bluuebunny/crossref_metadata_2025 huggingface dataset:
SPARC: Abundance, Chemical Content and Regional Distributions of Nerve Fibers in the Human Gastric Muscle and Mucosa
Authors: Stebbing Martin J. , Di Natale Madeleine , Fakhry Josiane , Hunne Billie L. , Ward Sean M. , Sasse Kent C. , Furness John B.
Published: April 2020

Disorders of gastric motility and sensation, including gastroparesis, are common, debilitating and currently poorly treated. To develop new treatments for these disorders, a thorough understanding of the gastric innervation and its pathology in humans is required. We have developed methods to identify and quantify the density of different classes of nerve fibers to the gastric muscle and mucosa. To do this we employed immunohistochemistry using characterized antibodies and a grid‐based quantitation method combined with high resolution confocal microscopy in resection tissue and biopsies from the gastric fundus and corpus. In the muscle we have identified nerve fibers immunoreactive for a range of neurochemical markers including neuronal nitric oxide synthase (NOS), vasoactive inhibitory peptide (VIP), tachykinins (TK), gastrin releasing peptide (GRP), neuropeptide Y (NPY), vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH). Neuronal NOS and VIP were commonly colocalized in putative inhibitory nerves innervating the external musculature. We saw extensive labelling for VAChT and TK, both markers of the excitatory fibers innervating the muscle. GRP and NPY were also present in subgroups of excitatory nerve fibers. The density of inhibitory fibers (NOS) was greater than excitatory fibers (TK) in all external muscle layers. TH was found in a proportion of fibers in the muscle, suggesting direct sympathetic innervation of the muscle occurs in human stomach. These TH fibers also contained NPY but were separate from NPY immunoreactive excitatory muscle motor nerves.

Prominent in mucosal innervation were fibers immunoreactive for VIP and GRP and a separate population containing VAChT. Another population of fibers containing NPY and TH were found around mucosal glands and also around arteries supplying the mucosa. For all common fiber types in the mucosa there was a gradient of densities such that most fibers were found in the bottom third of the mucosa and few fibers were found in the top third of the glands near the lumen. Rare CGRP containing fibers were also seen in the mucosa but were not present in every section suggesting they are rare and unevenly distributed.

We conclude that the signature chemistries of nerve fibers that innervate the gastric muscle and mucosa in human are similar to those present in other species, including rat. An exception is the prominent direct innervation of the gastric muscle by extrinsic sympathetic axons which is often absent in small mammals. This parallel chemistry will allow us to extrapolate circuits determined experimentally in rat to human and to identify cell body types in the myenteric ganglia.

Support or Funding Information

This work was supported by NIH (SPARC) grant ID # OT2OD023847 (PI Terry Powley) to John Furness and Martin Stebbing.
Sprint Interval Training and Endurance Exercise Performance: Influence of Constant vs Variable Time of Day of Training and Time of Assessment
Authors: Ewell Taylor Russell , Harms Kole Jerel , Abbotts Kieran Shay Struebin , Bell Christopher
Published: April 2020

In light of the many demands competing for our attention in today’s modern lifestyle, scheduling healthy behaviors, such as daily exercise, at or around the same time of day can be difficult. Accordingly, the purpose of this study was to compare the endurance benefits of short‐term sprint interval training (SIT) when SIT was completed at a constant or variable time of day. The influence of the time of day of assessment relative to the time of day of training was also examined. SIT consisted of six sessions of 4‐to‐7 x 30‐second sprints on a stationary cycle ergometer completed over two weeks. Endurance exercise performance was assessed via a laboratory‐based cycle ergometer time trial equivalent to 10‐km. Two habituation time trials were completed prior to pre‐SIT assessment. Twenty healthy adults (age: 23 ± 7 years, body mass index: 24.7 ± 3.0 kg/m2, maximal oxygen uptake: 44 ± 9 ml/kg/min (mean ± SD)) were randomly assigned to one of two training conditions: (1) Constant (6 males, 3 females), in which all training and pre/post time‐trials occurred at a fixed time of day that was not necessarily the same time for each participant; or, (2) Variable (7 males, 4 females), in which all training was scheduled such that each session never occurred within 6‐hours of the time of day of the previous session; post‐SIT time trials for the Variable group were completed at the same time of day as pre‐SIT time trials. A second post‐SIT time trial (Post2) was scheduled for all participants to occur at a time of day a minimum of 6‐hours earlier/later than the time of day of the first post‐SIT time trial. SIT improved time trial performance (main effect P=0.019) but only when the time trial did not occur at the same time of day as training (i.e. Post2); the magnitude of improvement was not influenced by whether the training was completed at a constant or variable time of day (Constant: Pre 1153 ± 173, Post 1136 ± 179, & Post2 1123 ± 154 s vs. Variable: Pre 1186 ± 159, Post 1166 ± 158, & Post2 1149 ± 130 s; group x training interaction P=0.646). SIT did not influence body mass or composition, as determined by dual energy X‐ray absorptiometry (all P>0.34). At the start of each SIT session, oral temperature was measured. The co‐efficient of variation appeared lower for the Constant compared with the Variable group (0.6 ± 0.3 vs. 1.1 ± 0.7%; P=0.099). Our preliminary data suggest that SIT improves endurance exercise performance when assessed at a variable time of day, irrespective of whether training is scheduled at a constant or variable time of day. From a public health perspective, scheduling regular exercise at or around the same time of day does not appear to be important for obtaining endurance benefits.
ADAM17 on glutamatergic neurons inhibits GABAergic signaling in a salt‐sensitive hypertension model
Authors: Xu Jiaxi , Lazartigues Eric
Published: April 2020

