bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Various Concentrations of Trimethylamine‐N‐oxide on Serum and Cecal Metabolites in Female SD Rats Fed a High‐Fat Diet
Authors: Lim Taehwan , Lee Kiuk , Kim Ryun-Hee , Hwang Keum Taek
Published: April 2020

Trimethylamine‐N‐oxide (TMAO) has been shown to be a putative promoter of cardiovascular disease. Trimethylamine (TMA) precursors, such as choline and L‐carnitine, in foods can be metabolized to TMA by some gut microbiota and further converted to TMAO in liver. Recently, gut microbiota has been found to play an important role in nutrient metabolism. It can metabolize not only nutrients but also bile acids, which could affect gut liver axis and modulate metabolism. However, the effects of various concentrations of TMAO on microbial metabolites including TMA, TMAO, individual bile acids, and short chain fatty acids in rats have been little studied. The aim of this study was to evaluate effects of dietary TMAO on levels of metabolites in serum (TMA, TMAO, and bile acids) and cecum (TMA, TMAO, bile acids, and short chain fatty acids) of female SD rats fed a high‐fat (HF) diet. Sixteen female SD rats were fed HF diet (AIN‐93G diet providing 45% of energy from fat) with or without various concentrations of TMAO (0.05%, 0.15%, and 0.3%; T0.05, T0.15, and T0.3, respectively) (w/w) in autoclaved tap water for 8 weeks. Levels of TMA, TMAO, and bile acids were determined by UPLC‐Synapt G2‐Si. Short chain fatty acid profile was determined by GC‐FID. Serum TMA and TMAO levels were significantly (p<0.05) higher in the T0.3 than control group (CON), while cecal TMA level showed the opposite response. Glycocholic and tauro‐alpha‐muricholic acids were significantly (p<0.05) higher in the serum of the T0.3 than that of the CON. Taurocholic and taurodeoxycholic acids were significantly (p<0.05) the highest in the cecum of the CON. Valeric and isovaleric acids were significantly (p<0.05) higher in the cecum of the T0.05 than that of the CON.

Support or Funding Information

This research has been supported by National Research Foundation of Korea (NRF‐2017R1D1A1B03028407) funded by the Ministry of Education of Korea.
Chronic Atrial Overload Contributes to the Slow Decay of Intramural Fibrosis in the Human Atrium: Assessment by High‐Resolution 3D Histology
Authors: Ravelli Flavia , Masè Michela , Cristoforetti Alessandro , Avogaro Laura , D’Amato Elvira , Tessarolo Francesco , Piccoli Federico , Graffigna Angelo
Published: April 2020

Background

The intramural distribution of fibrosis is considered one of the most important factors in the formation of a substrate for atrial fibrillation. Chronic atrial stretch is known to play a mechanistic role in this structural remodeling process. However, the distribution of fibrosis through the entire atrial wall is poorly characterized as well as its relationship with chronic atrial stretch. Based on these premises, this study aimed at assessing the intramural profile of fibrosis in the human atrium in patients with different degrees of atrial dilatation and arrhythmic profiles by high‐resolution 3D histology.

Methods and Results

Serial vibratome‐cut longitudinal slices, encompassing the whole atrial wall at a resolution of 5 μm, were obtained from right atrial appendage samples from cardiac surgery patients. Serial slices were stained in Picrosirius red, imaged by polarized light microscopy, and analyzed with a custom‐made computer program. In all patients the intramural fibrotic content followed a progressive decreasing profile along tissue depth, passing from the epicardium to the endocardium. On average it decreased from 69 ± 12 % to 34 ± 16 % within the first 20% of atrial tissue depth, undergoing a further slight decrease to 21 ± 14 % at 40% of tissue depth. Finally, in deeper mid‐wall to subendocardial layers, the average fibrotic content slightly varied around low values of 10–13%. Distinct intramural fibrotic profiles were observed in sinus rhythm patients with normal atria with respect to patients with dilated atria, who showed, respectively, rapid and slow exponential decays (decay constant: 80.9 ± 24.4 μm versus 171.2 ± 54.5 μm, p<0.005). Similar to patients with atrial dilatation, patients with atrial fibrillation showed a slow fibrotic decrease with decay constant of 142.9 ± 41.7 μm. The decay rate of intramural fibrosis correlated with the degree of atrial dilatation (r=0.69, p<0.001).

Conclusions

The quantification of the intramural profile of fibrosis in the human atrium is feasible by combining slice‐to‐slice histology with computerized image analysis. Our results showed a deeper penetration of fibrosis in the atrial wall closely related with the presence of atrial dilatation. The stretch‐related intramural penetration of fibrosis may contribute to epiendocardial dissociation of electrical activity and the formation of an arrhythmic substrate.

Support or Funding Information

This work was supported by Fondazione Cassa di Risparmio di Trento e Rovereto.
Tempol Treatment Inhibits Inflammatory Process and Improves Morphological Aspects in Early and Late‐stage of Prostate Cancer Progression in the Ventral Lobe of TRAMP Model.
Authors: Rossetto Isabela Maria Urra , Kido Larissa Akemi , Santos Felipe Rabelo , Montico Fabio , de Almeida Lamas Celina , Cagnon Valéria Helena Alves
Published: April 2020

