bluuebunny/crossref_metadata_2025 huggingface dataset:
Vertebral dimensions vary among prehensile and nonprehensile primate tails
Authors: Organ Jason M. , King Heather L.
Published: June 2021

Recent morphological studies of the primate tail have established that the mechanical structure of the prehensile tail, capable of suspending the body weight of the animal, differs from that of the nonprehensile tail: prehensile tail caudal vertebrae have relatively more expanded transverse and hemal processes (reflecting better developed tail musculature) and vertebral bodies are capable of resisting relatively higher bending (and torsional) stresses (i.e., they are more robust). However, these characterizations are based upon analysis of three functionally analogous vertebrae in each tail (one from each of three tail regions), taken to represent the entire tail. This study uses simple caliper measurements of caudal vertebrae in nine New World monkey species to demonstrate: 1) transverse process expansion is greatest in the distal region of the prehensile tail, but proximally this measure does not discriminate among tail types; 2) hemal process expansion is greatest in the prehensile tail in all tail regions, and this measure does discriminate among tail types; and 3) vertebral bodies are more robust in the prehensile tail in all regions. These results are consistent with previous analyses and support the use of the three functionally analogous vertebrae to characterize the morphology of the tail as a whole. Supported by NSF BCS‐0550676.

Grant Funding Source: NSF
Changes In Femoral Bone Geometry Compensate The Lower Bone Mass And Mineralization Degree In Ovariectomized Wistar Rats
Authors: Fonseca Helder , Moreira-Gonçalves Daniel , Ferreira Rita , Amado Francisco , Mota Maria Paula , Duarte José Alberto
Published: June 2021

Bone strength depends on a set of interdependent factors ranging from tissue properties to whole bone structural organization, and estrogen appears to influence several of these factors. Our goal was to determine the effects of estrogen loss on both tissue and geometrical femur properties and to appreciate their expression in whole bone strength. Twenty five Wistar rats were ovariectomized (OVX, n=13) or sham operated (SHAM, n=12) at 5 months and sacrificed at 14 months of age. Another group of rats (5 months) was sacrificed as baseline control (BSL, n=7). Serum bone resorption markers were higher while femur calcium content was lower in OVX compared to SHAM. Histomorphometry revealed a lower cortical bone thickness (Ct.Th) and trabecular volume in OVX proximal femur as well as a lower Ct.Th at the diaphysis. However, bone width, bone length, and femoral neck length were higher in OVX than in SHAM and all these variables correlated with femur ultimate load (UL) following biomechanical testing. There were no differences in femoral neck and mid‐diaphysis UL between OVX and SHAM. However, bone tissue intrinsic properties were lower in OVX compared to SHAM. Estrogen loss leads to a decrease in bone tissue quality resulting in lower intrinsic bone tissue properties. Yet, compensatory geometrical adaptations also occur that prevent bone strength from declining. FCT grants PTDC/DES/103047/2008 and SFRH/BPD/78259/2011.
Successful computational prediction of residues important for function in DNA polymerase III alpha subunit
Authors: Parasuram Ramya , Sharma Rajal , Beuning Penny J , Ondrechen Mary Jo
Published: June 2021

The process of DNA replication is carried out with high efficiency and accuracy by DNA polymerases. The replicative polymerase in E. coli is DNA pol III, which is a 10‐subunit complex that coordinates simultaneous replication on the leading and lagging strands. The 1160‐residue pol III alpha subunit copies DNA accurately, making one error per 105 nucleotide incorporations. The goal of this research is to determine the residues that contribute to this high fidelity and activity of the polymerase subunit. Homology modeling and the computational methods of Theoretical Microscopic Anomalous Titration Curve Shapes (THEMATICS) and Partial Order Optimum Likelihood (POOL)have been used to predict functionally important amino acid residues. Site‐directed mutagenesis and primer extension assays have been used to validate these predictions. We and others have shown that the top 11 residues predicted by POOL, which includes the three catalytic aspartates, are important for function. Fluorescence binding assays, thermal denaturation assays and steady‐state kinetic assays are being performed to better understand the contribution of these residues to the function of the polymerase. Work supported by NSF‐MCB‐0843603, NSF‐MCB‐0845033 and Research Corporation for Science Advancement.
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).
The Role of a Second Shell Hydrophobic Interaction in Trypsin‐Fold Serine Protease Function
Authors: Batt Anna , Rafidi Hanine , Baird Teaster
Published: June 2021

