bluuebunny/crossref_metadata_2025 huggingface dataset:
Senescence in Myoblasts; Role in Age‐Related Muscle Dysfunction
Authors: Shigdar Shahjahan , Staunton Caroline , Vasilaki Aphrodite , McArdle Anne
Published: June 2021

Loss of skeletal muscle mass and function are critical factors in the development of frailty although the mechanisms by which this loss occurs are poorly understood. The number of senescent cells increases with age in rodents (Krishnamurthy et al., 2004) and primates (Kreiling et al., 2011) and it is proposed that the presence of these cells plays a role in age‐related muscle dysfunction. We hypothesise that resident satellite cells become senescent and exert detrimental effects on mature muscle fibres via the senescence associated secretory phenotype molecules (SASP).

Initial studies have investigated whether a C2C12 mouse muscle myoblast cell line could be induced into senescence, whether key senescence associated molecules (such as p16, p21, and p53) are upregulated and determined the nature of the SASP.

C2C12 myoblasts were treated for 24 hours with the topoisomerase II inhibitor, Etoposide, to cause DNA damage. Myoblasts were examined for up to 5 days following treatment for cell viability, Senescence Associated β‐Galactosidase (SAβ‐Gal) activity, immunocytochemistry and western blot analysis for p16, p21, and p53.

Treated myoblasts exhibited a significantly altered morphology consistent with a senescence phenotype, increased SAβ‐Gal activity, and cyclin‐dependent kinase inhibitor levels compared with untreated cells.

These data suggest that myoblasts can be induced into a senescent phenotype. Further work will characterise the SASP of the cells and determine the effect on mature myotubes co‐cultured with senescent myoblasts.

Support or Funding Information

Work generously funded by the British Society for Research into Ageing and the Rosetrees Trust.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nicotine Acutely Induces Hyperglycemia and Hepatic Steatosis by Altering the Sympathetic Outflow
Authors: Ruiz Henry h. , Peddibhotla Swetha , Ramalingam Latha , Moustaid‐Moussa Naima , Shin Andrew C.
Published: June 2021

Cigarette smoking is a major risk factor for cardiovascular diseases, cancers, and metabolic syndrome. Studies have shown that smoking induces insulin resistance and hyperglycemia. Nicotine, the major constituent of cigarette, is thought to be responsible for the impaired insulin sensitivity and glycemic control. However, the underlying mechanisms are poorly understood. To first examine the acute effects of nicotine on glucose metabolism, we injected chow‐fed mice with either saline or nicotine (1.5mg/kg) intraperitoneally. Interestingly, a single injection of nicotine increased blood glucose by 50% within the first 15 min that lasted at least for 1h. Further, treatment with nicotine led to impaired glucose tolerance compared to controls. Lipolysis from white adipose tissue (WAT), primarily driven by the sympathetic nervous system (SNS), can provide glucogenic substrates to liver to promote gluconeogenesis. However, mice unable to mobilize lipids from WAT due to lack of adipose‐specific adipose triglyceride lipase (ATGL) similarly elevated blood glucose following nicotine injection. On the other hand, deletion of tyrosine hydroxylase (the rate‐limiting enzyme for catecholamine synthesis) only in the periphery completely abolished the rise of glucose in nicotine group, suggesting that the sympathetic drive to peripheral organs other than WAT may be responsible for nicotine‐induced hyperglycemia. Indeed, nicotine injection increased the sympathetic outflow to liver by 20%, but not WAT, as measured by norepinephrine (NE) concentrations 1h after injection. Hepatic glycogen content was markedly reduced in nicotine‐treated animals, probably due to the SNS‐driven glycogenolysis in liver. Surprisingly, a single nicotine injection in high‐fat (HF)‐fed obese mice not only induced severe hyperglycemia, but also hepatic steatosis compared to saline‐treated HF controls. NE content in liver was significantly lower (>two‐fold) in nicotine‐treated HF group compared to HF control group. While the reasons are not clear, we speculate that unlike in chow‐fed mice, nicotine in HF‐fed mice may suppress the sympathetic drive to liver resulting in less beta oxidation and/or reduced triglyceride secretion, hence fatty liver, whereas it may raise the sympathetic outflow to WAT to increase lipolysis that in turn can promote hepatic gluconeogenesis, resulting in hyperglycemia.

