bluuebunny/crossref_metadata_2025 huggingface dataset:
Chloride Channel Inhibition Reduces Tail Regeneration in the Developing Vertebrate Ambystoma Mexicanum
Authors: Franklin Brandon Michael , Voss Randal , Osborn Jeffrey
Published: June 2021

Regeneration is dependent on ion signaling which may be activated by endogenous bioelectric fields (EF) in response to the wounding. EF modulation by electrical stimulation (ES) or pharmacological ion channel manipulation alters regenerative capacity. Satellite cell proliferation during ES in the invertebrate, P. clarkii was dependent on K+ and Ca2+ flux (unpublished results). We hypothesize that regeneration in the vertebrate, Ambystoma mexicanum are mediated by intact cellular ion channel activity. Ambystoma hatchlings were raised in welled plates and Holtfreter's solution (2 ml). Tails were amputated 2mm proximal of the tip and imaged on days 0, 2, 4, and 9. Ion channel regulation of regeneration was assessed by inhibiting K+ channels with tetraethylammonium (TEA), Na+/K+ ATPase with ouabain (OB), and Cl− channels with DIDS. Uncut controls measured drug effects on normal growth. Tail tip growth determined regenerative capacity in cut hatchlings. OB and TEA had no effect on regeneration. Cl− channel blockade (DIDS) decreased regeneration from 0.276±0.003 to 0.193±0.002 mm/day (p<0.05) but did not alter uncut, control growth. Thus, ion channels may be critical to vertebrate regeneration by modulating proliferation via cell volume control. Specific ion channel gene expression will further assess the effects of bioelectric fields on vertebrate regeneration capacity.
Zebrafish as a model to analyze macromolecule absorption in intestinal enterocytes
Authors: Cocchiaro Jordan L , Navis Adam , Bagnat Michel , Rawls John F
Published: June 2021

Intestinal absorption and transcellular transport of macromolecules is important for neonatal passive immunity and development of oral tolerance to dietary antigens. The purpose of this study was to develop tools for live analysis of macromolecule absorption in the zebrafish intestine. Using a novel method for zebrafish gavage, we directly introduced macromolecules including immunoglobulin (IgG), dextran, and transferrin (Tnf) into the intestinal lumen of zebrafish larvae and performed live confocal microscopy to examine uptake and sorting within enterocytes. All macromolecules were initially absorbed into apical vesicles but subsequently partitioned within a larger supranuclear vacuole. In transgenic zebrafish expressing Lamp2‐RFP fusion, we found that Lamp2 was targeted to the vacuole membranes, and IgG and Tnf accumulated within these Lamp2‐ringed structures. Initial colocalization of macromolecules within the same apical vesicles suggested a non‐specific bulk endocytic mechanism; however, pretreatment of larvae with a panel of endocytosis inhibitors prior to gavage revealed that IgG and Tnf absorption were specifically blocked by chlorpromazine and phenylarsine oxide, inhibitors of clathrin‐mediated endocytosis. These results provide a zebrafish system to study the physiology and environmental regulation of macromolecule absorption and transport in the intestine. Research funded by NIDDK F32‐DK094592 to JLC and NIH RO1‐DK073695 awarded to JFR.
Mice lacking protein kinase Cα show enhanced membrane localization and activity of the epithelial sodium channel (ENaC) in the renal cortical collecting duct
Authors: Bao Hui‐Fang , Yue Qiang , Thai Tiffany L , Klein Janet D , Sands Jeff M , Eaton Douglas C
Published: June 2021

ENaC insertion into the apical membrane of collecting duct principal cells is dependent on recruitment by the phosphatidylinositol lipids PIP2 and PIP3. The presence of PIP2 at the membrane is determined by membrane targeting of myristolyated C kinase anchoring protein (MARCKS). Phosphorylation and membrane removal of MARCKS by protein kinase C (PKC) negatively regulates ENaC as shown using PKC activators in a cell culture model. To test whether PKCα influences ENaC activity in vivo, we tested the regulation of ENaC in PKCα −/− mice. Cortical collecting duct tubules were dissected and split open and principal cells were subjected to whole cell patch clamp. In the absence of PKCα, open probability (Po) of ENaC was increased three‐fold vs wild type SV129 mice (0.52 ± 0.04 vs 0.17 ± 0.02). The number of channels per patch was also increased. We observed an increase in membrane accumulation of α, β, and γ subunits of ENaC in the cortical collecting ducts of PKCα−/−. To confirm this increase, one kidney from each animal was perfused with biotin and membrane protein pulled down with streptavidin. The unbiotinylated kidney was used to assess total protein. While total ENaC protein did not change in PKCα−/− mice, membrane localization of both α and β ENaC was increased. These results suggest negative regulation of membrane accumulation and Po of ENaC by PKCα in vivo. This work was supported by NIH grants R37‐DK037963 and T32‐DK07656.
Characterization of myogenesis in C2C12 myoblasts using Flow Cytometry
Authors: Yang Zhipeng , Mo Chenglin , Isaacson Janalee , Romero‐Suarez Sandra , Vallejo Julian , Alsousi Asmaa , Wetmore Lori , Brotto Marco
Published: June 2021

Impaired myogenesis is linked to the muscle wasting and weakness characteristic of sarcopenia and muscular dystrophies. In the past, fusion index, a key biomarker of the myogenic differentiation process, has provided valuable data, but has required labor intensive techniques that often lack precision. Flow Cytometry (FC) provides an accurate and efficient way to characterize cell size, complexity and DNA content with forward scatter (FSC), side scatter (SSC), and propidium iodide staining (FL2), respectively. We propose that as myogenesis progresses, with the accompanying increase in fusion events, FC can be used to detect increasing mitochondrial biogenesis, DNA content and myosin heavy chain in C2C12 mouse skeletal myoblasts. For a more precise comparison of these parameters, we found it useful to synchronize cell cycle at G0/1 through serum deprivation for 24 hours. While still a developing methodology, our preliminary studies, demonstrate that FC could be a valuable method to quantify variations in cell size, character and DNA content in myoblasts and myotubes. This method promises to provide a more reliable characterization of myogenic differentiation to advance this field of research. Support: NIH‐National Institutes of Aging P01 AG039355 and Missouri Life Sciences Research Board (MB).
Interleukin 6 alters myogenic regulatory factors expression in cultured mammalian satellite cells
Authors: Macumber Jessica Ashley , Thornton Kara Jean , Doumit Matthew E , Murdoch Gordon K
Published: June 2021

