bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Anatomy and Histology Reveal the American Alligator (Alligator mississippiensis) Exhibits Regenerative Capacity of the Tail
Authors: Xu Cindy , Palade Joanna , Fisher Rebecca E. , Smith Cameron I. , Clark Andrew R. , Sampson Samuel , Bourgeois Russell , Rawls Alan , Elsey Ruth M. , Wilson-Rawls Jeanne , Kusumi Kenro
Published: April 2020

Reptiles are the only amniotes that maintain the capacity to lose and regrow entire appendages. Among reptiles, tail regeneration has been extensively studied in Lepidosauria, the subclass that includes lizards, but this study presents the first anatomical and histological evidence of tail regeneration in an archosaur, the American alligator (Alligator mississippiensis). All animals were wild caught juveniles or sub‐adults with an average total body length of 133.4±29.2cm (n=3). Although the duration of tail regrowth is unknown, the average length of the regenerated tail was 15.7±7.3cm. The regenerated tail was externally distinct from the original tail. The scales were compacted and lacked organization as well as dorsal scutes. Gross dissection, radiographs, and magnetic resonance imaging revealed that the caudal vertebrae were replaced by a ventrally‐positioned, unsegmented endoskeleton. This contrasts with lepidosaurs, where the regenerated tail is radially organized around a central endoskeleton, but shares commonality with urodele amphibians that regrow a tail with a clear dorsal‐ventral axis. Immunohistochemistry confirmed that this alligator structure was composed of cartilage, which formed a hollow rod with foramina distributed along the proximal‐distal axis. Furthermore, in contrast to the original tail, the regenerated tail lacked skeletal muscle and instead featured adipose and fibrous connective tissue supplied by a dense network of newly established axons and blood vessels. Despite substantial regrowth of the tail, the deposition of extensive connective tissue is reminiscent of mammalian wound healing or fibrosis. Using Herovici’s stain, it was shown that the regenerated tail is composed of both type I and type III collagen fibers. The lack of muscle contrasts with the lepidosaurs, which regrow functional skeletal muscle groups, but shares similarities with regenerated limbs in Xenopus adult frogs, which have a cartilaginous endoskeleton surrounded by connective tissue, but lacking skeletal muscle. Overall, this study of regeneration in the alligator identifies a distinct pattern of regrowth with features in common with lepidosaurs, amphibia, and wound repair in mammals. Further analysis will help to shed light on the conservation of regeneration in amniote vertebrates.

Support or Funding Information

This work was funded by the College of Liberal Arts and Sciences at Arizona State University to KK.
Ras activates the first step of GPI anchor biosynthesis in Candida albicans
Authors: SETHI SUBHASH CHANDRA , JAIN PRIYANKA , PRATYUSHA VAVILALA A. , GARAI PRAMITA , NAQVI NILOFER , SINGH SONALI , PURI NITI , KOMATH SNEHA SUDHA
Published: April 2020

Glycosylphosphatidylinositol (GPI) anchor biosynthesis is a multi‐step pathway found in all eukaryotes. GPI anchored proteins are involved in a variety of functions like adhesion, host cell attachment, invasion and biofilm formation in Candida albicans, a human pathogenic fungus. The first and committed step of the pathway i.e., transfer of GlcNAc from UDP‐GlcNAc to phosphatidylinositol (PI) to form GlcNAc‐PI, involves an enzyme complex (GPI‐GnT) of six subunits viz. CaGpi1, CaGpi2, CaGpi3, CaGpi15, CaGpi19 and CaEri1. A cross‐talk exists between GPI anchor biosynthesis, Ras signaling and ergosterol biosynthesis in Candida albicans, in which CaGPI2 controls hyphal morphogenesis through CaRAS1 while CaGPI19 seems to regulate azole drug response via co‐regulation of CaERG11. Interestingly, both CaGPI2 and CaGPI19 also negatively regulate each other. Further investigations on how Ras signaling cross‐talks with GPI biosynthesis in C. albicans suggest that Ras activates GPI‐GnT activity, which runs counter to what is reported in Saccharomyces cerevisiae. Out of the two Ras proteins of Candida albicans, CaRas1 alone activates the GPI‐GnT activity. Different forms of CaRas1 too can activate the GPI‐GnT in vivo. This evidence is further strengthened by the fact that CaRas1 physically interacts with CaGpi2, as evidenced by FRET. Furthermore, CaGpi2 appears to be important for virulence since a heterozygous mutant of CaGPI2 is unable to kill macrophage cells and the mutant is more susceptible for macrophage‐mediated killing. Current work is focused on identifying the domains of interaction between CaGpi2 and CaRas1. Understanding this interaction will enable us to target any or both of these molecules to control the virulence of this pathogen.

Support or Funding Information

SSK received funding from Department of Science and Technology (DST) India Grant SB/SO/BB‐011/2014 and an umbrella grant to the School of Life Sciences from the DST under Promotion of University research and Scientific Excellence Grant DST PURSE No. SR/PURSE/PHASE 2/11 (C), and Department of Biotechnology (DBT) India Grant BT/PR3749/BRB/10/986/2011. SCS received Junior and Senior Research Fellowships from University Grants Commission (UGC), India.
Pharmacological Modulation of Akt‐dependent Activation of eNOS/NO in Airway Cells
Authors: Gopallawa Indiwari , Lee Robert J
Published: April 2020

Rationale

Ca2+/calmodulin activates endothelial nitric oxide synthase (eNOS). However, other pathway(s) by which eNOS is activated is less studied in the airway cells. We hypothesized protein kinase B/Akt plays a role in the activation of eNOS and subsequent nitric oxide (NO) production.