The brain plays an important role in modulating sympathetic drive to individual organs, leading to changes in cardiac output, vascular resistance and blood pressure regulation. Within the central nervous system, A Disintegrin And Metalloprotease 17 (ADAM17) impairs the compensatory activity of angiotensin converting enzyme type 2 (ACE2) through ectodomain shedding during hypertension. We previously observed that activation of neuronal ADAM17, especially in excitatory neurons, contributes to the development of neurogenic hypertension through promoting sympatho‐excitation. It was also revealed that ADAM17 located on glutamatergic neurons plays a supportive role in Ang‐II‐induced neuronal activation. To investigate the contributing mechanism, deoxycorticosterone (DOCA)‐salt (1mg/g body weight sc + 1% saline po for 3 weeks) or sham treatment was given to mice harboring ADAM17 knockdown in glutamatergic neurons (A17G) and their control littermates (C57Bl6/J male mice, 14–16 week‐old, n=10). At the end of the treatment, mice were sacrificed and their brains were collected. Expression levels of NMDA receptors (Grin1), AMPA receptors (GluR1), as well as GABAa receptors and Gad67 was assessed by QRT‐PCR. Among these genes, mRNA levels for Grin1, GluR1, and GABAa receptors were shown to be unaffected in the hypothalamus of either A17G mice or controls. However, mRNA level of Gad67, a critical enzyme that catalyzes the formation of GABA, was found to be significantly reduced in DOCA‐salt‐treated control mice, while it was significantly upregulated in the hypothalamus of DOCA‐salt‐treated A17G mice, implying that activation of ADAM17 in glutamatergic neurons might have a negative impact on GABAergic signaling. It was reported previously that neuro‐inflammation, especially microglia activation, can increase glutamate activity and decrease GABA synthesis as well as its release. Through CD11b labeling, we observed that less microglia was activated by DOCA‐salt treatment in the hypothalamus of A17G mice, compared to controls. Meanwhile, the markedly reduced interleukin‐6 (IL‐6) level in the hypothalamus also suggested that DOCA‐salt‐induced neuro‐inflammation was blunted in mice lacking ADAM17 in glutamatergic neurons. In addition, the distribution of ADAM17 was also studied. Beyond glutamatergic neurons, assessment of ADAM17’s presence in other neuronal populations and brain regions revealed its expression in GABAergic neurons within the MnPO (median preoptic nucleus), PVN and BNST (bed nucleus of the stria terminalis). Although the mechanism still needs to be verified, ADAM17‐mediated shedding in glutamatergic neurons seems to blunt GABAergic inhibitory activity. This may support the increase of glutamatergic neuronal excitability and sympatho‐excitation during DOCA‐salt hypertension.

Support or Funding Information

Veterans Affairs Merit Award (BX004294), National Institutes of Health (HL093178 and COBRE P30GM106392) and LSUHSC‐NO Research Enhancement Program.
Role of Peroxisome Proliferator‐activated Receptor‐gamma in Type 2 Diabetes‐induced Testicular DNA Damage and Repair in Leptin Receptor‐deficient Obese Mice
Authors: Kilarkaje Narayana , Alfarhan Muneera Waheed , Al-Hussaini Heba
Published: April 2020

The DNA damage repair mechanisms under hyperglycemic conditions are a subject of intense research as unrepaired DNA damage may enhance the possibility of carcinogenicity in diabetic patients. In this study, we investigated the role of peroxisome proliferator‐activated receptor‐gamma (PPARγ) in diabetes‐induced alterations in testicular functions and DNA damage and repair. Leptin signaling intact lean (heterozygous; db+/db−) and leptin receptor‐mutant (db−/db−) obese male mice (7‐week‐old; n=10/group) received either pioglitazone (a PPARγ agonist) or 2‐chloro‐5‐nitro‐N‐phenylbenzamide (a PPARγ antagonist) for 84 days (12 weeks) to modulate the receptor activities. Semi‐quantification of 4‐hydroxynonenal and 8‐oxo‐dG levels meassured the oxidative stress, and the TUNEL assay measured apoptotic testicular cells. Western blotting technique quantified the levels of base‐excision repair (BER) pathway proteins‐8‐oxoguanine glycosylases 1&2, apurinic/apyrimidinic endonuclease, DNA polymerase δ, poly (ADP‐ribose) polymerase 1 and X‐ray repair cross‐complementing protein‐1. PPARγ activation increased the body weights and reduced the blood glucose levels, but inhibition of PPARγ activities did not significantly affect these parameters in obese diabetic mice. Inhibition of PPARγ activities improved testis and epididymis weights and sperm counts in obese diabetic mice. The obese diabetic mice showed seminiferous tubular degeneration, epithelial sloughing and tubular atrophy and, modulation of PPARγ activities did not significantly protect testicular structure and function. The lack of leptin signaling in obese diabetic mice increased oxidative stress and oxidative DNA damage, and stimulation of PPARγ activities reduced only lipid peroxidation, and inhibition of PPARγ activities inhibited both lipid peroxidation and oxidative DNA damage. The obese diabetic mice showed upregulated base excision repair proteins to repair the increased oxidative DNA damage. PPARγ stimulation inhibited the DNA damage repair indicating its detrimental effects, and the inhibition of PPARγ activities led to inhibition of DNA damage repair in conjunction with reduced oxidative DNA damage. However, regardless of these modulatory effects of PPARγ, the obese diabetic mice showed comparable levels of apoptosis in their testes. In conclusion, PPARγ activation although reduces blood glucose levels, in the absence of leptin signaling upregulates DNA damage and inhibits base excision repair mechanisms in the testis. The inhibited PPARγ activities in the absence of leptin signaling are beneficial to protect the testicular functions in obese diabetic mice.

