bluuebunny/crossref_metadata_2025 huggingface dataset:
Using HMGB1 Protein as a Surrogate Marker for Lung Cancer in High‐Risk Populations
Authors: Cole Breanna , Jeffords Eric , Simmons Glenn E.
Published: April 2020

Lung cancer is the leading cause of cancer‐related death, independent of gender or ethnicity, with a 5‐year overall survival of 17.1%. Although screening protocols for early detection exist, lung cancer is often diagnosed at an advanced stage. This is due, in part, to many patients experiencing barriers to healthcare access which contribute to delays in diagnosis. Supra‐normal biosynthesis of saturated (SFA) and unsaturated fatty (UFA) acids is a hallmark of cancer. In fact, several lines of evidence demonstrate that increased lipid desaturation by stearoyl‐CoA desaturase (SCD1) promotes lung cancer growth. We hypothesize that increased intracellular UFA, such as oleate (OA), promotes cancer cell growth by reducing cancer associated inflammation. To test this hypothesis we evaluated the effect of OA and inhibition of OA synthesis on proinflammatory High Mobility Group Box Protein 1 (HMGB1). Decreasing OA synthesis reduced intracellular HMGB1 expression, but increased its release from cells. Given our data, alterations in OA levels appear to have a substantial effect on localization of HMGB1. Further work is necessary to define the connections between SCD1, OA and HMGB1 release in lung cancer cells. This knowledge will provide insight into how fat regulates cancer development.

Support or Funding Information

The University of Minnesota School of Medicine New Faculty Start up and College of Saint Scholastica McNair Scholars Program.
New Late Jurassic Dinosaur (Ornithischia) from China Sheds Light on the Early Evolution of the Ornithopod Skeleton and Phylogeny of Ornithopoda
Authors: Spencer Marc R. , Forster Catherine A. , Poole Karen E. , Clark James M. , Xu Xing
Published: April 2020

Numerous partial to complete individuals of a new neornithischian dinosaur have been collected from the early Late Jurassic (Oxfordian) Shishugou Formation in northwestern China. The new taxon includes an ossified clavicle and, uniquely, a patellar sesamoid and trilobate maxillary and dentary teeth. Coeval taxa from the Shaximiao Formation in Sichuan, China (Hexinlusaurus, Agilisaurus, Yandusaurus), regarded in many recent analyses as falling outside of Cerapoda (Marginocephalia + Ornithopoda), lack many characters distributed among ornithopods that are present in the new Shishugou taxon, including a large hooked coracoid process, a small quadratojugal foramen, narrow and elongate frontals, well‐developed epipophyseal ridges on the postzygapophyses of anterior cervical vertebrae, and a well‐defined and elongate femoral neck with a deep femoral capital sulcus. However, the new Shishugou taxon also retains some plesiomorphic character states relative to most ornithopods such as having five sacral vertebrae, and a short, narrow lesser trochanter on the femur. Recent analyses of neornithischian dinosaurs are not in agreement over the taxonomic composition of Ornithopoda or Cerapoda, or the distribution of characters along the neornithischian branch. Putative synapomorphies of Ornithopoda vary from study to study, as does the taxonomic composition of the clade. In some studies, many erstwhile “ornithopods” such as Thescelosaurus and Orodromeus have been pulled out of Cerapoda as basal neornithischians, while other studies recover a more inclusive monophyletic Ornithopoda. The current consensus of basal neornithischian and cerapodan phylogeny is poorly resolved. New taxa such as the Shishugou taxon that exhibit some, but not all, characters used in many studies to define Ornithopoda may help unclutter some of the resolution at the base of the cerapodan tree. The morphology present in the new taxon gives polarity to a suite of characters for basal neornithischian dinosaurs that may help provide a clearer diagnosis for Ornithopoda.

Support or Funding Information

National Science Foundation (NSF EAR 0922187, 1636753, NSF DDIG 1405834), National Geographic Society, The George Washington University, National Science Foundation of China (41120124002, 41688103)
Association between Frequency of Breakfast Consumption and Regional Body Fat Distribution
Authors: Pfeifer Michaela , Danyluk Alexandria , Smith Kevin L. , Sompalli Sreeja , Singh Prachi , Covassin Naima
Published: April 2020

Background

Dietary habits are critical determinants of health and especially of cardiometabolic risk status. Previous studies have examined the relationship between breakfast consumption and indices of obesity, with conflicting findings. Such discrepancy may be owed to the use of surrogate measures of adiposity such as body mass index. Furthermore, it is unknown whether frequency of breakfast consumption is associated with different patterns of regional body fat distribution.

Objective

In this study, we aimed at investigating the association between frequency of breakfast consumption and body fat distribution.

Methods

We compiled data from 184 individuals (64% males, mean age 35.8±12.6 years, ranging from 18 to 63 years old) who participated in research studies performed in the Mayo Clinic Cardiovascular/Sleep Laboratory from 2005 to 2019 and underwent whole body dual‐energy X‐ray absorptiometry (DXA) for body composition. Breakfast eating habits were assessed by questionnaire and frequency of consumption was determined as number of times per week (range 0 to 7). Responses were recoded as low frequency (0 to 2 times/week), middle frequency (3 to 4 times/week), and high frequency (5 to 7 times/week). Measures of height and weight were collected to compute body mass index (BMI, kg/m2). Total body fat percentage, fat mass, and lean mass were obtained from DXA. Regions of interest were manually adjusted with the DXA software to quantify abdominal fat and lower body fat. Abdominal fat/total body fat and lower body fat/total body fat ratios were calculated.