Methods

To test if Akt activator SC79 activates Akt, A549 a type II alveolar epithelial cell‐line was transfected with a FRET‐based AktAR sensor and were treated with SC79 (10μg/ml). Cells were treated with SC79 ± phosphoinositide‐3‐kinase (PI3K) inhibitor LY294002(10μg/ml) and were harvested for Western blotting to detect phospho(p)‐eNOS. To test the production of NO, airway cells were loaded with DAF‐FM (5μM) ± eNOS inhibitor L‐NAME (10μM) and were stimulated with SC79. To test if SC79 might be protective against cadmium‐induced barrier functions and inflammation, cells were treated with cadmium( 100μM) or TNF‐α (0.1μg/ml) ± SC79 followed by Western blotting to measure tight junction protein (TJ) Zonula occluden (ZO‐1) and qPCR to measure IL‐8.

Results

Live cell imaging data show a significant increase in Akt activity (p<0.001) with SC79. AKT‐activation increased p‐eNOS via PI3K. Further, NO was induced with SC79 and was blocked by L‐NAME. Cadmium‐induced reduction in ZO‐1 protein levels and TNF‐α induced IL‐8 were both reversed by SC79.

Conclusion

Our preliminary data suggest that Akt plays a role in phosphorylating eNOS and NO production in airway cells. Further, Akt activators such as SC79 reversed IL‐8 and induced TJ proteins like ZO‐1. Future work will be directed towards testing the downstream mechanisms of eNOS/NO with Akt activators in patient‐derived primary airway cells.

Support or Funding Information

R01DC016309, R21AI137484
Mechanisms of Altered Bile Acid Homeostasis in Offspring Exposed to Maternal Obesogenic Diet
Authors: Thompson Michael D. , Kang Jisue , Hinrichs Holly , Derse Alaina , Ferey Jeremie , Xie Yan , Moley Kelle , Tarr Phil , Davidson Nicholas
Published: April 2020

Background

Non‐alcoholic fatty liver disease (NAFLD) affects approximately one‐quarter of the global population with increasing prevalence in the pediatric population. There is currently no standard medical therapy and no diagnostic study capable of predicting which patients will develop severe disease. Recent evidence in both humans and animal models show that maternal obesity is a risk factor for the development of NAFLD and progression to NASH. We have shown in a murine model of maternal obesity the offspring develop periportal fibrosis and inflammation with associated changes in bile acid (BA) homeostasis. In this study we define the mechanisms of changes in BA homeostasis.

Methods

Female C57Bl6 mice were fed chow (CON) or HF/HS diet for 6 weeks to induce obesity and bred with lean males. Offspring were fed chow diet from the time of weaning until tissue collection. BA pool size and levels of specific intrahepatic bile acid species were measured. RT‐PCR was performed to evaluate gene expression of factors involved in bile acid metabolism and signaling. 16S sequencing was performed for evaluation of offspring microbiome. Levels of stool BAs were quantified and timed BA excretion performed.

Results

Offspring from the maternal HF/HS (mHF/HS) lineage exhibited an increase in total BA pool size with associated changes in expression of genes involved in bile acid metabolism (increased Cyp7a1, Cyp27a1). Measurement of specific bile acids in liver showed a shift in the intrahepatic BA pool composition (decreased deoxycholic acid, increased muricholic acid) in offspring from the mHF/HS lineage. Decreased BA excretion was observed in mHF/HS lineage offspring. Fecal microbiome analysis showed a decrease in alpha diversity and shifts in the abundance of particular phyla (increased Bacteroidetes, decreased Proteobacteria) and genera (increased Barnesiella, decreased Clostridium). Measurement of cecal BA levels showed an increase in abundance of muricholic acid (FXR antagonist) and a decrease in abundance of deoxycholic acid (FXR agonist) in mHF/HS lineage offspring.

Conclusions

Maternal HF/HS diet exposure leads to pathologic changes in the offspring including periportal fibrosis and inflammation, which is associated with changes in BA homeostasis. Several factors are involved in the BA homeostasis changes including BA metabolism and excretion. Vertical transmission of gut dysbiosis may be a primary factor in driving changes in BA homeostasis in offspring exposed to maternal obesogenic diet.
Impact of DNA End Structure and Cellular Growth Phase on DNA Repair by Nonhomologous End‐Joining (NHEJ) in Saccharomyces cerevisiae
Authors: Fitzgerald O’Taveon R. , Rodriguez Nestor D. , Lewis L. Kevin
Published: April 2020

