bluuebunny/crossref_metadata_2025_split huggingface dataset:
The roles of the N‐terminal metal coordinating residues in DNA binding and oligomerization of Deinococcus radiodurans Dps‐1
Authors: Nguyen Khoa Huynh , Grove Anne 
Published: June 2021

The gram positive bacterium Deinococcus radiodurans is well known for its resistance against various environmental stresses. The exact mechanism for this resistance is still unknown. One protein that might contribute to this resistance is Dps (DNA Protection During Starvation). In some bacteria such as Escherichia coli, Dps protects the cells through two mechanisms: by binding and shielding the DNA and by preventing the formation of the toxic hydroxyl radicals. In Deinococcus, there are two Dps homologs: Dps‐1 and Dps‐2. The exact mechanism by which Dps‐1 binds DNA is still unclear; however, there is a metal binding site on the N‐terminal tail of Dps‐1 that might play an important role in DNA binding. Mutation of the residues that coordinate this metal lead to a reduction in DNA binding. Gel filtration reveals that these mutations also alter the oligomeric state of the protein. Furthermore, addition of divalent metals to bipyridyl‐treated wild‐type Dps‐1 restores DNA binding. Competition assays show that the ratio of iron to manganese can affect the ability of this protein to bind DNA. These results suggest that occupancy of the N‐terminal metal binding site is important for this protein to bind DNA and oligomerize.This work is supported by the National Science Foundation (NSF).
From Truncated Models to Full Protein QM/MM Analysis of the Reaction Catalyzed by Malate Dehydrogenase
Authors: Guterres Hugo , Bell Ellis 
Published: June 2021

Our previous work on computational analysis of the reaction catalyzed by malate dehydrogenase involved gas phase reaction simulation using geometry optimization calculations. These revealed that the proton abstraction preceded the hydride transfer due to the activity of the arginine residues around the active site. These gas phase simulations tested two truncated models, one including the arginine residues (R124, R130, and R196), whereas the other one did not. The initial transition states were compared to determine the initial catalytic reaction. A hybrid quantum and molecular mechanics (QM/MM)method has now been used with the whole gMDH dimer, in explicit solvent, to simulate the catalytic reaction with the substrate malate and the cofactor NAD. The simulation analysis helps to give insight into the role of specific regions of gMDH that contribute to the mechanism. The relative changes in the position of malate and NAD during the simulation were studied to determine their interactions with gMDH. In particular the minimum energy pathway for proton and hydride transfer were investigated. In silico characterization of the roles of arginine residues is being correlated with the effects of in vitro site directed mutations to ascertain the precise roles of individual arginines in binding and catalysis.This work is supported by NSF Grant MCB 0448905 to EB.
Protein splicing of a temperature‐dependent intein from an extreme thermophile
Authors: Colelli Kathryn M. , Reitter Julie N. , Mills Kenneth V. 
Published: June 2021

Inteins are intervening polypeptides that are excised during the process of protein splicing. Protein splicing is a self‐catalyzed process in which the intein is removed and the flanking polypeptides are ligated (N‐ and C‐ exteins). The Pyrococcus abyssi (Pab) PolII intein only splices at elevated temperatures. An NMR structure of the intein recently was solved in a collaborator's lab (Du, Z., et al. (2011) Structural and Mutational Studies of a Hyperthermophilic Intein from DNA Polymerase II of Pyrococcus abyssi. J. Biol. Chem. 286(44) 38638–38648.). The NMR data suggest increased rigidity of the intein in comparison to a mesophilic intein from Mycobacterium tuberculosis (Mtu), as well as a β‐hairpin found only in the structures of thermophilic inteins. We have replaced the β‐hairpin from the Pab intein with a linker inspired by the Mtu intein sequence to determine if the β‐hairpin plays a role in the structural stability/rigidity of the intein. We also have designed a genetic screening system that links protein splicing to cell growth on kanamycin in order to select for mutants that allow for splicing at lower temperatures.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245, the Camille and Henry Dreyfus Foundation (KVM), and the Arnold and Mae Beckman Foundation (KMC).
Protein Splicing of inteins from Synechococcus sp. PCC 7002 and Pyrococcus abyssi
Authors: Karanja Kevin K. , Urbanski Laura M. , Reitter Julie N. , Mills Kenneth V. 
Published: June 2021

