bluuebunny/crossref_metadata_2025_split huggingface dataset:
Urzymology: experimental access to the origins of catalytic activity and translation (967.8)
Authors: Carter Charles , Weinreb Violetta , Li Li , Collier Martha , Chandrasekaran Srinivas , Fried Howard 
Published: June 2021

Urzymology is the enzymological characterization of invariant cores, containing ~100 ± 40 amino acids, identified in enzyme superfamilies (1,2). Urzymes prepared from Class I and Class II aminoacyl‐tRNA synthetases (aaRS) accelerate cognate amino acid activation and tRNA acylation more than 105 times faster than necessary to support ribosome‐independent assembly of polypeptides and are therefore themselves highly evolved (3). Their high catalytic activities afford an experimental basis for combinatorial analysis of the effects of modular enhancements thought to have been involved in generating modern enzymes (4).Among the central questions now accessible via the study of Urzymes are the likely origin of Class I and II aaRS on opposite strands of the same ancestral gene (5); the origin of intramolecular communication (6,7); the modular construction of the Urzymes themselves, and hence their descent from even more primitive catalysts; and the suggestion that an operational RNA code preceded development of the canonical genetic code.Grant Funding Source: NIGMS 78227 and 40906
Identification of an antifungal peptide from hemolymph of an insect, Manduca sexta (976.4)
Authors: Alsouhail Qasim , Hiromasa Yasuaki , Kanost Michael 
Published: June 2021

Insects secrete antimicrobial peptides as part of the innate immunity response. These peptides are often cationic, low molecular peptides with diverse structures. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized a newly identified peptide, antifungal peptide‐1 (AFP1) from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). AFP1 was isolated by size exclusion chromatography from hemolymph plasma of larvae. Fractions containing activity against the yeast, Saccharomyces cerevisiae were analyzed my SDA‐PAGE and MALDI‐TOF MS/MS and found to contain a 41 residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome data bases. AFP1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to AFP1, each with 6 conserved Cys residues. A cDNA for AFP1 was cloned from cDNA prepared from fat body RNA. AFP1 was produced as a recombinant protein in Escherichia coli. Purified natural and recombinant AFP1 were active against S. cerevisiae, with IC50 of 15 micromolar, but they had no detectable activity against bacteria. Experiments to investigate activity of AFP1 against other fungi are ongoing. AFP1 mRNA level strongly increased after larvae were injected with yeast or with Micrococcus luteus. Our results indicate that synthesis of AFP1 is part of the innate immune response to infection in M. sexta.Grant Funding Source: Supported by NIH grant GM41247.
Modeling the Alcohol Binding Site of the NMDA Receptor Using the GluA2‐receptor Structure (LB110)
Authors: Ali Moid , Gundamraj Shivani , Kaur Tejas , Morris Sarah , Puri Simar , Singh Ricky , Singh Vipul , Smith‐Feinburg Lee , Wang Leah , Tuinstra Robbyn , Peoples Robert 
Published: June 2021

According to the National Institute of Alcohol Abuse and Alcoholism, about 18 million people have an alcohol abuse disorder. Alcohol binds to the N‐Methyl‐D‐aspartate Receptor (NMDA) receptor, inhibiting cognition, short‐term memory formation, motor coordination, and overall regular CNS function. The Brookfield Academy SMART (Students Modeling A Research Topic) Team used 3D printing technology to model the alcohol binding site on the NMDA receptor. This receptor is an ion channel in CNS neurons. Binding of the neurotransmitter, glutamate, allows the passage of calcium and sodium ions through the channel, thus controlling multiple intracellular signaling pathways. Alcohol inhibits the gating of the receptor, preventing the flow of ions, leading to the symptoms of intoxication. The NMDA receptor is a heterotetramer, containing two GluN1 and two GluN2A subunits. Alcohol binds to the transmembrane domain of the receptor, interacting with the amino acids Gly638, Phe639, Phe639, Leu819, and Met818 (of subunit GluN1) and Met 823, Phe636, Leu824 and Phe637 (on GluN2A). Site‐directed mutagenesis studies have identified the importance of these residues. Mutations in the same position on different subunits can drastically modulate the inhibition of the receptor by alcohol. Further understanding of the NMDA receptor mechanisms could lead to treatment for long‐term alcohol abuse.Grant Funding Source: The SMART team program is supported by a grant from NIH‐CTSA.
Bioactive lipid metabolism by carboxylesterase 1 (CES1) in macrophages (LB154)
Authors: Mangum Lee , Borazjani Abdolsamad , Crow John , Ross Matthew 
Published: June 2021

