bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Myelination in the Prefrontal Cortex by the Gut Microbiota: Implications for Health and Disease
Authors: Hoban Alan , Stilling Roman , Desbonnet Lieve , Shanahan Fergus , Dinan Timothy , Cryan John , Clarke Gerard 
Published: June 2021

BackgroundMultiple lines of evidence suggest a potential role for the gut microbiome in multiple sclerosis, a degenerative demyelinating disorder. For example, germ‐free mice, which are microbiota deficient, appear to be protected against the development of experimental allergic encephalomyelitis (EAE). The gut microbiota can regulate gene expression at the level of the CNS. In this study, we aimed to establish whether germ‐free mice have alterations in genes of relevance to CNS myelination.MethodsUsing qRT‐PCR, we evaluated expression levels of myelin basic protein (Mbp), myelin oligodendrocyte protein (Mog), myelin oligodendrocyte basic protein (Mobp), proteolipid protein 1 (Plp1) and myelin associated glycoprotein (Mag) in relevant brain regions in conventionally raised, germ‐free and germ‐free colonised mice.ResultsGerm‐free mice displayed increased expression of all 5 myelinating genes only within the prefrontal cortex. Colonisation of the germ‐free animals immediately post‐weaning normalised the expression of these myelin structural protein genes. Germ‐free mice also displayed a 2 fold increase in expression of Egr2/Krox20, a transcription factor within the PFC known to be involved in mechanisms regulating myelin formation.ConclusionThe gut microbiota can regulate myelin structural protein gene expression in the CNS. Moreover, our results suggest that the microbiota can be successfully targeted later in life to modulate myelination patterns. This raises the possibility that targeting the gut microbiota during critical time windows could be a viable approach to promote remyelination or to prevent further demyelination in disorders such as multiple sclerosis.
Mitosis in Secretory Ameloblasts
Authors: Hogg Russell , Smith Timothy , Sanchez Adriana , Bly Karlie , Gografe Sylvia 
Published: June 2021

Our objective is to test the dictum that secretory ameloblasts cannot divide. Dental biology textbooks often present this idea without citation or evidence; its source seems to be studies of rodent incisors from the 1960's. However, rodent incisors are ever‐growing, and not acceptable models for enamel epithelia in other mammals. There are two reasons to expect that ameloblasts divide. First, recent studies show that tensile forces induce mitosis in epithelia, and enamel epithelia likely undergo tension as enamel is deposited and the surface area of the growing crown expands. Second, we now know that ameloblasts are subject to the same biorhythms that synchronize proliferation of osteoblasts. If ameloblast division is also coordinated by the brain and occurs at night, it is unlikely to have been observed since most experimental protocols occur in daylight.To test the hypothesis, we first measured enamel area from surface and enamel‐dentine junction in Saimiri sciureus tooth sections. We also measured enamel rod width at both sites, as a proxy for ameloblast width. We found an average 9% greater area at the tooth surface, but no difference in enamel prism width. We also searched for mitotic figures in tooth buds of perinatal Saguinus spp. with random times of death, but found none. In conclusion, increasing tooth surface area from deep to superficial and no change in cell size suggests that ameloblast mitosis is likely. However, we did not find any mitotic figures, possibly because of sample size and lack of control over time of death.
Osteocyte Morphology in the Primate Craniofacial Skeleton
Authors: Smith Leslie , Ren Yinshi , Feng Jerry , Dechow Paul 
Published: June 2021

Frost's “mechanostat” theory suggests MES varies throughout the skeleton in order to maintain strength in low strain regions such as the cranial vault. More spherical osteocytes have been shown to produce more nitric oxide than oblong osteocytes undergoing the same stress in vitro. In this study we look at osteocyte shape differences in a high and low strain region of the human craniofacial skeleton: the zygoma (Z) and the browridge (BR), respectively, and between the BRs of a chimp and human. We hypothesize that BR osteocytes will be more spherical in shape than those in the Z, and that osteocytes in the robust chimp BR will be more spherical than those in the human. Rectangular samples were extracted from the BR and Z regions of a 36 year old human female and an adult female chimp BR. Samples were stained with FITC, embedded in MMA and captured using confocal microscopy. Osteocytes from each cortical region were analyzed in Imaris for differences in sphericity. T‐tests were used to determine significance. Osteocytes in all three regions were more spherical (average sphericity greater than 0.63) and less ellipsoidal. There were no significant differences found between either group. These results suggest that differences between postcranial and calvarial osteocytes that are useful in maintaining bone mass in regions of low strain may not exist between subregions of craniofacial bone.Funding: National Science Foundation Physical Anthropology HOMINID program (NSF BCS 0725141)
Vestibular and Proprioception Deficits in Adults with Controlled Diabetes Mellitus II Causes Postural Control Instability
Authors: Rosario Martin , Dunbar Donald , Ortiz Alexis 
Published: June 2021