Tempol (4‐hydroxy‐TEMPO) is a SOD mimetic and member of the nitroxide compounds family. It is used as a co‐adjuvant or protective factor in cancer treatment due to its anti‐oxidant properties. However, in prostate cancer (PCa) Tempol effects are still poorly understood. Its known that PCa progression requires a sustained inflammation, which generates a broad spectrum of reactive nitrogen and oxygen species, leading to uncontrolled cellular proliferation. We aimed to evaluate the Tempol effects in different stages of PCa progression (a) in vivo using a transgenic adenocarcinoma of the mouse prostate model (TRAMP); (b) and in vitro using human PCa cell lines with different genetic background. For the early‐stage analysis, 8‐weeks‐old mice (TPL1 group) were treated orally with 50 mg/kg of Tempol during 4 weeks. For the late‐stage analysis, 16‐weeks‐old mice were treated with 50mg/Kg (TPL2 group) or 100mg/Kg (TPL3 group) of Tempol during the same period. All control groups received water as vehicle. The ventral lobe of the prostate was collected and submitted to the histopathological, immunohistochemical and Western blotting analysis. PC‐3 and LnCaP cells were exposed to different doses of Tempol (0.5, 1.0 and 2.0 mM) and cell viability assay was performed. In vivo, Tempol treatment promoted a great improvement in the prostatic epithelium in all the analyzed cancer stages, reducing significantly premalignant (high‐grade prostatic intraepithelial neoplasia) and malignant lesions (well differentiated prostatic adenocarcinoma). The PCNA immunostaining confirmed the cellular proliferation decrease between the control and treated groups. Tempol also reduced COX‐2 and NFkB, especially in the early‐stage, whereas iNOS increased in this period. In the late‐stage, both doses of Tempol reduced NFkB levels concomitant with IkB‐α increase, suggesting an inhibitory effect of Tempol on NFkB pathway in association with its anti‐oxidant role. In vitro, Tempol decreased cell viability of both cell lines, being the 1.0 mM dose the most efficient and nontoxic concentration. In conclusion, Tempol had a remarkable action in the prostate, delaying cancer progression in the ventral lobe of TRAMP model and inhibiting PC‐3 and LnCaP growth. This SOD mimetic presented great capability of inhibiting the inflammation process, and consequently the cell proliferation. Its action was effective in different stages of PCa progression, particularly in the early stage, indicating that Tempol treatment can be explored as a preventive approach. Finally, for the late PCa stage, Tempol may be considered as an interesting adjuvant therapy.

Support or Funding Information

São Paulo Research Foundation, FAPESP, process 2018/21647‐6 and FAEPEX/UNICAMP, process 2202/19
Targeted proteomics analysis revealed the imbalanced allele‐specific protein expression of carboxylesterase1 in human livers
Authors: Her Lucy , Shi Jian , Wang Xinwen , Zhu Hao-Jie
Published: April 2020

Carboxylesterase 1 (CES1) is the primary hepatic hydrolase and is responsible for metabolizing many medications. Its clinical significance has been well demonstrated in various clinical trials with methylphenidate, clopidogrel, oseltamivir, and ACE inhibitors. CES1 expression varies markedly among individuals, which is a major contributing factor to the interindividual variability in the pharmacokinetics and pharmacodynamics of CES1 substrate drugs. Identifying genetic variants responsible for CES1 expression regulation could lead to the optimization of pharmacotherapy of medications metabolized by CES1. The analysis of allele‐specific expression is a powerful tool to determine regulatory genetic variants because this assay is less susceptible to the confounding effect of nongenetic factors (e.g. inducers) compared to those conventional gene expression‐based approaches (e.g. GWAS). In the present study, we developed a targeted proteomics method to measure the allele‐specific protein expression (ASPE) of CES1 in human livers using a heavy stable isotope‐labeled QconCAT internal standard. The CES1 nonsynonymous variant S75N (rs2307240) was utilized to differentiate the protein expressions of the two CES1 alleles. The QconCAT construct contained the sequences of both the wild type peptide (FTPPQPAEPW
SFVK) and its S75N variant (FTPPQPAEPW
NFVK), which ensures that the amounts of the two peptides released from the QconCAT internal standard are equal. Another essential feature of the QconCAT protein is that the 75S and 75N peptides are flanked with several native CES1 amino acids, such that the digestion efficiency of the QconCAT peptides will be the same of the native CES1. The method was successfully applied to the determination of the ASPE of CES1 in 32 S75N heterozygous liver samples. The ASPE ratios of 75S to 75N ranged from 0.80 to 1.47, indicating the presence of cis‐regulatory genetic variants of the CES1 gene. Further studies have been carried out to determine the variants responsible for the imbalanced ASPE of CES1 in human livers.

Support or Funding Information

This work was supported in part by the National Heart, Lung, and Blood Institute [Grant R01HL126969]
Chronic Alcohol Drinking Increases L‐Type Voltage‐Gated Calcium Channel Cav1.2 in the Substantia Nigra
Authors: Rodriguez Eric A. , Yamamoto Bryan K
Published: April 2020

