bluuebunny/crossref_metadata_2025_split huggingface dataset:
Adrenergic activation of cardiac preganglionic neurons in the Nucleus Ambiguus
Authors: Aiba Isamu 
Published: April 2020

Cholinergic neurons within the nucleus ambiguus (NAm) are the major parasympathetic preganglionic neurons innervating heart. This study characterized the excitability of NAm neurons using in vitro acute brainstem slice preparations obtained from young adult mice. Cholinergic premotor NA neurons were identified based on their expression of tdTomato fluorescence in the Chat‐Cre:floxed‐tdTomato mouse line. Retrograde labeling of pericardiac axon terminals study confirmed cholinergic NAm neurons with large soma as preganglionic cells. In current‐clamp recordings, a brief hyperpolarizing current evoked rebound action potentials (APs). Low voltage‐activated, T‐type calcium current (ICaT) is required for this rebound discharge pattern of NAm neurons. Voltage‐clamp recordings detected a significant T‐current in NA neurons, and quantitative in situ hybridization analysis identified Cav3.1 and 3.2 channels as the major contributors. These preganglionic neurons are strongly activated by adrenergic agonists in vitro. Bath application of adrenergic agonists, epinephrine (10 μM) and norepinephrine (20 μM), induced spontaneous action potentials in these neurons. Inhibition of synaptic ionotropic receptors did not prevent the adrenergic activation, suggesting a minor contribution of synaptic mechanism. Instead, adrenergic stimulation significantly increased the intrinsic excitability of NA neurons; input resistance of NA neurons was significantly increased and single evoked rebound discharges (normally composed of 1–2 APs) converted to regenerative oscillations with burst of >10APs lasting up to a few seconds. Pharmacological tests suggest that persistent sodium current is an important contributor to the activated action potential generation. Together this study identified basic intrinsic membrane excitability mechanism of NAm and its activation by adrenergic agonists.Support or Funding InformationU01 NS090340 (JLN) AHA Career development award (IA)
Characterization of Modified‐DNA Polymerase Fidelity
Authors: Seto Madison K. , Barrett Susanna E. , Weiden Aurora G. , Leconte Aaron M. 
Published: April 2020

Recently, several modified‐DNA (M‐DNA) polymerases have been identified which can synthesize long M‐DNAs. While these discoveries have enabled new applications of M‐DNA, these enzymes typically have poor fidelity, possessing error rates several orders of magnitude worse than native enzymes. However, to date, efforts to both quantify error rate, as well as experimental approaches towards understanding the origin of the poor fidelity of M‐DNA polymerases, have been limited. Here, we use a high‐throughput sequencing assay to characterize the error rate of leading M‐DNA polymerases, which showed that our enzyme, P1, has an error rate 1.6 times lower than SFM4‐6, the most frequently used Taq mutant for 2′M‐DNA synthesis. We also characterize the importance of individual mutations on fidelity, beginning to experimentally address the origins of fidelity, and have found that reverting even one mutation in a mutant yielded a fidelity that was 2.4 times greater than its parent, which will help us improve future M‐DNA polymerase rational design.Support or Funding InformationNational Science Foundation
A novel and convenient bacterial platform for production of encapsidated double‐stranded RNA targeting white spot syndrome virus in shrimp
Authors: Wuthisathid Kitti , Chaijarasphong Thawatchai , Chotwiwatthanakun Charoonroj , Somrit Monsicha , Sritunyalucksana Kallaya , Itsathitphaisarn Ornchuma 
Published: April 2020