Quiescent muscle satellite cells (SCs) express paired box transcription factor 7 (Pax 7) and Sprouty1 (Spry1), expression of these transcription factors decreases upon activation. In contrast, activated SCs increase expression of myogenic regulatory factors (MRFs) such as; myogenin and myogenic differentiation factor (MyoD) as they differentiate and mature. Hence, myogenic activation of SCs and thus myogenesis can be characterized by quantifying mRNA expression of these marker genes using quantitative RT‐PCR. Further, inflammation at the site of myotrauma is known to induce hypertrophic and hyperplastic muscle growth. We hypothesize that following myotrauma in mammals, cytokines such as interleukin 6 (IL6), promote myogenesis through SC activation. We observed decreases in both Pax 7 (p=0.09) and Spry1 (p=0.08) expression, an increase in myogenin (p=0.0014) expression and altered MyoD (p=0.04) expression when cultured SCs were treated with IL6 in vitro. These results support the hypothesis that cytokines mediate the recruitment of quiescent SCs and thereby facilitate the repair of damaged muscle tissue and have a direct promyogenic effect. The role of cytokines in myogenesis may yield feasible means of enhancing muscle growth and repair in response to trauma, pathology or sarcopenia. We acknowledge the UI SRGP FY2012, USDA Hatch Grant NC1184 and IBC Grant FY2013 for funding.
A gender comparison of electromyography (EMG) during repetitive arm work with and without mental stress
Authors: Crenshaw Albert , Fahlstrom Martin , Lyskov Eugene
Published: June 2021

EMG of the trapezius muscle was monitored throughout a 45 min arm work task that included without (on one occasion) and with stress (another occasion) for 8 males and 8 females. The work task involved raising and lowering the arm, constituting one cycle, at a rate of 30 cycles per min. The stress session included periodic implementation of the Stroop color word task and mathematical equation solving concurrent with the work. EMG signal response was on/off representing raising (contracting) and lowering (relaxing) the arm, respectively. The root mean square (RMS) was calculated, and overall RMS and RMS during the arm lowering phase (RMS‐relax) were determined over time. The overall RMS increased significantly over time, and the increase was greater for females than males. However, these data were not dependent on work type (i.e. with vs. without stress). RMS‐relax also increased significantly over time with females increasing greater than males; there was no difference due to type of work. Thus, over the 45 min work task females showed more fatigue (based on RMS increase) and less relaxation (based on RMS‐relax) than males; stress had no impact on these data.
Monocarboxylate transporter expression at the onset of skeletal muscle regeneration
Authors: Smith Dameon , Washington Tyrone
Published: June 2021

Monocarboxylate transporters (MCTs) are involved in lactate transport across the cell membrane and this has important implications for energetic efficiency of skeletal muscle. MCT expression is affected by mechanical overload, chronic electrical stimuli, and voluntary wheel running. However, the cellular regulation of MCT protein expression at the onset of skeletal muscle regeneration is unknown.

PURPOSE

To determine protein and gene expression of MCTs at the onset of skeletal muscle regeneration. Male C57/BL6 (12 weeks old) mice were randomly assigned to a control (uninjured) or bupivacaine (injured) group. Bupivacaine was injected into the tibialis anterior (TA) of the injured group and phosphate buffered saline (PBS) was injected into the TA of the uninjured group. The TA was extracted 3 days post‐bupivacaine injection. A 12% decrease in TA muscle mass to tibia length (2.43 ± 0.12 mg/mm vs. 2.14 ± 0.19 mg/mm, p < 0.02) was observed. IGF‐1 and myoD gene expression increased 5.0‐fold (p < 0.05) and 3.5‐fold (p < 0.05), respectively during regeneration. MCT1 protein decreased 32% (p < 0.03); however, gene expression was not altered. There was no difference in MCT4 protein or gene expression. CD147 and PKCθ protein increased 64% (p < 0.03) and 79% (p < 0.02), respectively. In conclusion, at the onset of skeletal muscle regeneration MCT1 protein is decreased and this appears to be regulated post‐transcriptionally.
Effects of High Dose of Methionine on Plasma Oxidative Stress and Lipid Profiles in Rats Treated With Ethanol
Authors: Kim SunYoung , Min Hyesun
Published: June 2021

The aim of this study was to investigate the effect of excess dietary methionine (Met) on oxidative stress and plasma lipid profiles in ethanol‐fed rats. High dose of Met supplementation was found to increase plasma levels of thiobarbituric‐reactive substances (TBARS), homocysteine (6‐folds increase), TG, total cholesterol, and HDL than those of rats fed ethanol alone. However, plasma levels of cysteine, cysteinylglycine, and glutathione remained unchanged by Met supplementation in ethanol‐fed rats. Plasma alanine aminotransferase decreased slightly by Met supplementation, but this change did not reach to statistical significant level. In addition, hepatic levels of glutathione and TBARS remained unchanged by Met supplementation. Hepatic levels of S‐adenosylmethionine (SAM), S‐adenosylhomocysteine (SAH), and SAM/SAH ratio were increased by Met supplementation. These results indicate that Met supplementation induces oxidative stress in plasma, but not in liver in ethanol‐fed rats. In conclusion, high dose of Met appears to have a modest protective effect on alcohol‐induced hepatic oxidative stress possibly by increasing the levels of SAM and SAM/SAH ratio in liver. (This research was supported by Basic Science Research Program through NRF fund, 2010–0024002.)
Protective role of Selenium and Adiponectin deficiency in chronic inflammation induced colon cancer.
Authors: SAXENA ARPIT , KAUR KAMALJEET , Fletcher Emma , Larsen Bianca , Hegde Shweta , FAYAD RAJA
Published: June 2021