Support or Funding Information

Supported by Kuwait University grant # YM05/17 and CGS.
Role of arrestin1 in regulation of GPCR signaling in platelet activation
Authors: Chaudhary Preeti Kumari , Kim Sang Gu , Kim Soochong
Published: April 2020

The arrestins are a class of soluble proteins that function in concert with the GPCR kinases to stop or arrest GPCR‐mediated intracellular signaling. Although β‐arrestins function in the desensitization of most GPCRs, the functional differences of β‐arrestin1 (arrestin2, BARR1) versus β‐arrestin2 (arrestin1, BARR2) in the regulation of GPCR signaling in platelets and the mechanisms of desensitization of platelets to GPCR agonists by β‐arrestins have not been understood. In this study, we identify the role of BARR2 in the regulation of platelet function and GPCR signaling in platelets. We used mice lacking BARR1 and BARR2 to evaluate the functional role of BARR2 in platelet activation. Platelet aggregation, dense‐granule secretion, and fibrinogen receptor activation induced by 2‐MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in BARR2 −/− platelets compared to WT platelets. In contrast to BARR2 −/− platelets, AYPGKF‐, thrombin‐, and U46619‐induced platelet aggregation and secretion were significantly inhibited in BARR1 −/− platelets compared to WT platelets, while 2‐MeSADP‐induced platelet aggregation and secretion were minimally affected in BARR1 −/−platelets. However, CRP‐induced platelet aggregation and secretion were not affected in both BARR1 −/− and BARR2 −/− platelets. These results suggest that BARR2 functions as the predominant regulator in the termination of GPCR signaling in platelets. In addition, platelet aggregation was restored in response to second challenge of 2‐MeSADP and AYPGKF in BARR2 −/− platelets whereas re‐stimulation of agonist failed to induce aggregation in WT and b‐arrestin1 −/− platelets, indicating BARR2 contributes to P2Y1, P2Y12, and PAR receptor desensitization. Furthermore, 2MeSADP‐ and AYPGKF‐induced Akt and ERK phosphorylations were significantly potentiated in BARR2 −/− platelets. Finally, BARR2 −/− mice show an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery, indicating BARR2 is critical for thrombus formation in vivo. We conclude that BARR2 plays an important role in regulation of platelet function through selective GPCR desensitization.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) Grant of the Korean government (NRF‐2016R1D1A1B01010310) and the Global Research and Development Center (GRDC) Program through the NRF funded by the Ministry of Education, Science and Technology (2017K1A4A3014959).
Effect of Obestatin and its Fragment Analog Nt8U on the Epididymal and Brain Tissue of DIO‐C57BL/6 Mice
Authors: Shilpa T.S. , Manjappara Uma V.
Published: April 2020

Imbalance in energy intake and energy expenditure due to change in lifestyles and genetic issues results in obesity which is a serious health problem. The entero‐endocrine system plays a major role in nutrient signaling. Appetite inducing signals and satiety peptides in the entero‐endocrine system have a definite role in management of obesity. Obestatin, a relatively new gastro‐peptide produced post translationally from preproghrelin gene and its N‐terminal fragment analog Nt8U (13 amino acids) have been shown to reduce food intake, gain in body weight, fat pads and also circulating lipids in normal adult male mice. In the present study, it is aimed to study the effect of these peptides on the epididymal and brain tissue of diet induced obese (DIO) mice. In an attempt to decipher their mechanism of action, it is intended to explore the changes these peptides bring about in the mouse brain at the genomic level. Mice fed with 60% (calorie by fat) high fat diet for a period of 19 weeks were administered obestatin and Nt8U intraperitoneally for 8 days and the mice maintained on HFD for 3 more weeks. Food intake and gain in bodyweight were monitored daily. Blood lipid parameters, plasma proteins and serum leptin levels were estimated after the administration regimen. No reduction in food intake or significant reduction in weight gain upon administration of the peptides. The leptin levels were drastically reduced from 21.7ng/dL in the saline treated mice to 10.4 and 8.9ng/dL in obestatin and Nt8U treated mice respectively. Triglyceride, cholesterol and HDL‐C levels were not altered after 8 days injection period but there was decrease in triglyceride levels by 30% in the Nt8U administered group and by 11% in the obestatin administered group 3 weeks after the treatment. Obestatin and Nt8U reduced epididymal fat by 7.5%. Estimation of serum enzymes SGPT, SGOT, ALP and LDH, weight and appearance of the organs showed the vital organs were unaffected by the treatment. mRNA from epididymal and brain tissue were extracted and subjected to Next Generation Sequencing (NGS). Analysis of NGS data revealed that, the genes Olr1, Alox 15, Gykl1 were upregulated in epididymal tissue and Cbr2, Btk in brain tissue. And, the genes Casq 1, Slc6a2, AldoB and Omp were down regulated in brain tissue. These differentially regulated genes modulate enzymes mainly involved in lipolysis and sequestration of triglycerides in the adipose tissue. In conclusion, administration of obestatin and Nt8U has a definite fat lowering effect as seen from reduced leptin levels. Reduction in triglycerides and the set of genes being differentially regulated strongly imply that there is alteration in lipid metabolism.