DNA within the cell is constantly bombarded by endogenous sources of damage (replication errors, cellular metabolism) and exogenous sources (radiation, chemical exposure), resulting in the formation of more than 100,000 DNA lesions per cell daily. There are several potential consequences of this damage, including stimulation of a cell stress response, changes in gene transcription, induction of controlled cell death (apoptosis), and the activation of DNA repair pathways. Mutations causing impaired DNA repair are associated with multiple human genetic disorders, which frequently lead to an increased cancer risk. A double‐strand break (DSB) is the most deleterious type of DNA damage, as it promotes mutations, chromosomal rearrangements, and other genomic changes that can lead to cell death. The two primary pathways that repair DSBs are Homologous Recombination (HR) and Nonhomologous End‐joining (NHEJ). This project is focused on the NHEJ pathway in Saccharomyces cerevisiae, which is mediated by three main protein complexes: Yku70‐Yku80 (Yku), Mre11‐Rad50‐Xrs2 (Mrx), and Dnl4‐Lif1‐Nej1 (Dnl). Our experiments use plasmid NHEJ assays to measure the efficiency and accuracy of DSB repair in mutant strains of yeast deficient in DNA repair. This is done by transforming plasmids containing a DSB in one of two selectable markers into cells and utilizing the NHEJ pathway to repair the plasmid. The results are then quantified by measuring cellular growth on selective media to determine the degree of reduced repair efficiency. Preliminary results indicated that the Mrx DNA end‐bridging complex is situationally required for efficient NHEJ, while the Yku and Dnl complexes are always required. The requirement for Mrx was dependent on the cell growth phase, as it was lower in cultures with high levels of G1 cells. Mrx was always required for efficient repair of ends with 3′ single‐stranded overhangs, but not for DSBs with 5′ single‐stranded overhangs. Our hypothesis is that Mrx may play a protector role at DSB ends containing 3′ overhangs, shielding them from nucleases such as Exo1 or Sgs1‐Dna2 that may preferentially target this end structure. In addition, using an improved version of the plasmid assays, we have identified three new S. cerevisiae genes that affect NHEJ repair efficiency. Current experiments are investigating the role of chromatin remodeling proteins Arp5, Arp6, and Arp8, along with DNA end‐processing proteins, to assess their role in NHEJ repair.
Four Weeks of Acute Intermittent Fasting Enhances Body Composition and Decreases Stress Levels in Healthy Individuals: A Pilot Study
Authors: Bains Gurinder , Moh Michael , Lohman Everett , Daher Noha , Silver Sasha , Zamora Francis , Berk Lee
Published: April 2020

Introduction

The obesity epidemic affects nearly 78 million adults in the United States. Weight loss and improvement of body composition through physical activity and dietary modifications, can help decrease the risk for obesity‐related disease. Weight management programs are numerous, with an estimated 45 million Americans undertaking a diet each year. There are several different types of diets: such as those that emphasize consumption or an increase/decrease in protein, carbohydrates, fat, or amount of total daily calories. A novel, modified alternative method to caloric restriction is Intermittent Fasting.

Purpose

To determine the acute effect of four weeks of Intermittent Fasting on body composition, stress levels, sleep quality, and quality of life.

Methods

Sixteen participants, 9 males and 7 females, with mean age 34.0 ± 11.7 years, completed a four‐week session of Intermittent Fasting. Participants were excluded if they had Diabetes or Metabolic Syndrome. The participants fasted for 16 hours from the time of their last meal of the evening to the start of their first meal the following day. Once the fast was broken, the participants were required to consume their normal daily caloric intake within an 8‐hour period, with no requirements on the number of meals. Participants did not make changes to their activity levels throughout the four weeks. Body composition was determined using the InBody 770 (InBody USA, Cerritos, CA). The Pittsburgh Sleep Quality Index and the Cohen Perceived Stress Scale were administered to determine sleep quality and self‐awareness of stress levels. Quality of Life was assessed using the 36‐Item Short Form Survey Instrument.

Results

Following four weeks of Intermittent Fasting, there were significant reductions in mean body fat percentage (26.2±2.5 vs. 25.4±2.5, p=0.01), visceral fat (cm2) (98.2±15.3 vs. 94.1±15.6, p=0.003), body fat mass (lbs) (47.5±6.6 vs. 46.2±6.9, p= 0.02), trunk fat (lbs) (25.2±3.5 vs. 24.2±3.5, p=0.001) and stress levels (14.1±1.9 vs. 11.0±1.4, p=0.03). There were no significant acute changes in sleep quality and overall quality of life.

Conclusion

Intermittent Fasting can be an effective option to help lower body fat and more importantly visceral fat. This in turn, could potentially aid in decreasing obesity‐related diseases. Our pilot study demonstrated potential health related benefits from an acute Intermittent Fasting program that could be incorporated into whole person wellness programs. Further research is needed to expand these positive findings over different time segments.
Building a Community of Practice for the Assessment of Biomolecular Visual Literacy
Authors: Procko Kristen , Beckham Josh T. , Dean Diane M. , Dries Dan R. , Engelman Shelly , Franzen Margaret A. , Jakubowski Henry , Novak Walter R.P. , Porter Sandra , Roberts Rebecca , Roca Alberto I. , Shor Audrey C.
Published: April 2020

The ability to understand, interpret, and create molecular images is a critical learning objective in biochemistry and molecular biology courses. Yet biomolecular visualization skills are rarely explicitly taught. Over a two‐year period, the BioMolViz group organized seven one‐day workshops to explore and establish best practices for the generation, revision, and finalization of biomolecular visualization (BMV) assessments. Our process utilizes a BMV framework (biomolviz.org) to build targeted assessments that address specific learning goals and objectives. We then revise the assessments through a peer review process and integrate a final analysis of images for accessibility to those who are colorblind. Although there is a wealth of information to guide the creation of multiple‐choice questions, we have found that there are few resources for the generation and evaluation of other types of assessments. For example, BMV assessments are more likely to be open‐ended and specifically target molecular visualization skills (e.g. require the student to create an image of DNA or identify a structural feature on a given image of a protein). We invite participants to help us address this gap and refine our method for generating assessments by attending our upcoming three‐day NSF‐funded summer workshops. Activities will include revising existing assessments and creating instruments of interest to new participants. In addition to producing high‐quality BMV assessments aligned with the framework, these workshops will offer a unique professional development for instructors interested in promoting visual literacy by building a community of practice for the instruction of BMV skills.

