bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of TREK1 two pore domain potassium channels by citalopram
Authors: Al‐Moubarak Ehab , Veale Emma L , Mathie Alistair
Published: June 2021

TREK‐1, two pore domain potassium (K2P) channels are responsible for background currents that regulate neuronal excitability. In this study we investigated the effect of the antidepressant, citalopram, on TREK1 channels. tsA‐201 cells were transiently transfected with wild type and mutated K2P channels. The whole cell patch clamp technique was used to obtain current recordings. Homology models for TREK1 were constructed using Modeller. Citalopram was docked to these models using the MOE program. Citalopram (100 μM) significantly inhibited WT TREK1 by 73 ± 2 % (n=8). Docking of citalopram into the TREK1 homology model suggests that it resides in the central cavity of the channel and its binding may involve residues from both the pore regions (T142 & T251) and regions of M2 and M4 (L174 & L289). These residues were mutated to alanine and the inhibitory effect of citalopram was reduced significantly in each case. The E306A gain of function mutation prevented citalopram inhibition. S‐citalopram (n=5) and R‐ citalopram (n=6) were equally effective at inhibiting TREK1. Citalopram (100 μM) was also able to inhibit TREK‐2 (57 ± 3 %, n=8) and TRESK (47 ± 4 %, n=7) but only inhibited TASK3 by 12 ± 5 %. (n=7). These data may explain the association observed between subjects with TREK1 gene variants and citalopram treatment resistance (Perlis et al 2008, Neuropsychopharm 33: 2810). Supported by BBSRC and Pfizer.
Age‐Related Serum Alkaline Phosphatase Trends
Authors: Frisch Frank , Wilson Zechariah , Arbuckle Cody , Sternlicht Eric , Caporaso Fred
Published: June 2021

Biochemical markers of bone turnover have been shown to be useful as inexpensive and noninvasive tools for monitoring skeletal health. Because duel energy absorptiometry and other indicators of bone mineral density reflect changes to bone accumulated over months, we sought to assess bone health and to explore potential sex differences in bone metabolism. Bone‐specific alkaline phosphatase (ALP), 25‐hydroxy‐and 1, 25‐dihydroxyvitamin D, calcium, phosphate, and parathyroid hormone (PTH) in 359 men and women (n=77 and 282, respectively) between the ages of 18 and 80 years old. Subjects were placed in one of three age groups (20–50, 51–70 and >;71 years of age for men and 18–40, 41–60, and >;60 years of age for women, and into a self‐identified ethnicity category. As expected, we found a downward trend in bone densities as subjects got older. We also found that ALP values were significantly lower in the older cohort of males and significantly higher in the older cohort of females when compared to their respective younger cohort (p<0.01). We propose an index utilizing PTH and ALP ratios that may reflect the metabolic direction for bone health. Given the complexities of hormonal, nutrient and activity levels of elderly subjects, these data suggest that the mechanisms that contribute to bone remodeling may be different in males when compared to females. This research was funded by Irvine Health Foundation.
Iterated micro‐trauma stimulates tendon healing via a gene expression pattern similar to early mechanical loading. Study on rat Achilles tendons.
Authors: Eliasson Pernilla , Hammerman Malin , Aspenberg Per
Published: June 2021

The objective of this study was to compare the gene expression response in healing unloaded rat Achilles tendons after micro‐trauma with previous findings after mechanical loading. 44 rats were given Botox into the calf muscle to reduce loading, and the Achilles tendon was transected. The healing tissue was traumatized by needling with a thin needle. qPCR for 18 genes, known to be regulated by loading, was performed 15 min and 3 h after a single needling episode. Mechanical properties of the tendon were measured after 4 consecutive days with needling, and showed increased strength of the tendon callus by 46%. 14 genes were significantly different from controls. 3 were regulated after 15 min, including up‐regulation of early growth response 1. 11 genes were regulated after 3 h; mainly genes involved in inflammation and coagulation. Trauma induced almost the same genes as loading, but there were differences in the level of increase. Inducible nitric oxide synthase was up‐regulated 38 times after trauma compared to 6 times after loading. A similar pattern was seen for other genes involved in inflammation. In conclusion, there was a striking similarity between the gene expression response to needling and mechanical loading. This suggests that the response to loading in early tendon healing can, at least in part, be a response to micro‐damage. This study was funded by the Swedish Research Council.
Understanding Signaling Regulation of Gut Smooth Muscle Contraction from Genetics Evidence
Authors: Gao Yun‐Qian , He Wei‐Qi , Qiao Yan‐Ning , Zhang Cheng‐Hai , Zhao Wei , Zhu Min‐Sheng
Published: June 2021

Multiple signal pathways have been implicated in regulation of gut smooth muscle contraction, but their physiological importance and functional contribution in gut are not clear. By use of knockout mice with smooth muscle‐specific deletion of myosin light chain kinase (Mlck) gene and myosin regulatory light chain (Myl9) gene, we found that the mutant mice abolished gastrointestinal motility within 21 days after birth, along with profound reduction of myosin light chain phosphorylation. This result emphasized the importance of calcium/calmodulin‐dependent mechanism in gut motility control, rather than calcium/calmodulin‐independent mechanism. This conclusion is also supported by the evidence from ZIP kinase knockout mice showing that deletion of Dapk3 gene did not affect smooth muscle contractility directly. Deletion of MYPT1 leads to elevation of myosin light chain phosphorylation and enhanced contractile response to KCl and agonists. Interestingly, the MYPT1 knockout mice also display significant reduction of gastrointestinal motility which may be partially restored by mechanical stimulation. This effect may be attributable to altered contractile phenotype of gut smooth muscle and reduced Cajal cells network in the smooth muscle. The loss‐of‐function evidence from knockout mice provides a scheme of signal regulation of gut motility in the context of smooth muscle contraction.
Cohesin‐dependent association of tRNA genes with nuclear pore complexes in budding yeast
Authors: Chen Miao , Gartenberg Marc R.
Published: June 2021