Objectiveindividuals with controlled type two diabetes (cDM) often are unaware of balance impairments, although these changes are occurring and may become impairments. Thus, the objective of this study is to make an individual comparison and characterization of each system involved during standing balance (vision (Vi), proprioceptive (Pro) and vestibular (Ve) tasks in people with DM and healthy‐control individuals and identify the most affected system. MethodsFourteen participants with DM (age 59 ± 5, BMI 29 ± 3 kg/m2and 8 ± 6 years since diagnosis) and eighteen healthy participants (age 57 ± 5, BMI 27 ± 3 kg/m2) participated in this study. Postural stability during still standing for 20 seconds was assessed using a pressure mat by measuring center of pressure (CoP). Static postural control was evaluated during eight sensory conditions that perturbed or stimulated the visual (Vi), proprioceptive (Pro) and vestibular (Ve) systems.ResultsA Mann‐Whitney U Test was used to compare CoP data between DM and normal subjects. When pitching the head with the eyes open while standing on the foam pad, CoP displacement was significantly greater in DM subjects, indicating increased body sway and less balance control (CoP displacement: 39.65 ± 18.6 cm2 in DM vs. 24.33 ± 9.5 cm2 in controls, P= 0.009).ConclusionsThe conditions in which the Ve and Pro systems were challenged, the group with DM had a greater CoP displacement when compared to the control group. This suggests that in patients with DM without a diagnosis of neuropathy, postural control changes are already detected making them unstable by affecting their balance.
Retinoic Acid and Retinal Ganglion Cell Survival after Optic Nerve Injury
Authors: Duprey‐Diaz Mildred , Blagburn Jonathan , Blanco Rosa 
Published: June 2021

Retinoic acid (RA) is a potent transcriptional activator that may be involved in neuronal plasticity and regeneration in the adult brain. We have shown that the enzyme that synthesizes RA, and the RA receptors, are present in frog retinal ganglion cells (RGCs) and are upregulated after axotomy. The focus of this work is to understand how RA signaling affects RGC survival and its involvement in the activation of intracellular signaling pathways after optic nerve injury. The optic nerve was cut and animals were followed from 1‐6 weeks. Surviving RGCs were retrogradely labeled and counted. Western blot analysis of experimental and control retinas was carried out to measure levels of pro‐survival and pro‐apoptotic proteins, and of activated signaling pathway components, in non‐treated retinas and retinas treated with RA signaling agonists and antagonists immediately after axotomy. Inhibition of endogenous RA signaling significantly decreased the number of RGCs surviving after axotomy; conversely, RA agonist treatment improved RGC survival. RA signaling antagonists abolished axotomy‐induced activation of mitogen‐activated protein kinase (MAPK),phosphoinositide 3‐kinase (PI3K) and signal transducer and activator of transcription 3 (STAT3) in the retina. Application of specific RA receptor subtype agonists into the eye caused an additional increase in the activation of STAT3, but not of the PI3K pathway. We conclude that RA signaling is important for RGC survival after nerve injury, and that further understanding of the role of RA is important for its potential future therapeutic use.Supported by NIH‐GM 093869 and RCMI‐G12RR03051.
Scorpion Toxin BeKm‐1 Blocks Low K+‐induced Endocytosis of Cell‐surface Human Ether‐a‐go‐go Related Gene (hERG) Channels
Authors: Han Xi , Guo Jun , Li Wentao , Zhang Shetuan 
Published: June 2021