Support or Funding Information

This work was supported by the National Science Foundation under award numbers DUE‐1022793, ‐1323414, ‐1503811, IUSE‐1712268, and RCN‐UBE‐1920270. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the National Science Foundation.
Metabolic Reconstruction and Modeling From Sequenced Genomes
Authors: Karp Peter D. , Caspi Ron , Ong Wai Kit
Published: April 2020

We present new developments in the bioinformatics algorithms that reconstruct the metabolic network of an organism from its sequenced genome, and that convert that metabolic network into a quantitative metabolic model. These algorithms are implemented in the Pathway Tools software. The Pathway Tools metabolic reconstruction process begins with an annotated genome; its first step infers the subset of metabolic reactions in the MetaCyc database that are catalyzed by enzymes within the genome. Reactome inference is based on the enzyme name and/or EC numbers within the genome annotation. The second step uses the inferred reactome to infer the subset of MetaCyc metabolic pathways present in the organism using a pathway scoring algorithm. Recent improvements to these algorithms are based on a large increase in the number of EC numbers in recent years, growth in the reaction (+30%) and pathway (+22%) content of the MetaCyc database, and development of a new pathway scoring algorithm that makes use of key reactions that must be either present or absent, and pathway taxonomic range information. The resulting metabolic reconstruction is available in the form of a Pathway/Genome Database (PGDB) that can be queried and visualized via an extensive set of online tools. The BioCyc.org website contains such PGDBs for 14,700 organisms.

The MetaFlux component of Pathway Tools enables creation and execution of quantitative steady‐state metabolic flux models. Several steps are used to refine a PGDB to a quantitative steady‐state metabolic flux model. First, we must identify a set of nutrients that support growth of the organism, and we must identify a set of biomass metabolites that form the molecular building blocks of the organism. Next we ensure that the reactome within the PGDB can synthesize each of the biomass metabolites from the provided nutrients. Often the reactome contains gaps due to incompleteness in the genome annotation. We present a new taxonomic gap‐filling algorithm with significantly improved accuracy: when evaluated on randomly degraded variants of the Escherichia coli metabolic model, it shows an average F1 score of 99.0, compared to 91.0 for the previous Pathway Tools gap filler, and compared to 80.3 for a basic gap filler. Once gaps have been filled in the reaction network, MetaFlux can execute the model, using flux‐balance analysis to compute flux rates for all reactions in the model. Those fluxes can be interpreted by visualizing them on a whole‐organism metabolic network diagram. MetaFlux can also simulate the effects of single and double‐gene knock‐out mutants on the model. And MetaFlux can execute groups of models that simulate the growth of organism communities.

Support or Funding Information

This work was supported by the National Institutes of Health under grants GM080746 and GM075742. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
GlycoSense&[trade]: A Flow Cytometry‐Based Technology for Rapid and Simplified Glycan Profiling
Authors: Saunders Matthew , Woods Robert J. , Yang Loretta
Published: April 2020

Lectenz Bio has developed an innovative technology called GlycoSense™ that detects binding between glycans and glycan‐specific reagents that are conjugated to spectrally‐unique beads. By combining the individual bead‐based reagents into a multiplex array, a profile of key glycan features can be obtained in a few minutes on a basic cytometer after an incubation step. We illustrate the performance of the method using glycoprotein standards and demonstrate its utility by monitoring changes in terminal glycosylation and linkages upon treatment with glycoenzymes. The GlycoSense™ approach addresses the unmet need for a rapid and robust tool to analyze protein glycosylation. In addition to established carbohydrate‐detection reagents such as antibodies and lectins, this approach can employ the engineered proteins (known as Lectenz®) developed by Lectenz Bio.

Support or Funding Information

NIH U01CA221213, R43GM123863
Assessing anti‐cancer potential of novel staurosporine analogs
Authors: Hurysz Brianna , Mowery Patricia
Published: April 2020

Staurosporine, a compound isolated from Streptomyces bacteria, is known to inhibit cell growth and induce apoptosis in cells, though it is a pan‐kinase inhibitor and therefore too toxic to be an anti‐cancer agent. However, staurosporine analogs with increased specificity have been tested in clinical trials. While most groups focus on modifying the sugar portion of staurosporine, we have explored the biological activity of novel analogs consisting of a central five‐membered ring structure with different substituents located at the 3 and 4 position. These molecules are structurally similar to the five‐membered pyrrolinone group and bis‐indole complex contained within staurosporine. Our most potent first generation analogs had a low micromolar IC50 value (concentration that killed 50% of histiocytic lymphoma U‐937 cells) and provided initial structural rules for effective cell targeting. We now report on a second generation of compounds with nanomolar potency. Unlike staurosporine, preliminary direct kinase binding data showed that our most potent analogs weakly bind to protein kinase C, indicating a unique mode of action. Compound analysis by the National Cancer Institute against a panel of 60 different cancer cell lines should allow us to narrow the cellular target. Using fluorescent microscopy analysis, we are confirming that the compounds induce cell death via apoptosis. Thus, our new findings aid the cytotoxicity studies in explaining the impact of our compounds on cancer cells.
Regulating ER protein homeostasis by differentially processing mRNAs
Authors: Li Weihan , Crotty Kelly , Ruiz Diego Garrido , Peschek Jirka , Walter Peter
Published: April 2020