Results

Low frequency of breakfast consumption was reported by 14% of the subjects (n=26), while 18% (n=34) reported middle‐frequency of consumption. The proportion of men/women (p=0.1) and BMI values (p=0.78) were similar across breakfast eating categories. Compared to those who had breakfast with high‐frequency, those who ate it less frequently were younger (p=0.038). In age, sex, and BMI‐adjusted analysis, low‐frequency of breakfast consumption was significantly associated with greater abdominal fat mass (standardized beta=0.11, 95% CI 0.09‐ 1.29, p=0.024) and higher abdominal fat/total body fat ratio (standardized beta=0.16, 95% CI 0.003–0.024, p=0.013). Total body fat percentage (p=0.26), fat mass (p=0.26), and lean mass (p=0.22) were not associated with frequency of breakfast consumption. Similarly, no relations were seen with lower body fat (p=0.85) or lower body fat/total body fat ratio (p=0.51).

Conclusion

Our study showed that infrequent breakfast consumption is selectively associated with greater abdominal fat mass. As accumulation of fat in the abdominal depot is a more robust predictor of adverse outcomes than total adiposity, these findings suggest that abdominal fat may mediate the increased cardiometabolic risk previously observed in conjunction with infrequent breakfast consumption.
Lanthionine Synthetase C‐like Protein 2: A potential therapeutic target for treating neuropathic pain
Authors: Christy David , Maixner Dylan W. , Weng Han-Rong
Published: April 2020

Glial activation and over‐production of pro‐inflammatory cytokines and chemokines in the spinal cord are critical mechanisms underlying the genesis of neuropathic pain. While pro‐inflammatory signaling molecules have been extensively investigated, much less is known about anti‐inflammatory signaling pathways and systems. Studies in recent years have demonstrated that lanthionine synthetase C‐like 2 protein (LANCL2) is expressed in cells related to the immune function, like T cells, macrophages, endothelial and epithelial cells, and dendritic cells, suggesting its potential as a target for immunoregulation. In this study, we determined the role of LANCL2 in the genesis of neuropathic pain. Specifically, we show that LANCL2 protein is expressed in spinal dorsal horn and down‐regulated in rats with neuropathic pain. Knockdown of spinal LANCL2 induces mechanical allodynia and thermal hyperalgesia, which is concomitantly accompanied with increases in extracellular signal‐regulated kinase (ERK) activity, tumor necrosis factor alpha (TNF‐α) production, and the activation of astrocytes and microglia in the spinal dorsal horn. Activation of spinal LANCL2 by intrathecal administration of abscisic acid alleviates mechanical and thermal hypersensitivity induced by nerve injury. Pre‐emptive pharmacological activation of LANCL2 prevents the down‐regulation of LANCL2 protein expression, and significantly attenuates the development of mechanical allodynia and thermal hyperalgesia in rats induced by nerve injury. These behavioral changes were accompanied with the suppression of ERK activity and TNF‐α production, and astrocyte and microglia activation in the spinal dorsal horn of rats with nerve injury. Our study provides evidence that LANCL2 may be a potential molecular target for the development of analgesics for neuropathic pain and the management of a wide range of conditions involving neuroinflammation.

Support or Funding Information

NS064289; NS107569
Protection of Nitrogen Fixation from CO by CowN in G. diazotrophicus
Authors: Chong Kiersten , Garcia Chloe , Lee Terrence , Strul Max , Bretzing Kevin , Owens Cedric
Published: April 2020

Nitrogen fixation is the process in which atmospheric dinitrogen is reduced to ammonia. Nitrogen fixation occurs naturally in certain bacteria, such as Gluconacetobacter diazotrophicus, via the enzyme nitrogenase, a multisubunit protein with an iron protein subunit (FeP) and a molybdenum‐iron protein subunit (MoFeP). Nitrogenase is inhibited by carbon monoxide (CO). Under some soil conditions, CO levels are sufficiently high to inhibit nitrogenase and prevent nitrogen fixation. However, G. diazotrophicus expresses a protein called CowN that prevents the inhibition of nitrogenase by CO. This research attempts to understand the mechanism of how CowN prevents inhibition of nitrogenase. We expressed G. diazotrophicus CowN heterologously in E. coli and purified the protein to homogeneity. In vitro studies with FeP, MoFeP, and CowN in the presence of CO showed that CowN effectively restores nitrogenase activity for CO concentrations up to 0.1 atm. The activity of CowN exhibits Michaelis‐Menten‐like kinetics with a Km of approximately 8 μM. Our experiments further show that CowN, which exists in a monomeric and oligomeric state, is only active as a monomer. Further work will aim to elucidate how CowN binds to nitrogenase and if CowN protects nitrogenase by directly preventing CO access to the active site.

Support or Funding Information

This research was supported by the Chapman Center for Undergraduate Excellence, USDA‐NIFA (Grant no. 2015‐67012‐22895) and the National Science Foundation (Grant no. 1905399).
Choline Acetyltransferase Dysfunction Caused by Mutations Associated with a Congenital Motor Disorder
Authors: Rodgers David W. , Batra Joytica , Cai Yiying , Espinosa Paul , Whitaker Abigail , Feibes Helen , Grospitch Ashley M.
Published: April 2020

Choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine, and over 20 missense mutations that reduce enzyme activity have been found in patients with a severe neuromuscular disorder known as congenital myasthenic syndrome with episodic apnea (CMS‐EA). Our structural studies showed that the disease‐causing mutations are widely distributed over the enzyme despite generally producing similar decreases in catalytic function. We report here that two of the CMS‐EA mutations (L102P and P103A) alter the conformation of the ChAT active site, likely accounting for their effects on activity. Further, we find that the core residue packing in ChAT is poor, leaving an unusually large number of cavities. Partially filling one of the cavities near the L102P site by making a second mutation restores the native conformation of the active site as well as enzyme activity. We propose that the poor core packing in ChAT mediates the effects of CMS‐EA mutations on enzyme activity by transmitting structural changes to the active site. This work raises the possibility of using one or more cavity‐binding ligands to stabilize the enzyme to a number of the CMS‐EA causing mutations. We have identified a ligand that enhances the activity of the L102P mutant and are currently defining its binding site.