Studies report that 77 % of methamphetamine (Meth)‐dependent adults in the United States also have alcohol use disorder. This comorbidity is associated with negative cognitive and behavioral effects. In addition, the serial exposure of alcohol and then methamphetamine (Meth) to adult male Sprague Dawley rats is neurotoxic and marked by a loss of tyrosine hydroxylase‐positive dopamine (DA) cells in the substantia nigra pars compacta (SNpc) that is absent after either drug alone. Dopamine cell loss was associated with caspase‐3 mediated apoptosis after Meth exposure to alcohol drinking rats; however, the effect of alcohol that sensitizes DA cells to Meth‐induced apoptosis remains unknown. Adult male Sprague Dawley rats had access to either two bottles of water or a choice of 10% alcohol and water every other day over 28 days. Rats with access to alcohol every other day had significantly increased L‐type voltage gated calcium channel Cav1.2 protein in the substantia nigra (SN) (33 ± 1.4 %) at 1 day after the last day of alcohol drinking compared to water drinking only controls (p<0.05). The increases in Cav1.2 were region‐specific since no such changes were detected in tissue from other dopaminergic regions including the dorsal striatum, ventral tegmental area, and nucleus accumbens. Furthermore, the cyclooxygenase‐2 selective and prostaglandin synthesis inhibitor, nimesulide (7.5mg/kg i.p., b.i.d.) administered on days when alcohol was not available, blocked the increases in Cav1.2 at 1 day after the alcohol drinking paradigm. Nimesulide administered only during the alcohol drinking paradigm also blocked the loss of DA cells in the SN pars compacta measured 1 week after exposure of Meth (10 mg/kg q 2 hrs X 4 injections). Although previous studies by others showed chronic alcohol exposure increases Cav1.2 in other brain regions, this is the first evidence of increases in the SN and that the increases occur in a prostaglandin synthesis‐dependent manner. The results are consistent with the excitotoxic effects produced calcium dysregulation and suggest that prostaglandins and disruption of calcium homeostasis produced by voluntary alcohol drinking sensitize DA cells in the SN to the neurodegenerative effects produced by the serial exposure to alcohol and Meth.

Support or Funding Information

This work is supported by the National Institutes of Health [DA042737].
Function of Mte1/ZGRF1 in Homologous Recombination
Authors: Durham Jesse
Published: April 2020

Fanconi anemia (FA) is a human genetic disorder that causes organ defects, physical abnormalities, bone marrow failure, and increased risk of malignancy. These phenotypes result from an impaired response to DNA damage due to the dysfunction of a cluster of FA proteins. Among these, human FANCM (budding yeast ortholog, Mph1) functions in DNA replication fork (RF) repair and DNA double‐strand break (DSB) repair by homologous recombination (HR). In a recent study, Mte1 in Saccharomyces cerevisiae was found to protect the Rad51‐made D‐loop, essential for HR, from disassociation by Mph1 (Xue et al. 2016). Our preliminary data showed that Mte1 interacts with Rad51 in vitro. However, the biological role of this interaction remains unknown. The goal of this project is to examine the effects of 2 different binding site mutants of Mte1, where mutations are hypothesized to affect binding of Rad51. Quick change site directed mutagenesis will be used to create the 3 mutants by changing 2 different phenylalanine side groups to alanine at positions 156, 169, and 156/169 combined. The Rad51 interaction of each mutant will be evaluated via in vitro pull down assay. The D‐loop binding activity of each mutant will be evaluated via electrophoresis mobility shift assays (EMSA). Mutation effects on Rad51‐made D‐loop formations can direct further studies investigating proteins required for HR. Evidence suggest that human ZGRF1 is functionally related to Mte1 due to N‐terminus conservation between the two. However, little is known about the role and function that ZGRF1 plays in HR in humans.

Support or Funding Information

Research supported by NIH R21 #ES028792 awarded to XX. JD supported by NIH R25 Bridge to Doctorate Program (GM102783)
Crystal Structure of SMYD2 Reveals Simultaneous Binding of PARP1 Peptides to the Active Site and an Unexpected Secondary Binding Site
Authors: Zhang Yingxue , Martin Kaitlyn , Spellmon Nicolas , Perry Emerson , Cao Tianxin , Muzzarelli Kendall , Smith Sheila , Brunzelle Joseph , Kovari Ladislau , Jin Jianping , Stemmler Timothy , Li Chunying , Yang Zhe
Published: April 2020

SMYD2 is a protein lysine methyltransferase with a broad substrate specificity and able to methylate both histone and non‐histone proteins such as histone H3 and H4, p53, retinoblastoma‐associated protein 1, and PTEN. However, how SMYD2 substrate specificity is precisely controlled and balanced over such a large number of targets remains elusive. Poly(ADP‐ribose) polymerase‐1 (PARP1) is another known SMYD2 substrate, acting as a first responder to DNA damage. SMYD2 mono‐methylates PARP1 at lysine 528 within the auto‐modification region. Methylated PARP1 positively regulates the poly(ADP‐ribosyl)ation activity and enhances cellular poly(ADP‐ribose) formation after oxidative stress. In this study, we determined the crystal structure of SMYD2 in complex with a K528‐containing PARP1 peptide. Unexpectedly, two peptide binding sites were identified from the structure, one corresponding to the known substrate binding site and the other a new secondary binding site. We found that the point mutations of the secondary binding site completely abolished binding of the PARP1 peptide to both sites in the isothermal titration calorimetry (ITC) analysis. The crystal structures of the corresponding SMYD2 mutants also showed disturbed binding, in which the mutation did not cause any significant structural changes but no peptide binding was observed. However, in the context of PARP1 protein, the same mutation enhanced the interaction in the ELISA assay, which was partially correlated with its substrate‐specific effects on the enzyme activities. The mutation of the secondary binding site mutation enhanced SMYD2 activity on histone H3 protein but attenuated the methylation of PARP1 protein, and there was no significant effect on histone H4 protein. Overall, our study revealed a new peptide binding site in SMYD2 that may play a regulatory role in the SMYD2 substrate selectivity.
Differential Cannabidiol Pharmacology Across Amygdala Subnuclei
Authors: Winters Nathan Daniel , Patel Sachin
Published: April 2020