In several co‐crystal structures of trypsin and its macromolecular inhibitors, the backbone carbonyl of Phe41 accepts a hydrogen bond from the P2′ backbone amide in the inhibitor (a “first shell” interaction) suggesting that this hydrogen bond interaction is significant. Lys60 and Tyr39 form a “cage” around Phe41(a “second shell” interaction), likely keeping the backbone carbonyl of Phe41 optimally positioned for hydrogen bonding. Two trypsinogen variants, in which Tyr39 was replaced with a phenylalanine (Y39F‐Tg) and an alanine (Y39A‐Tg), were constructed to evaluate how alteration and removal of the cage around Phe41affect activity. It is hypothesized that the removal will allow Phe41more conformational freedom and thereby weaken macrosubstrate/inhibitor binding. Initial kinetic characterization and inhibition studies of the variants have been carried out using small (1–3 residues), commercially available peptide substrates and macromolecular inhibitors. Preliminary data suggest that wild‐type, Y39F‐Tn, and Y39A‐Tn all show similar affinities for the small peptide substrates (KM = 13 ± 2 μM) while affinity for macromolecular inhibitors appears to be lower for the variants than for wild‐type. [This work was supported by NSF Award MCB‐ 0643988‐02 and a Research Infrastructure at Minority Serving Institutions Fellowship, NIMHD Award P20MD000544 (ARB)]
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.
Regulatory Dynamics of the Transcriptional Network Controlling the Cold Shock Response in Saccharomyces cerevisiae
Authors: Dahlquist Kam D , Fitzpatrick Ben G , Rohacz Nicholas A , Sherbina Katrina
Published: June 2021

We are using mathematical modeling to describe the interactions between transcription factors that control the environmental stress response of cold shock in Saccharomyces cerevisiae. We inferred a network of twenty‐one transcription factors that regulate one another from timecourse microarray data from wild type yeast subjected to 15, 30, and 60 minutes of cold shock at 13°C. The concentrations of the individual transcription factors were modeled using ordinary differential equations, which we solved as a system to determine the dynamical behavior of the model. Each equation includes a production rate, a degradation rate (which we computed using published half‐life data), and weights that denote the influence of the connected transcription factors. We estimated parameter values using the DNA microarray data. Model/data comparisons were performed with least squares. Model predictions fit the data well within two standard deviations of the experimental error. We then compared simulated model data where the transcription factors Cin5, Gln3, Hmo1, and Zap1 were removed in silico with experimental microarray data from the corresponding yeast deletion strains. This revealed the relative influence of these transcription factors on the dynamics of the transcriptional response to cold shock in yeast. This work was supported by NSF award 0921038 (KDD, BGF, KS) and the McLaughlin Chair of Biology (KDD, NAR).
WWOX/WOX1 is essential in UV irradiation/frostbite‐induced membrane bubbling
Authors: Chen Szu-Jung , Huang Shenq-Shyang , Chang Nan-Shan
Published: June 2021