Budding yeast tRNA genes, tDNAs, are located throughout the genome yet large families of the genes cluster together at the nucleolus. Formation of the tDNA clusters is condensin‐dependent while their nucleolar localization is microtubule‐dependent (Haeusler et al., Genes & Development, 2008). Previously we found that tT(AGU)C, the tDNA that creates a boundary at the heterochromatic HMR locus, situates infrequently at the nucleolus (Valenzuela et al., Molecular and Cellular Biology, 2008). Using fluorescence microscopy, we show that tT(AGU)C colocalizes with nuclear pore complexes (NPCs) and that this association occurs primarily in M phase of the cell cycle. Building on recent mapping of nucleoporins to tT(AGU)C (Ruben et al., PLoS ONE, 2011), we show by chromatin immunoprecipitation that Nup60 is enriched at tT(AGU)C and other tDNAs in M phase. Association of these loci with NPCs requires tDNA gene sequences, Nup60 and cohesin. DNA circles bearing tT(AGU)C also colocalize with NPCs, indicating that the association requires only the tDNA and not other more global features of chromosome structure. Taken together, these results reveal an unexpected yet general phenomenon of cohesin‐dependent tDNA‐NPC association at M phase. Finally, using RT‐qPCR we find that the NPC‐tDNA association correlates with an increase in nascent tRNA levels at M phase. We speculate that the NPC‐tDNA association facilitates cell‐cycle dependent expression of tDNAs. This research was supported by grants from NIH (GM51402).
Therapeutic potential of Liuwei dihuang wang, a traditional Chinese Medicine, for spinal muscular atrophy in vitro and in vivo
Authors: Lo Yi‐Ching , Tseng Yu‐Ting , Liang Wei‐Fang , Jong Yuh‐Jyh
Published: June 2021

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by abnormally low cellular levels of the survival motor neuron (SMN) protein, resulting in motor neuron degeneration, muscle atrophy and loss of motor function. Currently, there is no effective treatment for SMA. Liuwei dihung wan (LDW) is a traditional Chinese medicine used for neurosis, diabetics and renal disorders. The aim of this study is to evaluate the therapeutic potential of LDW for SMA using inducible SMN‐knockdown NSC34 motor neuron cells and SMA‐like mice (SMNΔ7). Protein expression was detected by western blots. Cell viability was determined by MTT and LDH assay. SMA‐like mice were assessed daily for survival and body weight. Motor function was measured by hind‐limb suspension test. In SMN‐knockdown NSC34 motor neurons, LDW increased SMN expression and cell viability. In SMA‐like mice, LDW improved survival, body weight and hind‐limb scores. Moreover, LDW increased SMN expression in brain, spinal cord and gastrocnemius muscle of SMA mice. In conclusion, LDW could enhance SMN expression, diminish loss of motor neurons, and improve motor function and survival of SMA mice, suggesting LDW is a potential candidate for SMA treatment. This study was supported by grant (no. NSC 99–2320‐B‐037–023‐MY3) provided from National Science Council of Taiwan.
Inhibition mechanisms of multiple drugs on norepinephrine, dopamine, and serotonin transporters
Authors: Choi Kee‐Hyun
Published: June 2021

Monoamine transporters (norepinephrine, dopamine, and serotonin transporters (NET, DAT, and SERT, respectively)) are localized to presynaptic sites, where they terminate neurotransmitter transmission and maintain presynaptic neurotransmitter storage. Due to the essential roles of neurotransmitters in brain, monoamine transporters are important therapeutic targets. In particular, triple‐acting agents simultaneously inhibiting NET, DAT, and SERT are under clinical evaluation for treating depression. Given their therapeutic values, in the present study, pharmacological properties of NET, DAT, and SERT were investigated using selective inhibitors of each transporter (nisoxetine, vanoxerine, and fluoxetine, respectively) as well as non‐selective inhibitors. Binding affinities were measured by competitive affinity assays using radioactive ligands, while inhibitor potencies were determined by fluorescence‐based uptake assays. The IC50 values of inhibitors in uptake assays were compared with the Kd values in binding assays, revealing substantial differences between potency and binding affinity (IC50 >;>; Kd) for SERT, but not for NET or DAT. It suggests additional critical steps other than binding might be necessary to exert inhibitory effects on SERT. Multiple drugs on individual transporter were also examined, resulting in additive effects.
5‐HT2C Receptor agonist anorectic efficacy potentiated by 5‐HT1B Receptor agonist co‐application; an effect mediated via augmented pro‐opiomelanocortin neuron activation
Authors: Doslikova Barbora , Garfield Alastair , Shaw Jill , Evans Mark , Burdakov Denis , Billups Brian , Heisler Lora
Published: June 2021

A key component of the appetite‐regulating neural network is the 5‐HT2C receptor (5‐HT2CR); agonists of which suppress food intake and were recently approved for obesity treatment by the US

Food and Drug Administration. Appetite is primarily mediated via activation of hypothalamic arcuate nucleus (ARC) pro‐opiomelanocortin (POMC) neurons which are disinhibited via a 5‐HT1BR mediated suppression of local inhibitory inputs. We investigated whether 5‐HT2CR agonist anorectic potency could be significantly enhanced by co‐administration of a 5‐HT1BR agonist and associated with augmented POMC neuron activation. The combined administration of subanorectic concentrations of 5‐HT2CR and 5‐HT1BR agonists produced a potent 45% reduction in food intake and significantly greater in vivo ARC neuron activation. The chemical phenotype of activated ARC neurons was assessed by calcium imaging in POMC‐EGFP brain slices, revealing that combined agonists activated significantly more POMC neurons (46%) compared to either drug alone (approximately 25% each). Single‐cell electrophysiological analysis demonstrated that 5‐HT2CR/5‐HT1BR agonist co‐administration did not significantly potentiate the firing frequency of individual ARC POMC‐EGFP cells compared to drugs alone. These data indicate a functional heterogeneity of ARC POMC neurons by revealing distinct subpopulations of POMC cells activated by 5‐HT2CRs and disinhibited by 5‐HT1BRs. Thus, co‐administration of a 5‐HT1BR agonist potentiates the anorectic efficacy of 5‐HT2CR compounds by increasing the number but not magnitude of activated ARC POMC neurons and is of therapeutic relevance to obesity treatment.
Quercetin Relaxes Cholecystokinin‐ and KCl‐Induced Tension in Guinea Pig Gallbladder Strips
Authors: Kline Loren , Karpinski Edward
Published: June 2021