Cannabidiol (CBD) is one of the primary phytocannabinoid constituents of Cannabis and is increasingly utilized to treat a variety of conditions, such as anxiety and epilepsy. Despite the growing use of CBD, the clinical and off‐label applications of CBD and related products have greatly preceded the state of pre‐clinical and clinical research regarding the pharmacological properties of CBD. As such, an empirical and mechanistic understanding of the physiological and therapeutic effects of CBD remains poorly lacking. CBD exhibits remarkably complex polypharmacology and engages a variety of targets in the central nervous system, rendering it a challenge to discern the precise molecular mechanisms by which CBD exerts its purported effects. Here, we explore the effects of CBD on the physiology of distinct cell types in the amygdala, a subcortical brain structure critical for the processing of fear and anxiety, and in some cases linked to epilepsy. Preliminary data shows that CBD decreases passive membrane excitability of pyramidal neurons in the basolateral nucleus, exhibited as a decrease in action potential firing in response to square wave depolarizing steps. Additionally, CBD decreases spontaneous release of GABA onto a population of cells in the central nucleus, without affecting the frequency of spontaneous glutamate release, resulting in a shift in the excitation/inhibition ratio. Ongoing studies are aimed at further characterizing these findings in distinct amygdala cell types, identifying the molecular targets of CBD that mediate these effects, and determining the behavioral relevance of CBD effects on amygdala physiology.
Developing a Blood Test to Support Treatment of Surgically Induced Type I Diabetes
Authors: Dailey Allyson , Kanak Mazhar , Couch Robin
Published: April 2020

In this pilot‐scale investigation, serum was collected from nine study participants, each of which having underwent a procedure of total pancreatectomy with islet autotransplantation. The procedure was a success with 6 of the patients (i.e. they are not dependent on insulin injections), whereas the insulin‐producing islet cells did not successfully autotransplant in 3 of the patients (and thus they are dependent on insulin injections). Each of the nine patients have provided four serum samples; one obtained prior to surgery, one immediately following surgery, one 1 hr following islet autotransplantation, and one 7 days following islet autotransplantation. Following organic extraction, the serum extracts were analyzed for metabolite content using liquid chromatography quadrupole time of flight mass spectrometry (LC‐QToF). A differential comparison of the serum metabolites present in each of the samples was used to define molecular features that may serve as biomarkers of patient outcome. Subsequent tandem mass spectrometry (LC‐MS/MS), along with comparison to molecular libraries and purchased authentic standards, will identify the molecular features, and permit an assessment of their suitability for use in a predictive diagnostic test to determine patient suitability/compatibility with islet autotransplantation.

Support or Funding Information

4‐VA Funds
Effects of diet and torpor on skeletal muscle relaxation in hibernating animals
Authors: Krishnan Jishnu K.S. , Rice Sarah , Mikes Monica , Hunstiger Moriah , Drew Kelly , Oliver S. Ryan
Published: April 2020

Arctic ground squirrels (AGS) undergo extreme temperature fluctuations during the hibernation season (body temperature, Tb, as low as −3 °C), where they mostly remain physically inactive. During interbout arousals, once Tb reaches ~16°C, hibernators recruit skeletal muscle (SkM) for shivering thermogenesis to attain Tb of ~37°C. Dietary polyunsaturated fatty acids (PUFA) are known to affect both SkM function and metabolism. Recent studies in cardiac muscle of hibernators have shown an increased level of ω‐6 in ω‐6:ω‐3 PUFA ratio are correlated to sarco/endoplasmic reticulum Ca‐ATPase (SERCA) activity with respect to torpor and hibernation status. SERCA catalyzes the re‐uptake of Ca2+ back into the sarcoplasmic reticulum (SR), which was previously bound to actin/myosin filaments thus completing the calcium cycle and eliciting muscle relaxation. We hypothesized that diet (increased ω‐6:ω‐3 PUFA ratios) and torpor status play essential roles in the regulation of the SERCA pump, which may improve SkM performance during hibernation. An Ex‐vivo functional assay was used to characterize performance alterations in SkM (diaphragm) collected from AGS fed the following diets (ω‐6:ω‐3, 5:1 control diet versus ω‐6:ω‐3, 1:1 test diet) and collected at 3 timepoints of torpor (early torpor, late torpor, and arousal). Additionally, the muscles were assessed for their response to hypothermic temperature stress (4 °C; 15 °C; 25 °C and 37 °C) to determine functional resilience. To our knowledge this is the first study correlating torpor status, diet, and effects of hypothermic temperature stress on SkM functional performance in hibernators (AGS). Data suggest: 1) diet and torpor significantly alter relaxation kinetics (calcium re‐absorption), but not force development (rate of calcium release), and 2) diet, torpor status, and inherent temperature resilience (4°C; 15°C) affect the muscles ability to produce force. This evidence supports the interpretation that functional properties of SkM can be altered by membrane lipid composition, ambient temperature, and hibernation status to control performance.

Support or Funding Information

This work was supported by the INBRE IDeA Faculty Pilot Grant (University of Alaska Fairbanks ) from NIH NIGMS grant number P20GM103395.
In Vitro Reconstitution of Centralspindlin‐Mediated Pebble/RhoGEF Transport
Authors: Warecki Brandt , Tao Li
Published: April 2020

Cytokinesis is the last step of cell division. Failure in regulation of cytokinesis is the cause of several human diseases, including cancer. Appropriate formation of the cleavage furrow during cytokinesis requires accurate regulation of the small GTPase Rho. Multiple experiments have demonstrated that Rho activity is modulated at the central spindle by RhoGEF (Pebble in Drosophila) and the conserved motor complex centralspindlin. However, perhaps due to the necessity of these proteins in in vivo systems, the precise mechanism for how centralspindlin and RhoGEF cooperate to activate Rho at the central spindle remains poorly understood. Here, we use the baculovirus expression system to generate recombinant Drosophila versions of the centralspindlin components Pavarotti/Kinesin‐6 and Tumbleweed/RacGAP as well as Pebble/RhoGEF. Using these purified proteins, we reconstitute key events in cytokinesis and test models for Rho activation that have been based on in vivo studies. We observe Pavarotti/Kinesin‐6‐mediated microtubule bundling and show that the recombinant centralspindlin holoenzyme recruits Pebble/RhoGEF to microtubules in vitro. Furthermore, we demonstrate that centralspindlin transports Pebble/RhoGEF along microtubules in a plus‐end directed fashion. Taken together, our studies support a model in which centralspindlin motor activity delivers Pebble/RhoGEF to the central spindle where Pebble/RhoGEF activates Rho, leading to cleavage furrow formation and proper cytokinesis.