White spot syndrome virus (WSSV) is a highly infectious virus that can cause 100% mortality in farmed shrimp in the matter of days, leading to significant economic losses to shrimp aquaculture worldwide. To mitigate the adverse impact of WSSV, effective antiviral strategies are necessary. Currently, double‐stranded RNA (dsRNA), which can silence viral gene expression through an RNA interference (RNAi) pathway, is among the most promising therapeutic tools for WSSV, but its farm‐scale utilization is held back by high costs and the lack of delivery approach. Recently, virus‐like particles (VLPs) have been applied as nanocarriers to deliver dsRNA into shrimp tissues, resulting in prolonged half‐life of the encapsidated dsRNA and stimulation of shrimp innate immunity. However, recombinant VLPs and dsRNA are traditionally expressed in two different E. coli strains: a protease‐deficient BL21(DE3) strain and an RNase III‐deficient HT115(DE3) strain, respectively. The ability to produce both VLPs and dsRNA simultaneously in a cell will reduce the costs and steps associated with the production, but attempts at VLP‐dsRNA coexpression in each of these strains resulted in degradation of either VLP or dsRNA due to the presence of proteases or RNaseIII, respectively. Here, to streamline VLP‐dsRNA production and allow simultaneous expression of VLP and dsRNA in the same cell, P1 transduction was employed to generate a novel E. coli strain which is both protease‐ and RNase III‐deficient. The newly engineered E. coli strain is able to express both MrNV‐VLP and VP28 dsRNA efficiently. Therefore, this new expression platform will pave the way for development of effective and low‐cost production of VLP‐encapsulated dsRNA for protection against viral infection in farmed shrimp.Support or Funding InformationThe authors would like to thank the following institutions for supporting research and scholarship to graduate student: Thailand Graduate Institute of Science and Technology (TGIST), National Science and Technology Development Agency (NSTDA) and Mahidol University.
Hypoxia‐induced annexin A6 expression underlies the Metabolic Vulnerability and response of TNBC cells to EGFR‐Tyrosine kinase inhibitors
Authors: Williams Stephen De’Shawn 
Published: April 2020

PURPOSETriple‐negative breast cancer (TNBC), and the basal‐like TNBC subtype, accounts for ~20% of all breast cancer (BC) cases, and is particularly prevalent in younger patients of African‐American and Hispanic descent. The importance of annexin A6 (AnxA6) as a tumor suppressor in various cancers has been widely investigated, but the mechanisms by which AnxA6‐induced basal‐like triple negative breast cancer progression remain largely unknown. Physiological changes such as hypoxia, metabolic acidosis, and oxidative stress in the tumor microenvironment (TME) during cancer progression endow cancer cells with malignant properties, ultimately leading to metastatic dissemination that remains the major cause of mortality in cancer patients. By linking the altered expression status of AnxA6, with changes in metabolic output of the tumor cells and the efficacy of the otherwise ineffective epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs), these metabolic vulnerabilities can be targeted to attenuate the growth and/or motility of this hard‐to‐treat breast cancer subtype.DESIGN METHODSAnxA6‐high (BT‐549) and AnxA6‐low (MDA‐468) TNBC cell lines were cultured in hypoxic conditions (94%N2; 1%O2; 5%CO2; 37°C) using a Bactrox Hypoxia Chamber (Terra Universal Inc., Fullerton, CA). TNBC cells were treated with EGFR‐TKI Lapatinib (Biovision Inc., Milpitas, CA).RESULTSWe demonstrate that exposure of AnxA6‐high (BT‐549) and AnxA6‐low (MDA‐468) TNBC cell lines to acute hypoxia (1% O2; ≤24 hrs.) is associated with down‐regulation of AnxA6 while chronic hypoxia (1% O2; >24 hrs.) led to a consistent up‐regulation of AnxA6. AnxA6, FABP4, and HIF‐1α downstream genes GLUT1 and PGK‐1, mRNA levels were upregulated in chronic hypoxic conditions. AnxA6‐dependent metabolic studies reveal that down regulation of AnxA6 in TNBC cells increased lactate production, glucose consumption, ATP‐synthesis and lipid metabolism under hypoxic conditions. In addition, chronic hypoxia sensitized TNBC cells to EGFR‐TKIs.CONCLUSIONThese data suggest that the transition from AnxA6‐high to AnxA6‐low may be dependent on hypoxia in the TME and consequently, TNBC cell survival and resistance to EGFR‐targeted therapies.Support or Funding InformationThe project is supported by NIH 1SC1CA211030 (AS)
Uncovering Dysregulated Micro‐RNA Expression in Senescent Human Vascular Smooth Muscle Cells: Impacts on Cellular Communication and Vascular Aging
Authors: Nguyen Diem Duong Ngoc , Chilian William , Pung Yuh Fen 
Published: April 2020