The human ether‐a‐go‐go related gene (hERG)‐encoded potassium channel conducts an important current for the repolarization of cardiac action potentials. hERG channel dysfunction leads to abnormal cardiac electrical activities characterized by a prolonged QT interval. We have previously shown that exposure of hERG‐expressing HEK cells to a 0 mM K+medium induces vigorous internalization of hERG but not ether‐a‐go‐go (EAG) channels from the plasma membrane. To investigate the mechanisms of 0 mM K+‐induced hERG endocytosis, we used a chimerical channel model constructed by combining hERG and EAG, as well as drugs that bind to either the internal pore mouth of the hERG channel (E‐4031) or the extracellular edge of the pore entrance (BeKm‐1). Using Western blot analysis and whole‐cell patch clamp, our data show that replacing the S5‐S6 linker of hERG with that of bovine EAG prevented 0 mM K+‐induced hERG endocytosis. Moreover, BeKm‐1 prevented 0 mM K+‐induced hERG internalization. Under the whole‐cell voltage clamp configuration, acute exposure of hERG‐expressing HEK cells to 0 mM K+ solution eliminated hERG conductance within 5 minutes, and the hERG conductance recovered slowly upon re‐exposure to 5 mM K+ solution. The 0 mM K+ exposure‐induced conductance loss was completely prevented by the application of BeKm‐1 (1 µM). We conclude that a conformational change in the outer pore‐mouth of hERG occurs upon removal of extracellular K+, which triggers hERG internalization. BeKm‐1 prevents such conformational change and the subsequent internalization of hERG channels under 0 mM K+ culture conditions.(Supported by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Ontario)​
Cholesterol Trafficking for Steroid Biosynthesis in MA‐10 Mouse Tumor Leydig Cells
Authors: Jagannathan Sathvika , Chebbi Seimia , Hullin‐Matsuda Francoise , Kobayashi Toshihide , Papadopoulos Vassilios 
Published: June 2021

The hormone‐sensitive and rate‐limiting step in steroid biosynthesis is the movement of cholesterol from intracellular sources to the inner mitochondrial membrane (IMM). Despite the numerous studies on cholesterol trafficking in steroidogenesis, the exact source and mechanism by which cholesterol is transported to IMM remains to be elucidated. D4 is the fourth domain of perfringolysin O protein, which has the ability to bind membranes containing greater than 30 mol% cholesterol of total lipid concentration with high affinity. mCherry‐tagged D4 was used to visualize cholesterol trafficking in MA‐10 cells. Confocal microscopy showed D4‐mCherry localized at the plasma membrane, but upon 45 minutes treatment with dibutyryl‐cAMP (dbcAMP) a significant reduction in plasma membrane labeling was observed. Functional inhibitors of proteins involved in cholesterol import into mitochondria and metabolism, blocked steroid formation and slowed down the movement of D4‐mCherry from the plasma membrane. Recombinant D4‐GFP protein readily bound the outer leaflet of the plasma membrane even after 120 minutes of dbcAMP stimulation, indicating that cholesterol was trafficked from the inner leaflet of the plasma membrane. D4‐mCherry also localized the late endosomes upon dbcAMP stimulation suggesting a route for the cholesterol from plasma membrane to mitochondria. These data suggest that the bulk of the steroidogenic pool of cholesterol, mobilized by cAMP for acute steroidogenesis, likely originates from the inner leaflet of the plasma membrane.Source: CIHR
Inhibition of Human Acetylcholinesterase by Enantiomers of V‐Type Chemical Warfare Nerve Agents and Analogs
Authors: Boeri Michael , Kasten Shane , Cerasoli Douglas 
Published: June 2021

Chemical warfare nerve agents are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Analytical scale methods using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer were developed for the separation and detection of individual stereoisomers of VX, VR, and VS, and the analogs V1 thru V4. Separation was evaluated with respect to the chromatography parameter resolution (Rs) with a value of 蠅1.7 indicating complete separation. The ChiralCel OD‐H column yielded Rs values >1.7 for each V‐type compound with mobile phase modifier concentrations 蠄2%. Methods that provided the highest Rs values were scaled up to the semi‐preparative level, allowing for isolation and analysis of individual stereoisomers at >99% purity. Ellipticity (Θ) values of each enantiomer were determined by circular dichroism (CD) spectroscopy. VX, V1, V2, and V3 all had a ΘMAX at 221nm, while VR, VS, and V4 had a ΘMAX at 226nm. For all agents, the first eluting isomer had a negative value at ΘMAX [(‐)Θ221‐226], while the second eluting isomers all had positive values at ΘMAX [(+)Θ221‐226]. Individual enantiomers and racemic mixtures of each agent were then incubated with recombinant human acetylcholinesterase to determine their inhibition rate constants (ki). The (+)Θ221‐226 enantiomer was found to be an average of 2‐3 orders of magnitude more inhibitory than the opposite enantiomer of the same agent.The views expressed in this poster are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.This research was supported by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.
An Optimized Viral Delivery System to Expand the Genetic Code in Mammalian Cells
Authors: Zheng Yunan , Chatterjee Abhishek 
Published: June 2021