Support or Funding Information

NIH INBRE P20GM103466‐17
Exercise training upregulates Dicer to modulate hippocampal miRNA expression in 5xFAD mice
Authors: Dungan Cory M. , Valentino Taylor R. , Vechetti Ivan J. , Zdunek Christopher J. , Bachstetter Adam D. , Lin Ai-Ling , McCarthy John J. , Peterson Charlotte A.
Published: April 2020

Changes to cerebral miRNA expression have been implicated in the progression of Alzheimer’s disease (AD), as miRNAs that regulate the expression of genes involved in amyloid beta (Aβ) processing are dysregulated in those that suffer from AD. Exercise training improves cognitive function and reduces Aβ plaque burden, however, the mechanisms are not fully understood. Using our progressive weighted wheel running (PoWeR) exercise program, which utilizes an unbalanced running wheel to promote physiological adaptations similar to those seen in humans, we assessed the effect of 20 weeks of voluntary exercise training on changes in cognitive function and hippocampal miRNA expression in female 5xFAD mice; a model that expresses human APP and PSEN1 transgenes with a total of five AD‐linked mutations. Two month old female 5xFAD mice were PoWeR trained for 20 weeks, running ~7km/day, while groups of female wild‐type (B6SJLF1/J) and 5xFAD served as sedentary controls. At the end of the training period, mice performed cognitive (Morris water maze, novel object recognition) and physical (rotor rod and balance beam) function tests. The hippocampus was removed to assess miRNA abundance via Nanostring and gene expression with RT‐qPCR. Exercise training improved cognitive and physical function, in addition to having a significant effect on the expression of the miRNA processing enzyme, Dicer. Specifically, Dicer was lower in sedentary 5xFAD hippocampus, when compared to sedentary WT mice, but was robustly elevated following exercise. Correspondingly, ~45% of the miRNAs that were dysregulated in sedentary 5xFAD mice were restored towards wild‐type levels following exercise. Specifically, miR‐29, which is a validated target of the APP processing protein, BACE1, was significantly higher in exercised 5xFAD mice. Accordingly, BACE1 expression was lower in these mice. Based on these results, we conclude that exercise training alters cerebral miRNA expression via Dicer to affect APP processing.

Support or Funding Information

NIH AG049806 to J.J.M and C.A.P.

NIH P30 AG028383 to C.M.D.
Exposure of Mice to the Scent of a Predator Blunts, but Does Not Block Torpor
Authors: Lee Yang , Chan Cordelia , Swoap Steven John
Published: April 2020

2,3,5‐trimethyl‐3‐thiazoline (TMT) is a chemical compound extracted from red fox urine and can be used to artificially simulate the presence of a predator. The purpose of this study was to test the hypothesis that TMT would block entry into torpor in the lab mouse. We first demonstrated that TMT induces fear in the mouse. Exposure to TMT induced a freeze response (67.2 ± 6.7% of time without movement over 10 minutes), as compared to 6.7 ± 1.7% without movement when exposed to water. When fed mice were exposed to TMT during the dark or light phase, body temperature dropped by 1.4 ± 0.2°C and 1.0 ± 0.2°C, respectively, over the first hour after exposure. Exposure to TMT for 30 minutes increased circulating corticosterone, 377 ± 33 ng/ml, as compared to 29 ± 4 ng/ml when exposed to water. To determine whether TMT influences daily torpor, mice were calorically restricted and exposed to either water or TMT. Mice were exposed one hour before the start of torpor, determined by the bout of the previous day. Exposure to TMT blunted torpor by increasing the minimum body temperature from 29.2 ± 0.3°C (water) to 30.1 ± 0.6°C (TMT) and by decreasing the amount of time the mice spent under 32°C from 431 ± 48 minutes (water) to 292 ± 78 minutes (TMT). These results establish that mice perceived the scent of TMT as a physiologically stressful stimulus and that torpor is blunted but not blocked in the presence of that stressor.
Cre Recombinase: a genomic manipulation tool
Authors: Sampe David Richard , Adkins Kira , Greenman Magdeline , Her Jennifer , Johnson Hattie , Kaur Bavneet , Keebler Anna , Skebba Ben , Yang Liliah , Makky Khadijah
Published: April 2020

Comprehending human biology necessitates an understanding of molecular mechanisms that contribute to human physiology. For example, to determine why drug users relapse after stress, researchers must identify the neurological pathways involved in the addiction, and understand how certain stimuli trigger relapse. When using Cre recombinase and the LoxP DNA sites (from P1 bacteriophages), no limitations exist for knocking genes in or out and any genetic disease can be researched. By using the Cre‐LoxP system, researchers can manipulate gene expression in specific tissues by excising, inverting, inserting, and translocating sections of DNA. Cre, a 38kD protein with 341 amino acids, has 14 alpha helices and five strands of beta sheets. Cre has two domains, the N‐terminus (amino acids 20–129) and the C‐terminus (amino acids 132–341). The active site of Cre contains the residues of Arg173, His289, Arg292, Trp315, and Tyr324 which catalyzes the interactions with LoxP. This interaction facilitates the genome change and affects whether or not certain genes are expressed. The Brown Deer High School SMART (Students Modeling A Research Topic) Team has designed a model of Cre Recombinase using 3D printing technology to investigate structure‐function relationships. The Cre‐LoxP system has become a routine lab practice. For example, in animal models the interface of stress triggers and relapse is being researched using CRE‐delivery approaches to express genes in pathways that may be critical to stress and relapse. Using the Cre‐LoxP system in drug addiction studies is only one example of the system’s utility in understanding human physiology and disease mechanisms.
Fatty Acid Regulation of Virulence in Vibrio cholerae: A Structural Analysis of Unbound, Ligand‐Bound, and DNA‐Bound ToxT
Authors: D’Ausilio Morgan E. , Morriello Cole , Osika Kristin , Poh Belinda , Provoost Ashleigh , Schwarz Caitlin , Schwarz Maureen , Volchenkov Anton , Wu Allen , Kull F. Jon
Published: April 2020