Overexposure to extreme cold, along with incidental UV irradiation, causes tissue damage and death ‐ the so called frostbite. Time lapse imaging analysis showed that post UV irradiation, COS7 fibroblasts undergo tiny, fluctuating membrane blebbing and then explode with a big bubble from the nucleus. Cold shock at 4oC significantly enhances the UV‐induced bubbling event. UV energy appears to be absorbed by the nucleus, so as to punch off a tiny nuclear pore that allows pressure release from the nucleus and subsequent plasma membrane exploding, as evidenced by using ECFP‐tagged nuclear protein. Unlike apoptosis, these COS7 cells exhibit little or no DNA fragmentation, cell shrinkage and phosphatidylserine expsoure. Blebbistatin, a blebs formation inhibitor, failed to prevent cells from bubbling up. Intriguingly, bubbling of Wwox gene knock out MEF (Wwox−/−) cells are significantly reduced, suggesting a crucial role of tumor suppressor WWOX/WOX1 in the bubbling event. Mechanistically, WWOX/WOX1 dissociates from TRAF2 for mitochondrial and nuclear relocation that contributes, in part, bubbling and death. [Supported by NSC and NHRI, Taiwan, and DoD, USA]
Community based lifestyle intervention improves body weight, anthropometric, and fitness parameters
Authors: Raatz Susan K , Johnson Luann K , Scheet Angela J , Langei Christine M , Westereng Rebecca A
Published: June 2021

Lifestyle modification of nutrition, physical activity and behavior is a proven methodology for weight loss and health improvement. We examined a community based lifestyle intervention (CBLI) program on anthropometric, fitness and biologic outcomes in 41 (2 men, 39 women) overweight and obese (BMI = 32.0 ± 0.5 kg/m2) adults (age 49.3 ± 1.9 years). We hypothesized that CBLI would improve weight loss, body composition, chronic disease risk biomarkers, and fitness levels. Participants were randomly assigned to an 8 week CBLI (n=23) led by a personal fitness trainer and registered dietitian at a local fitness center, or to a control group (n=18) which received one class session at baseline. At 8 weeks, CLBI had increased weight loss (p=.03), total fat loss (p=.0004), trunkal fat loss (p=.006), and improved flexibility (p=.03) compared to controls. CBLI participants reported improved Weight Loss Efficacy scores (p=.04) compared to controls. All participants reduced total cholesterol from baseline (p=.03 and p=.0005, control and CBLI, respectively). As baseline levels were within normal limits, no changes were observed in glucose, insulin or hsCRP. Community based lifestyle intervention is an appropriate format for weight management. This work was supported by the USDA, Grand Forks Park District, and Altru Health System.
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

Objective

According 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.

Methods

A 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.

Results

We 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.

Conclusion

In 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
Effects of social stress on locus coeruleus activity and cognitive flexibility
Authors: Chaijale Nayla , Curtis Andre L , Wood Susan K , Snyder Kevin , Luz Sandra , Bhatnagar Seema , Valentino Rita J
Published: June 2021

The locus coeruleus (LC)‐norepinephrine (NE) system has been implicated in hyperarousal, a core feature of stress‐related psychiatric disorders. Because chronic social stress (CSS) is a common stressor, we characterized the consequences of CSS on LC‐NE system using the resident‐intruder model. The goal of this study was to determine whether CSS alters LC neuronal activity and AST performance. Sprague Dawley rats were implanted with a multiwire bundle into the LC and baseline activity was recorded before day 1 and 7 of CSS. LC activity decreased after 5 days of CSS (Day 1: 2.04±0.05 Hz, Day 7: 1.26±0.06 Hz; n=25), but not in controls (CTRL; Day 1: 2.70±0.05 Hz, Day 7: 2.61±0.03 Hz, n=12). Additionally, after CSS LC neurons showed a slow oscillation (0.1Hz). Rats were then tested in AST after CSS and LC activity was recorded. CSS rats performed better in intradimensional set shifting (IDS): 36.7±4 and 23.8±4 trials to criterion for CTRL (n=11) and CSS (n=10) respectively (p<0.05). Recordings during simple discrimination showed that CTRL LC activity (4.8±0.5Hz; 14 cells, 2 rats) were higher than CSS (3.2±0.3Hz; 27 cells, 3 rats) throughout the task (p<0.05). A repeated measures ANOVA revealed an effect of group, an effect of time and a group by time interaction (p<0.005). Together, the data suggest that CSS may improve the formation of set and that CSS alters the rate and pattern of LC neuronal activity. DARPA grant‐58077 LSDRP.
The Potential of a Cellulose Nanocrystal and Water Soluble Chitosan Complex as an Oral Drug Delivery Carrier
Authors: Colacino Katelyn Rose , Wang Hezhong , Hong Jung Ki , Roman Maren , Lee Yong Woo
Published: June 2021