Selenium (Se), a nutrition supplement and a well known antioxidant, has been shown to inhibit tumorigenesis in several experimental cancer models. Adiponectin (APN), an adipocytokine secreted by the adipocytes has been shown to play a protective role in chronic inflammation induced colon cancer (CICC). Inflammation and colon cancer were induced in both APN knockout (KO) and C57B1/6 wild type (WT) mice by 2% dextran sodium sulfate (DSS) and 1,2‐Dimethylhydrazine (DMH) treatment respectively and were divided into 8 treatment groups (n=9 per group): 1) No treatment + normal diet (N); 2) Group 1 + Se; 3) treatment with three cycles of DSS + N; 4) Group 3 + Se; 5) weekly doses of DMH (20mg/kg of mouse body weight) for twelve weeks + N; 6) Group 5 + Se; 7) a single dose of DMH followed by 3 cycles of DSS + N; 8) a Group 7 + Se. Mice were observed for diarrhea, stool hemoccult, and weight loss and were sacrificed on day 192. Tissues were collected from the colon for genomic and proteomic studies. Se aided in the reduction of the physical signs and the histopathological scoring associated with CICC with APNKO mice having worse score when compared to WT mice. Se concentration, Gpx activity, Gpx protein expression and tumor cell apoptosis was found to be significantly higher in the colon of the mice given Se diet. Other proteomic and genomic studies also indicate the protective role of Se in the progression and the symptoms associated with CICC.
GIP Regulates Pept1 via Akt and EPAC Signaling Pathways in Intestinal Epithelial Cells
Authors: Coon Steven , Schwartz John , Singh Satish
Published: June 2021

Background

GIP, an incretin hormone, is important in the pathogenesis of obesity and diabetes. GIP increases dipeptide transport in both murine jejunum and intestinal cells grown in vitro. GIP activates both the cAMP and PI3 kinase pathways but the mechanism and intermediates involved in Pept1 regulation are not known.

Objective

To elucidate the intracellular signaling pathways that regulate GIP‐stimulated dipeptide transport.

Methods

Transport and signaling pathways for GIP were determined by growing IEC6 cells on filter inserts and measuring Pept1 uptake. 3H‐GlySar (50 uM) and GIP (100 nM ) were added to the apical and basolateral side, respectively.

Results

Akt inhibitor 3 which inhibits Akt, a downstream intermediate of the PI3 kinase pathway, decreased Pept1 activity by 75% (110±10 compared to 486±39 pmol/min/mg‐protein for GIP stimulated control). To probe the cAMP pathway, we found that the EPAC agonist (8‐pCPT‐2′‐O‐Me‐cAMP‐AM, 20 nM) increased Pept1 activity (520±41 pmol/min/mg‐protein) while a PKA agonist (8‐brcAMP, 50 nM) failed to increase Pept1 activity when compared to untreated (no GIP) control (240±39 pmol/min/mg‐protein). Western blots documented significant increases in apical expression of Pept1 upon GIP stimulation.

Conclusion

GIP augmented Pept1 transport is consistent with a model where membrane targeting of the protein is induced by Akt and via EPAC activation by cAMP.
Subsets of fibroblasts show resistance to apoptosis independently of their interstitial lung disease origin.
Authors: Selman Moises , Vicens Vanesa , Mendoza Criselda , Montaño Martha , Ramos Carlos , Romero Yair , Molina Maria
Published: June 2021

It has been suggested that fibroblasts from idiopathic pulmonary fibrosis (IPF) are resistant to apoptosis; however, studies in other interstitial lung diseases (ILD) are scanty. We compared the levels of TNFα‐IFNγ‐Fas induced apoptosis/autophagy of fibrobroblasts from IPF, hypersensitivity pneumonitis, ILD of autoimmune diseases, and control lungs. Flow cytometry showed two types of fibroblasts in all samples: small, “spindle” shaped, and large “stellate” fibroblasts (mostly myofibroblasts). Independently of the type of ILD, large fibroblasts were significantly more resistant to TNFα‐IFNγ‐Fas induced apoptosis compared to small ones (~10% versus ~40%), and also presented decreased autophagy activity measured by LC3 conversion. Non‐stimulated large fibroblasts showed significant upregulation of antiapoptotic genes (XIAP and HMOX‐1) and downregulation of proapoptotic proteins (TNFR1, TRAIL), and after TNFα‐IFNγ‐Fas treatment the upregulation of other antiapoptotic proteins (HSP70, HSP27 and Claspin). Ultrastructural morphology after TNFα‐IFNγ‐Fas revealed profuse mitochondria in large IPF fibroblasts while numerous apoptotic bodies were observed in small cells. These findings indicate that there is a subset of fibroblasts (likely myofibroblasts) that, independently of the type of ILD, expressed antiapoptotic proteins which may confer resistance to apoptosis.