Support or Funding Information

This research is funded by Council of Scientific and Industrial Research (CSIR) India, Department of Biotechnology (DBT), India. The presenting author is grateful to Indian Council of Medical Research (ICMR), India for providing Senior Research Fellowship.
Protective Effect of Jujube (Ziziphus jujuba Mill.) Extracts on Ethanol‐induced Liver Injury in Human HepG2 Cells
Authors: Yu Jaeeun , Hong Seonghwa , Choe Hyeonjeong , Lee Junsoo
Published: April 2020

Jujube has a long history of usage as a fruit and remedy. The main biologically active components are reported as vitamin C, phenolic acids, flavonoids, triterpenic acids, and polysaccharides. Oxidative stress plays a crucial role in the progression of alcoholic liver diseases and substances of antioxidant property are of special interest for therapeutic purposes. The purpose of this study was to investigate the protective effect of jujube extracts against ethanol‐induced liver injury in HepG2 cells. HepG2 cells were pretreated with methanol extract of jujube (25–100 μg/mL) and then hepatotoxicity was stimulated by addition of 3% ethanol. With response to ethanol‐challenge, increased amount of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) release were observed, whereas jujube pretreatment significantly reduced the levels of AST and ALT in HepG2 cells without appreciable cytotoxic effects. This protective effect was likely associated with antioxidant potential of jujube extracts, as evidenced by the attenuation of reactive oxygen species (ROS) and malondialdehyde (MDA) production and restoration of the depleted glutathione (GSH) levels in ethanol‐induced HepG2 cells. Considering these properties, quantification of the bioactive compounds is very important. Contents of phenolic acids and flavonoids were confirmed by LC‐MS/MS. Vanillic acid was the highest in jujube extracts followed by benzoic acid, rutin, and coumaric acid. Thus, we concluded that these active compounds may provide a pivotal mechanism for its hepatoprotective action. Further studies are needed to identify the mechanism and active compounds.
Tumor Exosomes Mediate the Horizontal Transfer of DNA Gene Mutation
Authors: Domenis Rossana , Cifù Adriana , Fabris Martina , Niazi Kayvan R. , Soon-Shiong Patrick , Curcio Francesco
Published: April 2020

INTRODUCTION

Tumor‐derived exosomes (TEXs) are crucial in shaping the local tumor microenvironment to promote cancer progression by advancing tumor metastasis. Recently, we have demonstrated that the activation of Toll‐like receptor 4 (TLR4) boosts the immunosuppressive ability of exosomes released by tumor cells. In addition to RNA and proteins, TEXs also transport dsDNA that reflects the mutational status of their parental cells. The purpose of this study is to verify whether exosomes shed by TLR4‐activated tumor cells are able to induce a malignant transformation of normal recipient cells through the horizontal transfer of mutated DNA.

METHODS

SW480 tumor cells were treated with LPS (1μg/ml) and TEXs were isolated from cell supernatants by polymer precipitation‐based method. TP53R273H and KRASG12V mutations have been identify by droplet digital PCR on purified exoDNA. Normal colon epithelial cells (CCD841) were used as recipient cells and internalization of Did‐labelled vesicles was evaluated by immunofluorescence. Vitality/proliferation (Trypan Blue dye exclusion test), migration capacity (Scratch assay) and mutational status (Droplet digital PCR) of CCD841 cells were monitored after the treatment with exosomes.

RESULTS

SW480‐derived exosomes carried dsDNA fragments in size range of 2–10 kbp containing TP53R273H and KRASG12V mutated sequences. Exosomes were efficiently internalized by CCD841 cells within 6 hours and elicited activation of signaling pathways that induced an increase in cell proliferation and migration capacity. Copies of TP53 R273H mutated DNA and mRNA were found within recipient cells up to one month without exosomal treatment. The activation with LPS stimulated the packaging of mutated DNA into exosomes. Consequently, mutated DNA was found in greater amounts in recipient cell treated with exosomes released by TLR4‐activated cells.

CONCLUSIONS

The activation of TLR4 modulates the packaging of mutated genes in exosomes, which are efficiently transferred in normal recipient cells. Although we observed that mutated TP53 gene is transcribed in recipient cells, further studies are needed to assess whether mutated gene are integrated into genome.

Support or Funding Information

This project was supported by Grant Interreg ARTE (J22F170001005)
Plakophilins 2 and 3 are involved in desmosome formation in the intestine
Authors: Nagler Simon , Ghoreishi Yalda , Waschke Jens , Schlegel Nicolas
Published: April 2020

The integrity of desmosomes is crucial to maintain intestinal epithelial barrier (IEB) function in health and disease. In addition to adherens junctions, desmosome‐dependent intercellular adhesion between cells is provided by the cadherins, Desmoglein2 (DSG2) and Desmocollin2 (DSC2) in the intestine. Both are tethered to the intermediate filament system via different adaptor proteins, such as the Plakophilins (PKPs). The specific role of the different isoforms of PKP 1–3 has been recognized in different organs. However, there is a wide lack of literature regarding the existence of the PKPs and their critical role in the intestine. Accordingly, it remains unclear whether PKPs play a specific role for IEB stabilization. Therefore, the aim of this study was to perform a basal characterization of intestinal PKPs.

To analyze the expression pattern of the different PKP isoforms, immunostaining, qRT‐PCR and Western blotting were used. In healthy human mucosa that derived from surgical specimens, PKP2 and PKP3, but not PKP1, were present at the cell borders of tissue sections. In co‐staining experiments with DSG2, both PKP2 and PKP3 were found to be present at desmosomal junctions. The expression of PKP2 and PKP3 but not PKP1 was confirmed on the RNA level by qRT‐PCR and on the protein level by Western Blot analysis. Similar results were obtained in enteroids generated from human mucosa and in Caco2 cells which are commonly used as models to investigate the mechanisms underlying changes of IEB showed a similar expression pattern of PKPs. Interestingly, in Caco2 cells that are deficient for DSG2, a compensatory upregulation of PKP2 RNA and total protein levels was detected when compared to Caco2 WT cells whereas no changes were evident for PKP3 in DSG2‐deficient cells. Stimulation of Caco2 WT cells with Tumor necrosis factor α (TNFα) resulted in a loss of IEB as revealed by measurements of transepithelial resistance. This was associated with a loss of DSG2, PKP2 and PKP3 at the cell borders. However, while total protein levels of PKP2 and DSG2 were unaltered under these conditions, PKP3 protein levels were upregulated.