Support or Funding Information

Research reported was supported by National Institutes of Health award NS079818 and by AMF‐Telethon award 18508.
Effects of Growth Factors and FFAR Agonists on CCN1 Expression in Breast and Prostate Cancer Cells
Authors: Balijepalli Pravita , NIVISON MARY P. , Meier Kathryn E.
Published: April 2020

CCN1/Cyr61 is a secreted extracellular matrix‐associated “matricellular” protein capable of regulating a broad range of cellular activities. Several studies have established that CCN1/Cyr61 is rapidly induced at the transcriptional level when cancer cells are incubated with growth factors that facilitate adhesion signaling, thereby enhancing cancer cell migration and survival. However, the role of CCN1 induction in the proliferative responses to growth factors has not been fully delineated. In this study, we examined the changes in expression levels of CCN1 in response to lysophosphatidic acid (LPA) and epidermal growth factor (EGF) in human prostate and breast cancer cell lines. We also studied the role of free fatty acid receptors (FFARs) in this context. FFAR agonists have been previously reported to show inhibitory effects on growth factor‐mediated proliferation and migration in human prostate and breast cancer cells. PC‐3 and MDA‐MB‐231 cells were serum‐starved for 24 hours and treated with 10uM LPA, 10nM EGF, and +/‐ 1uM TUG‐891. Following the incubation, whole‐cell lysates were collected, and subsequent immunoblotting was performed. LPA and EGF induced CCN1 protein by 4 hours. TUG‐891, an FFA4 agonist, inhibited CCN1 induction by LPA. In parallel, the cells were fixed with 4% paraformaldehyde and CCN1 was visualized using confocal immunofluorescence microscopy. The localization of CCN1 following its induction is consistent with its secretion and subsequent binding to ECM components. These results suggest that CCN1 plays roles in the sequence of downstream responses to growth factors in breast and prostate cancer cells.

Support or Funding Information

Supported by the ASPET David Lehr Award and by the WSU College of Pharmacy and Pharmaceutical Sciences.
Unusual Properties of a Human‐specific and Microglia‐specific Siglec‐11 Transcript Variant
Authors: Chen Dillon Y. , Hane Masaya , Varki Ajit
Published: April 2020

CD33‐related sialic acid‐binding immunoglobulin‐type lectins (Siglecs) are often found on innate immune cells and can modulate their reactivity. Previous studies showed that expression of the inhibitory Siglec‐11 in microglia is unique to humans and exists as an alternate splice form missing the exon encoding the last (5th) Ig‐like C2 set domain of the extracellular portion of the protein. Moreover, the brain‐enriched glycan polysialic acid (polySia/PSA) has been considered as a ligand of Siglec‐11. Here, we show that the human recombinant soluble microglial form hSiglec‐11(4D)‐Fc binds better to immobilized polySia than the tissue macrophage form hSiglec‐11(5D)‐Fc or chimpanzee cSiglec‐11(5D)‐Fc. We found that both intact and cleaved Siglec‐11 are in the culture medium, suggesting their ability to be secreted. Specifically, intact Siglec‐11 is found in exosomes and the microglial splice variant has reduced proteolytic cleavage and enhanced incorporation into exosomes. The exosomes bind to human neuroblastoma in a polySia‐dependent fashion. Mechanistically, cysteine palmitoylation is observed in the intracellular region and may regulate Siglec‐11 localization on lipid rafts and secretion on exosomes. Taken together, we demonstrate that hSiglec‐11(4D) is specifically expressed in human brain microglia and is released on the exosomes and/or proteolytic products, which have the potential to affect brain functions at a distance.

Support or Funding Information

National Institute of General Medical Sciences (NIGMS) R01GM32373
Tetanus Neurotoxin (TeNT) Traffics from PNS to CNS: Molecular Structure of the TeNT Receptor Binding Domain
Authors: Tiffany Karen A. , Bote Kayla , Fueger Kaitlyn , Gibeau Benjamin , Going Julia , Kunz Josslyne , Milton Grace , Novack Maya , Phelps Katelyn , Tiffany Benjamin , Voeller Henry , Sundeen Grace , Barbieri Joseph T.
Published: April 2020

The World Health Organization reported that in 2017 almost 31,000 newborns died from exposure to tetanus neurotoxin (TeNT), which is produced by the bacterium Clostridium tetani, despite there being an effective vaccine available. When an individual is exposed to TeNT, the toxin travels through the bloodstream to the peripheral nervous system (PNS) where it is trafficked to the central nervous system (CNS). TeNT cleaves vesicle‐associated membrane protein‐2 (VAMP2), a membrane protein in the CNS, blocking the release of neurotransmitters which signal muscle cells to relax, thus resulting in spastic paralysis. TeNT has two separate domains, A and B. The A domain is a protease and the B domain contains both an N‐terminal translocation domain (HCT/T) and a C‐terminal receptor binding domain (HCR/T). HCR/T is made up of 450 residues, arranged into six alpha helices and 33 beta sheets. HCR/T has two important carbohydrate‐binding pockets. The Arg1226‐containing “R” pocket binds sialic acid on b‐series gangliosides (i.e. GT1b), while the Trp1289‐containing “W” pocket binds to sugar groups on a‐series gangliosides (i.e. GM1a). Functioning “R” and “W” pockets are essential for TeNT toxicity. The Cedarburg High School SMART (Students Modeling A Research Topic) Team has designed a model of HCR/T using 3D printing technology to investigate TeNT structure‐function relationships. Further investigation into how TeNT moves from the PNS into the CNS can help elucidate new methods to cure or prevent tetanus, especially in newborn infants in developing countries where the mortality rate is significant.

