, and others. Multiple biomarkers in serum have been identified that are related to the presence of various cancers as well as the stage, metastatic potential and prognosis. Efforts to quantify changes in these biomarkers will enhance the understanding of the activation of cancer‐related pathways. The measurement of a single biomarker can have limited utility in such a complex disease as cancer, while multiplex profiling can lead to a better understanding of cancer biology and consequently to better diagnostic and therapeutic methods.

Luminex® Screening Assays are highly flexible bead‐based multiplex assays. They allow up to 100 biomarkers, selected from a total of about 200 analytes, to be simultaneously profiled in cell culture supernates, serum or plasma samples. We measured a total of 200 analytes from a variety of cancer cell supernates and serum samples from individuals with a variety of cancers or apparently healthy individuals. The biomarkers tested included cancer antigens, cytokines, chemokines, growth factors, MMPs, protease inhibitors, and other markers. Starting with only 50 µL of each sample, we obtained quantitative results for biomarkers of cancer growth and metastasis, angiogenesis, matrix remodeling, cell adhesion, metabolic function, and cell death.
Activation of Cdc42 is critical for sustained Ca2+ oscillations stimulated by antigen crosslinking of IgE/FcεRI complexes in RBL mast cells (1013.3)
Authors: Wilkes Marcus , Baird Barbara , Holowka David
Published: June 2021

Antigen stimulation of mast cells via FcεRI, the high‐affinity receptor for IgE, triggers a signaling cascade that requires Ca2+ mobilization for exocytosis of secretory granules during an allergic response. To characterize the role of Rho GTPases in FcεRI signaling, we utilized a mutant RBL cell line, designated B6A4C1, which is deficient in antigen‐stimulated Cdc42 activation that we showed previously to participate in these processes. To monitor Ca2+ responses in transfected cells, we co‐expressed the genetically encoded Ca2+ indicator, GCaMP3, together with mutant Rho proteins. In response to antigen, B6A4C1 cells exhibit attenuated Ca2+ oscillations that are restored to wild type RBL‐2H3 levels by expression of constitutively active Cdc42 G12V or a GEF for Cdc42, DOCK7. The capacity of Cdc42 G12V to reconstitute this response is lost when the conserved di‐arginine motif located at the C‐terminus of Cdc42 is mutated to di‐glutamine. To investigate the signaling step that is most directly activated by Cdc42 in this process, we evaluated its role in stimulated IgE receptor endocytosis, a Ca2+‐independent process that is defective in B6A4C1 cells. We find that Cdc42 G12V reconstitutes this response, implicating the importance of Cdc42 in a step upstream of phospholipase Cγ activation in Ca2+ responses, possibly in stimulated synthesis of phosphatidylinositol 4,5‐bisphosphate.

Grant Funding Source: National Institute of Allergy and Infectious Diseases Grant R01AI022449
2016: International year of pulse crops ő programming and potential impact on global nutrition research funding (1014.10)
Authors: Rueda Janice
Published: June 2021

In collaboration with Pulse Canada, Grains & Legumes Nutrition Council Australia and CICILS/IPTIC, the American Pulse Association has successfully achieved the designation by the United Nations of 2016 as the International Year of Pulses (IYP). Pulse crops, which include dry beans, dry peas, lentils and chickpeas, are rich in protein, essential micronutrients and phytochemicals and are integral parts of the diets of people around the world. Efforts are underway to organize events and activities of the IYP for 2016. Strategic planning details of the Health, Nutrition & Food Security and Sustainability & Production working groups will be shared, and the potential impact of the IYP on new global health and nutrition research and funding opportunities will be presented.
A village health worker‐led intervention to promote and support exclusive breastfeeding in rural Zimbabwe is feasible, acceptable and effective (1015.8)
Authors: Matare Cynthia , Mbuya Mduduzi , Stoltzfus Rebecca , Dickin Katherine , Humphrey Jean
Published: June 2021

Exclusive breastfeeding (EBF) is a pillar of child survival but in Zimbabwe, less than 6% of infants are exclusively breastfed for 6 mo. Peer counseling has been shown to improve rates of EBF.

We pilot‐tested a Village Health Worker (VHW)‐based EBF education intervention and assessed acceptability, within‐household reach, motivators triggered, learning outcomes, and EBF intentions and behavior. The intervention delivered key messages at four times: late gestation, and <1 wk, 1 mo, and 3 mo post‐partum.

Nurses trained 20 VHWs on the EBF intervention over 4.5 days. The modules were delivered to 43 women; 13, 5, 11 and 14 at the respective time points, above. Researchers conducted a knowledge survey before the VHW visit and semi‐structured in‐depth interviews with the mothers 1 wk after the visit. The interviews were translated into English, transcribed and analyzed using thematic analysis.

The intervention was acceptable and directly or indirectly reached other influential household members who shape infant feeding practices. The intervention improved EBF knowledge and led to changes in infant feeding behaviors and intentions, motivated mostly by desire for a healthy baby. Some mislearning around maternal diet determining breastmilk production also occurred.

We conclude that targeted messages within home‐based education modules delivered by VHWs to promote EBF are promising and should be tested at scale.

Grant Funding Source: Supported by the Bill and Melinda Gates Foundation (OPP1021542) and DFID, UK
Semi‐automated detection of milk duct dilatation recorded by ultrasound (1016.5)
Authors: Geddes Donna , Fiorentino Jamie , Keating Adrian
Published: June 2021

In lactating women milk is stored predominantly in the alveoli of the breast and is made available to the infant or the breast pump by milk ejection. During breastfeeding or breast expression milk ejection can be detected by visualization of duct dilation and milk flow with ultrasound imaging. Currently, analysis of the ultrasound video is performed manually, is time consuming and subject to error. This pilot study tested an algorithm which determined the average intensity within a region of the video across all video frames, with the aim of determining if changes in this averaged intensity within the duct corresponded with milk flow. The region of milk flow was identified visually from the ultrasound, which detected the movement of high intensity flecks within the milk. Ultrasound intensity within a duct was measured in 7 ultrasound videos. Intensity was also measured in an area of skin and subcutaneous fat for comparison, as no correlation with milk flow is expected in these regions. Percentage changes in intensity (mean (range)) were 22% (11‐37%), 21% (14‐27%) and 112% (60‐251%) for skin, subcutaneous fat and ducts respectively. The increase in intensity at the beginning of milk ejection was 83% (60‐140%). Preliminary results suggest that targeted measurement of ultrasound intensity may be useful in accurately measuring the timing of milk ejection.

Grant Funding Source: Medela AG