Human carboxylesterases (CES) have an emerging role in lipid metabolism. CES1 in macrophages is a hydrolase with broad endogenous substrate specificity, including cholesteryl ester, triacylglycerol, and 2‐arachidonoylglycerol (2‐AG), which may be relevant to cardiovascular disease and other pathologies related to lipid metabolism dysfunction. 2‐AG is an endogenous ligand of the cannabinoid receptors (CB1 and CB2) which are involved in the regulation of numerous processes, including lipogenesis, inflammation, and cholesterol flux in macrophages. To characterize the cholesterol homeostatic functions of CES1, its expression was stably knocked‐down in a THP‐1 monocyte/macrophage line (CES1KD/THP‐1) and the extent of in situ 2‐AG catabolism and cholesterol uptake and efflux from foam cells was evaluated. Expression levels of several genes involved in cholesterol and 2‐AG homeostasis were also determined. Perturbation of CES1 function altered in situ 2‐AG levels as well as the expression of multiple genes involved in cholesterol uptake and metabolism through both compensatory upregulation (CES3) and downregulation (ABCA1, CYP27A1, CD36, SRA) mechanisms. CES1KD/THP‐1 foam cells exhibited altered free and esterified cholesterol mass compared to control cells, whereas cholesterol efflux was unchanged. These finding suggest a complex central role for CES1 in macrophage cholesterol homeostasis.Grant Funding Source: Supported by NIH 1R15ES015348‐02
Photoprotective effects of reinforced galactooligosaccharides supplementation against skin damage in hairless mice (LB357)
Authors: Hong Ki‐Bae , Jeong Min Geum , Kim Jae Hwan , Park Yooheon , Suh Hyung Joo 
Published: June 2021

Ultraviolet B (UVB) radiation has been proposed as a main environmental stressor of skin damage because of its mutagenic and immunosuppressive effects as well the generation of reactive oxygen species (ROS). Photoprotective effects of probiotics have a potential to prevent UV‐induced skin damage and have drawn much attention recently. Galactooligosaccharides (GOS) are an important dietary prebiotics and generally defined as non‐digestible food ingredients that promote the function or viability of probiotics via their fermentation, but the effects of reinforced GOS between the immunomodulatory and anti‐inflammatory activities are still unclear. The aim of the present study was to investigate whether the oral administration of high content GOS by using enzyme reaction process to modulate the growth of beneficial bacteria and the immune‐suppressive effect of UVB and skin wrinkle development in male hairless mice. The oral administration of GOS was effective to alleviate skin damage by reducing the expression of primary matrix degrading enzyme as compared to control group, and modulate the water holing capacity and trans‐epidermal water loss. These result supports that a potential protective effect of high content GOS on skin damage induced by UVB exposure, and it may be mediated by the inhibition of ROS and modulation of MMP expression.Grant Funding Source: Supported by Leading Industry Development for Economic Region, MOTIE, Republic of Korea
Trans fatty acids content of vegetable oil spreads and margarines in the USA (LB385)
Authors: Balentine Douglas , Garsetti Marcella 
Published: June 2021

Historically vegetable oils spreads and margarines (“spreads”) contained significant amounts of trans fatty acids (TFAs) from partially hydrogenated vegetable oils (PHVOs). Our goal was to survey the US marketplace and determine the amount of TFAs and the use of PHVOs in spreads.We sampled 43 spreads in 2011 and 46 spreads in 2013 and we measured TFAs by capillary gas chromatography. We searched for PHVOs in the ingredient declaration.From 2011 to 2013, the mean TFAs content of spreads per 14g serving decreased from 0.51 g to 0.30 g (p=0.11). As a comparison, it was 1.7 g per 14 g serving in 2002 [1]. TFAs per 14 g serving decreased both in soft spreads (from 0.29 g to 0.13 g; p<0.05) and in stick products (from 1.62 g to 0.98 g; p=0.16). In 2013, 6 out of 11 manufacturers were no longer using PHVOs. Out of the 9 spreads found with PHVOs in 2013, 6 were private label and 4 were stick products.Compared to 2002, the average TFAs content of spreads was markedly reduced by 70% by 2011 and 82% by 2013 to an average of 0.30g per 14g serving. Most manufactures of branded products, notably Unilever and GAF, provide products without PHVOs. Some private label spreads and some stick products still contain significant amounts of PHVOs. At the end of 2013, 80% of the national volume of branded spreads was free of industrial trans fats. [1] Satchithanandam et al. Lipids 2004;39(1):11‐18.Grant Funding Source: Support for the analytical work was provided by Unilever
Identifying determinants of conformational stability in GTPases (LB607)
Authors: Sridharan Vishwajith , Isom Daniel , Dohlman Henrik 
Published: June 2021