The endoplasmic reticulum (ER) protein folding capacity is balanced with the protein folding burden to prevent accumulation of un‐ or misfolded proteins. The ER‐membrane resident kinase/RNase Ire1 maintains ER protein homeostasis. Ire1 forms oligomers upon activation and initiates two distinct mRNA processing programs. First, in both metazoans and Saccharomyces cerevisiae, Ire1 catalyzes the non‐conventional cytoplasmic mRNA splicing of XBP1 (metazoans) or HAC1 (S. cerevisiae)—thereby initiating a transcriptional response that increases the ER folding capacity. Second, in metazoans and Schizosaccharomyces pombe, Ire1 selectively degrades ER‐localized mRNAs—thereby post‐transcriptionally reducing the ER’s protein folding burden. Thus, Ire1 orthologs in S. cerevisiae and S. pombe are specialized to only one of the two functional outputs, while Ire1 in metazoans can perform both. Here, we show that the respective Ire1 orthologs have become specialized for their functional outputs by divergence of their RNase specificities. Using a novel screening method in bacteria, we found that Ire1’s RNase specificity is regulated by rewiring a salt bridge at Ire1’s dimer interface. In addition, RNA structural features separate the splicing substrates from the decay substrates. Using these insights, we de novo engineered non‐conventional mRNA splicing in S. pombe, which confers S. pombe with both Ire1 functional outputs.
Dynamic changes of intracellular Ca2+ content of rats brain after traumatic brain injury and its correlation with the concentration of calcitonin gene‐related peptide
Authors: Xue Wen , Duan Hubin , Qiao Xiaohong , Hao Chunyan , Zhang Xiaoyi , Du Yanrong , feng zhigang , Guo Zhiyong , Zhen Junping , Li Youliang
Published: April 2020

Objective

To investigate the dynamic changes of intracellular Ca2+ content of rats brain after traumatic brain injury (TBI) and its correlation with the concentration of calcitonin gene‐related peptide (CGRP)

Methods

The rats model with TBI was prepare by Feeney’s methods. 144 male Wistar rats were randomly divided into Sham operation group(Sham group, 36 rats) and traumatic brain injury group (TBI group, 108 rats ). Above two groups subdivided into six groups by 2h,6h,12h,24h, 48h and 72h after TBI( 6 rats in each group. The calcium content in the rats brain cells were detected by laser scanning confocal microscope, and the CGRP concentration in plasma of rats were got by enzyme‐linked immunosorbent assay.

Results

There were significant differences of calcium fluorescence intensity in the rats brain cells between the Sham group and the TBI group (P<0.05). In the meantime there were also significant differences of the CGRP concentration in plasma of rats between the Sham group and the TBI group (P<0.05). After the injury, calcium fluorescence intensity in the rats brain cells began to rise within 2 hours, reached its peak in 24 hours, and decreased in 48 hours (P<0.05). In the TBI group, the CGRP concentration in plasma of rats increased at 2h, then decreased at 24h,48h, and increased slowly at 72h after TBI (P<0.05).

Conclusion

There was a negative correlation between CGRP concentration in rats plasma and the intracellular calcium content of rats brain. Calcium overload played an important role in the brain secondary damage after TBI.

Support or Funding Information

• This study was supported by the National Natural Science Foundation Youth Fund (30600637), China Postdoctral Science Foundation Grant (2019T120195), China Postdoctral Science Foundation Grant (2014M561207), the Shanxi Province Basic Research Program ( 2010021034‐4, 201601D011119), the Shanxi Scholarship Council of China (2011‐096 and Key Projects No.4), Department of Human Resources and Social Security of Shanxi province fund grant, The science and technology research projects of health department of Shanxi Province(2016D011119), The Teaching Reform Program of Shanxi Higher Education, The Education Reform Project of Postgraduate Students in Shanxi Province. The Project of “131” Leading Talents of Shanxi Higher Education, The Education Reform Project of Shanxi Medical University, the fund grant of the eleventh college of shanxi medical university(Lvliang Hospital).
Engaging Metabolic Pathways: Infographics to Promote Creativity and Multimodal Learning in Biochemistry
Authors: Love-Rutledge Sharifa Tahirah , Robinson Joy
Published: April 2020

In this poster, we will discuss an infographic exercise that enables students to better understand and utilize the fundamental concepts of biochemistry. The biochemistry class is typically a capstone experience for undergraduates toward the completion of a biochemistry degree. Students typically enter this course from multiple biochemistry or biotechnology tracks which leads to variation in background knowledge and experience with synthesizing information. Thus students enter the biochemistry class with a wide array of experiences leading to an uneven playing field for instructors. Understanding metabolic pathways is a fundamental part of the biochemistry classes but due to their various backgrounds students struggle with studying the material. Our exercise requires that students collaboratively create a metabolic map that highlights regulatory points, evaluate their results through peer feedback, and finally design an infographic on the effects of a metabolic disorder on energy homeostasis. This infographic exercise encourages students to review the steps in metabolic pathways, apply concepts, analyze information, and enable them to construct new knowledge. This activity has the goal of using the ASBMB required core concept of energy transformation by developing skills in accessing, comprehending, and communicating science.
Anticlastogenic and Antidyslipidemic Activities of Jatropha Gossypifolia Leaf Extract on Female Rats Exposed to Cypermethrin
Authors: Faokunla Opeyemi Olubunmi , Femi-olabisi Fehintoluwa Joy , Olorunyolemi Imoleayo Moses , Badejo Omotolani Adeola
Published: April 2020