SEPAR & PAPIIT 214612
Cordyceps sinensis Cs‐5 enhances sexual functions in normal and impotent models
Authors: Yang Jie‐Ying , Zhang Yan , Dong Yun‐Zi , Tan Ning‐Zhi , Lu Ji‐Hong , Zhu Jia‐Shi
Published: June 2021

Cordyceps sinensis (Cs) has been widely used for anti‐fatigue and anti‐aging. Cordyceps sinensis Cs‐5, a mycelia fermentation product of Cs, has shown its benefits in anti‐fatigue and immune modulation. In this study we examined the Cs‐5 effects on improving sexual functions in rat and mouse models. (1) Male SD rats (2 mo) underwent bilateral orchiectomy (OE). After 2‐wk recovery, they were randomized into 4 groups (n=14 each), receiving vehicle or Cs‐5 (0.5 or 1.5g/kg) by gavage, or testosterone (2mg/kg) via s.c. injection for 3 days. Erection latency (EL) was measured on Day 3. Cs‐5 shortened EL in OE rats compared to OE controls (p=0.03 & <0.01). (2) Normal ICR male mice (8 mo) were randomized into 3 groups (n=16 each) based on the baseline capture latency (CL), and received vehicle or Cs‐5 at 0.5 or 1.0 g/kg for 22 days. Two hours after treatment on Days 3 and 22, mice were mated with 3 females for 1 male in a single cage, and their sexual behaviors were monitored through infrared track system and recorded for 20 min. Cs‐5 (0.5g/kg) significantly shortened CL of mice on both Days 3 and 22, compared to controls (p<0.01); and increased the percentage shortening of CL at 0.5 and 1.0g/kg on Day 3 (p<0.01) and the copulation/capture ratio at 1.0 g/kg on Day 22 (p=0.02). In conclusion, Cs‐5 enhances sexual function by shortening EL in OE rats, and by shortening CL and increasing copulation/capture ratio in normal mice.
Plant‐originated extract of Cucurbita Maxim Sweet prevents foregut damage induced by entero‐salivary nitrites recirculation impair via suppression of inflammatory mediators’ expression
Authors: Pshyk‐Titko Irena , Zajachkivska Oksana , Yashchenko Antonina
Published: June 2021

Cucurbita maxim sweet seed extract (CSE), Ukrainian plant hybrid kavbuz from watermelon and pumpkin, contents high amount of beta‐carotene and bioflavonoids. Studies in vitro showed that CSE exhibits a variety of actions via antioxidant and vasotropic effects, yet the mechanism remains still unknown. In this study, to investigate the mechanism responsible for these actions, we examined the effect of CSE on changes in inflammatory mediator's expression in foregut during modeling of entero‐salivary nitrites recirculation (mESNR), an important functional cytoprotective system in foregut.

Rats were used with dose‐depended treatment without/with CSE in dose 50–200 mg/kg under mESNR by acid blocks atropine (3 mg/kg), ranitidine (100 mg/kg), L‐NAME (10 mg/kg) and their combination; oral, esophageal, gastric lesions were determined by histology score index (HIS); IL‐1beta, IL‐8 via ELISA.

ESNR impairment caused nonerosive hemorrhagic lesions in foregut mucosa, as well as dystrophic changes in muscular layer, accompanied by increased IL‐1beta, IL‐8 syntesis.CSE potently prevented cytolytic response to changes of ESNR, with concomitant decreasing of IL‐1beta, IL‐8; these effects of CSE were significantly abrogated by pretreatment of L‐NAME.

CSE prevents mESNR‐induced foregut lesions via suppression of inflammatory mediators such as IL‐1beta, IL‐8, the important pathogenic factors in foregut.
Effect of Enalapril and Losartan on Testosterone induced Benign Prostatic Hyperplasia in rats
Authors: PATEL SANDIP BHIKHUBHAI , PATEL VRUSHANT , CAPTAN HEMANT
Published: June 2021

The aim of the present investigation is to establish the therapeutic potential of drugs affecting Renin Angiotensin System (RAS) on Testosterone induced Benign prostatic hyperplasia (BPH) in rats.

Animals were distributed in 5 groups (6 rats each). Group 1 receives only vehicles. Four other groups injected with Testosterone (3mg/kg) to produce BPH, Group 2 was model group, Group 3 animals were treated with Enalapril at a dose of 10mg/kg, Group 4 animals were treated with Losartan at a dose of 10mg/kg, Group 5 animals were treated with Finasteride at a dose of 5mg/kg. The drugs were administered once a day for 21 days consecutively. On 22nd day, blood samples were collected from the Retroorbital plexus, centrifuged to obtain serum for determination of various parameters. Then animals were sacrificed, prostates, Testis were weighed and histopathological studies of prostate were carried out.

Enalapril and Losartan treatment showed significant inhibition of prostate enlargement, prevent the reduction in Serum Testosterone levels, reduced the level of Serum Prostate Specific Antigen, reduced the level of Serum Prostatic Acid Phosphatase, protection of histoarchitecture of prostate when compared with model group.

These results suggest that ACE inhibitor and Angiotensin‐II antagonist has a definite inhibitory effect on BPH and might be an alternative medicine for treatment of human BPH.
Desensitization of the P2Y1 receptor in platelets
Authors: Blatt Tasha , Bergmeier Wolfgang , Nicholas Robert
Published: June 2021

Understanding thrombosis is critical for effective treatment of patients at risk for heart attack and stroke. Simultaneous activation of the Gq‐coupled P2Y1 receptor and the Gi‐coupled P2Y12 receptor is required for ADP‐promoted platelet aggregation— deletion or inhibition of either receptor results in the loss of ADP‐induced aggregation. Unlike the P2Y12 receptor, the P2Y1 receptor in both mouse and human platelets undergoes profound and rapid desensitization in response to sub‐aggregating levels of ADP. We hypothesize that this rapid inactivation is specific to the P2Y1 receptor and is physiologically relevant for proper regulation of platelet hemostasis. To test the specificity of desensitization, we compared the rate of desensitization after agonist addition of the 5HT2A and TxA2 receptors, which are also Gq‐coupled, to that of the P2Y1 receptor. These data reveal that only the P2Y1 receptor rapidly desensitizes in response to its cognate agonist. To determine the residue(s) responsible for P2Y1 desensitization, we transplanted retrovirally infected bone marrow cells expressing P2Y1 receptor Ser/Thr mutants into P2Y1−/− mice to examine the desensitization of the P2Y1 receptor ex vivo. These mice will also be used to examine the physiological consequences of expressing a non‐desensitizing P2Y1 receptor by measuring thrombus formation in vivo in response to arterial vessel wall injury.