Cellular functionalities are contingent upon the ability of proteins to maintain or transform their shape (conformation). The molecular forces that govern protein folding have long been known. These include hydrophobic interactions, hydrogen bonding, and disulfide bonds. However, how proteins maintain their conformation remains largely unanswered. In this study, twenty G proteins from Saccharomyces cerevisiae were cloned, purified from E. coli, and tested for conformational stability. The proteins vary in Tm from 35 to 60°C. This is paradoxical considering their highly analogous crystal structures and identical catalytic activity. We find that thermal stability correlates with percentage of Cys, Met, or Pro residues in the primary sequence. We also find that the stabilities group by their respective family members, with the following trend observed for least to most thermostable G proteins: Rho/Cdc42 < Ran < Large G‐proteins < Ras < Rab < Sar/Arf. Both these results suggest that thermal stability has a greater dependence on primary sequence than tertiary structure. Ultimately, a more detailed understanding of protein stability can help in engineering enzymes for new molecular therapeutics.Grant Funding Source: Supported by National Science Foundation REU Site Award 1156840 to UNC‐Chapel Hill
Normoglycemia sensitizes MDA‐MB‐231 breast cancer cells to metformin through an AMPK‐dependent mechanism (LB610)
Authors: Zordoky Beshay , Bark Diana , Soltys Carrie , Sung Miranda , Dyck Jason 
Published: June 2021

Background: Metformin has been shown to have strong anti‐proliferative effect in many breast cancer cell lines, mainly due to the activation of the energy sensing kinase, AMP‐activated protein kinase (AMPK). MDA‐MB‐231 cells are aggressive and invasive breast cancer cells that are known to be resistant to several anti‐cancer agents as well as to the anti‐proliferative effect of metformin. As metformin is a glucose lowering drug, we hypothesized that normoglycemia will sensitize MDA‐MB‐231 cells to the anti‐proliferative effect of metformin.Methods: MDA‐MB‐231 cells were treated with increasing metformin concentrations in hyperglycemic or normoglycemic conditions. The growth inhibitory effect of metformin was assessed by MTT assay. The expression of several proteins involved in cell proliferation was measured by western blotting.Results: In agreement with previous studies, treatment with metformin did not inhibit the growth of MDA‐MB‐231 cells cultured in hyperglycemic conditions. However, metformin significantly inhibited MDA‐MB‐231 growth when the cells were cultured in normoglycemic conditions. In addition, we show that metformin‐treatment of MDA‐MB‐231 cells cultured in normoglycemic conditions and not hyperglycemic conditions caused a striking activation of AMPK, and an AMPK‐dependent inhibition of multiple molecular signaling pathways known to control protein synthesis and cell proliferation.Conclusion: Our data show that normoglycemia sensitizes the triple negative MDA‐MB‐231 breast cancer cells to the anti‐proliferative effect of metformin through an AMPK‐dependent mechanism. These findings suggest that tight normoglycemic control may enhance the anti‐proliferative effect of metformin in diabetic cancer patients.Grant Funding Source: Supported by Canadian Institutes of Health Research and Alberta Innovates ‐ Health Solutions
Neonatal and adult cardiomyocytes differ in their susceptibility to the proapoptotic protein bnip3 (LB657)
Authors: Lee Youngil , Lee Hwa‐youn , Gustaffson Asa 
Published: June 2021