Vascular aging is highly associated with cardiovascular morbidity and mortality and vascular smooth muscle cell (VSMC) senescence is one of its key contributors. However, intracellular and extracellular signaling and communication of senescent VSMCs are not fully elucidated. Understanding micro‐RNA (miRNA) regulation during VSMC senescence may elucidate the molecular mechanistic link between vascular aging and early adverse vascular remodeling. This study aimed to identify dysregulated miRNA expression within senescent VSMCs as well as senescent VSMC‐derived exosomes. Healthy and senescent human VSMCs (hVSMCs) were cultured in vitro and exosomes secreted by these cells were isolated via ultracentrifugation. Subsequently, cellular and exosomal miRNAs were extracted and used for library preparation of whole‐genome small RNA next‐generation sequencing (NGS). Post‐sequencing and differential expression analyses compared the miRNA profiles between healthy and senescent hVSMCs as well as their derived exosomes. Thereafter, NGS data were validated using real‐time polymerase chain reaction (qPCR). Additionally, target prediction and pathway analysis were performed. In senescent hVSMCs, eight significant differentially expressed mature miRNAs were identified (n= 4, q <0.05). Bioinformatic analyses showed correlation between upregulated miRNAs and activated ubiquitin proteasome pathway, a senescence hallmark. Meanwhile, downregulated miRNAs might associate with cell cycle arrest at G1/S phase via the TGF‐β/SMAD signaling pathway, promoting hVSMC entry into senescence. On the other hand, three significant differentially expressed miRNAs were found in senescent hVSMC‐derived exosomes. Data suggested that the upregulated exosomal miRNA was involved in the formation of advanced glycation end‐products, known to play a role in vascular complications. Concurrently, downregulation of exosomal miRNAs promoted cellular senescence, thus advanced aging. Interestingly, there was a consistent observation of hsa‐miR‐155‐5p downregulation in both senescent hVSMCs and their secreted exosomes. Further analyses with the other dysregulated miRNAs suggested hsa‐miR‐155‐5p involvement in the TGF‐β/NF‐κB signaling pathway. The dysregulated miRNAs in senescent hVSMCs and senescent hVSMC‐derived exosomes offered valuable insights on the mechanisms involved in hVSMC intracellular and extracellular signaling during senescence, thus contributing to our understanding of vascular aging in humans.Support or Funding InformationThe study was funded by Ministry of Higher Education Malaysia grant, FRGS/1/2015/SKK08/UNIM/03/2 to YFP.
Allosteric Modulators of CDK4 for Lung Cancer Therapeutics
Authors: MORRIS May Catherine , Bihel Frederic , Diot Sebastien , Peter Clovis , Bouclier Celine , Bricard Jacques , Pellerano Morgan , Hellal Malik , Prével Camille 
Published: April 2020

Lung cancer is the leading cause of cancer‐related death worldwide. CDK4/Cyclin D kinase is a key cell cycle regulator and constitutes an attractive pharmacological target for development of anticancer therapeutics, in particular in KRAS‐mutant lung cancer. Although several ATP‐competitive inhibitors targeting protein kinases have been developed as anticancer therapeutics, including several recently FDA‐approved CDK4 inhibitors, these compounds suffer from limited efficacy, selectivity and toxicity due to their conventional mechanism of action. Alternative strategies to overcome limitations associated with conventional kinase inhibitors include targeting protein/protein interactions and conformational transitions which are essential for kinase activation.Using a CDK4‐specific conformational biosensor that discriminates against ATP‐pocket binding compounds and that we implemented to screen a small chemical compound library of original small molecule scaffolds, we identified and validated a druggable and patentable compound that tampers with CDK4 function. This compound inhibits Rb phosphorylation, blocks proliferation of several cancer cell lines, and does not bind the ATP pocket of CDK4 nor any of 456 other kinases. An ongoing hit‐optimization process led us to identify derivatives exhibiting greater anti‐proliferative activity than Abemaciclib or Palbociclib in the A549, KRAS‐mutated NSCLC cell line. Interestingly, these non‐ATP CDK4 inhibitors only have a cytostatic effect, and do not induce cytotoxicity even at high doses. Further studies aimed at characterizing the mechanism of action of this compound have highlighted an unexpected and atypical mechanism of inhibition targeting CDK4. Our compounds constitute attractive leads as next generation of CDK4‐specific inhibitors and lung cancer therapeutics.Support or Funding InformationThis work was supported by the SATT Connectus, Strasbourg France and awarded the 2019 Best Academic Project of MATWIN (https://matwin.fr/en/cdk4ppi‐awarded‐2019‐best‐academic‐project‐by‐the‐matwin‐international‐board/)
Embryonic hyperglycemia causes a reduction in photoreceptor cells and increased oxidative stress in the developing retina
Authors: Titialii Kayla , Morris Ann 
Published: April 2020