Support or Funding Information

National Institutes of Health Clinical and Translational Science Award (NIH‐CTSA UL1RR031973)
Extract From Yellow Passion‐Fruit (passilora edulis) Inhibits Prostate Cancer Cell Proliferation By TLR4/NFkB Pathway Regulation
Authors: Kido Larissa Akemi , Baseggio Andressa Mara , Rossetto Isabela Urra , Lamas Celina Almeida , Montico Fabio , Cagnon Valeria Helena Alves , Marostica Mario Roberto
Published: April 2020

The yellow passion‐fruit (passiflora‐edulis) is rich in piceatannol (PIC), a polyphenolic and resveratrol analog known by its beneficial effects on health such as anti‐inflammatory anti‐cancer properties. The present study aimed to investigate the passion‐fruit extract (PFE) mechanistic of action in prostate cancer (PCa) cell lines with different genetic backgrounds and in the transgenic adenocarcinoma of the mouse prostate (TRAMP). For in vitro experiments, LNCaP (androgen‐responsive) and PC‐3 (androgen‐independent) cells were treated with different doses of PFE (125–500 μg/mL) and used for cell viability assay and western blotting analysis. For in vivo experiments, TRAMP mice were gavaged with 20mg/Kg of PIC contained in the PFE during 4 or 10 weeks of treatment. The ventral prostate was collected for light microscopy, immunohistochemistry and western blotting analysis. Because inflammation is intrinsically evolved with prostate alterations, like hormonal imbalance and cell survival, we focused on if PFE could affect these pathways. PFE treatment caused a significant reduction of cell viability in both cell lines tested, as well as decreased nuclear factor kappa B (NFκB) and toll‐like receptor 4 protein level. Interestingly, LNCaP cells were more sensitive to the PFE action in all experiments and we verified that the androgen receptor was markedly reduced by the treatment in these cells. PFE delayed CaP progression in the TRAMP model in both periods of treatment, significantly decreasing high‐grade prostatic intraepithelial neoplasia and well‐differentiated adenocarcinoma. These results were confirmed by the decrease of proliferating cell nuclear antigen immunostaining. Also, treatment with PFE reduced inflammation markers such as tumor necrosis factor‐alpha (TNF‐α), NFκB, and modulated negatively cell cycle regulatory protein levels and signal transducer and activator of transcription 3. Therefore, the results herewith showed that PFE was able to modulate disease progression through the inhibition of the inflammatory pathways, delay of the cell cycle, especially in the hormone‐dependent profile, demonstrating its chemopreventive and therapeutic potential in PCa.

Support or Funding Information

Sao Paulo Research Foundation ( Number: 2017/01573‐5)
Matrix Metalloproteinase‐9 Promotes Membrane Protrusive Activity in Ovarian Cancer Cells
Authors: Cook Nelson A. , Finnegan Katie D. , Kallenbach Reid A. , Lines Caleb L. , Ellerbroek Shawn M.
Published: April 2020

Matrix Metalloproteinase‐9 (MMP9) is an extracellular endopeptidase associated with ovarian cancer development and progression. To further understand MMP9 contributions to metastatic behavior of an established ovarian cancer cell line (OVCA433), we utilized CRISPR to generate seven cell clones null for MMP9 expression. Analysis of the pooled clones revealed MMP9‐null cells exhibited identical phenotype and growth compared to parental OVCA 433 cells. Cell adhesion and transwell migration towards type I collagen was also not impacted by MMP9 loss. MMP9‐null cells were significantly delayed, however, in non‐directional filamentous actin‐based membrane protrusions on a variety of matrix substrates in response to Epidermal growth factor stimulation. Further, over or re‐expression of MMP9 in OVCA433 or MMP9‐null cells, respectively, was sufficient to drive both lamellipodial and filopodial membrane protrusions on glass. These results indicate MMP9 directly contributes to ovarian cancer protrusive activity.

Support or Funding Information

Wartburg Undergraduate Research Grants
FDA‐approved PARP Inhibitors are Cytoprotective against Cisplatin Injury in Renal Proximal Tubular Cells
Authors: Kim Jinu , Ha Ligyeom , Noh Mi Ra , Jang Hee-Seong , Padanilam Babu J.
Published: April 2020

Cisplatin is a potent chemotherapeutic drug used to treat many solid cancers. However, its dose‐limiting side effect is nephrotoxicity, leading to acute kidney injury and chronic kidney disease. We previously reported that genetic or pharmacologicial inhibition of poly (ADP‐ribose) polymerase 1 (PARP1) protects against cisplatin nephrotoxicity.