(Supported by NIH grant HL54889)
β‐arrestin dependent signaling of metabotropic glutamate receptor 1
Authors: Hathaway Hannah A , Pshenichkin Sergey , DiRaddo John O , Gelb Tara , Grajkowska Ewa , Wroblewski Jarda T
Published: June 2021

Metabotropic glutamate 1 (mGlu1) receptor is a G protein‐coupled receptor expressed mainly in the central nervous system. Classically, stimulation of mGlu1 receptors activates Gαq and increases production of the second messengers IP3 and DAG. In addition, our research has demonstrated that mGlu1 receptors activate a distinct, G protein‐independent signal transduction cascade. This mechanism is mediated by β‐arrestin recruitment to the C‐terminal tail of agonist‐occupied receptors. This receptor‐β‐arrestin complex acts as a scaffold for various signaling proteins. We have shown that selective activation of the mGlu1 receptor‐mediated β‐arrestin signaling cascade is cytoprotective, both in cerebellar granule neurons and in CHO cells transfected with mGlu1 receptors. However, the signal transduction mechanism remains unclear. The objective of this study was to determine how molecular changes in the mGlu1 receptor dictate β‐arrestin‐dependent signaling. Molecular biology methods were used to produce mGlu1 receptor variants with changes in the C‐terminal tail. The influence of these changes on β‐arrestin signaling was measured by cell viability assay and phospho‐ERK ELISA, and changes in G protein‐dependent signaling were measured by phosphatidylinositol hydrolysis. Here we report that variations in the C‐terminus of mGlu1 receptors affect β‐arrestin‐dependent signaling. NIH Grant NS37436
Determination of the site of ligand binding on the Sigma1 Receptor
Authors: Schrock Joel M , Islam Mohammad M , Kim Felix J , Grinnell Steven G , Pasternak Gavril W
Published: June 2021

The Sigma1 Receptor (Sig1R) is a 26 kilodalton integral membrane protein which shares no structural homology with any traditional receptor family. Despite the description of Sig1R effects in many different signaling pathways, the mechanism of Sig1R action has not been determined. Historically, the distinction between Sig1R agonists and antagonists has been made based on the pharmacological response in specific systems, which has added to the difficulty in determining the mechanism of Sig1R action. Our laboratory has focused on determining the site of ligand binding on the Sig1R in order to better define Sig1R agonists and antagonists. Using circular dichroism, Sig1R antagonists but not agonists stabilize the C‐terminal tail of the Sig1R, indicating binding. In order to further define the binding sites of Sig1R agonists and antagonists, [125I]1‐(4‐Iodophenyl)‐3‐(2‐adamantyl)guanidine (IPAG, a Sig1R antagonist) was synthesized. IPAG bound with high affinity to both the full‐length Sig1R as well as the C‐terminal tail of the Sig1R, indicating that the binding site of IPAG is in the C‐terminal tail. Antagonists potently lowered binding of [125I]IPAG to the C‐terminal tail, while Sig1R agonists did not compete [125I]IPAG binding with high affinity, potentially indicating different binding sites for Sig1R agonists and antagonists.
The influence of methadone on toll‐like receptor 4 and human mu opioid receptor expression
Authors: Dodson Summer , Das Subhas , Davis Randall , Stevens Craig W.
Published: June 2021

This research discusses the potential interaction of toll‐like receptor 4 (TLR4), an innate immune receptor found both peripherally and in the CNS, and the human mu opioid receptor (hMOR) in the presence of methadone and/or lipopolysaccharide (LPS). Opioid dependency increases the likelihood of infection and inflammation and studies have found that methadone maintenance programs yeild improved immune function in participants. This well documented immunologic dysfunction of opioid dependent individuals has led to the investigation into the role of TLR4 in this system. It is important to include the effects of methadone both alone and in the presence of LPS due to opportunistic infection in abusers. HEK Blue 4 cells overexpressing TLR4 protein have been used in this research primarily as a tool to identify TLR4 activity. 100 μM methadone significantly inhibits TLR4 activation in the presence of 100 ng/mL LPS. Methadone at 100 μM (+/− LPS) upregulates hMOR transcript in this cell line. CHME‐5 microglial cells also show a link between methadone and TLR4 mRNA expression. 10 μM methadone increases TLR4 mRNA after 30 minutes. 100 μM methadone with LPS has higher expression at 30 minutes then declines. This research begins to illustrate the complex relationship of TLR4 and methadone in neuroimmunomodulation. Supported in part by an Oklahoma Center for the Advancement of Science and Technology (OCAST) HR10–31 to CWS.
Feedback control of upstream 5‐hydroxytryptamine (5‐HT) in the dorsal raphe nucleus (DRN) by downstream 5‐HT2A and AMPA receptors in the prefrontal cortex (PFC)
Authors: Zhou Zhu , Chan Patrick G. , Sun Jingjing , Uchizono James A.
Published: June 2021