Support or Funding Information

NIH K01 DK099463Presidents' Collaborative Research Initiative Grant, Texas Tech University System

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NOD‐like Receptors: Novel Regulators of the Microbiota‐Gut‐Brain Axis in Mice
Authors: Pusceddu Matteo M. , Wong Kyle Akio , Stokes Patricia , Sladek Jessica A. , Gareau Melanie G.
Published: June 2021

Background

The crosstalk between the gut and the brain has revealed a complex communication system responsible for maintaining a proper gastrointestinal (GI) homeostasis as well as affect emotional mood and cognitive functions. The immune system, which is recognized to maintain the symbiotic relationship between the host and the microbiota, plays a pivotal role in the regulation of the microbiota‐gut‐brain (MGB) axis. Specifically, nucleotide binding oligomerization domain (Nod)‐like receptors (NLR), which sense bacterial pathogens and initiate the innate immune response, are essential in maintaining GI homeostasis. Moreover, their expression in the brain implicates them as potential prime mediators of the MGB axis. The hypothalamic‐pituitary adrenal (HPA) axis is a critical component of the stress response, and has been shown to be closely correlated to the MGB axis.

Hypothesis

We hypothesized that deficiency in NLRs, (i.e. Nod1/Nod2), will dysregulate the MGB axis, including mood, cognitive behavior, and GI physiology.

Methods

Adult (6–8 weeks of age) male and female Nod1/Nod2 double knockout (NodDKO) and wild type (WT; C57Bl/6) mice were utilized. Mice were subjected to behavioral tests (novel object recognition [NOR] task, light/dark box, open field test) following exposure to an acute stress (water avoidance stress [WAS]) to assess recognition memory, anxiety‐like behavior, and general health, respectively.

Results

Deletion of Nod1 and Nod2 receptors reduced cognitive performance in both male and female mice following WAS compared to WT controls. In contrast, only male NodDKO mice exhibited anxiety‐like behavior vs. WT controls. These changes in behavior were accompanied by altered mRNA expression levels of genes for stress (↓corticotropin releasing factor receptor [CRFR]1; ↑CRFR2), cognition (↑brain‐derived neurotropic factor [BDNF]) and inflammation (↑tumor necrosis factor [TNF]α; ↓interleukin [IL]10) in the hippocampus and prefrontal cortex of NodDKO mice in a sexually dimorphic manner. Stress‐induced impairment in colonic physiology (↑short circuit current [Isc] and ↑conductance [G]) were also observed in NodDKO mice in addition to altered mRNA expression for pro‐inflammatory cytokines (↑IL6) and antimicrobial peptides (↓RegIIIγ).

Conclusion

Our study suggests that innate immune NLRs are important for the regulation of mood, cognitive memory and GI physiology in response to acute stress in part via an impaired HPA axis, demonstrating a critical role in the maintenance of the MGB axis and homeostasis.

Support or Funding Information

This work was funded by NIH 1R01AT009365‐01 (MGG), 5R21MH108154‐01 (MGG).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An apple a day: pectin is an important source of formate in the rat.
Authors: Brosnan John T. , Clow Kathy A. , Brosnan Margaret E.
Published: June 2021

Since formate plays an important role in one‐carbon metabolism we examined the potential role of pectin as a source of formate. Rats were fed purified, AIN 93‐based diets in which the source of fibre was either 5% pectin (PF rats) or 5% cellulose (CF rats). The intestinal formate content was much higher in the PF rats than in the CF rats, with ileum>caecum> large intestine. Formate was primarily found in intestinal contents rather than in tissue. The gut output and the hepatic uptake of methanol were significantly elevated in the PF rats, as was the hepatic uptake of formate. Both plasma formate and methanol were markedly higher in PF rats. Chow‐fed rats, treated with a mixture of antibiotics (vancomycin, gentamycin, rifampin) for 7 days, had markedly decreased formate levels and a complete suppression of intestinal formate output compared to chow‐fed rats without antibiotics. Hepatic uptake of formate continued in the antibiotic‐treated animals. There is a requirement for the intestinal microbiome in the maintenance of normal circulating formate levels.