In summary, for the first time, we provide evidence that PKP2 and 3 but not PKP1 are expressed in human intestinal epithelial cells as well as in human enteroids and Caco2 cells. Compensatory upregulation of PKP2 following loss of DSG2 suggests a functional role for PKP2 to stabilize desmosomal integrity. Furthermore, since TNFα treatment was accompanied with an upregulation of PKP3, a potential role of PKP3 during inflammatory processes can be hypothesized.
Nrf2‐Activating Phytochemicals, Sulforaphane and Licochalcone A, Stimulate Cell Growth‐Regulating Kinases in HCT116 Human Colorectal Cancer Cells
Authors: Jang Chan Ho , Oh Jisun , Kim Jong-Sang
Published: April 2020

Some phytochemicals have been reported to modulate phase II detoxifying/antioxidant enzyme expressions via both Keap1 (Kelch‐like ECH‐associated protein 1)‐dependent and independent regulations of a nuclear factor E2‐related factor 2/antioxidant response element (Nrf2/ARE) system. Based on multiple studies, various kinase signaling pathways are also responsible for regulation of Nrf2/ARE activity differently depending on cell types, microenvironmental factors, treatment doses, etc. Sulforaphane (SFN) and licochalcone A (LicA) are known as potent Nrf2/ARE inducers that can modulate several kinases including phosphoinositide 3‐kinase (PI3K)/Protein kinase B (Akt) and p38 mitogen‐activated protein kinase (MAPK) in propagating cancer cells. The purpose of the present study was to investigate the molecular mechanism through which SFN and LicA can activate Nrf2/ARE system and upregulate heme oxygenase‐1 (HO‐1) expression in HCT116 human colorectal cancer cells. Results revealed that SFN and LicA induced ARE‐transcriptional activity and subsequently increased HO‐1 expression through PI3K and p38 MAPK signaling pathways in HCT116 cells. In addition, HCT116 cell proliferation was significantly promoted when Nrf2/ARE activity was increased by either SFN‐induced PI3K activation or LicA‐induced p38 MAPK activation. These observations indicate that SFN and LicA regulated the Nrf2/ARE/HO‐1 system via phosphorylation of PI3K and p38 MAPK, respectively, in proliferation of HCT116 colorectal cancer cells. In conclusion, the present study highlights the working mechanism of potent Nrf2/ARE activators that possibly perturb the activities of various kinases in the growing cancer cells.

Support or Funding Information

This work was supported by the National Research Foundation of Korea grant (Grant No. 2017R1A2B4005087) funded by the Ministry of Science and ICT, Republic of Korea.
Targeting the Homomeric α7 Nicotinic Receptor and Acetylcholine Binding Protein with Lophotoxin by X‐ray Crystallography and Mass Spectrometry
Authors: Hernandez Gisela Andrea Camacho , Schnarkowski Franziska , Harel Michal , Ho Kwok-Yiu , Campeau Anaamika , Sankaran Banumathi , Gonzalez David , Fenical William , Taylor Palmer
Published: April 2020

Lophotoxin is a member of a cyclic diterpenoid family of toxins, devoid of nitrogen, isolated from soft coral Lophogorgia. Previous work suggested irreversible binding of lophotoxin to nicotinic acetylcholine receptors (nAChRs), utilizing electric organs of Torpedo californica; Inhibition is thought to be mediated by an electrophilic addition of the toxin with a hydroxyl of a conserved tyrosine (Tyr 190) of this family of receptors. According to molecular modeling, an alpha‐beta unsaturated carbon in lophotoxin forms a covalent bond with a tyrosine hydroxyl group that will irreversibly inhibit nAChRs. Binding of this unique family of cyclic diterpinoid terpene toxins is associated agonist site of the nAChR, since binding competitively inhibited by reversible agonists, such as epibatidine, or by competitive antagonists, such as MLA. To investigate receptor interactions of lophotoxin with neuronal nicotinic acetylcholine receptors (nAChRs) and related pentameric ligand‐gated ion channels, we employed HEK cells transfected with cDNA’s encoding one of two receptor subtypes: α7‐nAChR and 5HT3AR, along with a fluorescent reporter, and a receptor extracellular domain surrogate, the soluble, acetylcholine binding protein (AChBP). We found that lophotoxin not only inhibited α7‐nAChR function and AChBP ligand binding, as expected, but surprisingly also the function of the serotonin gated ion channel (5HT3A). X‐ray crystallographic studies show the bound lophotoxin to reside in the interfacial site underneath a partially closed C‐loop. Mass spectrometry of the linked peptides show a covalent attachment to peptides bordering the C‐loop bearing a covalent linkage to the tyrosine in the C‐loop. Hence, the binding orientation and atomic residues involved in this marine toxin can be delineated through detailed protein chemistry and crystallographic resolution of the structure. The irreversible binding mode of interaction can serve as a model for potential irreversible inhibitors. Lophotoxin’s irreversible action provides a means for longer term modulation of α7receptor signaling.