The objective was to explore an in vivo feedback pathway that may potentially control 5‐HT in the DRN. While 5‐HT levels in the DRN have been well studied, pathways that modulate this DRN pool from downstream cascade interactions have been difficult to elucidate. This work supports at least one pathway that appears to contribute to the feedback dynamics of 5‐HT levels in the upstream DRN. Neurosurgical implantation of microdialysis two probes were placed into the DRN and PFC from which 5‐HT measurements were simultaneously recorded; DOI (5‐HT2A agonist) and NBQX (AMPA antagonist) were also delivered via these probes to the PFC and DRN, respectively. Three different regimens were tested: I) aCSF, DOI(100 μM), DOI(500 μM), DOI(800 μM); II) aCSF, DOI(100 μM); III) DOI(100 μM)+NBQX(50 μM), DOI(100 μM+NBQX(150 μM), DOI(100 μM)+NBQX(300 μM). In Regimen I, DOI, at all three concentrations, increased DRN and PFC 5‐HT levels. In Regiment II, DOI pharmacodynamics were determined as a positive control and resulted in time‐course, increased 5‐HT levels in the DRN and PFC. Regimen III showed NBQX gradually decreased DOI‐induced increased 5‐HT DRN levels. Pharmacodynamic evidence supports the possibility of a 5‐HT2A/AMPA feedback control circuit that originates in the PFC and modulates DRN 5‐HT levels. This work was supported by Formurex® and the University of the Pacific.
Weight and width: percent fat does not improve association between adiposity and insulin
Authors: Fedewa Michael Vernon , Das Bhibha M. , Schmidt Michael D. , Evans Ellen M.
Published: June 2021

The aim of this study was to compare the independent associations of waist circumference (WC), BMI, and DEXA (%FAT) with fasting insulin among college freshmen (N=232; 18.5±0.5y, 69.8% female, 68.1% Caucasian). WC, BMI, and %FAT were 74.0±8.1 cm, 23.2±3.4 kg/m2, and 28.4±8.7%, respectively. Insulin (INS) was obtained from conventional clinical methods (M=21.9±22.1 pmol/L) and log transformed to achieve normal distribution. As expected, BMI and WC were moderately associated with %FAT via DEXA (R=.66 and .65, respectively) after controlling for sex. In a sex‐adjusted linear regression model, BMI accounted for 7.1% of the variation in INS (p<0.001, R=.27). The addition of WC improved the prediction of INS, as explained variance increased by 3.8% (p<0.05). However, the further addition of %FAT did not significantly improve the prediction of INS (ΔR2=1.3%, p>;0.05). Our results suggest that conventional clinical anthropometric measures, BMI and WC, provide adequate predictive value when used to measure the association between adiposity and fasting insulin, and including %FAT does not significantly improve the association with metabolic risk in college freshmen.

Grant Support: USDA 2008–55215‐18825
Auxiliary Cavβ subunits of the high voltage L‐type Ca2+ channels modulate the low voltage activated cardiac Cav3.2 T‐type Ca2+ channel currents
Authors: Woon Marites Tolentino , Markandeya Yogananda S , Reynolds Courtney R , Balijepalli Ravi C
Published: June 2021

Ca2+ signaling and contraction are initiated by the activation of high voltage L‐type Ca2+ channels (LTCC) in cardiomyocytes. However, pathological hypertrophic Ca2+ signaling results from the re‐expression of low voltage T‐type Ca2+ channel (TTCC) current. But the precise regulation of the cardiac TTCC isoforms, Cav3.1 and Cav3.2, in cardiac hypertrophy is unclear. While the Cavβ subunits (isoforms β1, β2, β3 and β4) of the LTCC are required for membrane trafficking and functional regulation of the Cav1.2 subunit of LTCC, auxiliary subunits for the TTCCs have not been identified. We investigated if the β subunits of the LTCC regulate the function of low voltage activated cardiac TTCCs. Co‐immunoprecipitation performed in transiently expressing HEK293 cells or hypertrophic mouse cardiomyocytes demonstrated that the β1, β2 and β3 but not the β4 subunits co‐IP with Cav3.2 channel protein. Whole cell patch clamp analysis in transiently expressed HEK293 cells demonstrate that β1 and β2 subunits caused a significant increase in ICav3.2 density but not ICav3.1. In contrast the β3 and β4 did not alter the ICav3.2 or ICav3.1 density. In addition, the co‐expression of β1 caused a rightward shift in the V1/2 of ICav3.2 activation. We also found that the expression of the β1 and β2 subunits was increased in ventricular myocytes in mouse model of pressure overload induced cardiac hypertrophy. We conclude that an increased expression of the β1 and β2 subunits may regulate the ICav3.2 and contribute to the altered Ca2+ signaling in pathological cardiac hypertrophy. Research support NHLBI R01HL105713 to RCB.
Lifecourse socioeconomic position and dietary intakes and patterns in the Jackson Heart Study
Authors: Hickson DeMarc Anthony , Gao Yan , Talegawkar Sameera , Liu Jiankang , Manjourides Justin , Roux Ana V Diez , Tucker Katherine L
Published: June 2021

Prior work examining the associations of socioeconomic position (SEP) and diet has used individual and neighborhood‐level measures at one point in adulthood and do not consider SEP over the life‐course. Using data from 4,771 Jackson Heart Study participants (21–95 years of age; 63.6% women), we examined the relation of childhood SEP (a summary score of parental education and childhood resources), adulthood SEP (a summary score of income, education and wealth), and neighborhood SEP (median household income) with dietary intakes and patterns. In multi‐level models adjusted for age, sex and total energy, lower childhood, adulthood and neighborhood SEP were generally associated with lower intakes of protein, fiber, whole grains and fruits and vegetables. Independent associations were most consistent for adulthood SEP followed by neighborhood and childhood SEP. Adulthood and neighborhood SEP were inversely, though not independently, associated with a Southern dietary pattern. Unexpectedly, childhood and adulthood SEP were positively associated with a Fast Food dietary pattern, though the associations were not independent of the other SEP measures. Adulthood SEP was positively and independently associated with a Prudent (or healthy) diet pattern. These findings suggest that SEP at different points throughout the life‐course contribute differently to dietary intakes and patterns in African Americans.
Exercise‐induced PCr recovery kinetics and tissue oxygenation: The role of free radicals and aging
Authors: Hart Corey R. , Layec Gwenael , Trinity Joel D. , Liu Xin , Kim Seong‐Eun , Groot H. Jon , Jeong Eun‐Kee , Richardson Russell S.
Published: June 2021