Though cypermethrin (Cyp) has contributed considerably to human welfare, their adverse effects on non‐target organisms are significant. The lipophilic nature of this pyrethroid exacerbates its toxicity by facilitating rapid access to the various tissues which in turn increase its interaction with the central nervous system, for which pesticides have high affinity. This study was designed to investigate the anticlastogenic and antidyslipidemic effects of Jatropha gossypifolia (JG), alpha‐lipoic acid (αLA) and vitamin C (VC) on plasma lipoprotein fractions in female rats. Eighty female rats were divided into ten groups of eight animals each and treated orally for 30 days. Groups 1 and 2 were given corn oil and cypermetrhin respectively, groups 3,4,5 and 6 were co‐administered with Cyp+JG50 mg/kg, Cyp+JG100 mg/kg, Cyp+αLA50 mg/kg and Cyp+VC50 mg/kg respectively. Groups 7, 8, 9 and 10 were given only JG50 mg/kg, JG100 mg/kg, αLA50 mg/kg and VC50 mg/kg respectively. From this study, cypermethrin administration resulted in an array of dyslipidemia as evidenced by significant increase in the level of cholesterol in plasma (64.05%), vLDL+LDL (571.88%), ovary (313.97%), uterus (147.49%); and triglyceride in plasma (88.69%), vLDL+LDL (71.51%), uterus (27.27%); however, a significant decrease was observed in ovary triglyceride (70.24%). Also, a significant increase was observed in the mean polychromatic erythrocyte (429.00%) compared to the control. Co‐administration with the methanolic leaf extract of JG, αLA and VC was able to attenuate the observed cypermethrin‐induced dyslipidemia and clastogenicity. Toxicological study from this study revealed that the plant extract is therapeutically safe for consumption at the doses under investigation. It can thus be concluded that the methanolic extract of Jatropha gossypifolia leaf could help regulate the dyslipidemic effect of cypermethrin on lipid parameters in organs and plasma lipoprotein fractions. The anticlastogenic effect of the plant could also be linked to the various phytoconstituents embedded in the plant.
Charge Neutralization Tunes Dynamic Arrest of Initially Disordered Reflectin Proteins
Authors: Levenson Robert , Gordon Reid , Lee Tyler , Malady Brandon , Morse Daniel E.
Published: April 2020

The intrinsically disordered reflectin proteins fill the reflective Bragg lamellae of iridescent cells in squid. In vivo, phosphorylation of the cationic reflectins leads to protein condensation and hierarchical assembly, driving osmotic dehydration of the lamellae and causing enhancement of intensity and tuning of the color of reflected light. In vitro, purified monomeric reflectin protein can be driven to cyclably and tunably assemble by pH‐neutralization or addition of salt, forming spheres of low polydispersity and reproducible size. Analysis of reflectin assembly by dynamic light scattering, x‐ray scattering, and transmission electron microscopy shows that the calibration between charge‐neutralization and assembly size is enabled by the rapid dynamic arrest of particle growth, as controlled by an electrostatic switch spatially distributed across the reflectin chain. Confocal microscopy of fluorescently labeled micron‐sized reflectin assemblies shows that they exhibit internal dynamics that rapidly slow following assembly, suggesting that assembly occurs through a transient liquid‐liquid phase separation that undergoes gelation to form stable protein‐dense condensates. Electron paramagnetic resonance (EPR) analysis shows the initially disordered reflectin monomers form ordered secondary structure that may be critical in the arrest of growth and stabilization of particles. These results provide new insights into the assembly of these unique intrinsically disordered proteins and the biophotonic systems they form, and suggest pathways for the creation of novel tunable biomaterials.

Support or Funding Information

This research was supported by the U.S. Department of Energy, U.S. Army Research Office, and Institute for Collaborative Biotechnologies.
Ameliorative Effect of The Methanolic Leaf Extract of Jatropha gossypifolia on Cypermethrin‐Induced Heamatological Alterations of Female Wistar Rats
Authors: Olorunyolemi IM , Faokunla OO
Published: April 2020

Cypermethrin (Cyp), a synthetic pyrethroid developed for agricultural and public health applications has become harmful to the environment and mammalian life owing to indiscriminate uses. Thus, pesticide safety and how exposure may affect human health in general and hematological status in particular are of paramount concern. This study was aimed at investigating the ameliorative effects of the methanolic leaf extract of Jatropha gossypifolia (JG) on cypermethrin‐induced alterations on the haematological status of female wistar rats. Eighty female rats divided into ten groups of eight animals each were treated orally for thirty days. Groups 1 and 2 were given corn oil and cypermethrin (20 mg/kg) respectively. Groups 3, 4, 5 and 6 were co‐administered with Cyp+JG50 mg/kg, Cyp+JG100 mg/kg, Cyp+ALA50 mg/kg and Cyp+VC50 mg/kg respectively. Groups 7, 8, 9 and 10 were given only JG50 mg/kg, JG100 mg/kg, ALA50 mg/kg and VC50 mg/kg respectively. The animals were sacrificed after the 30th day. The whole blood was collected and used for haematological analyses. Results obtained from this study showed that cypermethrin administration significantly (p<0.05) decreased concentration of RBC, HCT and HGB; WBC, LYM and %LYM; and PLT, while it significantly increased MCH, MCHC and MCV; and RDW_CV. Co‐administration with the methanolic leaf extract of JG, ALA and VC were able to attenuate the adverse effects of cypermethrin exposure. Groups administered with only the plant extract, ALA and VC were therapeutically safe at the doses under study. It can be concluded from this study that the methanolic leaf extract of Jatropha gossypifolia and ALA protects against haematological alterations.
Inhibition of Toll‐Like receptors alters the ability of tributyltin to increase production of interleukin 1 beta by human immune cells
Authors: Alcala Aliyah , Whalen Margaret
Published: April 2020