Quercetin, a flavonoid, relaxed cholecystokinin octapeptide‐ (CCK) and KCl‐induced tension in a concentration dependent manner. An in vitro technique was used to determine which system(s) mediated the relaxation. Paired t‐tests were used; differences between mean values of p<0.05 were considered significant. Adding quercetin prior to CCK or KCl produced a significant (p<0.001) decrease in the amount of tension (0.75+0.04 vs. 0.43+0.04 g CCK; 0.8+0.07 vs. 0.5+0.04 g KCl). When the PKC inhibitors bisindolymaleimide IV and chelerythrine Cl‐ were used together, a significant (p<0.001) reduction in the quercetin‐induced relaxation (45.7+4.3 vs. 27.6+3.4%) was observed. To determine if PKA mediated the quercetin‐induced relaxation, PKA inhibitor 14‐22 amide myristolated (PKA‐IM) was used. PKA‐IM significantly (p<0.05) decreased the amount (40.4+3.7 vs. 34.5+3.3%) of quercetin‐induced relaxation. The use of 2‐APB also significantly (p<0.001) reduced the amount of quercetin‐induced relaxation (51.2+3.5 vs. 14.8+3.6%). L‐NMMA significantly (p<001) decreased the quercetin‐induced relaxation (47.1+5.1 vs. 36.8+4.1%). KT5823 had no effect on the quercetin‐induced relaxation. Quercetin blocked extracellular Ca2+ entry which affected downstream events such as activation of PKC, PKA, intracellular Ca2+ release, and activation of NO synthase. The quercetin‐induced relaxation of CCK‐induced tension is mediated by multiple signaling pathways.
Abundant 5‐HT Release from EC Cells Disrupts Colonic Motility
Authors: Lavoie Brigitte , Tchitchkan Dmitri , Mawe Gary
Published: June 2021

Serotonin (5‐HT) that is synthesized by enterochromaffin (EC) cells in the intestinal mucosa can activate the ascending contractile and descending relaxant limbs of the peristaltic reflex. However, recent studies have demonstrated that peristalsis persists in the absence of mucosal 5‐HT, raising the question of whether mucosal 5‐HT actually influences propulsive motility. In this study we evaluated the actions of emetic drugs (ipecac, emetine and cisplatin), which are known to induce massive release of 5‐HT from EC cells, on propulsive motility in isolated segments of guinea pig distal colon. A Gastrointestinal Motility Monitor (GIMM; Catamount Research) system was used to record and analyze the rate of fecal pellet propulsive motility. Following a 30 minute equilibration period, pellet velocity was evaluated over three trials separated by 5 minute rest periods, and under control conditions was typically in the range of 1.8‐2.4 mm/sec. Drugs were then added to the solution for 10 min, and an additional 3 trials were recorded. Intraluminal infusion of ipecac (2‐10%), emetine (1‐10mM) or cisplatin (3µM‐1mM) to the bath slowed motility in a concentration‐dependent manner, and halted transit at high concentrations. Bath application of the 5HT3/5HT4 antagonist SDZ‐205‐557 hydrochloride (10µM) inhibited the effects of the emetic compounds, but intraluminal application of the antagonist had no effect. In summary, extensive activation of 5‐HT release from EC cells over an entire segment of the colon has a negative impact on propulsive motility. These findings support the concept that release of mucosal 5‐HT can influence propulsive motility in the intestines. Supported by DK62267
Altered Gastrointestinal Motility in a Mouse Model of Multiple Sclerosis
Authors: Spear Estelle , Lavoie Brigitte , Applebee Angela , Teuscher Cory , Mawe Gary
Published: June 2021

Multiple sclerosis (MS) patients often experience constipation, but the etiology of this symptom is unknown. To study this in an animal model, we tested the hypothesis that mice with experimental autoimmune encephalomyelitis (EAE) would exhibit altered gastrointestinal (GI) transit and they would have circulating antibodies directed against targets in enteric ganglia. EAE was induced in C57BL/6J mice by injection of complete Freund's adjuvant (CFA) and myelin oligodendrocyte glycoprotein. After somatic motor symptoms developed, small intestinal transit was measured by calculating the leading edge (LE) and geometric center (GC) 20 minutes after oral gavage of rhodamine dextran. Whole GI transit time was determined by oral gavage of carmine red and timing the appearance in fecal pellets. Antibody targets were evaluated by immunohistochemical staining of guinea pig intestine with EAE and control plasma. Small intestinal transit was significantly slower in EAE mice than in controls (LE, p<0.02; GC, p<0.01), and was not altered in CFA controls. The rate of whole GI transit was significantly longer in EAE mice versus healthy controls (p<0.05). Plasma from EAE mice yielded more intense immunoreactivity in myenteric ganglia than plasma from control animals, with immunostained structures including neurons and nerve processes. In summary, EAE is associated with slowed intestinal motility compared to healthy control mice, which could be representative of the bowel dysmotility exhibited by multiple sclerosis patients. The symptomology may involve the presence of enteric ganglion‐targeted antibodies in the blood of EAE mice. Funding: National MS Society
Proximity Ligation Assay of Interactions between β‐1 Integrin and Ca2+ Sensitization Proteins During the Contractile Response of Gastric Fundus Smooth Muscles to Cholinergic Stimuli
Authors: Xie Yeming , Perrino Brian
Published: June 2021

Contraction and relaxation of smooth muscle involves the regulation of myosin light chain (LC20) phosphorylation by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Rho‐associated, coiled‐coil containing protein kinase 2 (ROCK2), and myosin phosphatase target subunit 1 (MYPT1) are crucial for gastrointestinal smooth muscle contractile regulation via their inhibition of MLCP activity. β‐1 integrin is a trans‐membrane receptor that forms focal contacts and mediates the attachment of smooth muscle cells to the extracellular matrix and therefore plays an important role in cell adhesion, tension generation, and mechanotransduction in smooth muscle cells.