Support or Funding Information

The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE‐AC02‐05CH11231. The Pilatus detector was funded under NIH grant S10OD021832. The ALS‐ENABLE beamlines are supported in part by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169
Student Perception on Integration of 3D Complete Anatomy Software Application in Medical Curriculum
Authors: Nair Rakesh Ravikumaran , Lindsey Allison
Published: April 2020

Background

Anatomy is a discipline where spatial visualization is of importance. In medical school, anatomy is often taught to students via lectures with slides and cadavers. While slide images and cadavers can show the relationships between muscles to a limited extent, neither can show movement well or be manipulated by the student to isolate specific structure(s). The 3D Complete Anatomy application was utilized by students to study muscle movements, determine points of proximal and distal attachment, understand spatial relationships between structures, take self‐assessments, and more, at their convenience.

Objective

of this study is to examine student perceptions about learning anatomy using 3D Complete Anatomy application and to explore how these perceptions shape the learning experience.

Method

At Florida International University Herbert Wertheim College of Medicine, first year students were provided iPad Mini’s with the 3D Complete Anatomy software. Students were advised to review the Complete Anatomy app during the introduction of the period 1 Anatomy course. Students were also encouraged to use the Complete Anatomy app during their mandatory anatomy lab sessions. Students’ feedback will be assessed using approximately ten survey statements that they can indicate their level of agreement with. Their responses will be scored using a five‐point Likert scale. The statements will assess the app’s ability to enhance students’ learning, retention of knowledge, and overall ease of studying the material. We are in the processes of getting exempt from IRB.

Results

We expect the results to support our hypothesis that the 3D Complete Anatomy Application would improve first year medical students’ learning, retention of concepts, and ease of studying the material. Additionally, we expect the students to recommend the app for future classes to be used along with to the plastinated and prosected models during anatomy lab sessions. Final data analysis of the variables will be done after obtaining IRB exempt.

Conclusions

We expect that the majority of students will believe that the 3D Complete Anatomy application assisted their understanding of the spatial relationships between anatomical structures and mastery of the material. Additionally, we believe this resource will be among the tools the students used on a regular basis to effectively learn the material. We hope to be able to apply this knowledge of using supplemental 3D applications to enhance students’ understanding of spatial relationships to future anatomy classes.
Muscle‐specific Nrf2 deficiency exacerbates cancer cachexia‐induced skeletal muscle atrophy
Authors: Okutsu Mitsuharu , Yamada Mami
Published: April 2020

Skeletal muscle atrophy is a hallmark of several debilitating diseases such as cancer, thereby contributing to morbidity and mortality. Oxidative stress, which is caused by excessive production of reactive oxygen species and/or decreased antioxidant defense, plays an important role in skeletal muscle atrophy in cancer cachexia. Nuclear factor erythroid 2–related factor 2 (Nrf2) is a master regulator of antioxidant gene transcription. However, a potential role for Nrf2 in protection against muscle atrophy in cancer cachexia remains to be determined. To investigate this, C57BL/6 mice were given a subcutaneous injection in the left flank with 107 3LL lung cancer cells or PBS control. A grip strength test was performed at 25–27 days after 3LL injection using a grip strength meter. At 4 weeks after 3LL injection, soleus, plantaris, gastrocnemius, and extensor digitorum longus muscles were harvested to assess muscle weight and expression of select proteins. Muscle‐specific Nrf2 knockout (Nrf2mKO) and wild‐type littermates (WT) mice were also injected with 3LL cells and their skeletal muscles were harvested within the same time frame. Our results demonstrated that grip strength in 3LL injected C57BL/6 mice was significantly lower than in control mice. Gastrocnemius and extensor digitorum longus muscle weights in 3LL injected mice were significantly lower than control mice. Protein carbonylation, a surrogate of oxidative stress, was significantly increased in 3LL injected mice compared with control mice. Extracellular superoxide dismutase (EcSOD) protein expression was also increased in 3LL injected mice. Gastrocnemius and extensor digitorum longus muscle weight in 3LL injected Nrf2mKO mice were significantly lower than in 3LL injected WT mice. Loss of Nrf2 in skeletal muscle also blunted the 3LL injection‐induced increase in EcSOD protein expression. These findings indicate that endogenous muscle Nrf2 is a key protective molecule against atrophy likely by limiting the oxidative stress burden of cancer cachexia.

Support or Funding Information

This study was funded by Grant‐in‐Aid for Scientific Research (B) (15H03080) (18H03153) to Mitsuharu Okutsu.
Evaluating Combination Therapies for Treatment of Chronic Inflammatory Pain: a GABAA Approach
Authors: Woodhouse Kristen L. , Cook James M. , Li Jun-Xu
Published: April 2020

Approximately 20% of U.S. adults suffer from chronic pain. Current therapies for treatment of chronic pain are limited, ineffective for a large number of patients, and often produce undesirable side effects. Though not used clinically to treat pain, benzodiazepines alleviate pain when administered into the spinal canal. This analgesic effect is due to their action at α2 and α3 subunits of the GABAA receptor: since α2/α3 subtype‐selective GABAA positive allosteric modulators (PAMs) act specifically at these subunits, they produce analgesia without eliciting side effects typically seen with classical benzodiazepines, such as sedation or cognitive impairment. This study evaluates the analgesic efficacy of combinations of two α2/α3 subtype‐selective PAMs (KRM‐II‐81 and NS16085) with the NSAID acetaminophen and two opioids (fentanyl and buprenorphine) in a rat model of chronic inflammatory pain (complete Freund’s adjuvant). We used a fixed ratio approach to evaluate combinations of drugs, assessing both mechanical pain (von Frey) and thermal pain (Hargreaves), in order to visualize the onset and duration of action of each of these drugs alone and in combination. We found that combining KRM‐II‐81 (4.0 mg/kg) and buprenorphine (0.048 mg/kg) produced greater analgesia than either of these drugs alone at comparable doses. These data are among the first to systematically examine the performance of α2/α3 subtype‐selective GABAA PAMs in combination with other analgesics, and are an important step towards developing novel therapies for treating chronic pain.