Toll‐like receptors (TLR) are integral for activating the innate immune system responsible for initial detection of infection through recognition of pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). MAP Kinases are downstream signaling components that lead to the production of chemokines, type I interferons, and pro‐inflammatory cytokines, such as interleukin 1β (IL‐1β), in response to TLR activation. IL‐1β is a primary pro‐inflammatory cytokine necessary for an appropriate response to injury or infection. Dysregulation of IL‐1β levels leads to chronic inflammation, which is implicated in the development of diseases such as atherosclerosis, cancer, and autoimmunity. Tributyltin (TBT) is an environmental contaminant due to its use as a biocide in numerous products and in anti‐fouling paints. Previous studies have found that exposure to TBT, which is present in human blood, increases the secretion and intracellular concentrations (production) of the pro‐inflammatory cytokine IL‐1β in peripheral blood mononuclear cells (PMBCs) and this increase requires MAPK activation. The current study examines whether the upstream regulators of MAPK activation in immune cell production of IL‐1β, the TLR, are also required for TBT to induce increases in IL‐1β. TLRs were inhibited with compounds specific for selected TLRs and the ability of TBT (100, 50, and 25 nM) to cause increased production of IL‐1β after 24 h of exposure was examined using ELISA and western blot. Results indicate that when TLR are inhibited there is a decrease in TBT‐induced production IL‐β. This suggests that TBT utilizes the TLR to stimulate IL‐1β production.

Support or Funding Information

The National Science Foundation Tennessee Louis Stokes Alliance for Minority Participation (TLSAMP) Bridge to the Doctorate (BD) program
Crystallographic snapshots of TsrM, a radical S‐adenosylmethionine enzyme whose reaction is not so radical
Authors: Knox Hayley L. , Chen Percival Yang-Ting , Blaszczyk Anthony J. , Grove Tyler L. , Schwalm Erica L. , Wang Bo , Drennan Catherine L. , Booker Squire J.
Published: April 2020

TsrM methylates C2 of the indole ring of L‐tryptophan (Trp) as the first step in the biosynthesis of the quinaldic acid moiety of thiostrepton, a ribosomally synthesized and post‐translationally modified thiazolyl peptide natural product that exhibits potent in vitro activity against many Gram‐positive bacteria of urgent clinical relevance. TsrM is annotated as a member of the radical S‐adenosylmethionine (SAM) superfamily and exhibits hallmarks of these enzymes, such as a [4Fe–4S] cluster ligated by cysteines residing in a CxxxCxxC motif and a binding site for S‐adenosylmethionine. However, unlike all other characterized members of the radical SAM (RS) superfamily, TsrM does not catalyze a reductive cleavage of SAM to the almost universal 5′‐deoxyadenosyl 5′‐radical intermediate. Instead, recent studies suggest that it catalyzes its reaction via two polar SN2 displacements, an attack on the methyl group of SAM by a cob(I)alamin species and a subsequent attack of C2 of Trp onto methylcobalamin. Herein we report the first crystal structures of a class B RS methylase. The structure contains both the cobalamin and [4Fe‐4S] cluster cofactors bound and show the presence of unique ligands to each. We additionally have solved a structure with substrate analogs bound, which reveals a novel active site, suggesting that TsrM is only masquerading as a radical SAM enzyme.
Elucidating the role of the axial cysteine residue in NHase catalysis and the enzyme maturation
Authors: Ogutu Irene , Lankathilaka Wasantha K. P. , Holz Richard C. , Bennett Brian A.
Published: April 2020

Nitrile hydratases are metalloenzymes that contain trivalent Fe/or Co ions in their active site, coordinated in an N2S3 ligand environment. The sulfur atoms are post‐translationally modified and have three different oxidation states (αCys, Cys‐SOH and Cys‐SOO−) while backbone amines account for the nitrogen ligands and are deprotonated. Theoretical calculations assume that the αCys ligand is a thiolate, making it a strong π‐donor ligand and a good a nucleophile. A critical outstanding mechanistic question is whether the formation of a transient disulfide bond is essential for catalytic turnover. Such a transient disulfide bond was proposed based on QM/MM calculations between the αCys sulfur and the sulfur of the proposed sulfenic acid cyclic intermediate. However, more recent theoretical studies is self‐inconsistent in its conclusions regarding a transient disulfide formation. To examine the role of the αCys108 ligand in catalysis, it was mutated to A, M, S, and H in the Co‐type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase). ICP‐MS demonstrated reduced metal ion content for each mutant: <5% (αC108A), ~44% (αC108M), ~50% (αC108S) and ~55% ( αC108H) and catalytic activity also decreased to ~0.3%, ~0.8%, ~1.6%, and ~6.7%, respectively, towards acrylonitrile. The X‐ray crystal structure of αC108A confirmed the mutation and the decreased active site metal content. Therefore, the axial thiolate is important to properly tune the electronic properties of the active site metal ion, but is not essential to catalysis. These data provide insight into the critical mechanistic question of whether the axial αCys ligand forms a transient disulfide bond during turnover.