We investigated the role of free radicals on muscle oxidative capacity and O2 availability in activity matched young (Y) and old (O) people following exercise. 39 subjects (19 Y and 20 O) performed sub‐maximal plantar flexion exercise following the consumption of either a placebo (PL) or an antioxidant (AO) cocktail. PCr resynthesis was assessed by 31P‐MRS (magnetic resonance spectroscopy), while muscle re‐oxygenation and limb blood flow were evaluated by near‐infrared spectroscopy and ultrasound Doppler, respectively. The PCr recovery time constant in O (PL: 37 ± 6; AO: 38 ± 6 s) and Y (PL: 38 ± 6; AO: 36 ± 4 s) as well as the maximal rate of oxidative ATP synthesis in O (PL: 15 ± 2; AO: 14 ± 2 mM.min−1) and Y (PL: 13 ± 2; AO: 15 ± 3 mM.min−1) were not altered by age or AO. In contrast, muscle reoxygenation rate, although not affected by age, was slower in the Y following AO, as evidenced by the time constant (P < 0.05) and mean response time (PL: 49 ± 6; AO: 70 ± 10 s) of deoxy‐Hb recovery, while no difference was observed in the old. These data reveal that, when subjects are activity matched, muscle oxidative capacity is preserved with age. In addition, unlike the O, the compromising effect of the AO on O2 availability in the microcirculation of the Y, without affecting PCr recovery kinetics, implies that free radicals play a role in blood flow regulation and that O2 availability exceeds mitochondrial capacity in this population.
Calorie restriction effects on insulin signaling and glucose uptake in multiple insulin‐stimulated rat skeletal muscles in vivo
Authors: Sharma Naveen , Sequea Donel A , Castorena Carlos M , Qi Nathan , Cartee Gregory D
Published: June 2021

Calorie restriction (CR; ~60% of ad libitum, AL, food intake) has been shown to enhance whole‐body insulin sensitivity. Skeletal muscle accounts for the majority of insulin‐stimulated glucose disposal, however no previous studies have reported CR effects on both in vivo glucose uptake and insulin signaling in the same skeletal muscles. Accordingly we evaluated the effect of CR (initiated at 14‐wks of age) on the tibialis anterior (TA) and gastrocnemius (GN) muscles from 9‐mo old rats under euglycemic‐hyperinsulinemic clamp conditions. Glucose levels during the clamp were not significantly different for AL (120.7±2.7 mg/dl) versus CR (126.1±2.4 mg/dl) groups. Insulin levels during the clamp were also elevated to similar levels in AL (141.3±9.1 μU/ml) and CR (140.3±6.7 μU/ml) groups. Glucose infusion rates were 78% higher (P<0.05) in CR (31.7±1.6 mg/kg−1 × min−1) versus AL (17.8±0.8 mg/kg−1 × min−1) rats. Glucose uptake (radiolabelled 2‐deoxyglucose) was increased (P<0.05) for CR vs. AL rats in both the TA and GN. Insulin receptorTyr1162/1163 phosphorylation was unaffected by CR in either muscle. AktSer473 phosphorylation was increased (P<0.05) in both muscles of CR vs. AL rats, and there was a strong trend for increased AktThr308 phosphorylation in CR vs. AL rats for both the TA (P=0.052) and GN (P=0.052). These results suggest that CR leads to greater whole‐body insulin sensitivity attributable at least in part to CR effects on in vivo insulin‐stimulated glucose uptake secondary to greater Akt phosphorylation in multiple muscles, including the TA and GN.
Clavicular brown adipose tissue has a different origin from the perirenal depot that is indicative of muscle related lineage
Authors: Symonds Michael E , Pope Mark , Birtwistle Mark , Budge Helen
Published: June 2021

Multiple evidence from human studies indicates that the primary sites for brown adipose tissue in humans are the bilateral clavicular depots. In this study we aimed to identify an analogous region in the young sheep and whether it contained microRNA (MIR) specific to muscle or adipose tissue. This was compared with perirenal‐abdominal adipose tissue which represents the major fat depot in young sheep.

Four triplet‐bearing mothers were entered into the study and a randomly selected triplet euthanased at 1, 7 or 28 days of age for adipose tissue sampling. Gene expression for target genes was determined by quantitative reverse transcriptase polymerase chain reaction.

MIR 206 was highly abundant in skeletal muscle and was also detectable in BAT sampled from the clavicular but not perirenal depot at all sampling ages. This was despite an age related decline in UCP1 and related genes, whereas gene expression of RIP140 increased with age.

Our study suggests a different cell lineage in clavicular adipose tissue compared with other BAT depots in the young sheep. Despite showing beige characteristics with increasing age, retention of some muscular origins may mean it is more response to environmental challenges aimed at promoting BAT function. The young sheep, therefore, provides a novel model for examining the developmental process of adipose transformation in early life.
Analysis of connectivity and timekeeping hierarchy in the circadian clock neuron network of Drosophila
Authors: Shafer Orie Thomas , Yao Zepeng
Published: June 2021

The objective of this study was to determine the nature and functions of the neuronal connections underlying circadian timekeeping in Drosophila melanogaster. The maintenance of a synchronized clock network requires physiological interactions between distinct classes of clock neurons. Using a new approach to neural circuit interrogation in the adult fly brain we have characterized the connection between the critical ventral lateral pacemaker neurons (LNvs) and the dorsal lateral clock neurons (LNds). We show that it is a connection that induces cAMP increases in the LNds and that it has no measurable effects on Ca2+ levels. This connection is dependent on pigment dispersing factor receptor (PdfR), suggesting that the neuropeptide PDF mediates this connection. To investigate the function of this connection in the control of sleep/activity rhythms, we introduced a range of circadian period discrepancies between the LNvs and the dorsal clock neurons in both wild type and PDF/PdfR mutant and knockdown conditions. We find that the LNvs are capable of setting systemic period over a wide but finite range of periods and that this resetting requires PDF/PdfR. These results reveal the physiological basis and the limits of timekeeping hierarchy in the Drosophila brain. This work was supported by an NIH Pathway to Independence Award (NINDS R00NS62953).
Beat to beat pulse oximetry vs. standard averaging techniques during high altitude sleep
Authors: Wentz Robert James , Issa Amine , Taylor Bryan , Kasak Alex , Summerfield Doug , Johnson Bruce
Published: June 2021

Background

Pulse oximetry is often used as a screening technique for detection of sleep apnea. Most pulse oximetry companies use averaging algorithms to help reduce noise; however averaging also tends to attenuate high and low values. Most, if not all healthy individuals, develop periodic breathing at high altitude.