Protein splicing is a four‐step intramolecular reaction, in which an intein flanked by N‐ and C‐terminal exteins is removed, with ligation of the exteins. We are studying inteins that interrupt genes from Synechococcus sp. PCC 7002 (Ssp 7002), Trichodesmium erythraeum (Tery) or Pyrococcus abyssi DNA Polymerase II (Pab Pol II). The cyanobacterium Ssp 7002 has an intein that interrupts a putative dTDP glucose dehydratase. The Ssp 7002 intein has a highly conserved C‐terminal Asn, unlike an intein from Tery, which has 63% sequence identity with the Ssp 7002 intein but has a C‐terminal Gln. Both inteins splice with their native C‐terminal residue. We plan to study the effect of switching Gln for Asn (and vice versa), as well as the influence of conserved residues on the mechanism of splicing. The Pyrococcus abysii PolII intein has a conserved, catalytic His residue. Mutation of this residue to Ala or Gly prevents splicing. We will explore whether we can rescue activity in the His‐Gly mutant in vivo by incubation with imidazole derivatives.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.
Short‐chain fatty acids activate sympathetic neurons via a novel signaling pathway
Authors: Inoue Daisuke , Kimura Ikuo , Ozawa Kentaro , Takei Yosinori , Hirasawa Akira , Tsujimoto Gozoh 
Published: June 2021

Mammals maintain energy homeostasis by regulating sympathetic nervous system. We previously reported that GPR41, a Gi/o‐protein coupled receptor for short‐chain fatty acids (SCFAs), directly regulate sympathetic activity and energy expenditure at the level of ganglion (1). However, the signaling mechanism for GPR41‐mediated sympathetic activation in details remains to be elucidated. In this study, we examined the downstream signaling of GPR41 for sympathetic activation using primary‐cultured mouse sympathetic neurons. SCFA propionate increased tritium‐labeled noradrenaline secretion via GPR41. Pharmacological and RNAi experiments showed that propionate‐induced increase of noradrenaline secretion is mediated by Gβγ‐PLCβ3‐ERK1/2 pathway. Moreover, co‐immunoprecipitation experiments showed that activated ERK1/2 phosphorylates synapsin2, synaptic vesicle‐associated protein. Furthermore, siRNA for synapsin2 effectively suppressed propionate‐induced noradrenaline secretion. The results show a novel signaling mechanism for Gai/o‐coupled receptor GPR41‐promoted noradrenaline secretion from sympathetic nerve.This study was supported by research grants from the Japan Science and Technology Agency.
Skipping breakfast is associated with lower physical activity energy expenditure in young healthy women
Authors: Marquez Alexa Paulina , Forester Shavawn , Witbracht Megan , Campbell Caitlin , Gustafson Mary , Keim Nancy 
Published: June 2021

ObjectiveAccording to recent NHANES data, the prevalence of breakfast consumption has decreased in adults from 89% to 82% between 1971 and 2002. Skipping breakfast has been negatively correlated with physical activity but positively correlated with body weight and risk factors associated with obesity. Our objective was to assess physical activity and other health parameters in habitual breakfast skippers and breakfast eaters.MethodsA sample of 67 healthy women, 25 ± 0.6 y, were grouped according to their self reported breakfast eating habits, breakfast skippers (n=18) breakfast eaters (n=49). Breakfast was defined as consuming no food or liquid except water before 10am. Blood pressure (BP), BMI, body fat%, fasting blood lipids, and physical activity were measured.ResultsWe found no significant differences in BMI, body fat %, waist‐to‐hip ratio, resting metabolic rate, and blood lipids. However, physical activity was lower (p=0.007) and systolic BP was higher (p=0.04) in breakfast skippers compared to breakfast eaters.ConclusionIn our sample of young healthy women, physical activity was lower in those who skip breakfast. It appears that skipping breakfast and reduced physical activity occur across a wide age spectrum. Further research is needed to understand the mechanisms through which breakfast habits affect physical activity in the young adult population.Funding: USDA, CSRESS 2009‐35215‐05364
Is peak bone mass achieved following recovery from reduced energy availability?
Authors: Camp Kaleigh Ann , Swift Sibyl N. , Lima Florence , Greene Elizabeth S. , De Souza Mary Jane , Bloomfield Susan A. 
Published: June 2021