Pectins are polysaccharides, rich in galacturonic acid of which approximately 80% of the carboxyl groups are esterified with methanol. These methyl groups may be removed during digestion by means of a methylesterase. We suggest that methanol, so produced, can give rise to substantial quantities of formate which becomes available to rat tissues as a source of one‐carbon groups. These findings are also relevant to experiments with cobalamin‐deficient animals in which pectin is commonly used as a cobalamin‐binding agent.

Support or Funding Information

Supported by CIHR

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational Design and Experimental Evaluation of Novel Perezone Derivates as Anticancer Agents
Authors: Rodríguez Maricarmen Hernández , Sánchez Pablo Mendoza , Jimenez Elvía Mera , Vázquez María Inés Nicolás , Miranda‐Ruvalcaba René
Published: June 2021

Cancer is one of the leading causes of morbidity and mortality worldwide. The disease is characterized by uncontrolled growth and proliferation of abnormal cells with a high probability to develop metastasis. Although conventional treatments are effective, these methods exert severe side effects due to affect normal cells. For this reason, the development of compounds that target dysregulated proteins in cancer results in a research field of interest. Recently, it was demonstrated that perezone and its synthetic isomer; isoperezone are capable to activate the apoptotic pathway, however, the protein target which explains its pro‐apoptotic activity is unknown. For this reason, the aim of this study was to perform computational studies to propose the protein target to perezone and isoperezone to design and evaluate novel perezone derivates. Proteins regulators of apoptosis pathway which have been dysregulated in cancer were selected to perform docking studies and molecular dynamic simulations with perezone and isoperezone. The proteins murine doble minute 2 (MDM2) and Poly [ADP‐ribose] polymerase 1 (PARP‐1) were selected. A family of ester derivates was submitted to docking simulations with MDM2 and PARP1 proteins to select the most promising compounds. According to these results, the compound 4 (Isoperezone‐angelate; MDM2 ΔG=− 7.65 Kcal/mol and PARP1 ΔG=−9.67 Kcal/mol) and compound 12 (monoangelate of hidroxiperezone; MDM2 ΔG=−8.92 Kcal/mol and PARP1 ΔG= −8.48 Kcal/mol) represent the best candidates due to its high affinity to both proteins, these compounds were synthesized, purified and evaluated in vitro as an anti‐cancer agent against K562 and MCF‐7 breast cancer cell lines. Compound 4 (K562 IC50=4.4 μM and MCF‐7 IC50=0.12 μM) and compound 12 (K562 IC50=2.1 μM and MCF‐7 IC50= 5.2 μM) showed a better potency than perezone (K562 IC50=9.22 μM and MCF‐7 IC50=329.2 μM) on cancer cells. In conclusion, compound 4 and 12 are examples of drugs designed in silico that show improved biological properties against human cancer cell lines.

Support or Funding Information

UNAM‐DGAPA Posdoctoral Fellowship of MHR, LANCAD‐UNAM‐DGTIC‐165.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilization of Semi‐Solid Agar Inoculation in Microbial Co‐Culture: A Novel Protocol for Antibiotic Discovery
Authors: Slaughter Syara I. , Cadet Valerie E.
Published: June 2021

Microbial co‐culture has many important applications, not least of which being the identification of novel antibiotic factors. However, current co‐culture methods including culture overlay utilizing either the spread or streak plate methods, risk contamination of samples during isolation attempts. Since the bacteria must occupy the same physical space to obtain nutrients, mixed cultures are often obtained during selection of colonies of interest. Semi‐solid agar inoculation co‐culture is an innovative culturing technique which aims to physically separate co‐cultured bacteria to aid in the isolation process. Semi‐solid tryptic soy agar (TSA) with a 1% agar concentration was shown to gellify without difficulty and was able to withstand a variety of culturing methods including lawn culturing, streaking, and point inoculation. Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and environmental soil microbes inoculated into the semi‐solid agar were able to proliferate and disseminate throughout the entirety of the plate without difficulty. When cultured on top of the inoculated agar, the previously mentioned bacteria grew as expected. Additionally, the resultant colonies exhibited comparable morphology to the same bacteria grown on non‐inoculated agar at typical agar concentration. Due to the ability of secreted factors to diffuse throughout the semi‐solid agar, interactions between the microorganisms, or lack thereof, were readily seen. This technique could prove especially powerful in increasing bacterial yield from environmental samples and aid in antibiotic research and discovery for antibiotic‐resistant priority pathogens (ARPPs).