Poor blood glucose regulation has been linked to various long‐term metabolic disruptions in adults, such as tissue damage in the eye. Yet, little is known about the effects of embryonic hyperglycemia on retinal development. To address this question, our lab has established a protocol for inducing hyperglycemia in zebrafish embryos by submerging zebrafish embryos in fish water containing 50mM glucose +/− 10μM dexamethasone from 10 hours post fertilization (hpf) until 120 hpf. Following treatment, larval heads were either fixed for sectioning or used for RNA extraction and the remaining body was used to quantify glucose concentration. The fixed heads were processed for sectioning and immunohistochemistry to label and quantify cell types in the retina. Treatment with 50mM glucose + 10μM dexamethasone reliably produced hyperglycemic larvae, with at least 2.5‐fold increased whole‐body glucose concentration compared to control‐treated larvae. Our results show that hyperglycemic larvae had a significant reduction in rod and cone photoreceptors with abnormal morphology, and significant increase in reactive oxygen species (ROS) production in the retina. In addition, Müller glia cells showed abnormal morphology, but an increase in number. Finally, genes related to the pentose phosphate pathway were also up‐regulated in hyperglycemic eyes. Our results suggest that embryonic hyperglycemia results in abnormal photoreceptor development via ROS production, which is accompanied by an elevation in pentose phosphate pathway activity. As the population of women with uncontrolled diabetes during pregnancy continues to grow, it is imperative to pinpoint the effects of embryonic hyperglycemia. The similarities between humans and zebrafish will allow us to study these effects, which will aid in the development of therapeutic strategies.Support or Funding InformationGertrude F. Ribble Mini Grant, Department of Biology, University of Kentucky Diabetes Research Center, Washington University, Grant No. 5P30 DK020579
The Anatomy, Bioacoustics, and Neural Physiology of Dolphin Biosonar
Authors: Mulsow Jason , Houser Dorian S. , Finneran James J. 
Published: April 2020

Light is rapidly attenuated in sea water, and the habitats of dolphins and other toothed whales are characterized by reduced visual stimuli relative to terrestrial habitats. Sound is efficiently transmitted under water; however, and these species have undergone significant modifications to head structures that facilitate both passive and active (echolocation) acoustic sensing of marine environments.The nasal anatomy involved in the creation of dolphin biosonar signals is intricate. Structures immediately beneath the blowhole – termed the phonic lips – create short‐duration, directional pulses that travel through a fat‐filled melon (forehead) and into the water. The exact mechanism by which these “clicks” are generated in the phonic lips is not well understood. Recent electrophysiological measurements suggest that neural potentials may precede the production of every click. Thus, dolphins may actively mediate click generation on a click‐by‐click basis. It is not known if this is possible at the highest rates observed in dolphin echolocation (hundreds of clicks per second), as this would require very rapid motor activity. Dolphins also appear to control the direction of the outgoing biosonar beam independent of large head movements. This may be facilitated by deformations of the melon or manipulation of a complicated system of air sacs surrounding the click generation structures.One of the most obvious anatomical modifications to sound reception structures is the complete absence of external pinnae, and only a small remnant of the ear canal is apparent on the surface of the skin. Although lower frequencies (below 20 kHz in the case of bottlenose dolphins) may primarily be received in this region, higher‐frequency sound (up to approximately 140 kHz) is thought to primarily travel through the mandible. The mandible is partially hollow and filled with specialized “acoustic fats” that connect to the auditory bullae. The cochlea is similar in construction to those of terrestrial mammals; however, there are bony laminae present that result in increased basilar membrane stiffness that facilitates very high‐frequency hearing. Recent data suggest that these laminae may drastically increase the traveling wave speed along the high‐frequency basal end of the basilar membrane, and result in near simultaneous stimulation of the 40–140 kHz region that is critical to echolocation. Refinement for echolocation is further reflected in the neural anatomy of dolphins; e.g., the auditory nerve and brainstem are hypertrophied for rapid transmission of sonar signals. Ongoing research is aimed at determining how the auditory regions of the cortex process acoustic stimuli, but little is currently known about these phenomena.Support or Funding InformationFunded by the Office of Naval Research
The Diverse Sensory Specializations of Cetacea
Authors: Reidenberg Joy S. , Laitman Jeffrey T. 
Published: April 2020