Here we use proximity ligation assays (PLA) to examine the role of β‐1 integrin in gastric fundus smooth muscle contraction. Muscle contraction with bath‐applied carbachol (CCh) or neurally‐released ACh by atropine‐sensitive electrical field stimulation (EFS) significantly increased the interaction of MYPT1, ROCK2 and S19 phosphorylated LC20 with β‐1 integrin. Furthermore, PLA analysis of intact gastric fundus smooth muscle tissue shows that both bath‐applied CCh and neurally‐released ACh increase MYPT1‐T853 phosphorylation, in contrast to our previous findings obtained by western blot analysis of whole cell lysates showing that only CCh treatment increased MYPT1‐T853 phosphorylation. These results indicate that Ca2+ sensitization and contractile proteins interact with β‐1 integrin in focal contacts in response to cholinergic stimuli, indicating a potentially important role of β‐1 integrin in contributing to the contractile responses of fundus smooth muscles to cholinergic stimuli.
The Therapeutic Potential of Toll‐like Receptor 4 Antagonists in Preventing Liver Injury Induced by Biliary Obstruction and Subsequent Endotoxemia
Authors: Yokoyama Yukihiro , Oya Shingo , Kokuryo Toshio , Nagino Masato
Published: June 2021

Objective

The objective of this study was to elucidate the efficacy of toll‐like receptor 4 (TLR4) antagonists in preventing liver injury induced by biliary obstruction and subsequent intra‐portal lipopolysaccharide (LPS) infusion in rats.

Background

Biliary obstruction often leads to the development of bacterial translocation and subsequent endotoxemia. Both biliary obstruction and endotoxemia lead to a severe liver injury.

Materials and Methods

Rats were subjected to either a sham operation (Sham group) or bile duct ligation for 7 days (BDL group). Seven days after each operation, LPS (0.5 μg) was injected through the ileocecal vein. In other experiments, rats that had undergone BDL were pretreated, prior to LPS challenge, with intravenous TAK‐242, a TLR4 inhibitor.

Results

The expression of the TLR4 protein as well as the number of Kupffer cells in the liver were significantly increased in the BDL group compared with the Sham group. The expression of TLR4 co‐localized with Kupffer cells, which was confirmed by double immunofluorescence staining. Serum levels of liver enzymes and proinflammatory cytokines after intra‐portal LPS injection were significantly higher in the BDL group than in the Sham group. However, pretreatment with TAK‐242 strongly attenuated these changes.

Conclusions

These results imply that blocking TLR4 signaling effectively attenuates liver damage in rats with BDL and subsequent intra‐portal LPS infusion. TAK‐242 treatment may be used for patients who are susceptible to liver damage by biliary obstruction and endotoxemia.
A Convenient Human Hepatocyte Model for Molecular Mechanisms in Caffeine Preconditioning and Protection from Acetaminophen (APAP)‐Induced Hepatotoxicity
Authors: Gupta Priya , Sharma Yogeshwar , Bandi Sriram , Vishwanathan Preeti , Gupta Sanjeev
Published: June 2021

Long‐term low‐dose caffeine use may be tissue protective with superior outcomes in liver cirrhosis or other chronic diseases. However, molecular mechanisms underlying these effects of caffeine are unknown and suitable cell‐based models will be helpful for this purpose. Caffeine may affect cells through multiple pathways including interference with DNA damage/repair through ATM, ATR or DNA‐PK activity. Here, we focused on regulation by low‐dose caffeine of ATM‐related mechanisms. To induce hepatotoxicity, we cultured HuH‐7 cells with 10 mM APAP (IC50) for 16‐20h. The effects of no caffeine preconditioning or preconditioning over 5 d with 1‐10 mM caffeine were studied. Cell viability assays with MTT dye reduction indicated low‐dose caffeine preconditioning decreased but short‐term caffeine exposure increased APAP toxicity, p<0.05. Flow cytometry showed low‐dose caffeine preconditioning depleted cells in S and G2/M and enriched cells in G0/G1, indicating replicative stress. This residual cell population contained several‐fold greater numbers of Side Population cells with putative stem cell properties, p<0.05. Use of an ATM promoter construct indicated that caffeine interfered with ATM activity in HuH‐7 cells. Therefore, low‐dose caffeine preconditioning in HuH‐7 cells involved ATM‐related mechanisms, and offers convenient models for studies. Cytoprotection against APAP toxicity in caffeine‐preconditioned cells will allow insights into ATM signaling‐specific pathways, cell cycle controls, and stem‐like cells capable of withstanding injury.
Subcellular Zinc Deficiency Links with ER and Mitochondria Cell Death Signaling in Alcoholic Liver Disease
Authors: Sun Qian , Zhong Wei , Zhang Wenliang , Sun Xinguo , Zhou Zhanxiang
Published: June 2021