We previously demonstrated focal bone loss and disrupted metabolic hormone levels in sedentary (SED) and exercising (EX) rats after 12 wks of reduced energy availability (EA; energy intake minus exercise energy expenditure). We hypothesized that peak bone mass (total bone mineral content, totBMC) at 12 mo of age would be diminished by 12 wks reduced EA, even after 12 wks of recovery (REC). After acclimation to AIN‐93M purified diet, 5‐ mo‐old female rats (n=72) were randomized into SED and EX groups; half of each group was designated −25% EA. During REC, all rats were fed ad lib and exercise volume was halved. After 12 wks REC, fat mass was 30% lower in −25EA SED vs. ADLIB SED; however, there were no differences in lean mass. After 4 wks REC, totBMC (by DXA) in −25EA SED was 15% lower vs. ADLIB SED; after 12 wks REC it matched ADLIB SED values. TotBMC for −25EA EX was higher than −25EA SED rats at all time points. Serum IGF‐1 and leptin, reduced after 12 wks of − 25EA, rebounded quickly with regain of fat mass, except for lower leptin in −25EA SED. After 12 wks REC, mineralizing surface on mid‐shaft tibia (by histomorphometry) was low only in ADLIB EX. These data suggest that cortical bone integrity and metabolic hormones recover from stringent reductions in EA when energy balance is re‐established in both SED and EX conditions. Whether the same recovery potential exists for cancellous bone compartments after reduced EA has yet to be determined.Grant Funding Source : Department of Defense #WSIXWH‐06‐1‐0479
Traumatic Brain Injury (TBI) Induces Down‐Regulation of Olfactory Receptors That Are Ectopically Expressed In The Brain: Implications In TBI‐Mediated Tauopathy
Authors: Pasinetti Giulio Maria , Gordon Wayne , Dams-O'Connor Kristen , Zhao Wei , Ho Lap 
Published: June 2021

Traumatic brain injury (TBI) is a risk factor for Alzheimer's disease (AD). Tau‐related neuropathology is a key feature of both TBI and AD. We recently identified ectopic expression of selective olfactory receptors (OR) in peripheral blood mononuclear cells (PBMC) and in the brain. We demonstrated that reduced contents of certain ORs in PBMC may serve as biological surrogates (biomarkers) of TBI. Moreover, we found significantly reduced contents of OR biomarkers in post‐mortem brain specimens of cases with a history of TBI. Exploring the potential role of OR biomarkers in the regulation of abnormal tau processing, we found overexpression and activation of an OR biomarker in primary cortico‐hippocampal neurons significantly attenuates pathophysiological tau paired helical filament immunoreactivity. Thus, down regulation of certain OR biomarkers in the brain may causally promote pathophysiological mechanisms underlying TBI clinical complications and, possibly, increase risk of TBI patient to develop AD. Our evidence provides clinically accessible biomarkers for assessing the likelihood of TBI subjects to develop clinical complications, providing more sensitive outcome measurements for clinical interventions and, ultimately, the characterization of potential novel therapeutic targets to prevent TBI‐associated tauopathy.Supported by Department of Defense: Award W81XWH‐08‐2‐0026 to GMP
Changes in mouse serotonin and dopamine receptor labeling in the intermediolateral nucleus after spinal cord injury
Authors: O'Neill Brannan Elizabeth , Hochman Shawn , Sawchuk Michael , Zimmerman Amanda 
Published: June 2021

Autonomic dysreflexia (AD), a neurological condition associated with spinal cord injury (SCI), is a hypertensive crisis initiated following activation of peripheral pain fibers. As descending monoaminergic neuromodulatory transmitter systems project densely to thoracic spinal cord sympathetic preganglionic regions, lost connections after SCI may contribute to altered autonomic function. Using immunohistochemistry (IHC), this study examined the distributions of three receptors; serotonergic 5HT2A, and dopaminergic D2 and D3 in the intermediolateral nucleus (IML), the location of sympathetic preganglionic neurons (SPNs) in a mouse model of AD. Labeling was explored in control cords at ~P70, and in littermates 3 weeks following spinalization at high thoracic levels (T1/T2). Analysis focused on SPNs in the IML, efferent neurons that are presumably responsible for blood pressure increases.In controls, IHC showed labeling of processes adjacent to the IML for 5HT2A receptors and somatic/perisomatic labeling of SPNs for D2 and D3 receptors. In injured mice, D2 and D3 receptor expression significantly decreased below the injury, both close to the injury site (thoracic levels T2–T7) as well as caudally distant from the injury site (thoracic level T9–T12) (p<0.01). Though 5HT2A receptor expression was also decreased at more rostral thoracic regions (p<0.01), expression increased in a rostrocaudal gradient, with expression levels in the lower thoracic cord comparable to controls (p=.081). 5HT2A receptors can be constitutively active and facilitate sympathetic motor output. We hypothesize that their constitutive activity, unrestrained by descending inhibitory influences that include actions on D2 and D3 receptors, help support a substrate for afferent‐induced overactivation of SPNs in AD. This work is funded by the Department of Defense, SC090469.
Strategies for the Rejuvenation of Aged Muscle Stem Cells
Authors: Proto Jonathan , Lu Aiping , Hannigan Timothy , Wang Bing , Niedernhofer Laura , Robbins Paul , Huard Johnny 
Published: June 2021