Cetaceans (whales, including dolphins and porpoises) have many unique sensory adaptations that enable them to perceive and respond to stimuli underwater. These adaptations include audition, vocalization, vision, balance, chemoreception, and somatosensation.Cetaceans generate and receive sounds at both ends of the frequency spectrum, and use these sounds for both communication and echolocation. Toothed whales (odontocetes) specialize in high frequency sounds, while baleen whales (mysticetes) focus on low frequency sounds. High frequencies have short wavelengths, and thus enable odontocetes to detect minute features in the reflected echoes related to geometry, texture, density, vector, velocity, etc. Nasal structures generate two different frequencies simultaneously (stereo echolocation). Sounds are received in jaw fats and transmitted to the ears. These features help odontocetes target and catch prey, but can also be used for short‐range navigation and communication.The mysticete larynx generates low frequency sounds. Echoes from these large wavelengths can reveal large features at a distance, facilitating navigation through the ocean landscape during migration. As low frequencies also attenuate more slowly, they can be used for long distance communication. Low frequencies present a hearing dilemma, as wavelengths can be larger than the whale! However, using the large skull as an antenna allows mysticetes to perceive change along most of the wavelength, and relay that information through bony conduction to the ear. Males use highly patterned vocalizations (songs) that advertise sexuality. Songs can be shared with and repeated by new whale groups, thus indicating sophisticated cultural exchange.Vision is another sense that is adapted to life underwater. Cetacean eyes allow visual acuity both above and below water. These include changes to the lens and retina that accommodate light filtering at various depths, and differences in the refraction of light in water versus air. The sclera is also thickened, and may be an adaptation that prevents the eye from deforming under high pressures during diving.Much less is known about the senses of touch, balance, smell, or taste. Cetacean skin is very sensitive, but little is known about its specific somatosensory detection abilities (vibration, water flow, pressure, electromagnetic fields, etc.). Balance seems essential for navigating in three‐dimensional space, yet paradoxically, cetacean semicircular canals are reduced. Smell and taste appear useless to a predator that feeds underwater and swallows prey whole, yet olfactory bulbs and taste buds appear to persist at least in baleen whales. These unique features present open areas of inquiry in cetacean sensory ecology.
Interspecies transcriptome analyses identify genes that control the development and evolution of limb skeletal proportion
Authors: Cooper Kim , Saxena Aditya , Sharma Virag , Neufeld Stanley , Tran Mai , Gutierrez Haydee , Erberich Joel , Birmingham Amanda , Cobb John , Hiller Michael 
Published: April 2020

Despite the great diversity of vertebrate limb proportion and our deep understanding of the genetic mechanisms that drive skeletal elongation, little is known about how individual bones reach different lengths in any species. Here, we directly compare the transcriptomes of homologous growth cartilages of the mouse (Mus musculus) and bipedal jerboa (Jaculus jaculus), which has extremely long metatarsals of the feet and ‘mouse‐like’ arms. When we intersected gene expression differences in metatarsals of the two species with expression differences in forearms, we found that about 10% of all orthologous genes are associated with disproportionate elongation of jerboa feet. Among these, Shox2, has gained expression in jerboa metatarsals where it is not expressed in other vertebrates that have been assessed. This transcription factor is necessary for proximal limb elongation, and we show that it is sufficient to increase mouse distal limb length. Unexpectedly, we also found evidence that jerboa foot elongation occurs in part by releasing latent growth potential that is repressed in mouse feet. In jerboa metatarsals, we observed higher expression of Crabp1, an antagonist of growth inhibitory retinoic acid, lower expression of Gdf10, an inhibitory TGFβ ligand, and lower expression of Mab21L2, a BMP signaling inhibitor that we show is sufficient to reduce limb bone elongation. By intersecting our data with prior expression analyses in other systems, we identify mechanisms that may both establish limb proportion during development and diversify proportion during evolution. The genes we identified here therefore provide a framework to understand the modular genetic control of skeletal growth and the remarkable malleability of vertebrate limb proportion.Support or Funding InformationThis work used the Extreme Science and Engineering Discovery Environment(XSEDE) at SDSC, which is supported by National Science Foundation grant number ACI1548562. This work was also supported by Natural Sciences and Engineering Research Council grant RGPIN/355731‐2013 to JC and by a Searle Scholar Award from the Kinship Foundation, a Pew Biomedical Scholar Award from the Pew Charitable Trusts, a Packard Fellowship in Science and Engineering from the David and Lucile Packard Foundation, and the National Institutes of Health under award number R01AR075415 awarded to KLC.