PARP1 inhibitors including olaparib, niraparib, and rucaparib are currently FDA approved for treatment of ovarian cancer. In this study, we tested the effect of the above FDA‐approved PARP inhibitors in cisplatin nephrotoxicity. Human HK‐2, pig LLC‐PK1, and mouse HUMPT renal proximal tubular cells (PTC) were subjected to one cycle of treatment with high‐dose cisplatin (1 to 1.5 mM) or four cycles of treatment with low‐dose cisplatin (1 to 3 μM). Each cycle consisted of 6 hours of treatment with cisplatin and 18 hours of recovery in cisplatin‐free medium. The FDA‐approved PARP inhibitors (300 pM to 1 μM) or the same volume of dimethylformamide (vehicle) were added immediately before the first treatment cycle and incubated continuously. The 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay performed to determine cell viability, lactate dehydrogenase (LDH) release assay to determine cell membrane disruption, and propidium iodide (PI) staining to determine necrotic cell death.

All of the PARP inhibitors improved cell viability after the high‐dose cisplatin treatment in renal PTC. Increased percentages of LDH release and PI‐positive cells observed after one cycle of treatment with high‐dose cisplatin were significantly abrogated by the PARP inhibitors. Moreover, combined treatment with the PARP inhibitors showed an additive effect on cell viability in renal PTC treated with high‐dose cisplatin. The PARP inhibitors also enhanced cell viability after the four cycles of treatment with low‐dose cisplatin in renal PTC.

Thus, our findings suggest that the FDA‐approved PARP inhibitors can protect efficaciously against death of PTC during cisplatin nephrotoxicity and could be developed as potential therapeutic agents to improve cisplatin nephrotoxicity.

Support or Funding Information

This study is supported by NIH grants DK‐116987, DK‐120533, AHA grants 15GRNT25080031 (BJP), 15POST25130003 (HSJ), and NRF grant NRF‐2019R1F1A1041410 (JK).
SRSF3 and hnRNP K Antagonistically Regulate Splicing of Large Exons
Authors: Kawachi Toshihiko , Masuda Akio , Takeda Jun-ichi , Ito Mikako , Hamaguchi Tomonari , Ohno Kinji
Published: April 2020

Mammalian internal exons are usually between 50 and 200 bp long. Their average length is ~150 bp, and only 5% of them are larger than 300 bp. Although previous studies showed that splice site recognition becomes less efficient with increasing exon size, the mechanism how large exons are correctly spliced is not well understood. To investigate the role of RNA binding proteins (RBPs) in the splicing of large exons, we analyzed 842 publicly available RNA‐seq datasets from GEO, in which 48 canonical splicing factors were individually knocked down. MISO analysis was performed to detect exons that were specifically included or excluded by knockdown of an RBP, and average lengths of these exons were calculated. Our analysis revealed that SRSF3 is the top RBP that promotes inclusion of the largest exon set (390 exons per analysis, 337 bp on average).

Interestingly, most of the SRSF3‐dependent large exons are annotated as constitutive exons in the public database. Consistently, these exons have high splice site strengths. Motif analysis revealed that the SRSF3‐dependent large exons are greatly enriched with C‐rich sequences, which are preferentially recognized by SRSF3. In addition, our analysis also detected the enrichment of C‐stretches, which are potential binding sites for hnRNP K. ENCODE eCLIP shows that HNRNPK extensively binds to the SRSF3‐dependent large exons. To examine whether hnRNP K is involved in the regulation of SRSF3‐dependent splicing of large exons, we knocked down Hnrnpk along with Srsf3‐knockdown using C2C12 myoblasts. RNA‐seq analysis revealed that Srsf3‐silencing alone induced skipping of 374 exons, of which average length is 395 bp. Co‐silencing of Srsf3 and Hnrnpk rescued skipping of 142 exons by Srsf3‐silencing, of which average length is still long, 458 bp. These results indicate that substantial number of large exons require SRSF3 to prevent their skipping by hnRNP K.

To investigate the molecules associated with SRSF3 and hnRNP K in the splicing of large exons, we next conducted proteomic approach that defines the native protein complexes in the chromatin fraction. Cells expressing FLAG tagged SRSF3 or hnRNP K were lysed under the physiological salt concentration, and isolated chromatin fractions were immunoprecipitated with At‐FLAG antibody. Mass spectrometry analysis of the immunoprecipitants revealed that SRSF3 is mainly associated with U1 snRNPs, U2AFs, and SF1, and to a lesser extent with hnRNPs, while hnRNP K is preferentially associated with hnRNPs and U1 snRNPs, but not with U2AF or SF1. Thus, SRSF3 and hnRNP K are involved in early but distinct stages of spliceosome assembly. Together, our analysis identified the antagonistic splicing regulation of SRSF3 and HNRNPK on C‐rich large exons.
Regulation of transient receptor potential canonical 4 activity by phospholipase C δ1
Authors: Kang Hana , Ko Juyeon , Myeong Jongyun , Kwak Misun , So Insuk
Published: April 2020

Transient receptor potential canonical (TRPC)4 and TRPC5 channels are non‐selective calciumpermeable cation channels that maintained by phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) and inactivated due to its depletion. Phospholipase C (PLC) is an enzyme that cleaves phospholipids and the PLCδ is the most calcium‐sensitive form among the isozymes that stimulated by physiological concentration of Ca2+. Here, we investigated the regulation mechanism of TRPC4 by the Ca2+‐PLCδ‐PI(4,5)P2 signaling cascade. In order to identify the interaction between ion channel and PLCδ protein, we implied co‐immunoprecipitation and fluorescence imaging including Föster Resonance Energy Transfer. We evaluated the activity of channels with electrophysiological recording in HEK293 cells expressing TRPC channels. Here, we demonstrate that TRPC4 directly interacts with PLCδ1. We also found that PLCδ isozymes are activated by the calcium through opened channels but not by the cytosolic calcium increase. Our study established the PLCδ1 inhibits overall TRPC4’s currents activity, but mainly regulates the basal TRPC4 currents to have a characteristic low basal activity. These regulation of PLCδ1 on TRPC4 activity occurs depend on PI(4,5)P2 hydrolysis. Resultantly, PLCδ1 is considered to be a negative regulator of TRPC4.