Induction of hepatic zinc deficiency and apoptotic cell death are consistently observed in patients with alcoholic liver disease. This study aimed to determine how chronic alcohol exposure affects subcellular zinc homeostasis and whether organelle zinc deficiency drives ER and mitochondrial cell death signaling. Rats were pair‐fed with control or ethanol liquid diet for 5 months. Alcohol exposure reduced zinc level in whole liver and isolated ER and mitochondria. Zinc transporter proteins were detected in both isolated ER and mitochondria, and alcohol exposure increased ER ZIP 13, an export transporter. Alcohol exposure induced cell death signaling from both ER as indicated by increased ER stress marker proteins (p‐eIF2α, ATF4, and CHOP) and mitochondria as indicated by increased mitochondrial Bax and cytosolic cytochrome c. To define the role of zinc deficiency in generation of cell death signaling, hepatoma cells were treated with zinc chelator, 3 µM TPEN. Zinc deprivation induced caspase‐3 activation and apoptosis in association with increased ER stress marker proteins and reduced mitochondrial membrane potential (MMP). To determine whether zinc deficiency‐induced apoptosis is dependent on oxidative stress, antioxidant N‐acetyl cysteine (NAC) was added at the time of TPEN treatment. NAC completely quenched TPEN induced oxidative stress, but only partially prevented TPEN‐induced ER stress, MMP reduction, caspase 3 activation and apoptosis. This study demonstrated, for the first time, that chronic ethanol exposure induced ER and mitochondria zinc dyshomeostasis which triggers ER and mitochondrial cell death signaling independent on oxidative stress.
Lactobacillus rhamnosus Cultured Milk Isolate may Reduce Adipogenesis
Authors: Kotala Justin , Onyango E
Published: June 2021

Obesity has reached epidemic levels around the world. There is a need for a better understanding of the different factors in the digestive tract environment that may modulate adipogenesis. Our goal is to understand the influence of potential probiotics on the differentiation and subsequent lipid storage of adipose cells. This study was performed to test the in vitro effects of a Lactobacillus rhamnosus isolate from “amabere amaruranu”, a traditional Kenyan cultured milk, on 3T3‐L1 cell line. Cultures of 3T3‐L1 cells were treated with bacterial isolate cell extract, filtered supernatant from overnight bacterial culture or with a PBS control. Cells were allowed to differentiate in the presence of different dose levels of appropriate treatments for 10 days. Cells were collected, lysed and the resultant lysate analyzed for adipogenic‐related transcription factors using western blot. Protein expression levels indicated that the treatment with bacterial isolate cell extract, but not the bacterial culture supernatant, downregulated the expression of various adipogenic‐related transcription factors in a dose‐dependent manner. The highest dose of the bacterial cell extract downregulated peroxisome proliferator‐activated receptor‐γ, sterol regulatory element‐binding protein 1, and adipose triglyceride lipase by 1.6, 1.5, and 1.8 fold, respectively. The cell extract did not affect the level of expression of CCAAT/enhancer binding protein‐α. In conclusion, bacterial cell extract from, Lactobacillus rhamnosus, from “amabere amaruranu” cultured milk showed anti‐adipogenic properties, and could be used for anti‐obesity management. This study shows that the L. rhamnosus isolate warrants further studies in vivo.
Mechanism of Ceruloplasmin‐Copper Uptake by, and Overexpression in, Mammalian Cells
Authors: Vargas Rebecca , Islas Leonel , Gaite Michaella , Berger Devon , Ramos Danny , Mar David , Kidane Theodros , Cuajungco Math , Linder Maria
Published: June 2021

Ceruloplasmin (Cp) is an abundant multifunctional copper‐binding protein in the blood plasma best known for its ferroxidase activity. Using purified 64Cu‐labeled Cp, we have now established that it also delivers copper directly to cultured cells. Here, we further examined whether this process occurs at the cell surface, involving a reductase, and attempted to identify a new copper transporter and overexpress human Cp. Uptake of copper from 64Cu‐Cp (purified from mouse plasma) by mouse embryonic fibroblasts that do and do not express Ctr1, was not prevented by endocytosis inhibitors; and holoCp appeared to convert to apoCp during uptake. The only known copper reductase expressed was identified as Steap2 by qPCR. Successive siRNA treatments did not reduce cell surface reductase activity, and other approaches are being applied. Identification of copper uptake transporter in the Ctr1‐/‐ fibroblasts was sought using biotinylation‐targeting with mouse Cp, and cross‐linking with formaldehyde. No clear candidates have as yet emerged. To provide a reliable source of 64Cu‐Cp for future studies, attempts were made to clone and express human Cp in Sf9 insect cells using baculovirus (which did not succeed); overexpression in human cell lines using either a conventional or lentivirus construct is now in the process. We conclude that uptake of copper from Cp occurs at the cell surface and may require a reductase that delivers Cu1+ not just to Ctr1 but also to another transporter.
Contribution of Membrane Progesterone Receptors α and β to Ventilatory Chemoreflex in Female Mice.
Authors: Boukari Ryma , Rossignol Orlane , Marcouiller François , Bairam Aida , Joseph Vincent
Published: June 2021

We hypothesized that the membrane progesterone receptors (mPR) α and β which are expressed in the brainstem, modulate ventilatory chemoreflex. Adult female mice were instrumented with an osmotic pump for chronic infusion of small interfering RNA (siRNA) against mPRα or mPRβ in the fourth ventricle. Two weeks later, respiratory recordings were performed using whole body plethysmography under normoxia (30 min) and then in response to hypoxia (12% O2), hypercapnia (5% CO2) or hypoxic‐hypercapnia (5% CO2 + 12% O2), 10 min each. Compared to control experiments (vehicle solution without siRNA), the deletion of mPRα decreased the ventilatory response to hypercapnia (early phase, at 1 min of exposure). Whereas mPRβ deletion suppressed the ventilatory response during the early phase of exposure to hypoxia and hypoxic‐hypercapnia. In mice treated with the siRNA against mPRβ, the ventiltory responses to hypoxia and hypoxic‐hypercapnia were observed after a delay of 5.5±0.7 and 5.1±0.5 min compared to 0.2±0.1 and 0.7±0.4 min in controls. Specific deletions of mPRα and mPRβ have been verified by immunohistochemistry. We conclude that mPRα and β modulate the ventilatory chemoreflex in adult female mice. Founded by CIHR (MOP‐102715).
Time Course of Altered Ventilatory Activity Evoked by Intratracheal Lipopolysaccharide (LPS) Administration in Spontaneously Breathing Adult C57BL/6 Male Mice
Authors: Rice Keeley , Ahmad Mohammed , Hyder Muzammil , Conyers Jonathan , Solomon Irene
Published: June 2021