The present study evaluates the potential use of polyelectrolyte‐macroion complexes (PMCs) between water soluble chitosan (WSC), a cationic polysaccharide, and cellulose nanocrystals (CNCs), anionic, rod‐like nanoparticles, as carriers in oral drug delivery applications. CNCs were prepared by sulfuric acid hydrolysis of dissolving pulp and carboxylated to increase surface charge. PMCs were prepared with WSC purchased from Qingdao Olympic Chemistry Company at a 1:1 charge ratio. Human epithelial colorectal adenocarcinoma (Caco‐2) cells were purchased from American Type Culture Collection and employed for measurements of transepithelial electrical resistance (TEER) and differences in tight junction protein expression. Field‐emission scanning electron microscopy images showed PMCs to be spherical structures with a diameter in the micron range. WSC and PMC treatment significantly decreased TEER across the Caco‐2 cell monolayer in a similar fashion. Additionally, immunofluorescent staining revealed the expression of tight junction proteins such as ZO‐1 and occludin significantly decreased after treatment with WSC and PMCs. These data provide evidence that WSC‐CNC PMCs alter TEER and permeability across Caco‐2 cell monolayers similarly to WSC. Our results suggest that WSC‐CNC PMCs are a viable option as carriers for oral drug delivery. (This work was supported by NSF # DMR0907567)
Selective breeding of mice for high voluntary exercise alters adaptive plasticity of metabolic phenotypes in skeletal muscle
Authors: Meek Thomas H , Smith Lemar I , Eisenmann Joey C , Shyy John Y.-J. , Garland Theodore
Published: June 2021

The response to selection on behavior necessarily entails associated changes in lower‐level (subordinate) traits. We studied mice from 4 replicate lines that had been selectively bred for high levels of voluntary exercise (High Runner or HR) that run ~3‐fold more wheel revolutions/day than those from 4 non‐selected control (C) lines, and have evolved higher baseline endurance and VO2max. We examined hindlimb skeletal muscle metabolic capacity in mice housed with or without wheel access for 8 weeks. Triceps surae muscles were assayed at 37C for Vmax of hexokinase (HK), citrate synthase (CS), carnitine palmitoyl‐transferase (CPT), and β‐hydroxyacyl‐CoA dehydrogenase (HAD). Wheel access increased heart ventricle mass in both HR and C lines, but only HR showed increased enzyme activities as well as higher phosphorylation of 5ˈ‐AMP‐activated protein kinase. Analysis of wheel‐access groups indicated the greater training effect in HR could be explained statistically by their greater wheel running for ventricle mass, CS, and HAD, but for HK and CPT, HR mice had greater adaptive plasticity of the response to voluntary exercise. Thus, HR do not have inherent baseline differences in metabolic capacity, but they have evolved greater adaptive plasticity for some traits. Supported by NSF IOB‐0543429 and IOS‐1121273 to TG.
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.
Structure and mechanism of the CRISPR associated complex for antiviral defense (CASCADE)
Authors: Lawrence C. Martin , Lintner Nathanael G. , Kerou Melina , White Malcolm F.
Published: June 2021

The CRISPR/Cas prokaryotic adaptive immune system archives fragments of virus‐derived DNA within a hosts CRISPR loci. The CRISPR loci are then transcribed, processed, and loaded into 2 distinct complexes, CMR and CASCADE, that respectively target destruction of complementary invading RNA and DNA in subsequent infections. We report structural and functional characterization of the evolutionarily conserved components of CASCADE. Cas7 (aka Csa2), the dominant CASCADE protein, co‐purifies with Cas6‐processed CRISPR‐RNA (crRNA) and smaller amounts of Cas5a. TEM studies of the nucleoprotein complex reveal that Cas7 assembles along crRNA with right handed helical symmetry. The crystal structure of Cas7 unexpectedly revealed a decorated RNA‐recognition motif (RRM) and two additional domains present as insertions in the RRM. Biochemical and ribonuclease protection assays, mutational analysis, particle symmetry and modeling studies collectively indicate that the crRNA is bound nonspecifically along the inside surface of this central Cas7 helix, which is capped at the 5′ and 3′ ends by Cas5a and Cas6, respectively. Importantly, we identify similarities between CASCADE and the RecA/DNA complex that strongly suggest mechanistic similarities between CASCADE mediated recognition of invading DNA by crRNA, and RecA catalyzed strand invasion and D‐loop formation during DNA double strand break repair. Supported by the National Science Foundation (MCB‐0920312).
Thermodynamic Comparison of Dinucleotide bulges in DNA and RNA
Authors: Lockwood W Charles , Grover Neena
Published: June 2021