Bnip3 is a BH3‐only protein that is upregulated during hypoxic condition and is known to cause cell death in many different cell types. Studies have found that overexpression of Bnip3 in neonatal cardiac myocytes (NCM) induces cell death in the absence of a hypoxic insult. Here, we compared the sensitivity of NCM and adult cardiac myocytes (ACM) to Bnip3‐mediated cell death under a normoxic condition. Freshly isolated NCM and ACM were infected with an adenovirus encoding either β‐galactosidase (β‐gal) or Bnip3 for 24 hours. TMRM and Yo‐Pro1 were used to detect the changes in mitochondrial membrane potential and apoptotic cell death, respectively. Interestingly, we found that overexpression of Bnip3 failed to induce significant cell death in ACM, whereas significant cell death was observed in NCM. Ultrastuctural analysis of ACM overexpressing Bnip3 revealed normal mitochondrial architecture. Instead, Bnip3 overexpression appeared to selectively activate mitochondrial autophagy under normoxic conditions. Moreover, overexpression of Bnip3 significantly augmented oxidative stress‐mediated cell death in ACM, suggesting that Bnip3 required the presence of additional stress to activate cell death in ACM. Bnip3 is known to induce ROS production and to investigate the mechanism underlying the difference in susceptibility to Bnip3‐mediated cell death between ACM and NCM, we examined levels of the antioxidant, manganese superoxide dismutase (MnSOD) in these cells. We found that MnSOD expression was significantly higher in ACM compared to NCM. Also, susceptibility to Bnip3‐mediated cell death was restored in ACM in the presence of 2‐ME (100μM), a pharmacological MnSOD inhibitor. Taken together, these results suggest that ACM are resistant to Bnip3 due to its enhanced antioxidant capacity and that additional stress is required to switch from Bnip3’s pro‐autophagy to pro‐death function. Supported by NIH R01 HL087023Grant Funding Source: NIH R01 HL087023
A novel method to detect in vivo oxidant production reveals increased susceptibility to high‐fat diet induced penile oxidative stress in GSNOR knockout mice (LB688)
Authors: La Favor Justin , Burnett Arthur 
Published: June 2021

Excessive production of reactive oxygen species (ROS) has consistently been described as a cause of erectile dysfunction. However, direct measurement of ROS in the penis has remained elusive due to the short half life of ROS and limited accessibility to appropriate tissue beds in humans or animals. Binding of nitric oxide to a cysteine thiol is termed S‐nitrosylation, which can mediate function of many proteins. S‐nitrosylated glutathione reductase (GSNOR) is a major mediator of de‐nitrosylation, which can impact reactive oxygen/nitrogen homeostasis. The objective of this study was to optimize a new technique to measure in vivo ROS production in the mouse penis, and to investigate the effect of a high‐fat diet on penile ROS production in wild type (WT) and GSNOR knockout (KO) mice. WT and GSNOR KO mice were fed a standard chow or high‐fat diet for 3 or 6 weeks (n=3/group). A linear microdialysis probe (3mm membrane, 20kDa cutoff) was inserted into the shaft of the penis of mice under ketamine/xylazine anesthesia, allowing sampling of extracellular fluid. To measure extracellular ROS, probes were perfused with saline containing 100 µM Amplex Ultrared and 1 U/ml horseradish peroxidase. Dialysate fluorescence was measured at the outlet with a Turner Designs (TD‐700) fluorometer at 510Ex/590Em. Mean hydrogen peroxide (H2O2) concentrations were: WT (chow = 0.664, 3wk HFD = 0.666, 6wk HFD = 1.202 µM), GSNOR KO (chow = 0.735, 3wk HFD = 1.349, 6wk = 1.389 µM). Following 3 weeks of HFD, mean penile H2O2 was increased 103% in GSNOR KO vs. WT mice. It can be concluded that lack of GSNOR accelerates the development of oxidative stress in the penis in response to a high‐fat diet.Grant Funding Source: Supported by a trainee grant from the Sexual Medicine Society of North America
Virtual Care in an Outpatient Subspecialty Substance Use Disorder Treatment Program
Authors: Levy Sharon , Deister Diana , Fantegrossi John , Green Leslie , Lunstead Julie , Martinez Tamara , Riccardelli William , Schizer Miriam , Schram Jesse , Schram Patricia , Weitzman Elissa 
Published: June 2021

Objectives:
This article reports on the experience of an outpatient adolescent substance use disorder treatment program transitioning to virtual care during the COVID-19 pandemic. We describe the processes used to make the transition, including development of a safety protocol for patients seen virtually, present clinical volume data before and after the transition, and we describe a range of patient experiences through 3 clinical vignettes.

Methods:
Using data from the electronic health record, we generated counts of the total number of scheduled and completed appointments between December 2019 and June 2020. We used simple proportions to calculate the completed visit rate.

Results:
Both the absolute number of scheduled appointment and the percent of appointments completed increased with the initiation of virtual care, supporting the acceptability of this modality. Several patients experienced clinical improvements in conjunction with greater engagement, though challenges were also noted.