Intratracheal (IT) administration of LPS, which is often used as model of acute lung injury/acute respiratory distress syndrome in rodents, has been reported to induce time‐dependent changes in expression of pro‐inflammatory cytokines in respiratory‐related medullary regions. Little is known about the time course of alterations in ventilatory activity following IT LPS administration. Therefore, we examined the effects of IT LPS administration (0.5 mg/kg) on ventilatory behavior (recorded using whole body plethysmography) at 2‐, 6‐, 24‐, 48‐, 72‐, 96‐, and 120‐hr in unanesthetized spontaneously breathing adult C57BL/6 male mice. Before LPS, ventilatory activity was characterized by a regular pattern of breathing with spontaneous periodic short duration apneic pauses (as previously described in this strain) and periodic sighs. After LPS, multiple time‐dependent ventilatory behaviors were observed, with the most common patterns consisting of: (1) periods of dysrhythmic breathing characterized by clusters of typically 2‐3 (but as many as 6) breaths of varying magnitude in rapid succession, with each cluster being followed by an apneic pause of varying duration; (2) brief periods of regular stable breathing that exhibited increased frequency at 2‐hr, a return to pre‐LPS levels at 6‐hr, and an increase again at and after 24‐hr, with tidal volumes that were unchanged or reduced, especially at later time points; (3) numerous prolonged apneic pauses; and (4) an increased incidence of sighs. These findings indicate that IT LPS produces time‐dependent modulation of ventilatory activity, which may reflect the LPS‐induced differences in the cytokine expression profile.
Mesenchymal Stem Cell Survival after Intraspinal Transplantation
Authors: Barbero Margaret , Gransee Heather , Zhan Wen‐Zhi , Sieck Gary , Mantilla Carlos
Published: June 2021

Following spinal cord hemisection at C2 (SH), removal of descending excitatory drive to phrenic motoneurons results in ipsilateral diaphragm muscle paralysis with partial spontaneous recovery of rhythmic phrenic activity over time. Brain‐derived neurotrophic factor (BDNF) plays an important role in neuroplasticity following SH. Intraspinal transplantation of mesenchymal stem cells engineered to produce BDNF‐GFP (BDNF‐MSCs) at the time of injury promotes functional recovery of all animals, despite low levels of MSC survival at 14 days post‐SH. The goal of the present study was to determine the survival and localization of BDNF‐MSCs after intraspinal transplantation. BDNF‐MSCs were injected intraspinally at C4 (2 sites, total 2 x 105 cells in 4 µl) in adult male Sprague‐Dawley rats. After one and three days, the cervical spinal cord was fixed, excised, and cut longitudinally. Survival of transplanted BDNF‐MSCs was confirmed in all animals at 1 day post‐transplantation (n=5) and in five of 6 animals at 3 days post‐transplantation. BDNF‐MSCs remained localized at the site of injection in the white matter at 1 and 3 days, with an average volume of 1.1 ± 0.3 µl. There was no localization of MSCs in the vicinity of phrenic motoneurons identified by retrograde labeling with cholera toxin subunit B. Understanding the time course of stem cell survival after transplantation is important in defining windows of opportunity during which the beneficial effects of BDNF on respiratory function after spinal cord injury are executed.

Supported by HL96750 and Mayo Clinic Center for Regenerative Medicine.
The Unfolded Protein Response Regulates Apoptosis of Alveolar Epithelial Cells (AECs) by Modulating Autocrine Angiotensin (ANG)II and ANG1‐7
Authors: Nguyen Hang , Dang MyTrang , Ono Shinji , Morimoto Konosuke , Uhal Bruce
Published: June 2021

STUDY OBJECTIVE

Our prior work showed that ER stress‐induced apoptosis of AECs is regulated by the autocrine ANGII/ANG1‐7 system. MG132 or surfactant protein C (SP‐C) BRICHOS domain mutation G100S induced apoptosis in human AECs by activating cathepsin D and by decreasing the protective angiotensin‐converting enzyme 2 (ACE‐2). This study tested the hypothesis that ER stress‐induced apoptosis of human AECs might be mediated by UPR pathways that in turn modulate the autocrine ANGII/ANG1‐7 system of these cells.

METHODS

A549 cells were challenged with the proteasome inhibitor MG132 or the SP‐C mutant G100S. UPR markers and ANG system components were measured by Western blotting. Apoptosis was measured by caspase activation and nuclear fragmentation.

RESULTS

MG132‐ or G100S mutation‐induced ER stress activated the UPR pathways (phospho PERK, phospho eIF2α, ATF4, phospho IRE1, spliced XBP1, ATF6), which led to an increase of cathepsin D and a decrease of anti‐apoptotic ACE‐2. Blockade of UPR pathways with UPR chaperone 4‐PBA or siRNAs (siATF6, siIRE1, siPERK) prevented both the changes in cathepsin D and ACE‐2 and the AEC apoptosis caused by MG132 or G100S.