Bulges are non‐helical regions in RNA that provide sites for protein, ligand and metal ion interactions. In crystal structures, non‐canonical interactions of bulges are often the sites for interactions with metal ions. To fully elucidate RNA interaction in the bulge regions, we are also examining the corresponding DNA analogs. Dinucleotide bulges in DNA and RNA were modeled from Group II intron and HIV‐2 TAR RNA to understand the sequence and ion binding properties. Thermal denaturation experiments were performed on the wild type and modified constructs containing dinucleotide sequences in 1 M KCl and various concentrations of Mg2+ ions. DNA analogs typically are less stable than the RNA analogs in 1 M KCl and do not gain additional stability upon binding to Mg2+ ions. Comparative thermodynamic analysis will be presented for various DNA and RNA constructs. This work was funded by NSF Grant MCB‐0950582 to NG.
Acute nutrient consumption attenuates intramuscular 26S proteasome activity during energy deficit
Authors: Carbone John , Margolis Lee , Cao Jay , McClung James , Young Andrew , Pasiakos Stefan
Published: June 2021

The effects of dietary protein and feeding during energy deficit (ED) on cellular regulators of muscle proteolysis are not well documented. This study examined intramuscular caspase‐3 and 26S proteasome responses to feeding during sustained ED in adults consuming varying levels of dietary protein. Using a randomized*block design, 32 men and 7 women were assigned to either standard (0.8 g·kg−1·d−1; n =13), moderate (1.6 g·kg−1·d−1; n =14), or high (2.4 g·kg−1·d−1; n =12) protein diets for 31 days. A 10 day weight maintenance period was followed by 21 days of ED, during which energy intake was restricted to 60% of total daily energy expenditure. Enzyme activities were assessed using fluorescence‐based assays and muscle samples obtained on days 10 and 31 following an overnight fast (FAST) and 120 min after consuming a mixed meal (480 kcals, 20 g protein; FED). The activity of the 26S proteasome's β1, β2, and β5 subunits were 28%, 14% and 22% lower (P < 0.05), respectively, for FED relative to FAST regardless of energy status and dietary protein. Caspase‐3 activity remained steady in response to feeding and was not influenced by energy and protein manipulations. These data demonstrate that feeding attenuates ubiquitin‐mediated proteolysis, suggesting a novel cellular mechanism for preservation of muscle mass independent of habitual dietary protein intake and decrements in energy balance. Supported by USAMRMC, USDA ARS GFHNRC and EMU CHHS FRF
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

OBJECTIVE

To assess the use of a computerized burn resuscitation decision support system (BRDSS) in swine with smoke inhalation and severe burns.

METHODS

We 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.

RESULTS

Arterial 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.

CONCLUSION

BRDSS 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
Thermodynamics and Kinetics of the Interactions of Calmodulin with Orai
Authors: Wei Chin‐Chuan , Shakya Shubha , Jensen Drake , Bennett Katherine
Published: June 2021