Support or Funding Information

Funded by Divison of Research, Georgia Campus‐Philadelphia College of Osteopathic Medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PINX1 promotes malignant transformation of thyroid carcinomas through AKT, MAPK, and β‐catenin signaling activation
Authors: Kang JiHoon , Park Sunhoo , Myung Jae Kyung
Published: June 2021

Although a large proportion of thyroid cancers, specifically papillary thyroid carcinomas (PTCs), generally show indolent biological behavior, some patients present with aggressive clinical manifestations such as distant metastasis and recurrence. It was also shown that PTC could be progressed to more malignant subtype, anaplastic thyroid carcinomas (ATCs), through specific gene mutation and signaling activation. However, the key modulators regulating malignant transformation of PTCs still remain to be identified. In the current study, we found that PIN2/TERF1 interacting telomerase inhibitor 1 (PINX1) was highly expressed in ATC patient tissues and cell lines compared to PTC patient tissues and cell lines. To investigate the effects of PINX1 on proliferation and migration, we checked colony formation capacity, cell migration, and EMT markers expression after regulation of PINX1 expression. Knockout of PINX1 using specific siRNAs decreases cell proliferation and inhibits cell cycle progression in ATC cell lines, whereas PINX1 gene transfection increases cell proliferation and promotes cell cycle progression in PTC cell lines. Moreover, downregulation of PINX1 in ATC cells reduces cell migration and EMT‐related markers expression, while upregulation of PINX1 in PTC cells increases cell migration and EMT‐related markers expression. To verify whether PINX1 is involved in anaplastic progression, we investigated AKT, MAPK, and β‐catenin signaling activation which were reported to play major roles in anaplastic progression of thyroid carcinomas. Decrease of PINX1 expression repressed the phosphorylation of AKT, p38, and ERK and reduced the expression level of β‐catenin in ATC cell lines. Moreover, increase of PINX1 expression promoted the phosphorylation of AKT, p38, and ERK and upregulated the expression level of β‐catenin in PTC cell lines. These results showed that PINX1 might promote cell proliferation, migration and anaplastic progression of thyroid carcinomas through the activation of AKT, MAPK, and β‐catenin signaling pathways.

Support or Funding Information

This study was supported by a grant from the Korea Institute of Radiological and Medical Sciences (KIRAMS), funded by the Ministry of Science and ICT (MSIT), Republic of Korea. (No. 50476‐2018)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An active‐like Src conformation explains its dynamic regulation
Authors: Ahuja Lalima G , Meng Yilin , Kornev Alexandr P , Roux Benoit , Taylor Susan
Published: June 2021

Biological signaling by protein kinases plays an essential role in cellular function. The switch between inactive and active protein kinase conformations is central to their regulation. The active conformations of protein kinases are triggered and maintained by various post‐translational modifications. The inactive conformation of Src tyrosine kinase has a non‐phosphorylated activation loop and a rolled‐out αC helix. The R‐spine at the Src kinase core is disassembled via the RS3 residue when the αC helix is disengaged. Maneuvering of the C‐spine is crucial for obtaining a pseudokinase conformation of Src that could be crucial for exploring its scaffolding functions. Our Umbrella sampling methods allow for mapping the conformational landscape of the Src kinase domain and characterizing four defining states; the inactive, active conformations and two intermediates. Mutation of the C‐spine V281F allows for the closing of the two kinase lobes in the absence of ATP. An allosteric region at the β3‐αC loop allows for engaging the αC helix and the RS3 residue of the R‐spine via the L297F mutation. The L297F/ V281F double mutant allows for stabilization of a unique catalytically dead kinase conformation in the active‐like state. These studies allow for exploration of active‐like Src kinase states that define both the underlying kinase activation switch and the dynamic allostery in the molecular communication at the kinase core.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Downregulation of autophagy‐regulatory proteins contributes to induction of replicative senescence in Hs68 cells
Authors: Lee Michael , Hwang Sung‐Hee
Published: June 2021