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R01DA047967
Synthesis, characterization and antibacterial activity of Co(II) complexes with tricyclic fused chromeno‐pyrimido‐pyrido systems
Authors: Berisha Vjollca , Koskoviku Mimoza , Hashani Ismet , Govori Sevdije
Published: April 2020

Two novel ligands 7‐iminochromeno[4,3‐d]pyrido[1,2‐a]pyrimidin‐6(7H)‐ones have been prepared. The ligands and their Co(II) metal complexes have been characterized by IR, 13C‐NMR, 1H‐NMR, Mass spectra and Elemental analysis. Antimicrobial activities of the ligands and their complexes have been screened against the Escherichia coli ATTC 8739 and Staphylococcus aureus ATTC 25923. The results show that Co (II) complexes possess higher antibacterial activities than ligands towards the bacterial species.
Exercise Mitigates Bone Loss via ROS‐NLRP3 Inflammasome Axis In High Fat Diet Fed Mice
Authors: Behera Jyotirmaya , Ison Jessica , Tyagi Suresh C. , Tyagi Neetu
Published: April 2020

Emerging evidence suggests that western diet‐induced obesity is associated with chronic low‐grade inflammation. Chronic inflammation is also linked with increased bone loss and osteopenia phenotype. However, the mechanistic basis of obesity‐associated bone loss via induction of inflammasome is not yet well understood. The recent study also found that irisin is expressed by various tissue including skeletal muscle during exercise. It is also currently unknown whether irisin is secreted by bone tissue upon exercise and possible involvement in the regulation of NLRP3 inflammasome activation during obesity in mice. To test the hypothesis, we employed 8‐weeks age‐matched male/female C57BJ/L6 wild‐type (WT) mice were grouped as fellows according to their diet and exercise (Ex) category: standard nonfat diet NFD (6%); (ii) NFD+Ex; (iii) HFD (42%); (iv) HFD+Ex. A nonfat diet (6%) and a high‐fat diet (40%) were given for three months. The selected mice were exercised for this 12‐week duration on a treadmill, with controlled speeds of 7 meters/min for the first week, 9 meters/min for the second week and 11 meters/min the following weeks for a total of 330 meters every day. After every 110 meters reached, the mice were given rest of 10 minutes. We tested protein/mRNA expression of oxidative stress, inflammasome metabolic indicators Glucose Transporters 1 and 4 (GLUT‐1, GLUT‐4), irisin, IL‐6, and IL‐1β in primarily isolated bone marrow mesenchymal cells (BMMSCs), plasma and bone from all the groups by Western Blot, real‐time PCR, and Immunohistochemistry. The data demonstrated that high levels of irisin protein expression were induced in both plasma and murine femoral bone tissues of HFD+EX mice. The mechanistic study shows that cellular oxidative damage and NLR3 signaling was indeed down‐regulated and resulted in decreased secretion of IL‐6 and IL‐1β in bone marrow mesenchymal cells (BMMSCs) and plasma of HFD+EX mice. Interestingly, HFD+EX mice showed higher trabecular, cortical bone formation and bone strength in vivo in compared to HFD mice. Lentiviral FNDC5/recombinant irisin administration suppresses HFD induced ROS‐NLR3 inflammation and restores bone mass, whereas blockade of FNDC5/irisin by siRNA delivery prevents the physical exercise mediated bone formation in HFD mice. Further osteoblast‐specific FNDC5 (or Irisin) deficiency in mice completely blocks EX‐induced trabecular bone formation. In conclusion, our findings demonstrate that exercise‐induced irisin mitigates the bone loss in part via inhibiting ROS‐NLR3 signaling in HFD mice, and possibly provide a mechanism for therapeutic effect in metabolic osteoporosis.

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Acknowledgment

This work is financially supported by the National Institute of Health Grant AR‐067667‐NT is greatly acknowledged.
Optical Metabolic Imaging (OMI) of Toxoplasma gondii and Cryptosporidium parvum in a 3D system
Authors: Siamof Cerise Marie , Gallego-Lopez Gina , Knoll Laura
Published: April 2020

Both T. gondii and C. parvum are intracellular, apicomplexa parasites auxotrophic for specific metabolites. Cat intestine is the definitive host of T. gondii, and cats can produce infectious oocysts, which infect humans primarily through fecal‐oral transmission. This infection can cause fatal encephalitis in immunocompromised humans. C. parvum is another intestinal parasite with a wider range of hosts who can excrete infectious oocysts in feces. These oocyts can cause fatal diarrheal illness in cows and humans, and C. parvum infection has been linked to colon cancer in humans. How T. gondii and C. parvum change host cell metabolism has not been well‐characterized to date. Currently, there are limited treatment options available for C. parvum, and the organism has been difficult thus far to culture and propagate in vitro.

For this reason, mouse intestinal organoid models have been developed to simulate the conditions a parasite would encounter in vivo. It is hypothesized that the model for studying T. gondii in vitro can be easily transferred to C. parvum, given their similarities. Using mCherry fluorescent protein labelled T. gondii and cat intestinal organoids, we have modeled intestinal infection with T. gondii. The relative ease of molecular manipulations of T. gondii makes it the ideal organism with which to build a model for optical metabolic imaging (OMI) of C. parvum. OMI of mCherry‐labelled T. gondii in 2D human fibroblasts has been completed with inhibitors such as Mycocaloide B and Cytochalasin D, and the results showed changes in NADH and FAD metabolites in vitro, showing that infection with intracellular parasites impacts metabolism of host cells.