A decline in the regenerative capacity of adult stem cells during aging is well documented. We investigated whether reversing the aged phenotype is possible in muscle‐derived stem cells (MDSCs). Previous studies have demonstrated that MDSC‐based cell therapies are efficacious for muscle, bone, cartilage, and cardiac injury. We have isolated MDSCs from aged (24 month old) and young (14 day old) wild type (WT) mice. We screened a number of compounds for their effect on aged stem cell proliferation and myogenic differentiation. Treatment with an inhibitor of nuclear factor kappa B (NF‐kB) rescued the myogenic capacity of aged MDSCs. This led us to hypothesize that NF‐kB activation reversibly suppresses the function of aged MDSCs. To investigate this further, we isolated MDSCs from 30 month old mice haploinsufficient for p65 (p65+/−). Our results have found that aged p65+/− MDSCs are myogenic in vitro and have a higher resistance to oxidative stress than WT cells. Currently, we are comparing the muscle regeneration potential of aged WT and p65+/− MDSCs following injection into injured skeletal muscle of aged mice. Our results suggest that MDSC “aging” may be reversible, and that pharmacologic targeting of pathways such as NF‐kB may enhance the efficacy of cell therapies in aging patients. This work was supported by the Henry J. Mankin Endowed Chair for Orthopaedic Research and the Hirtzel Foundation.Grant Funding Source : Department of Defense
Genetic evidence for an involvement of the TOR complex 1 in the process of transcription elongation in Saccharomyces cerevisiae
Authors: Haller Jasmine Lee , Allison Mary Kathryn , Harper Jennifer Aileen , Outhay Malena Ann , Warsinske Hayley Catherine , Duina Andrea Angelo 
Published: June 2021

In eukaryotic cells, DNA is compacted into chromatin, a structure that includes the nucleosome – a particle consisting of histones and DNA – as its fundamental unit. The presence of nucleosomes across the regulatory and transcribed regions of genes constitutes a physical obstacle for factors that need to access DNA in order to ensure the proper expression of the underlying genetic information. In past studies aimed at better defining the interplay between chromatin and transcription, a histone H3 mutant – H3‐L61W – was isolated that was shown to deleteriously affect the elongation phase of transcription. Here we describe experiments showing that deletions of genes encoding two members of the TOR complex 1 (TORC1) – Tco89 and Tor1 – can suppress a subset of phenotypes conferred by the H3‐L61W mutation. Deletion of TCO89 causes the strongest suppressive effects and additional studies show that deletion of this gene in wild‐type histone H3 cells confers phenotypes indicative of defects in chromatin structure and transcription elongation. Taken together, these and other studies point to a possible role for TORC1 in affecting transcription elongation through effects on chromatin structure.This material is based upon work supported by the National Science Foundation under grant No. 0919241.
Intranasal rapamycin rescues mice from staphylococcal enterotoxin B‐induced shock
Authors: Krakauer Teresa , Buckley Marilyn 
Published: June 2021