Autophagy is reported to be involved in preventing cellular senescence. However, the extent to which autophagy contributes to senescence is presently unclear. Here, we investigated the role of autophagy in replicative senescence using Hs68 human foreskin fibroblasts. Human foreskin fibroblasts, Hs68 cells, at an initial passage of 15 were serially cultured for several months until they reached cellular senescence. Induction of replicative senescence in aged cells (at passage 40) was confirmed by senescence‐associated β‐galactosidase (SA‐β‐gal) activity that represents a sensitive and reliable marker for quantifying senescent cells. The expression level of p53, which is required for induction of senescence, p53 expression continued to increase up to passage 34. Notably, the gradual decrease in basal autophagy coincided with replicative senescence induction as determined by LC3 conversion assay. However, despite decreased basal autophagic activity in senescent cells, autophagy inducers could induce autophagy in senescent cells. RT‐PCR analysis of 11 autophagy‐related genes revealed that the decreased basal autophagy in senescent cells might be due to the downregulation of autophagy‐regulatory proteins, but not autophagy machinery components. Moreover, the senescence phenotype was not induced in the cells in which rapamycin was added to the culture to continuously induce autophagy from passage 29 until passage 40. Together, our findings suggest that reduced basal autophagy levels due to downregulation of autophagy‐regulatory proteins may be the mechanism underlying replicative senescence in Hs68 cells. In addition, alterations in autophagic activity offer an additional target for therapeutics that limit the process of senescence.

Support or Funding Information

Supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2016R1D1A1B03930193)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vitamin D Stimulates Serotonin Production via Induction of the Tryptophan Hydroxylase 2 Isoform in B14 Rat Medullary Neurons
Authors: Lucas Daniel A. , Sabir Marya S. , Mallick Sanchita , Whitfield G. Kerr , Haussler Mark R. , Jurutka Peter W.
Published: June 2021

The active form of vitamin D3, 1,25‐dihydroxyvitamin D (1,25D), and serotonin (5‐HT) are postulated to play significant roles in abnormal social behavior associated with psychological conditions including autism spectrum disorders (ASDs) and depression. Moreover, we have observed that 1,25D regulates synthesis of two isoforms of tryptophan hydroxylase (TPH), the rate‐limiting enzyme in the 5‐HT biosynthetic pathway, including induction of the tryptophan hydroxylase 2 (TPH2) gene in the central nervous system (CNS) and suppression of tryptophan hydroxylase 1 (TPH1) expression in some peripheral tissues. Interestingly, major characteristics associated with autism may include low concentrations of serotonin in the CNS and elevated levels of serotonin in the periphery. Thus, in addition to assessing the effect of 1,25D on TPH gene expression, we also examined, for the first time, the ability of 1,25D to stimulate production of 5‐HT in neuronal cells. Rat serotonergic medullary cells (RN46A‐B14; B‐14) were cultured to confluency and plated in six‐well plates. After allowing cells to attach in the plates overnight, the cells were cultured in differentiation medium, followed by treatment of differentiated B‐14 cells with ethanol vehicle or 10−8 M 1,25D. At time zero, a 200 μL sample of media from each well was collected to serve as a baseline for 5‐HT concentration. The cells were then incubated over the next three days, and additional 200 μL samples were collected from each well at the 24‐, 48‐ and 72‐hour time points. A 5‐HT ELISA assay was then conducted using the collected samples. The results of the ELISA revealed a statistically significant increase (57%, p < 0.05) in 5‐HT production following treatment with 1,25D for 24 hours versus vehicle, with subsequently larger percent‐increases in 5‐HT synthesis observed at 48 and 72 hours (64% and 103%, respectively, p < 0.05). These results suggest that 1,25D stimulates the production of serotonin, likely through the induction of the neuronal TPH2 gene, and the time‐course of this effect appears to lag behind the induction of TPH mRNA which peaks at 24 hours. In summary, this study illuminates a novel coordinated biochemical and regulatory interaction between two neuromodulators, vitamin D and serotonin, thereby identifying a potential connection between serum vitamin D levels and the occurrence of psychiatric disorders commonly linked to an imbalance in CNS serotonin production, including autism and depression.