T. gondii will be imaged in vitro with additional metabolic inhibitors, including atovaquone and sodium fluoroacetate. Then, OMI will be done of T. gondii in cat intestinal organoids, a 3D system, because the materials are already available and serve as a way to test the model prior to using it on C. parvum. It is anticipated that the previously obtained results will be seen again in the 3D system. This project will label C. parvum with mCherry fluorescent protein, propagate this microorganism in a 3D system and ultimately perform OMI. Thus, the model that has been successfully developed with T. gondii will be improved, then used to understand how infection with Cryptosporidium parasite influences host metabolism, particularly in organoid models. Ultimately, this better understanding will assist in the development of therapeutics for C. parvum, an area which is currently lacking.
Plasma potassium plays a major role in controlling the concentrating ability of the kidney
Authors: Al-Qusairi Lama , Zapf Ava , Grimm P. Richard , Welling Paul A.
Published: April 2020

Hypokalemia can induce diabetes insipidus (DI), characterized by loss of free‐water reabsorption, loss of urine concentrating ability, polyuria and polydipsia. Although the disorder has been described for decades, the pathophysiological mechanisms remain poorly understood. In addition, it still unclear if there is a sexual dimorphism.

In this study, we characterized hypokalemia‐induced DI over a spectrum of plasma potassium levels in mice fed a range of low potassium diets (LKD) between 0% and 0.11% K+, over 8 days compared to control diet (1%K+). We observed that 0%K+ and 0.026%K+ diets both induced hypokalemia, polyuria and a decrease in urine osmolality starting after 5 and 6 days, respectively. However, the 0.056%K diet induced only mild DI starting at day 7. Interestingly, the 0.11%K diet did not result in DI.

We then assessed the DI in males and females mice fed with LKD (0%K+) or control diet for 8 days. After 3 days of LKD, both males and females exhibited decrease in urine osmolality which was more sever in females (−21%) than in males (−12%). Profound diuresis was observed at day 5 and it was more enhanced in females (5.2 +/−0.58 mL/day) than in males (3.0 +/−0.43 mL/day), which might be a consequence of the more severe hypokalemia in females compared to males (2.6 +/−0.29 versus 3.5 +/−0.08 mmol/L) after 8 days of LKD.

Moreover, it has been shown that the urinary concentration defect occurs mainly in the inner medullary collecting duct (IMCD), involving a reduction in AQP2 channel expression. Recent studies localized AQP2 protein in the autophagosome in the IMCD of DI models suggesting the autophagic degradation of AQP2 under hypokalemia‐induced DI (Khositseth S., Sci rep 2015)(Kim WY., Sci rep 2019). We confirmed and extended this observation, finding that after 8 days of LKD, the ATG12‐Atg5‐Atg16 complex, the LC3II/LC3I ratio, and P62 were all increased in the medulla but no difference was detected by western blot in the cortex. However, we observed that AQP2 levels were more severely decreased in the cortex than in the medulla of the hypokalemic versus control mice suggesting that pathways other than autophagy are at play. Together, the data indicate: i) a tight relationship between loss of the renal concentrating ability and development of hypokalemia, ii) hypokalemia in response to dietary potassium deficiency is more sever in female mice than males, and as a consequence, females are more prone to develop DI than males, iii) the autophagy is induced in the medulla of the hypokalemic mice, iv) The hypokalemia‐induced decrease in AQP2 is correlated with induction of autophagy in the medulla but not in the cortex.

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NIH
β3‐adrenoreceptor as a new player in sympathetic regulation of the acid‐base homeostasis in the kidney.
Authors: Milano Serena , Gerbino Andrea , Carmosino Monica , Svelto Maria , Procino Giuseppe
Published: April 2020

Sympathetic nervous system has an important role in the regulation of renal function.

We previously showed that, in mouse, the β3‐adrenoreceptor (β3‐AR) localizes in most of the nephron segments and its selective agonism promotes a potent antidiuretic effect.

Here, we report the expression of β3‐AR in distal nephron intercalated cells (ICs). Co‐localization study of β3‐AR either with H+‐ATPase or Cl−/HCO3− exchanger pendrin in mouse kidney showed that the β3‐AR was expressed in H+‐secreting type A, in HCO3− secreting type B, and in non‐A non B ICs. To unveil a possible role of β3‐AR in systemic pH regulation, we analyzed the urine pH of wild type (wt) and β3‐AR knock‐out (ko) mice. We found that the urine pH of β3‐AR ko mice was significantly higher than wt mice. In line with these results, localization and expression analysis showed that the renal H+‐ATPase significantly decreased in β3‐AR ko mice compared to wt mice supporting the idea that H+ secretion is partially blunted in these animals. Next, we investigated in kidney cells (M1‐β3‐AR) the effects of β3‐AR stimulation on the activity of H+‐ATPase monitoring the intracellular pH with the pH‐sensitive dye BCECF.

Of note, exposure of M1‐β3‐AR cells to a selective β3‐AR agonist induced a 2.5 fold increase of H+‐ATPase activity compared with resting cells and this effect was prevented by a selective β3‐AR antagonist or by the H+‐ATPase inhibitor bafilomycin. Moreover, β3‐AR agonism promoted H+‐ATPase apical expression in M1‐β3‐AR cells. In addition, the PKA inhibitor H89 abolished the stimulatory effect of β3‐AR agonism, demonstrating the involvement of the cAMP/PKA pathway. We conclude that the modulation of H+‐ATPase activity in renal ICs by endogenous β3‐AR agonists may play an addition physiological role in hormonal control of systemic acid‐base homeostasis.

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Telethon (project # GGP15083)