Support or Funding Information

This research was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information & Communication Technology), and Future Planning (MSIP) of the Korean government (2018R1A4A1023822 to I.S.S.). H.Kang, J.Ko., J.Myeong., and M.Kwak. were supported by the BK plus program from the MSIP.
Subcellular Location‐Dependent Activation and Signaling of the Delta‐Opioid Receptor
Authors: Crilly Stephanie E. , Weinberg Zara Y. , Ko Wooree , Puthenveedu Manojkumar A.
Published: April 2020

G protein‐coupled receptor (GPCR) signaling from intracellular compartments is an emerging idea in the field. Classically, GPCRs have been thought to signal mainly from the plasma membrane. However, GPCRs localized to endosomes and the Golgi are a critical component of receptor signaling. GPCR location affects signaling at multiple levels, from generation of second messengers to transcriptional responses, and even physiological responses. One physiologically relevant receptor that shows location‐dependent signaling is the delta‐opioid receptor (DOR), an attractive alternative target for pain management. DOR is localized primarily to intracellular compartments, including the Golgi, in neuronal cells. Relocation of these receptors to the cell surface increases the ability of DOR agonists to relieve pain, suggesting that the subcellular location and signaling of this receptor are closely linked and important for receptor pharmacology. However, the mechanisms by which DOR relocation to the plasma membrane from Golgi membranes influences receptor pharmacology are poorly understood. Live cell imaging with nanobody and mini‐G protein biosensors indicates receptor localization influences ligand‐induced recruitment of active‐conformation biosensors. Additionally, we used cAMP and calcium sensors to study how DOR localization influences receptor signaling. Together, our data support a model in which DOR activation and signaling are spatially regulated. This spatial regulation may underlie the complexity of physiological effects associated with DOR activation and provide new ways to modulate receptor signaling.

Support or Funding Information

M.A.P. was supported by NIHGM117425 and NSF1517776. S.E.C was supported by NSF Graduate Research Fellowship DGE1256260.
Unfolding the Mysteries of Oxygen Sensing ‐ A Comprehensive Analysis of the Hypoxic Response in Zebrafish Gills One Cell at a Time via Single Cell RNA Sequencing
Authors: Pan Wen , Godoy Rafael , Cook David , Scott Angela L. , Nurse Colin A. , Jonz Michael G.
Published: April 2020

All vertebrates “sense” changes in O2 through specialized cells, called chemoreceptors, which interact with the peripheral and central nervous systems to maintain O2 homeostasis. Aquatic vertebrates are especially prone to fluctuations in environmental O2 availability. Therefore, it is vital for them to readily detect changes in PO2 and make appropriate respiratory and cardiovascular adjustments. Teleost fish, such as zebrafish (Danio rerio), have O2 chemoreceptors, called neuroepithelial cells (NECs), present in the gill epithelium and undergo membrane depolarization and vesicular recycling when exposed to acute hypoxia. NECs have also been shown to increase in number and size following chronic hypoxia. Here we present a comprehensive analysis of the effects of hypoxia on gill cells including NECs through a transcriptomic study. Using the transgenic line ETvmat2:GFP, zebrafish were acclimated to normoxia (PO2 = 160 mmHg) or severe hypoxia (PO2 = 35 mmHg ) and maintained for 14 days. Isolated cells from the distal ends of gill filaments, areas rich in NECs, were analyzed using single cell RNA sequencing (scRNA‐seq) analysis. Four normoxia (control) and four hypoxia‐treated biological samples were individually labelled before scRNA‐seq library construction using the 10X Genomics chromium technology. A total of 45,000 gill cells were consequently sequenced on a NextSeq500 (Illumina) sequencer with a read depth of 17,000 per cell. Our preliminary results separated 21 cell clusters and identified one cluster to be NECs based on gene expression. Highly differentially expressed genes in NECs included tph1a encoding tryptophan hydroxylase 1, the rate limiting enzyme in 5‐HT biosynthesis, and sv2 encoding synaptic vesicle 2 protein. Other differentially expressed genes in NECs are involved in mitochondrial oxidative metabolism and ATP synthesis. Moreover, differential expression analysis showed a clear shift in the transcriptome of NECs following 14 days of chronic hypoxia. NECs in the hypoxia group showed a high expression of genes involved in stress‐regulated polyubiquitination in contrast to the control group. The transcriptomic change implies a change in dynamics of the NEC population. We are the first group to conduct a single cell transcriptomic study in zebrafish gill cells. Our study is expected to provide a complete atlas for the hypoxic response in single gill cells and serve as a platform for future work on peripheral oxygen sensing.