Vibrio cholerae is an infectious bacterium that causes activation of transmembrane ion channels responsible for mediating the tonicity of cells in the small intestine. Activation of these ion channels results in osmotic movement into the small intestine, thus dehydrating cells and the infected individual. The transcription factor, ToxT, upregulates production of cholera toxin (CT) and the toxin coregulated pilus (TCP), key virulence factors in the progression of cholera. ToxT, a member of the AraC family of transcriptional regulators, has been crystallized in the presence of an unsaturated fatty acid (UFA) revealing a repressed form of the transcription factor in a conformation that prevents DNA binding. Two □‐helices in the DNA binding domain (DBD), □6 and □9, must be parallel to bind consecutive major grooves in the DNA. In the UFA‐bound structure, these helices are non‐parallel, supporting the model that ligand binding to ToxT prevents DNA binding. ToxT has been shown biochemically to be homodimeric when bound to DNA. However, crystallization of the DNA bound form of ToxT has been challenging due to instability of the complex. Recently, a natural variant of ToxT with increased solubility has been crystallized in the presence and absence of UFA. Crystallization of the apo form of ToxT required a lysine to alanine substitution at position 231 in the ligand‐binding pocket of the protein to prevent UFA binding. Although the structure of apo‐ToxT is highly similar to the UFA‐bound structure, the apo version was shown to be more flexible than ligand‐bound ToxT, supporting a model in which □6 and □9 in each subunit of dimeric ToxT are parallel and capable of binding to DNA. Using the structures described in this study, The Pingry School SMART (Students Modeling A Research Topic) Team used a 3D‐printer from the Milwaukee School of Engineering (MSOE) to model the apo structure and the dimeric DNA‐bound model of ToxT. These models support an in‐depth analysis of ToxT structural conformations in support of the proposed mechanism of regulation. A detailed structural understanding of ToxT may support future development of vaccines and therapeutics for cholera and other pathogens whose toxicity is regulated by AraC family members.
Evidence of Degradation of Endothelial Glycocalyx in African Children with Falciparum Malaria
Authors: Bush Margaret A. , Florence Salvatore M. , Kalingonji Ayam R. , Chen Youwei , Yeo Tsin W. , Anstey Nicholas M. , Rubach Matthew P. , Granger Donald L. , Mwaikambo Esther D. , Weinberg J. Brice
Published: April 2020

The microvasculature and endothelium play a pivotal role in pathophysiology of malaria. The endothelial glycocalyx (eGC) is a layer on the luminal side of vascular endothelial cells with involvement in homeostatic functions including regulation of vascular permeability, cellular adhesion and blood flow‐mediated nitric oxide formation. Breakdown products of eGC degradation include glycosaminoglycans (GAG) and syndecan‐1, a core protein. The objective of this study was to evaluate a possible role of eGC breakdown in the pathogenesis of malaria. We prospectively evaluated measures of endothelial glycocalyx integrity in children with severe and moderately severe malaria and healthy controls, and determined the relationship between glycocalyx integrity and markers of malaria severity, microvascular reactivity, and endothelial activation. At the Hubert Kairuki Medical Center in Dar es Salaam, Tanzania we studied 146 subjects, aged 2–11 years, including 60 healthy controls (HC), 49 patients with moderately severe malaria (MSM) and 37 patients meeting WHO criteria for severe malaria (SM). Ethics committee approvals were obtained prior to study initiation. We assessed eGC integrity biochemically by measuring plasma syndecan‐1 (ELISA) and total urinary glycosaminoglycan (GAG) breakdown products (dimethylmethylene blue assay). Angiopoietin‐2 has been reported to cause breakdown of eGC and was measured by ELISA. Quantitative measures of eGC were assessed using non‐invasive, side‐stream dark field (SDF) microscopy of the pinna and axilla. Levels of urinary GAG were increased in malaria patients compared with HC (mean±SEM 4.3±0.4 g/mol creatinine); MSM (12.4±1.0); SM (13.4±1.0); p<0.0001. Plasma levels of angiopoietin‐2 and syndecan‐1 were also elevated in MSM and SM compared with HC (p<0.0001), and significantly correlated with each other. For HC compared with MSM, imaging results showed eGC degradation [an increase in the perfused boundary region (mean±SEM 1.55±0.04 microns vs.1.68±0.05; p<0.05)], increased microvascular density (549± 34 μm/mm2 vs. 649 ±38; p<0.05)], and a decrease in the extent of RBC filling (79.1± 0.98% vs. 74.9±1.02%; p<0.01). In conclusion, these results provide evidence for degradation of the eGC that could contribute to pathogenesis of vascular dysfunction in malaria. Decreased nitric oxide formation, endothelial activation and increased adhesion of infected RBC are potential consequences of the eGC degradation and vascular dysfunction. Administration of an agent that prevents or repairs eGC degradation may be helpful as adjunctive therapy for malaria.