Co‐translational, site‐specific incorporation of unnatural amino acids (UAAs) by nonsense suppression has proven to be a powerful tool for investigating and engineering protein structure and functions. This methodology utilizes an engineered aminoacyl‐tRNA synthetase/tRNA (aaRS/tRNA) pair, orthogonal to the host cell, to deliver the UAA of interest in response to a nonsense codon. The ability to use this toolbox to investigate and manipulate protein function in human and other mammalian cells can be highly beneficial. The primary challenge is to efficiently co‐deliver all the genetic components, and a lack of understanding regarding optimal expression levels of these components. Viral vectors are ideal vehicles for delivery, potentially enabling application in a variety of mammalian cell lines, and even live tissues and animals.Using a unique viral delivery vector, and optimal expression of the genetic components, we have substantially enhanced UAA mutagenesis efficiency in mammalian cells. We identified the tRNA as the limiting component, while the traditional aaRS expression level was shown to be unfavorably high. The large and complex cargo could be efficiently packaged within our unique baculoviral delivery vector‐ showcasing the prowfess of this platform.
Sequential Phosphorylation of SIKE by TBK1
Authors: Bell J Ellis , Kerckhove Tanner , Kim Seung‐Hwan , Bell Jessica 
Published: June 2021

Our recent work (Marion et al. J. Biol. Chem. (2013) 288:18612) has demonstrated that SIKEcan be multiply phosphorylated by the protein kinase, TBK1, and that phospho‐mimetic mutants representing differently phosphorylated SIKE forms inhibit TBK1 utilization of its other substrate, IRF3, with significantly different inhibition constants. To investigate both the effects and time dependence of site‐specific SIKE phosphorylation we have combined computational (homology modeling using I‐TASSER and docking with TBK1 studies using the ClusPro server ) and experimental approaches ( CD, Thermal melts, Chemical denaturation and Multiwavelength Collisional Quenching studies) of WT SIKE and several phospho‐knockout or phosphor‐mimetic forms of SIKE. In time‐dependent phosphorylation experiments, SIKE with different levels of total phosphorylation was isolated by ion exchange chromatography and unique phosphorylation sites of the separated isoforms identified by fragmentation and MALDI‐TOF or tandem mass spectrometry as appropriate. To investigate the impact of phosphorylation on the structure and stability of SIKE we have used a combination of circular dichroism spectroscopy and multiwavelength collisional quenching to investigate the conformation and flexibility of SIKE and the effects of phosphorylation.. This work is supported NIH Grant: R21AI107447
Targeting 6‐Phosphofructo‐2‐kinase/Fructose‐2,6‐Bisphosphatase‐4 (PFKFB4) in Cancer
Authors: Telang S. , Trent J. , Chesney J. , Clark J. 
Published: June 2021