Support or Funding Information

This work was supported by (CHE‐1808711, RCH; CHE‐1308672, BB; CHE‐1532168 BB & RCH), the Todd Wehr Foundation, Bruker Biospin, and the National Institutes of Health/NIBIB National Biomedical EPR Center (P41‐EB001980)
Investigating Novel Turnover Mechanisms of Huntingtin in Yeast
Authors: Warner Amanda , Buchan Ross
Published: April 2020

Huntington’s disease is a devastating neurodegenerative disease which leads to symptoms of involuntary movements, memory loss, and ultimately death. The disease is characterized by a CAG repeat expansion mutation in the huntingtin (HTT) protein which leads to its aggregation inside the cytoplasm of striatum neurons. It is unknown whether these aggregates are directly toxic or if they reflect a cellular protection mechanism. Recently, endocytosis activity has been shown to reduce the toxicity, protein levels and propensity to aggregate of TDP‐43, a protein that forms potentially toxic aggregates in amyotrophic lateral sclerosis (ALS) patient motor‐neurons. Conversely, high expression of aggregation‐prone TDP‐43 can impair endocytosis function. Thus, there is a possibility that HTT aggregates, similar to TDP‐43 in ALS, will also inhibit endocytosis and be cleared by the endocytosis pathway. Using a Huntington’s disease yeast model, wild type and mutant HTT localize to the cytoplasm as well as the vacuole, an acidic organelle where proteins are trafficked by endocytosis for degradation. “WT” HTT with 25 CAG repeats is expressed diffusely throughout the cytoplasm while “pathological” HTT with 103 CAG repeats forms cytoplasmic aggregates, in addition to exhibiting diffuse cytoplasmic localization. Pathological HTT exhibits slight growth defects in vps34Δ and vps15Δ strains; these PI3K subunit genes are key to normal endocytic (and autophagic) function. Protein half‐life assays indicate that WT HTT has a slower turnover rate compared to the pathological HTT. Proteasome inhibition also slows WT HTT turnover but does not impact pathological HTT turnover rate. A genetic screen using the tet‐off regulatable promoter library will be conducted to determine for the first time what essential genes genetically interact with mutant HTT. Further microscopy and turnover assays will be conducted to determine if enhancement or suppression of endocytosis leads to clearance or accumulation of HTT in the vacuole respectively, and whether endocytosis activity represents a viable therapeutic target for Huntington’s disease.

Support or Funding Information

This project was funded by the University of Arizona Undergraduate Biology Research Program with funds from the Arnold and Mabel Beckman Foundation.
Identifying protein expression changes in acute myeloid leukemia
Authors: Caplan Mika , Hynes-Smith Willow , Swenson Samantha , Wittorf Karli , Gilbreath Tyler , Vahle Heather , Buckley Shannon
Published: April 2020

Acute myeloid leukemia (AML) is a devastating cancer affecting the hematopoietic system. It is characterized by infiltration of abnormal proliferating immature myeloid cells originating in the bone marrow. Although this disease represents only 35% of diagnosed leukemias, it accounts for nearly 50% of leukemia‐related deaths, making it the leading cause of leukemia‐related mortality. Disease initiation often occurs through common recurrent genetic aberrations resulting in the formation of oncogenic fusion proteins. Two common mutations include mixed lineage leukemia (MLL) rearrangements and an inversion of chromosome 16, known as Inv(16). Although many of these AML initiating mutations have been identified, the downstream effects leading to disease progression are still largely unknown. Previous research has relied on RNA sequencing and microarray techniques to study the downstream effects, providing data at the transcriptional level. While these studies have proven efficacious, they fail to capture the changes that occur at the proteomic level. To interrogate the effect of protein expression alterations in AML, we performed a quantitative mass spectrometry (MS) analysis using mouse models to compare three tumor types (Inv(16), MLL‐AF9, and MLL‐ENL) to untransformed cells from the tumor‐initiating population. In parallel, we performed RNA sequencing for the same populations. With these combined results, we identified 61 proteins whose expression was upregulated in AML tumors, but strikingly, were unaltered at the transcriptional level. These proteins are shown to be associated with mitochondrial function as well as RNA processing. In addition, analysis of patient expression data sets in adult and pediatric AML reveal that a number of the proteins differentially expressed have no significant RNA expression alterations. These studies identify a set of proteins that have not previously been associated with leukemia, and may ultimately serve as potential targets for therapeutic manipulation to hinder AML progression and help contribute to our understanding of this disease.

Support or Funding Information

National Institute for General Medical Science (NIGMS) INBRE ‐ P20 GM103427, NIH P20GM121316
Cytosolic Ca2+ modulates Golgi structure through PKC‐mediated GRASP55 phosphorylation
Authors: Ireland Stephen Curtiss , Ramnarayanan Saiprasad , Fu Mingzhou , Zhang Xiaoyan , Emebo Dabel , Wang Yanzhuang
Published: April 2020

It has been well documented that the endoplasmic reticulum (ER) responds to cellular stresses through the unfolded protein response (UPR), but it is unknown how the Golgi apparatus responds to similar stresses. In this study, we treated HeLa cells with ER stress inducers, thapsigargin (TG), tunicamycin (Tu) and Dithiothreitol (DTT), and found that only TG treatment resulted in Golgi fragmentation. TG induced Golgi fragmentation at a low dose and short time when UPR was undetectable, demonstrating that Golgi fragmentation occurs independently of ER stress. Further experiments demonstrated that Golgi fragmentation was through elevated intracellular Ca2+ and protein kinase Cα (PKCα) activity, which phosphorylates the Golgi stacking protein GRASP55. Significantly, activation of PKCα with other activating or inflammatory agents, including Phorbol 12‐myristate 13‐acetate (PMA) and histamine, modulates the Golgi structure in a similar fashion. Hence, our study revealed a novel mechanism through which increased cytosolic Ca2+ modulates Golgi structure and function.

Support or Funding Information

This work was supported by the National Institutes of Health (Grants GM112786, GM105920, and GM130331), MCubed and the Fastforward Protein Folding Disease Initiative of the University of Michigan to Y. Wang. S. Ireland is a graduate student who has been partially supported by the Mary Sue and Kenneth Coleman Endowed Fellowship Fund from the University of Michigan.