CONCLUSIONS

These data show that ER stress induces apoptosis in human AECs through UPR‐mediated pathways that in turn modulate the autocrine ANGII/ANG1‐7 system. They also demonstrate that ER stress‐induced apoptosis of human AECs can be prevented by inhibition of UPR‐specific pathways.
Effects Of Negative Pressure Wound Therapy And Elastomeric Materials On Bone Healing
Authors: Wang Rui , Wagner William
Published: June 2021

Negative pressure wound therapy has been used to accelerate clinical wound healing, however few have suggested applications for bone healing and potential pathways. In order to modulate the natural bone repair and make in vivo repair feasible, degradable materials for negative pressure wound therapy were designed. We fabricated and tested an electrospun composite of Type I collagen, chondroitin 6‐sulfate and Poly (1,8‐octanediol‐co‐citrate), termed BRM, that mimicked extracellular environment with collagen and elastin imbedded in a matrix of proteoglycans and glycoproteins. A closed chamber system was designed to generate and monitor negative pressure environments and conditions for osteoblast growth. When cultured on BRM and plastic dish with and without negative pressure, the cell proliferation was significantly greater (P<0.05) on BRM compared to electrospun collagen. With a negative pressure of ‐125mmHg, alkaline phosphatase activity was increased by Day 7 and osteoblast proliferation was increased by 1.3 folds on Day 17. The closed chamber system will be used to exam molecular pathways by the interactions of BRM and negative pressure on bone marrow derived stem cells. This study permits the evaluation of material design and feasibility of negative pressure to be explored for the accelerated bone healing.
Acute Alcohol Treatment and Cardiac Dysfunction in Obese Diabetic Mice: Role of PDE5 and MicroRNA‐21
Authors: Samidurai Arun , Salloum Fadi , Xi Lei , Das Anindita , Kukreja Rakesh
Published: June 2021

Objective

Excessive intake of alcohol under diabetic condition leads to increased stress and causes both transient and lasting structural changes including fibrosis in heart. We tested the hypothesis whether phosphodiesterase5 (PDE5) inhibitor, sildenafil prevents alcohol‐induced cardiac dysfunction in Type 2 Diabetic (T2D) db/db mice and the role of microRNA‐21(miR‐21) in fibrosis through regulation of ERK1/2‐MAP kinase signaling and sprouty‐1(Spry‐1).

Methods and Results

Adult male db/db mice were randomized to receive: saline as control (0.2 ml; i.p, n =9) or ethanol (1.5 g/ BID; i.p, n=5) or ethanol with sildenafil (0.7mg/kg/day; i.p, n=5) for 3 days. Echocardiography showed a decline in Ejection Fraction (EF) in mice treated with ethanol as compared to their baseline (P < 0.05). Treatment with sildenafil in combination with ethanol showed normal EF

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). Real time PCR using TaqMan microRNA probe showed 1.5 fold increase in miR‐21 level in the heart after alcohol treatment

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). Protein analysis showed down regulation of spry‐1, a target of miR‐21 and upregulation of ERK‐1/2 phosphorylation. Sildenafil treatment prevented the upregulation of miR‐21 and hence increased the level of spry‐1 and a concomitant decrease in ERK‐1/2 phosphorylation.

Conclusion

The induction of miR‐21 in ethanol treated db/db mice with a subsequent decrease in spry‐1 and an increase in Phosphorylation of ERK‐1/2 suggest a role of this signaling cascade in fibrosis and cardiac dysfunction. Therapeutic intervention using sildenafil prevented LV dysfunction caused by alcohol intake and trigger of fibrosis signaling pathway.

This study was supported in part by grants from National Institutes of Health (HL51045, HL79424, HL118808 to RCK).
Aromatic Amino Acid Network: Biosynthesis, Regulation and Transport
Authors: Dudareva Natalia
Published: June 2021

Aromatic amino acids are essential constituents of proteins in all living organisms, while in plants phenylalanine (Phe) and tyrosine (Tyr) additionally serve as precursors for thousands of specialized compounds that have a profound impact on plant growth, development, reproduction and defense. Plants direct 20‐30% of photosynthetically fixed carbon to the production of Phe and Phe‐derived compounds, which constitute approximately 30‐45% of plant organic matter. Humans are incapable of synthesizing Phe and must primarily obtain it directly or indirectly from plants. Phe biosynthesis in plants occurs via two alternative routes with either arogenate or phenylpyruvate as a key intermediate. Using petunia flowers as a model system, we have shown that plants predominantly synthesize Phe in plastids via the arogenate pathway while they can also utilize the microbial‐like phenylpyruvate pathway. Moreover, flux through the phenylpyruvate route is increased when the entry point to the arogenate pathway is limiting. Interestingly, the phenylpyruvate pathway utilizes a cytosolic aminotransferase that strongly favors Tyr as the amino donor. These results demonstrate that Phe biosynthesis is not limited to plastids and that there is an interconnection between aromatic amino acid catabolism and biosynthesis in planta. Another aspect of plant metabolism to be discussed is its compartmentalization and metabolite transport within the cell and how this intracellular transport contributes to the interconnections between and within metabolic networks.
Involvement of Gap Junctions Between Smooth Muscle Cells in The Sustained Hypoxic Pulmonary Vasoconstriction Development
Authors: Kizub Igor , Lakhkar Anand , Gupte Sachin
Published: June 2021

It is well known that inhaled hypoxia causes pulmonary artery constriction to maintain optimal ventilation‐perfusion matching in the lung. However, sustained hypoxic pulmonary vasoconstriction (HPV) leads to the development of pulmonary hypertension. The mechanisms of the sustained HPV remain unclear. The aim of this study was to determine the role of gap junctions (GJs) between smooth muscle cells (SMCs) in the sustained HPV development. The vascular tone was measured in isolated rings of bovine intrapulmonary arteries (BIPA) using isometric force measurement technique. Chronic hypoxia (PO2 = 10‐20 Torr) after 13 h elicited the sustained HPV in 3rd‐ and 2nd‐order BIPA precontracted with 30 mM KCl. The endothelium removal had no effect on the sustained HPV amplitude neither in 3rd‐ nor in 2nd‐order BIPA. Inhibition of GJs with 18β‐glycyrrhetinic acid (30 µM) and heptanol (3.5 mM) significantly decreased the sustained HPV amplitude both in deendothelised 3rd‐order and 2nd‐order BIPA. Similarly, a highly selective inhibitor Gap‐27 (100 µM) significantly reduced the sustained HPV amplitude in deendothelised 3rd‐order BIPA. Taken together, these data indicates that GJs between SMCs are involved to the sustained HPV development in bovine pulmonary artery, reflecting possibility of a novel pathway existing for signaling in SMCs during hypoxia that supports HPV. This study was supported by The Fulbright Program and the U.S. Department of State's Bureau of Education and Cultural Affairs to Kizub I.V., 2014
Prenatal Nicotinic Exposure (PNE) Promotes Pulmonary IL‐1β and 5‐HT and Gene/Protein Expression of IL1R1, NK1R, ADRA1, and TRPV1 in N/J ganglia Neurons via Acting on α7nAChRs in Rat Pups
Authors: Zhao Lei , Zhuang Jianguo , Zang Na , Lin Yong , Xu Fadi
Published: June 2021