Objective

To determine the influence of a typically used averaging technique on the capture of oxygen desaturations during a night at high altitude.

Methods

Data from a recent Mount Everest expedition are presented in 2 individuals wearing a Nonin 3150 that allowed beat by beat measures of SaO2 relative to their traditional averaging algorithm. Data include 2 complete nights comprised of 5 hours or greater of artifact free data at approximately 18, 000 feet.

Results

The table shows the difference in desaturation profiles between standard and beat to beat data. A desaturation was defined as a drop in SaO2 of 4% or greater.

Conclusion

During a night at high altitude beat to beat SaO2 data detects more desaturation events and shows ~2 % difference in nadir depth as well as a ~ 5 second faster detection time. This may be important when attempting to screen for patients with expected sleep apnea. Research supported by Mayo Clinic and TNF.
Back to scratch: hands‐on home cooking increases ethnic produce consumption among elementary school students from low‐income ethnically diverse families
Authors: Chen Qiong , Goto Keiko , Wolff Cindy , Bianco‐Simeral Stephanie , Gruneisen Kristin , Gray Katharine
Published: June 2021

Objective

To evaluated the effectiveness of a four month school intervention among K‐2 students.

Methods

Mixed methods of pre/post‐surveys and focus groups were used to evaluate the intervention. K‐2 students and their parents from four intervention and two control schools participated in the study. A total of 592 intervention students participated in classroom food demonstrations and tasting activities using seven locally grown food recipes primarily featuring Latino and Hmong cultures. Students received a food kit containing ingredients to take home for each recipe. Generalized estimating equations were used for survey data analysis. Data from parent focus groups were analyzed based on the principles of grounded theory.

Results

Both quantitative and qualitative results revealed that intervention students increased familiarity, preference, and consumption of the featured vegetables, and significantly increased their involvement in food preparation at home. Qualitative results showed that children's involvement in food preparation resulted in an increase in fruit and vegetable consumption. In addition, the intervention helped parents further appreciate local and/or ethnic produce from different cultural groups.

Conclusions

Promoting locally grown ethnic produce to K‐2 children is effective in increasing their involvement in food preparation and consuming a variety of vegetables at home.
Meal time dilemma; college students demand healthy but choose tasty foods
Authors: Frith Amy , Sato Sakura
Published: June 2021

College dining services and intrapersonal factors are crucial to college students’ food choices. The goal of this study was to identify college students’ perceptions of and reasons for choosing healthy or less healthy foods in dining halls. Perceptions of health characteristics of foods were collected by questionnaire from students (n=25) in a residential college campus. In another 88 students, self‐reported food consumption based on 21 food characteristics, types of foods, frequency of consumption and reasons for choosing those foods were collected for each meal. Students perceived healthy food to have these characteristics: fresh, fewer chemicals, more vitamins and minerals, high protein, and non‐processed. While many students (41%) reported choosing healthy foods “very often” or “always”, the majority reported that they demand healthy foods. Students (32%) primarily chose both healthier and less healthy foods for taste. Fewer students chose healthier foods for health (22%), and they chose less healthy foods because they were filling (13%), or it was a habit (7%). Females chose healthier food more often than males and freshman chose healthier food the least often. Future research should examine how to address the discrepancy of demand versus consumption of healthy foods, and to explore interventions utilizing promoting “tasty” with healthy foods.
Lactoferrin ameliorates Prostaglandin E2 (PGE2)‐mediated inhibition of Na‐glucose cotransport in enterocytes
Authors: Talukder Jamil , Muhammad Naairah , Williams Rosalyn , Johnson LaTia , Wright Jaleesa
Published: June 2021

Enhanced cyclooxygenase pathway metabolite, PGE2, inhibits SGLT1 mediated glucose absorption in chronically inflamed intestinal villus cells. Lactoferrin (Lf), a multifunctional glycoprotein is absorbed by receptor‐mediated transcytosis has been shown to exert anti‐inflammatory effects. This study was designed to investigate whether Lf would prevent PGE2 inhibition of SGLT1 mediated glucose absorption in enterocytes. [3H]O‐Methyl‐Glucose uptake was measured in 10 days postconfluent rat intestinalepithelial cells (IEC‐6) grown on transwell plates. The cells used were treated with phlorizin, PGE2, AH6809, and Lf. Na‐dependent glucose uptake, phlorizin, and immunoblotting studies established the activity and apical membrane localization of SGLT1. PGE2 inhibited SGLT1 in both concentration and time dependent manner with an inhibitory constant Ki of 50.03 nM and antagonized by prostanoid receptor inhibitor. PGE2 did not alter Na+/K+‐ATPase activity. In contrast, qRT‐PCR and western blot analyses revealed that SGLT1 specific transcripts and protein expression level were decreased 3‐fold by PGE2 treatment. Kinetic studies demonstrated that PGE2 inhibited SGLT1 by reducing the cotransporter expression which is reversed by treatment with Lf. We speculate that Lf ameliorates PGE2 inhibition of SGLT1 mediated glucose absorption by interfering with the signal transduction pathway in IEC‐6 cells.