Support or Funding Information

This research project is funded by Natural Sciences and Engineering Research Council of Canada (NSERC).
Investigating the Roles of Orexinergic System in Methamphetamine‐induced Addiction
Authors: Lee Chan , Bae Hui Jin , Jang Jung-Hee , Park Gyu Hwan
Published: April 2020

Methamphetamine (METH) is a powerful neurotoxic psychostimulant characterized to affects activity of dopamine transpoters leading to blockage of dopamine (DA) uptake into the synaptic vesicle as well as DA reuptake through the plasma membrane inducing synaptic DA release, which resulted in continuous excess extracellular DA levels in the synaptic cleft. Orexinergic system has been reported to play crucial roles in regulation of arousal, wakefulness, and motivated behaviors for drug abuse. In this study, we have investigated the roles of orexin and/or orexin receptors on METH‐induced addictive behaviors by conducting conditioned place preference (CPP) test using SB334867, an orexin‐1 receptor antagonist. The changes in the dopaminergic system integrity and orexin‐related signaling molecules were examined to elucidate underlying molecular mechanisms. C57BL/6 mice were administered with METH (1 mg/kg, i.p.) alternately on each other day for 8 days, allowed to undergo withdrawal period without injection of METH for additional 8 days, and then challenged again with METH. METH‐administered mice exhibited increased levels of orexin and orexin‐1 receptor in the brain regions of hippocampus, striatum, hypothalamus, and amygdala. The application of SB334867 significantly attenuated the acquisition, expression, and reinstatement of the METH‐induced CPP. These findings suggest that orexinergic system may play a role in the acquisition and relapse of METH‐induced addictive behaviors, thereby providing a novel therapeutic target for the METH addiction.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)
Sequence Analysis of Homologous Alu Elements: A Novel Methodology for Primate Phylogenetic Tree Construction
Authors: Sood Nitish , Mehra Mehul , Mittal Anav , Bates Christopher
Published: April 2020

Non‐human primates are essential model organisms across a broad spectrum of biomedical research, from drug discovery & development to insights into immunological responses. The study of phylogeny enhances understanding of the particular experimental advantages of each model organism. Despite considerable efforts invested into developing currently accepted practices of phylogenetic analysis, these strategies often involve significant expenditures of time and resources. To address these concerns, we developed a novel computational approach to primate phylogenetic tree construction using the Alu transposable element. Short Interspersed Nuclear Elements such as Alu have previously been validated as accurate tools for phylogenetic analysis, as they predominantly evolve in a unidirectional and homoplasy‐free manner. However, previous Alu‐based investigations into phylogenetic relationships have been limited to detecting specific Alu insertion polymorphisms as a proxy for evolutionary divergence. Here, we present a novel methodology for primate phylogenetic analysis using genetic diversity in homologous Alu sequences. Alu elements for 11 primate genomes, classified by subfamily, were extracted from the University of California Santa Cruz’s Genome Table Browser. Subsequently, multiple sequence alignments were performed to identify which Alu sequences were homologous among the 11 primates. Following identification, phylogenetic trees for each of 22 conserved subfamilies were constructed. These trees were then compared to randomly generated phylogenetic trees and found to be significantly (p < 1e‐10) more accurate on all tree distance metrics calculated (Robinson‐Foulds, Path difference, Subtree Pruning and Regrafting, Hamming distance, and Penny Hendy). The Alu‐based trees were then compared to published primate phylogenetic trees to confirm the validity of this technique. No significant difference in accuracy was found for the most accurate Alu subfamily tested, demonstrating its validity. We therefore propose that our approach of comparing homologous Alu sequences can serve as a supplement or alternative to current primate phylogenetic tree construction.
Maternal Stress Effects on Hatching Success and Larval Malformation Prevalence in Palmetto Bass.
Authors: Hanna James Patrick , Prangnell David I. , Matthews Michael D. , Huertas Mar
Published: April 2020

Striped Bass Morone saxatilis are an iconic North American fish. This species, and it’s hybrid with White Bass Morone chrysops, Palmetto Bass are intensively cultured for stocking into Texas water bodies. However, a large proportion of hatchery‐spawned Palmetto Bass larvae were recently plagued with malformations. Pug headedness is a common malformation that restricts the fish’s ability to intake food by misshaping the skull and mouth. Malformations such as this drastically reduce larval survival. Female Striped Bass broodfish used to produce Palmetto Bass are caught in the wild, hormonally induced to maturation, transported to the hatchery, and hand‐spawned by stripping. Consequently, we hypothesize that this extreme handling stress affects larval development and viability. Eight Striped Bass females were strip‐spawned, and samples of plasma, ovary, eggs, and hatchlings where collected to measure the concentrations of glucose, lactate and cortisol. Percentage of hatch, malformations and viability were also recorded for each spawn. A standard ELISA plate was used to measure cortisol and colorimetric protocols were used to measure glucose and lactate. Females that produced spawns with lower hatch (9.9 %) and larvae with elevated malformations rates (53.2%) showed significantly higher concentrations of plasma cortisol (290.7 ± 80.7 ng/mL) than females associated with low larval malformation rates (120.6 ± 16.0 ng/mL). Interestingly, females with low hatching rate had significantly less cortisol in ovarian tissue but more cortisol in their eggs than females with a higher performance. Spawns with higher malformation rates also had higher values of larval tissue cortisol (4.5 ± 1.0 ng/g) than spawns with lower malformation rates (0.2 ± 0.0 ng/g). This data points to a transfer of cortisol from maternal gonads to the spawn while under handling stress. Glucose and lactate had similar values in all individuals. Thus, only cortisol correlated with the presence of malformations in larvae. This study demonstrates the need to reduce handling stress in Striped Bass culture to minimize the production of malformed larvae. The impact of integrating saltwater baths, anesthetics, and reducing handling time, can be easily monitored with cortisol measurements and may improve production outcomes.
Attenuation of Aortic Remodeling with Low Doses of Neprilysin/Angiotensin II Receptor Blockers Sacubitril/Valsartan in a Fibrillin‐1‐Deficient Model of Marfan Syndrome
Authors: Bernatchez Pascal , Sauge Elodie , White Zoe
Published: April 2020

Introduction

Sacubitril is a neprilysin inhibitor indicated for heart failure patients with reduced ejection fraction (HFReF), which causes protective natriuretic peptide accumulation. To maximize efficacy and minimize side effects, sacubitril is administered in combination with valsartan, a blood pressure‐lowering angiotensin II receptor blocker (ARB). We and others have shown that ARBs are capable of blood pressure lowering‐independent effects on vascular and lung remodeling, but whether the sacubitril/ARB combination shows greater anti‐vascular and lung remodeling effects is unknown.