We have shown that PNE sensitizes bronchopulmonary C‐fibers (PCFs) in rat pups. Here we asked if PNE was able to increase the pulmonary mediators known to sensitize/active PCFs, and their corresponding receptors expressed in nodose/jugular (N/J) ganglion neurons in P11‐14 pups. If so, we further asked if the evoked changes depended on nicotinic acetylcholine receptors (nAChRs), particularly α7nAChR. We compared IL‐1β, substance P (SP), serotonin (5‐HT), and adenosine (AD) in BALF and IL‐1 type I receptor (IL1R1), NK1R, TRPV1, and adenosine A1 receptor (ADRA1) in the N/J ganglia in P11‐14 rat pups prenatally exposed to saline (Ctrl) and PNE alone or coupled with mecamylamine (MM), a non‐selective blockade of nAChRs and methyllycaconitine (MLA), a selective blockade of α7nAChR. We found that PNE significantly augmented IL‐1β and 5‐HT but not SP and AD in BALF and gene and protein expressions of IL1R1, NK1R, TRPV1, and ADRA1 in the N/J ganglia. Both MM and MLA abolished the responses of IL‐1β and 5‐HT in BALF. MM eliminated the gene and protein responses of N/J ganglion TRPV1, NK1R, IL1R1, and ADRA1 to PNE except little effect on IL1R1 gene, while MLA eliminated all of the gene responses. Our data suggest that PNE is able to elevate IL1R1, NK1R, ADRA1, and TRPV1 in the N/J ganglia, which, along with increased IL‐1β and 5‐HT releases in the lungs, likely contributes to sensitize/activate PCFs. These PNE‐induced modulations are greatly dependent on the activation of α7nAChRs (Supported by HL‐107462).
Metabolic Therapies Improve Mitochondrial Morphology and Function
Authors: Ari Csilla , Poff Angela , Held Heather , Landon Carol , Goldhagen Craig , Mavromates Nicholas , Kesl Shannon , Ward Nathan , Fiorelli Tina , Van Putten Ashley , Sherwood Jacob , Arnold Patrick , D'Agostino Dominic
Published: June 2021

In diseases where glucose utilization is impaired ketone bodies and TCA cycle intermediates serve as alternative fuel. Since these diseases are linked pathophysiologically to impaired mitochondrial function, we hypothesized that nutritional ketosis induced by ketogenic precursors or TCA cycle intermediate supplements promote mitochondrial biogenesis and preservation of existing mitochondria.

Exogenous ketone supplementation was used in Sprague Dawley (SD) rats and in SOD1 G93A mice. In SD rats, ketone ester and ketone salt‐MCT oil mixture was gavaged daily, whereas the Deanna protocol (DP) supplementation containing tricarboxylic acid (TCA) cycle intermediates was added to standard rodent chow fed to SOD1 G93A mice. Plasma was collected at endpoint and global metabolomic profiles were compared by Metabolon. Morphology, density and distribution of skeletal muscle mitochondria were studied by TEM and SEM.

Exogenous ketone supplementation and the DP supplementation dose‐dependently increased compounds associated with glycolysis and the TCA cycle. Elevations of intermediates of fatty acid oxidation (FAO) and FAO substrates indicated increased beta‐oxidation in response to the DP supplementation. Higher density of mitochondria and mitochondrial cristae could be observed in skeletal muscle in response to supplementation. These processes together likely result in improved ATP production, enhanced motor function and slower progression of diseases associated with metabolic dysfunction.
Histopathology and Mitochondrial Function in Liver‐Specific LRPPRC Knockout Mice
Authors: Cuillerier Alexanne , Cadete Virgilio , Deschênes Sonia , Burelle Yan , Consortium LSFC
Published: June 2021

The French‐Canadian variant of Leigh Syndrome (LSFC) is an inherited mitochondrial respiratory chain disorder prevalent in the Saguenay‐Lac‐St‐Jean region of Quebec. It is caused by LRPPRC mutation, a leucine‐rich pentatricopeptide repeat protein that regulates the stability of mitochondrial mRNAs encoding proteins involved in oxidative phosphorylation. Mutated LRPPRC, present at low levels in all tissues from patients, causes a severe decrease in cytochrome c oxidase (COX) activity primarily in the brain and liver, leading to unpredictable subacute crises of neurodegeneration and hepatic lactic acidosis, killing 80% of children. To gain better knowledge of the disease, we characterized phenotype and mitochondrial function of mice harbouring liver‐specific inactivation of Lrpprc (Lrpprc‐HKO). Lrpprc‐HKO mice are viable but display a general growth delay. Livers from 5 week‐old mice present loss of lobular organization, steatosis, cholestasis, necrosis/regeneration and severe reduction in COX/SDH staining ratio. Mitochondrial preparations present several functional abnormalities including: i) increased state 2 and lower state 3 respiration with substrates feeding complex I, II and IV, ii) lower capacity to oxidize fatty acids, iii) reduced H2O2 release and scavenging and iv) increased resistance to Ca2+‐induced PTP opening. These changes are associated with two primary molecular defects, which contribute to explain the phenotype: i) a severe loss of COX activity and ii) an ATPsynthase assembly defect. Studies are underway to mechanistically link these molecular defects with the observed mitochondrial dysfunction and identify further consequences of Lrpprc liver deletion.