Calcium dependent inactivation (CDI) is a key regulator of calcium release‐activated channels. Calmodulin (CaM) has been shown to interact with Orai1, evoking CDI. Though the calmodulin‐binding sequence (CBS) of Orai has been identified, the mechanism is not clear. Here we generated recombinant CaM and characterized its interaction with a peptide corresponding Orai CBS (Orai‐Pep) using isothermal titration calorimetry (ITC) and fluorescence. The peptide binding to CaM increases the extent of hydrophobicity as indicated by the increase of ANS fluorescence. ITC revealed that such a binding is moderate with Ka= 9.5 × 105M−1 and the reaction is exothermic with ΔH = −15.8 kJ/mol. To better define how CaM promotes CDI, we focused on the interaction of CaM and troponin C (TnC) chimeras with Orai‐Pep. 1TnC, in which the 1st EF‐hand of CaM is replaced with the corresponding EF‐hand of TnC, and 2TnC have one‐order lower binding affinity while 3TnC and 4TnC show similar binding strength to that of CaM. This data indicates that the 1st and 2nd EF‐hands of CaM are more important for binding, possibly through their interaction with Trp76 of Orai, in which its accessibility to acrylamide and KI decreases in the complex of 1TnC and 2TnC. Currently we are investigating the kinetics and heat capacity difference of CaM and chimeras upon peptide binding. This work is supported in part by National Science Foundation.
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.
Understanding protein‐protein interaction network: a case study with glutaminase interacting protein
Authors: Ovee Mohiuddin , Zoetewey David , Banerjee Monimoy , Bhaskaran Rajagopalan , Mohanty Smita , Zencir Sevil , Topcu Zeki
Published: June 2021

Glutaminase interacting protein (GIP) is a 124 amino acid long protein containing single PDZ domain. This protein intersects a number of important biological pathways, of which many are unknown. Its involvement in cancer pathways makes it a good target for drug design based on the structure. We resolved the solution structures of both free GIP and GIP in complex with C‐terminal peptide analog of glutaminase L to shed light on the mechanism of binding with the goal of future development of a potential inhibitor for GIP. To understand more of GIP's function, interactions with two target peptides were investigated using different biophysical methods. One of the peptide had the C‐terminus of brain‐specific angiogenesis inhibitor 2 (BAI2) and the other one used had a consensus PDZ class I binding motif. Both of the peptides showed moderate binding affinity toward GIP with BAI2 peptide having comparatively higher affinity. Elucidating the mechanism of interactions for different target partners would help to lay out the network of function for GIP. This research was financially supported by USDA PECASE Presidential Early Career Award for Scientists and Engineers award 2003–35302‐12930, NSF Grant IBN‐0628064, USDA Grant 2011–65503‐20030 and NIH Grant DK082397 to Smita Mohanty.
A putative Fe+3/phytosiderophore (PS) transporter isolated from oats (Avena sativa)
Authors: Davis George Thomas , Stocksdale Mark
Published: June 2021

The objectives of this project were: 1) to isolate and characterize a Fe+3/phytosiderophore (PS) transporter from Oats (Avena sativa), and, 2) develop a method for the synthesis of the oat phytosiderophore avenic acid. The putative transporter's functionality will be evaluated by complementation assays in yeast (Saccharomyces cerevisiae) using synthetic avenic acid (Stocksdale).

Total RNA was isolated from the roots of hydroponically grown, iron‐starved oats (var. Rodeo). Full‐length clones were obtained using 5′RACE and RT/PCR. Sequence analysis of the clone, AvsYS1, (Genbank Accession# FJ477297) shows>;85% similarity at both the nucleotide and amino acid level to HvYS1, a functionally confirmed Fe+3/ (PS) transporter from barley (Hordeum vulgare). AvsYS1 was subcloned into pYES2 (Invitrogen), a galactose‐inducible shuttle vector.

DDY4 ( a gift from Dr. David Eide) is a mutant S. cervisiae lacking a Fe+3 uptake ability. DDY4 will be transformed with pYES2/BS4YAT, pYES2 containing the AvsYS1 insert. To evaluate functionality, transformants will be grown in minimal media (plates and liquid) supplemented with FeCl3, synthetic avenic acid or complexed FeCl3/avenic acid. If AvsYS1 encodes a functional transporter we will be able to rescue DDY4 in a the medium is supplemented with FeCl3/avenic acid.

This research was supported by a USDA CSREES Grant, #2007–35318