Support or Funding Information

National Institutes of Health (R01 HL130783)
Evaluation of Sucrose‐enriched Diet Consumption in the Development of Risk Factors Associated to Type 2 Diabetes, Atherosclerosis and Non‐alcoholic Fatty Liver Disease in a Murine Model
Authors: Plazas-Guerrero Carolina Gabriela , de Jesùs Acosta-Cota Selene , Castro-Sánchez Francisco Humberto , Ríos-Burgueño Efrén Rafael , Sarmiento-Sánchez Juan Ignacio , Picos-Corrales Lorenzo Antonio , de Jesús Vergara-Jiménez Marcela , Osuna-Martìnez Ulises
Published: April 2020

Introduction

Overconsumption of sucrose has been associated with negative metabolic effects related to non‐communicable diseases. However, this relationship continues to be a controversial topic and further studies are needed to generate scientific evidence to help clarify existing questions and which can be used in the future for the implementation of prevention and control strategies of this important health problem.

Objective

The aim of this study was to evaluate the sucrose‐enriched diet consumption in the development of risk factors associated to type 2 diabetes, atherosclerosis and non‐alcoholic fatty liver disease in a murine model.

Methodology

Twelve healthy male Wistar rats were randomly divided into two experimental groups (n=6 each) which received one of two different diets for twenty five weeks. Control group (CG) was fed with a standard laboratory diet and tap water. Sucrose group (SG) received the same laboratory diet and water enriched with 50% w/v of sucrose. Body weight, food, macronutrient and total caloric intake were determined. After intervention period, blood samples, hepatic and aortic tissues were collected. Glucose, insulin, total cholesterol, triglycerides, very low‐density lipoprotein cholesterol, low‐density lipoprotein cholesterol and high‐density lipoprotein cholesterol levels were determined in serum. Insulin resistance was calculated and total hepatic cholesterol was measured. Histopathological analysis was made for liver and aortic tissues. Results of parametric tests were expressed as mean values ± standard errors of the mean and for non‐parametric tests, results were expressed as medians and ranks. A p value of < 0.05 was considered significant.

Results

Sucrose‐enriched diet‐fed rats showed a decrease in food, lipids and protein intake as well as in serum total cholesterol levels, an increase in carbohydrates intake, glucose, insulin, triglycerides, very low‐density lipoprotein cholesterol and high‐density lipoprotein cholesterol levels, the development of insulin resistance, a higher percentage of steatosis and a greater degree of non‐alcoholic steatohepatitis. No significant differences were found in body weight, total caloric intake, low‐density lipoprotein cholesterol and hepatic cholesterol between the experimental groups. In aortic tissue samples, both groups showed a slight wall thickening.

Conclusion

Our results show that sucrose‐enriched diet consumption during twenty five weeks contribute to the development of risk factors associated to type 2 diabetes, atherosclerosis and non‐alcoholic fatty liver disease in male Wistar rats.
Eicosapentaenoic Acid Prevents Obesity‐induced Metabolic Impairments through The Host‐genetic Dependent Effects of Resolvin E1
Authors: Pal Anandita , Al-Shaer Abrar E. , Guesdon William , Torres Maria J. , Armstrong Michael , Quinn Kevin , Reisdorph Nichole , Neufer P. Darrell , Shaikh Saame Raza
Published: April 2020

Eicosapentaenoic acid (EPA) is a n‐3 polyunsaturated fatty acid that is poorly consumed in the western diet. Increased EPA consumption has been reported to improve glucose and insulin homeostasis in rodent models. We demonstrate that administration of pure EPA ethyl esters to C57BL/6J male mice improved obesity‐induced glucose intolerance, hyperinsulinemia, and hyperglycemia. Analyses of National Health and Nutrition Examination Survey data also revealed fasting glucose levels of obese adults to be inversely related with EPA intake in a sex‐dependent manner. To investigate potential mechanisms by which EPA improved glucose homeostasis, we compared rodent models consuming a control and a high fat diet in the presence or absence of EPA. 16S rRNA sequencing demonstrated that EPA supplementation was not associated with an improvement in the murine gut microbiome composition. Subsequent untargeted and targeted mass spectrometry analyses revealed distinct modifications to the lipidome. Notably, EPA dramatically enhanced the levels of 18‐hydroxyeicosapentaenoic acid (18‐HEPE) in the heart, white adipose tissue, and liver. Therefore, we investigated if administration of the downstream bioactive metabolite of 18‐HEPE, resolvin E1 (RvE1) could improve hyperinsulinemia and hyperglycemia. We also determined if these effects were mediated through the RvE1 receptor ERV1/ChemR23. RvE1 administration to obese mice mitigated hyperinsulinemia and hyperglycemia in a manner that was dependent on ERV1/ChemR23, as revealed with ERV1/ChemR23 knockout mice. Finally, we assessed if the host genome altered the metabolic outcomes of RvE1. Secondary SNP analyses revealed extensive genetic variation in human EPA and RvE1 metabolizing genes. RvE1’s effects on fasting insulin and glucose were divergent in diversity outbred mice that model human genetic variation. In conclusion, increased intake of EPA ethyl esters prevent obesity‐induced metabolic impairments through RvE1 binding to ChemR23. The data also underscore the critical need for precision prevention studies that account for host‐genetic variants in the EPA‐RvE1 axis.