High glycolytic flux, even in the presence of oxygen (i.e. the Warburg effect), is a central neoplastic hallmark. Oncogenic activation and HIF‐1α over‐expression stimulate glycolysis by activating the 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatases (PFKFB1‐4) which synthesize fructose‐2,6‐bisphosphate (F2,6BP), a potent allosteric activator of the metabolic sensor, 6‐phosphofructo‐1‐kinase (PFK‐1). We recently found that PFKFB4 is highly expressed in multiple cancer cell types with disparate driver oncogenes, is strongly induced by hypoxia via HIF‐1α upregulation and that its suppression leads to decreased F2,6BP, glycolytic flux to lactate and the TCA cycle, increased apoptosis and inhibition of lung tumor growth in vivo. Importantly, compared with normoxia, silencing PFKFB4 in hypoxia significantly decreases survival and proliferation, indicating that PFKFB4 may be preferentially required for hypoxic survival and growth. Taken together, these data provide rationale for the development of small molecule inhibitors targeting PFKFB4.Using molecular modeling and receptor‐based virtual screening, we have identified a small molecule inhibitor of PFKFB4, termed 5MPN, that selectively inhibits recombinant PFKFB4 activity. We find that 5MPN: (i) decreases the F2,6BP, glucose uptake and proliferation of multiple cancer cells; (ii) selectively suppresses Ras‐transformed bronchial epithelial cell growth without affecting normal bronchial epithelial cells; and (iii) markedly reduces xenograft growth in vivo (IV and PO). Our data indicate that targeting PFKFB4 may be a viable therapeutic strategy against cancer and future studies will be aimed at the examination of 5MPN in combination with PFKFB3 inhibitors and other targeted anticancer agents.
Rin GTPase deficiency promotes neuroprotection following traumatic brain injury
Authors: Pannell Megan , Cai Weikang , Brelsfoard Jennifer , Carlson Shaun , Littlejohn Erica , Stewart Travis , Saatman Kathryn , Andres Douglas 
Published: June 2021

Traumatic brain injury (TBI) affects approximately 1.4 million people in the USA annually and remains a leading cause of death and disability. In addition to the cell death caused by the initial insult, TBI also leads to the activation of secondary cascades including excitotoxicity, free radical production, and glial activation. These mechanisms initiate detrimental consequences for months following injury, obstructing cognition and motor functioning.Ras family proteins act as GDP/GTP regulated molecular switches governing diverse cellular processes by allowing for the relay of extracellular stimuli into cellular signaling pathway. Rin was identified as a small G‐protein with expression limited to the central nervous system.Through the generation of a Rin knockout mouse, our lab has elucidated a model in which loss of Rin signaling aids in neuroprotection following TBI. While targeted disruption of Rin does not alter neural development, its loss promotes enhanced adult neurogenesis. Phospho‐specific immunoblotting suggests that Rin deficiency protects primary neuronal cultures from the effects of reactive oxygen species by increasing AKT signaling and blunting p38 MAPK activation. Furthermore, we have shown that the absence of Rin is neuroprotective against TBI, decreasing the amount of hippocampal neurodegeneration and improving cognitive performance after injury. Taken together, these data suggest that endogenous Rin regulates a neuronal pro‐apoptotic cascade and that inhibition of Rin signaling may have broad therapeutic potential by promoting neuronal survival and limiting post‐injury cognitive dysfunction.
Alternative Splicing Generates Novel Fads3 Transcript in Mice
Authors: Zhang Ji Yao , Qin Xia , Park Hikyu , Kim Ellen , Liu Guowen , Kothapalli Kumar , Brenna J Thomas 
Published: June 2021

IntroductionFatty acid desaturase (FADS) gene family consists of three genes (Fads1, Fads2 and Fads3). Fads1 and Fads2 catalyze critical steps in the biosynthesis of long chain polyunsaturated fatty acids (LCPUFA). Fads3 is the third member, found to have at least eight evolutionarily conserved alternative transcripts (AT), but with no clearly established function. Here we present identification of a novel Fads3 transcript in mice (Fads3AT9). Our main objective is to characterize Fads3AT9, its expression in mouse tissues and in undifferentiated and differentiated fat (white and brown) cells.Methods.Total RNA obtained from mouse tissues and fat cells were reverse‐transcribed into cDNA. The resulting cDNA was used as template for PCR‐reactions.Results.Sequencing analysis revealed complete absence of exon 2 resulting in an open reading frame of 1239 bp, encoding a putative protein of 412 aa with loss of 37 aa compared to classical Fads3 (Fads3CS). Fads3AT9 retains all the conserved regions characteristic of front end desaturase (cyto b5 domain and three histidine repeats). It is widely expressed in 12 different mouse tissues and fat cells, with strongest expression seen in kidney, and heart. Fads3CS had greater abundance than AT9 in all tissues.ConclusionsWidespread expression patterns in mouse tissues and fat cells suggest it may play an important role in the regulation and/or biosynthesis of LCPUFA from precursors.Funding Source:NIH R01 AT007003