Objective

Since ARBs are routinely prescribed to patients with Marfan syndrome, which is a fibrillin‐1 mutation‐induced connective tissue disease that causes aortic widening, dissection and lung emphysema, we sought to test the anti‐remodeling effects of low‐doses sacubitril/valsartan combination on the vascular and pulmonary complications associated with Marfan syndrome in mice.

Results

Treatment with low dose valsartan and sacubitril/valsartan (pure forms in drinking water) did not significantly lower blood pressure when compared with vehicle group in the C1039G fibrillin‐1 model of Marfan syndrome. There was a non‐significant inhibitory trend of Marfan‐associated aortic root widening with valsartan and especially sacubitril/valsartan, whereas only the latter induced a significant and near‐complete (74%) inhibition of Marfan‐associated ascending aorta widening (P<0.008). None of the treatment groups significantly reduced emphysema, loss of lung volume, change in hysteresis, tissue damping and resistance associated with Marfan syndrome.

Conclusion

Sacubitril/valsartan combination may be more clinically relevant than valsartan alone in settings of aortic dilation, even at low doses.

Support or Funding Information

Supported by Novartis Canada
Insulin Analog with Additional Disulfide Bond Forms a Structurally Secure, Thermodynamically Stable Molecule
Authors: Candler Noelle Riley , Horth Shelby Noelle
Published: April 2020

Insulin is a hormone secreted by the pancreatic cells. In vivo, normal insulin contains 3 disulfide bonds that account for structural instability and low melting point. In this experiment, an insulin analog was attempted to be made mechanically and thermodynamically more stable than human insulin (HI). It was hypothesized that the addition of a fourth disulfide bond (4SS‐insulin) would be more stable as previous attempts at other types of transformational change to the A‐chain and B‐chain were unsuccessful. By adding a fourth disulfide bond in positions A10/B4, the outcome would be a significant clinical application for the treatment of insulin‐dependent diabetes as scientists are constantly trying to find insulin analogs that would potentiate the effects of blood sugar lowering. To experiment, the fourth disulfide bond was successfully formed using yeast as a template. They tested fragility (fibrillation), low pH, and thermodynamic stabilization, finding that the fourth disulfide bond made insulin more mechanically and thermodynamically stable. Once this was complete, in vivo experimentation on rates demonstrated the effect on blood glucose and in vitro experimentation on lipogenesis in rat adipocytes. In vivo, 4SS‐insulin had a longer and more profound effect on blood glucose than HI. In vitro, 4SS‐insulin had a greater affinity for insulin receptors than HI. The Kohler High School MAPS Team used 3‐D modeling and printing technology to examine the relationship between a structurally secure fourth disulfide bond and the thermodynamic stability of an insulin analog in comparison to typical HI. The clinical applicability of this new biomedically engineered 4SS‐insulin is significant. Patients could be able to self‐inject less insulin less frequently, helping with affordability and improved glycemic control. Additionally, the storage of insulin becomes more convenient as 4SS‐insulin can withstand a 34.6 °Celsius increase in melting temperature.

PDB: 4EFX
Immuno‐Sympathoinhibitory targeted Electroacupuncture Treatment Enhanced Blood Pressure Lowering Responsiveness in Middle‐Aged Hypertensive Patients
Authors: Tjen-A-Looi Stephanie , Xie Lifang , Fu Liang-Wu , Nguyen Lan , Malik Shaista
Published: April 2020

We have shown that 8‐week electroacupuncture (EA) at P5‐6 and St36‐37 reduces blood pressure (BP) (70% effectiveness, BP decrease of ≥ −6 mmHg in peak or average systolic or diastolic) and norepinephrine in a group of male and female (38–75 year) patients with mild to moderate hypertension (HTN). During the 8 EA sessions, the onset of the BP reduction was observed by week 4, while these patients were not on antihypertensive medications. BP reduction with 30‐min EA once a week treatment at P5‐6+St36‐37 as such is defined as sympathoinhibitory. Interestingly, only 59% of middle‐aged and elderly (46–75 years) patients with HTN are responsive to this sympathoinhibitory treatment. To this end, we formulated a targeted treatment that addresses low grade chronic inflammation present in hypertensive patients. High sensitivity C‐reactive protein (hs‐CRP) is a marker of systemic chronic low‐grade inflammation. We hypothesized that immuno‐sympathoinhibition by EA treatment decreases BP and hs‐CRP in majority of middle‐aged and elderly patients with HTN. Participants were not on antihypertensive medications and randomly allocated to either sympathoinhibitory (n=12) or immuno‐sympathoinhibitory (n=9) EA treatment. After completion of the 8‐week course of treatment, the BP lowering responsiveness (≥ −6 mmHg) to the immuno‐sympathoinhibitory EA was 88% (P=0.016) while the sympathoinhibitory was 58% (P=0.042). The hs‐CRP appeared to be reduced in immuno‐sympathoinhibitory EA treated patients (−6.7±3.1 %, n=5) following 8‐week EA treatment in contrast to patients treated with sympathoinhibitory EA (0.03±0.15%, n=12). Thus, EA targeted towards the immune and sympathetic systems combined may be important in lowering the BP and chronic low grade inflammation in majority of middle‐aged and elderly hypertensive patients.

Support or Funding Information

Adolph Coors Foundation