Staphylococcal enterotoxin B (SEB) and related exotoxins produced by Staphylococcus aureus are potent activators of the immune system and cause toxic shock in humans. Currently there is no effective treatment except for the use of intravenous immunoglobulins administered shortly after SEB exposure. We evaluated the efficacy of intranasal administration of rapamycin in a lethal mouse model of SEB‐induced shock. The results show that intranasal rapamycin delivered as late as 17 h after SEB still protected 100% of mice from lethality. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB. Furthermore, only three doses of rapamycin were sufficient to block SEB‐induced shock. Intranasal rapamycin represents a novel use of an immunosuppressant targeting directly to site of toxin exposure, reducing dosages needed and allowing a wider therapeutic window.Source of support: Defense Threat Reduction Agency
Use of computerized decision support system for resuscitation of swine with inhalation injury and severe burns
Authors: Ivey Katherine Marie , Batchinsky Andriy , Baker William , Fenrich Craig , Cancio Leopoldo , Salinas Jose 
Published: June 2021

OBJECTIVETo assess the use of a computerized burn resuscitation decision support system (BRDSS) in swine with smoke inhalation and severe burns.METHODSWe designed BRDSS for fluid resuscitation of burn injuries. The system uses an internal fluid model to recommend hourly crystalloid rates based on a urine output (UO) target of 30–50mL/hr. A graphical display shows the subject's fluid status compared with Parkland [4mL/kg/% total body surface area (TBSA)] and Modified Brooke (2mL/kg/%TBSA) resuscitation formulas. Under intravenous anesthesia, female swine (n=7, 41.5 ± 1.8kg) received smoke inhalation injury and 40% TBSA flame burn. Resuscitation with lactated Ringer's was guided by BRDSS recommendations. Resuscitation was stopped when a maintenance fluid rate of 125mL/hr was achieved for at least 6 hours; this was considered an indicator of adequate resuscitation. Statistical analysis is by t‐test.RESULTSArterial base excess decreased from 0.5 ± 2.7 pre‐injury to −3.1 ± 2.2 post‐injury (p<0.05), and was restored to 0.8 ± 1.9 by 30 hours (p<0.05). Total 24‐hour resuscitation volume was 3.4 ± 0.3mL/kg/TBSA. Mean UO in the first 24 hours was 84.4 ± 15.4mL/hr.CONCLUSIONBRDSS successfully resuscitated all subjects in the study while achieving total 24‐hour resuscitation volumes between the Modified Brooke and Parkland formulas and maintaining adequate UO.SUPPORT: U.S. Army Institute of Surgical Research
The role of an extended beta‐sheet in stabilizing the structure of a thermophilic intein
Authors: Colelli Kathryn M. , Pusztay Jennifer M. , Reitter Julie N. , Mills Kenneth V. 
Published: June 2021

Inteins are intervening polypeptides that are excised during the process of protein splicing, by which the intein is removed and the flanking polypeptides are ligated (N‐ and C‐ exteins). The Pyrococcus abyssi (Pab) PolII intein only splices at elevated temperatures. An NMR structure of the intein recently was solved in a collaborator's lab; the NMR data suggest increased rigidity of the intein in comparison to a mesophilic intein from Mycobacterium tuberculosis (Mtu), as well as a β‐hairpin found in inteins from archaebacteria. We have shown that mutations within the hairpin, which is distant from the active site, can both decrease the rate of catalysis as well as globally destabilize the structure of the protein.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245, the Camille and Henry Dreyfus Foundation (KVM), and the Arnold and Mae Beckman Foundation (KMC).
The biogeochemical chlorine cycle in forest ecosystems
Authors: Pirovano Ashley , Leri Alessandra 
Published: June 2021

Organochlorine compounds, once thought to be solely anthropogenic, are now known to be produced naturally in forest ecosystems. One natural chlorination mechanism involves catalysis by the enzyme chloroperoxidase (CPO). Using hydrogen peroxide to oxidize inorganic chloride, CPO generates reactive chlorine intermediates (HOCl) capable of chlorinating non‐specific organic substrates. These reactive chlorine species are believed to be implicated in lignin degradation during plant matter decay, with organochlorine produced as a by‐product. However, certain lignin‐degrading microorganisms, including the fungus Phanerochaete chrysosporium, are known to dechlorinate anthropogenic organochlorine pollutants. To explore this paradox regarding the role of chlorine in lignin degradation, we investigated the function of different microorganisms in the biogeochemical chlorine cycle using synchrotron‐based Cl 1s X‐ray absorption near‐edge structure (XANES) spectroscopy. These tests provide evidence of chlorination through a CPO‐like mechanism by an enzyme extract of the lichen Parmotrema hypotropum and preliminary evidence of dechlorination by the fungus P. chrysosporium.Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE‐AC02–98CH10886.