Conclusions:
Virtual visits are a practical mode of treatment for adolescent substance use disorders, and more evidence is needed to understand the risks and benefits of this treatment modality.
The Use of Magnesium Sulfate and Peripheral Nerve Blocks
Authors: Zeng Jie , Chen Qi , Yu Cong , Zhou Ju , Yang Bin 
Published: June 2021

Objectives:
With the popularization of ultrasound, nerve blocks have been widely implemented in current clinical practice. Although, they have seen limited success due to their shorter duration and suboptimal analgesia. Magnesium sulfate as a local anesthetic adjuvant for peripheral nerve blocks could enhance the effects of local anesthetics. However, previous investigations have not thoroughly investigated the analgesic efficacy of magnesium sulfate as an adjunct to local anesthetics for peripheral nerve blocks. Thus, we attempted to fill the gap in the existing literature by conducting a meta-analysis.

Materials and Methods:
We performed of a quantitative systematic review of randomized controlled trials published between May 30, 2011 and November 1, 2018. Inclusion criteria were: (1) magnesium sulfate used as adjuvant mixed with local anesthetic for nerve blockade, (2) complete articles and published abstracts of randomized controlled trials, (3) English language.

Primary and Secondary Outcome Measures:
The primary outcome measures were time of onset, total duration of the sensory blockade, and Visual Analog Scale pain scores. The secondary outcomes were postoperative oral and intravenous analgesics consumption and the incidence of nausea and vomiting.

Results:
The 21 trials analyzed in this study included 1323 patients. Magnesium sulfate effectively prolonged the total duration of sensory blockade (mean difference [MD]=114.59 min, 95% confidence interval [CI]: 89.31-139.88), reducing Visual Analog Scale pain scores at 6 hours (MD=1.36 points, 95% CI: −2.09 to −0.63) and 12 hours (MD=1.54 points, 95% CI: −2.56 to −0.53) postsurgery. Magnesium sulfate also effectively reduced postoperative analgesic use within 24 hours postsurgery (standard MD=−2.06, 95% CI: −2.67 to −1.35). Furthermore, adjuvant magnesium sulfate significantly reduced the incidence of nausea and vomiting after transversus abdominis plane blockade (odds ratio: 0.39, 95% CI: 0.18-0.81).

Conclusion:
Adjuvant magnesium sulfate enhanced the anesthetic effects of local anesthetics and improved postoperative analgesia following the perineural blockade.
Testosterone is Sufficient to Impart Susceptibility to Isoflurane Neurotoxicity in Female Neonatal Rats
Authors: Chinn Gregory A. , Duong Katrina , Horovitz Tal R. , Sasaki Russell Jennifer M. , Sall Jeffrey W. 
Published: June 2021

Background:
Volatile anesthetic exposure during development leads to long-term cognitive deficits in rats which are dependent on age and sex. Female rats are protected relative to male rats for the same exposure on postnatal day 7. Here we test our hypothesis that androgens can modulate chloride cotransporter expression to alter the susceptibility to neurotoxicity from GABAergic drugs using female rats with exogenous testosterone exposure.

Methods:
Female rats were injected with testosterone (100 μg/animal) or vehicle on postnatal days 1 to 6. On postnatal day 7, the animals were randomized to either isoflurane exposure or sham. Spatial memory was assessed with the Barnes maze starting on postnatal day 41. Western blots were run from testosterone treated postnatal day 7 animals to measure levels of chloride cotransporters sodium-potassium-chloride symporter (NKCC1) and chloride-potassium symporter 5 (KCC2).

Results:
Exogenous testosterone modulated isoflurane anesthetic neurotoxicity in female rats based on poor performance in the probe trial of the Barnes Maze. By contrast, females with vehicle and isoflurane exposure were able to differentiate the goal position. These behavioral differences corresponded to differences in the protein levels of NKCC1 and KCC2 after exogenous testosterone exposure, with NKCC1 increasing (P<0.001) and KCC2 decreasing (P=0.003) relative to female controls.

Conclusions:
The expression of chloride cotransporters, NKCC1 and KCC2, is altered by testosterone in female rats and corresponds to a cognitive deficit after isoflurane exposure. This confirms the role of androgens in perinatal anesthetic neurotoxicity and supports our hypothesis that the developing GABAergic system plays a critical role in the underlying mechanism.