bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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.
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

INTRODUCTIONTumor‐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.METHODSSW480 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.RESULTSSW480‐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.CONCLUSIONSThe 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 InformationThis 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 InformationThis 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 InformationThe 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

BackgroundAnatomy 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.Objectiveof 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.MethodAt 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.ResultsWe 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.ConclusionsWe 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 InformationThis 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.Support or Funding InformationR01DA047967
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.Support or Funding InformationAcknowledgmentThis work is financially supported by the National Institute of Health Grant AR‐067667‐NT is greatly acknowledged.