Support or Funding Information

R01AT008375 (SRS), P30DK05635 (SRS), NIH/NCRR S10 RR026522‐01A1 (NR), R01DK096907 (PDN)
Defining roles of the sterol recognition region in Hedgehog cholesterolyation
Authors: Purohit Rahul , Vielmas Erika , Peng Daniel S. , Ondrus Alison
Published: April 2020

The Hedgehog (Hh) signaling pathway plays an essential role during embryogenesis and when dysregulated, can lead to human cancer (basal cell carcinomas) and the most lethal form of juvenile medulloblastoma. Binding of the Hh ligand to its receptor Patched (Ptch) relieves inhibition of the GPCR protein Smoothened (Smo) leading to activation of the Gli transcription factors and changes in target gene expression. The Hh ligand is decorated with two covalent modifications, N‐terminal palmitoylation and C‐terminal cholesterolyation. Hh proteins are synthesized as approximately 45 kDa full‐length species, whereupon a unique process yields the mature morphogen: an autocatalytic cleavage that splits the protein in two and attaches cholesterol to the newly formed C‐terminus. Of the two approximately equal molecular weight cleavage products, the dually lipidated N‐terminal fragment (Hh‐N/“Hog” domain) constitutes the classical Hh morphogen, whereas the C‐terminus (Hh‐C/“Hint”) is responsible for the cleavage reaction.

In order to elucidate the chemical mechanism of cholesterolyation, we have determined the roles of the previously uncharacterized sterol recognition region (SRR) in Hh proteins. Using bioinformatics, alanine scanning, and biochemical assays we have identified key motifs and specified residues of the SRR that are required for biochemical processing of Hh. These results provide a first in detail insights into the role of this unique small region of the Hh protein that is capable of cellular targeting and engaging cholesterol to generate an active Hh ligand.

Support or Funding Information

This work was supported by the Margaret E. Early Medical Research Trust.
Selective Stimulation of Vagal Pathways Using a Multi‐contact Circumferential Cuff Electrode
Authors: Shulgach Jonathan A. , Nanivadekar Ameya C. , Miller Derek , Fulton Stephanie , Sciullo Michael , Ogren John , Wong Liane , McLaughlin Bryan , Yates Bill J. , Horn Charles C. , Fisher Lee E.
Published: April 2020

Vagus nerve stimulation (VNS) has been used to treat epileptic seizures, depression, cardiovascular disease, and obesity; however, non‐selective vagal stimulation can produce adverse side effects, such as nausea and reduced blood pressure. The current study was designed to assess the potential for a precision, multi‐contact nerve cuff electrode to produce selective and discrete activation of vagus nerve fibers. We used an acute anesthetized ferret preparation (n = 6) to test the effects of an abdominal vagus nerve electrode (Micro‐Leads Neuro) on cellular responses (compound action potential, CAP) recorded from a 32‐channel microelectrode array (MEA) inserted into the nodose ganglion. We investigated the selectivity of nodose responses by varying: (1) electrode contact stimulation pair; (2) pulse width, using 0.1, 0.5, and 1.0 ms; and (3) pulse amplitude, steps of 20 μA. Stimulation was delivered at 2 Hz using the average response from 120 pulses per trial. Selectivity was quantified by the number of MEA channels responding to only one of the two cuff stimulation pairs. Across all animals, stimulation selectively evoked responses recorded by multiple different contacts in the MEA. In a similar experimental setup in three additional animals, multi‐contact stimulation of the abdominal vagus was used to drive changes in gastric myoelectric activity (GMA), as assessed by a set of four to six 4‐contact electrode planar arrays (Micro‐Leads Neuro) sutured to the serosal surface of the stomach. In one animal, selective stimulation drove changes in GMA without a concomitant change in heart rate (HR) or respiration rate (RR), while in two other animals, there was a selective change in HR or RR without a change in GMA. The present study supports the viability of producing selective responses from multi‐contact abdominal vagus nerve cuff electrodes and suggests the need to further investigate the effects of selective VNS on end‐organ therapeutic and off‐target effects, such as gastrointestinal motility, heart rate, blood pressure, inflammatory responses, and metabolic effects (e.g., blood cytokine levels and insulin release).

Support or Funding Information

This research was supported by funding from the NIH SPARC Common Fund Program (award #U18TR002205).
Aged Plasma and Plasma‐Derived Factors Drive Detrimental Effects in the CNS of Young Mice
Authors: Le David , Liu Angela , Campbell Meghan K. , Yang Cindy , Minami S. Sakura , Braithwaite Steven P. , Czirr Eva
Published: April 2020

Aging results in cognitive decline and molecular changes that contribute to the risk of developing aging‐associated disease. As the aging population is rapidly increasing, with those over 60‐years‐old set to double by 2050, there is urgent need for a better understanding of the aging process and development of therapeutics for diseases of aging. Recent publications have shown that some aging‐associated phenotypes can be driven by plasma and plasma‐derived factors. In these studies, we sought to identify and validate detrimental effects of aged plasma and plasma‐derived factors on cognitive, histological, and molecular readouts in order to discover novel therapeutic targets for age‐related diseases. Our data show that administration of aged human plasma into young immunodeficient NSG (NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ) mice results in a decrease in neurogenesis and mild impairment in cognition. These results indicate that factors in aged human plasma can drive detrimental effects in the brains of young NSG mice. Plasma proteomics have identified aging signatures and specific proteins that are potential drivers of these negative effects in aging, and beta‐2 microglobulin (B2M) has been shown to be a pro‐aging factor with deleterious effects on cognition and neurogenesis. To confirm B2M as a negative factor, we treated young mice (WT; C57BL/6J) with recombinant human B2M and assessed changes in behavioral performance and histological markers. We found that systemic administration of B2M resulted in cognitive impairment during contextual fear conditioning and decreased neurogenesis, suggesting that it is a driver of negative effects during aging. Our data elucidate molecular pathways that contribute to the aging process and provide promising targets for developing translational therapeutics.

Support or Funding Information

Studies were funded by Alkahest.