Support or Funding Information

This project was funded by the New College of Interdisciplinary Arts and Sciences at Arizona State University and the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nutrition, Microbiome, Aging, and Cancer
Authors: Zillow Anna L , Gabriela  , Riscuta  , Brown Tony L , Tulp Orien L , Einstein George P
Published: June 2021

According to SEER data, aging is one of the highest cancer risk factors for both sexes. While cancer is a multifactorial disease of the human body, two underlying mechanisms were commonly identified in elderly: 1) low chronic inflammation and 2) immunosenescence. Microbiota, mainly probiotics, can potentially positively influence these two mechanisms directly and also via microbially produced metabolites. Dietary interventions have the potential to modify microbiota and therefore the inflammatory status and eventually could influence the risk and progression of some chronic diseases including cancer. The challenge remains as to identify the subgroups who will benefit, the time and duration of each nutrition intervention, and early detection of any undesirable effects of those interventions.

The objectives of this study were to use meta analysis of multiple studies to: 1]To examine the association between diet and dietary components, microbiota composition and inflammatory status in health and disease of elderly; 2 ]To investigate how changes in the microbiota influence the inflammatory and immune status with age, and 3] To evaluate the relationship between inflammation and cancer. The results of this analysis indicate that 1) Inflammation increases with age and is a risk factor for developing cancer but the mechanisms are not entirely understood. 2) Gut microbiota is one important factor in age‐associated inflammation and cancer. 3) Aged microbiota may trigger inflammation and impair immune function. 4) Inflammation has been linked to both microbiota and cancer. Thus, microbiota can influence cancer not only by constituent activation of inflammation cascades, but also by triggering genotoxic pathways and tumor microenvironment perturbation. Accordingly, our study suggests that nutritional habits, such as prebiotics, foods containing probiotics and generally whole foods, can manifest a net increase of anti‐inflammatory markers in the body.

Support or Funding Information

Supported by Institutional Resources of the University of Science Arts and Technology, Montserrat, and the Einstein Medical Institute.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipid Membrane Interaction of Amyloidogenic Peptides
Authors: Dey Simli , Gupta Ankur , Bhowmik Debanjan , Das Anirban , Rawat Anoop , Maiti Sudipta
Published: June 2021

Membrane interaction appears to be a key step in the toxic pathway of many amyloidogenic proteins, such as Amyloidβ (Aβ40/42, associated with Alzheimer's disease) and hIAPP (associated with Type II diabetes). While the oligomers are generally considered to be more toxic than either the monomers or the fibrils, the exact identity of the toxic oligomer has remained a matter of debate. How much the local structure and composition of the membrane influences the binding of the oligomers is another controversial issue.

Here we use a combination of single‐molecule fluorescence techniques (e.g. single molecule photobleaching (smPB) and Fluorescence Correlation Spectroscopy (FCS)) and atomic force microscopy to quantitatively address these two issues. smPB measurements of freshly prepared hIAPP in physiological solution and on a supported lipid bilayer (composed of POPC: POPG: Cholesterol 1:1:1) allow us to measure the relative affinity of each oligomeric species. Our measurements show that both monomers and small oligomers (1–5 mers) have high affinity for the membrane, and dimers appear to have the highest affinity. However, FCS measurements show that the monomers have much lower affinity than the oligomers, Though FCS is unable to resolve the contribution of individual types of oligomers. It is likely that the smPB measurements are skewed by pre‐measurement photobleaching which is a known problem with this technique. We have now developed a method to measure pre‐bleaching, and are in the process of calibrating our results.

We have also addressed the influence of the local membrane structure on binding of hIAPP oligomers. This aspect may throw light on the initiation of these diseases in the human body. We use a ternary neutral lipid mixture (DOPC: Sphingomyelin: Cholesterol in 2:2:1 mole ratio) to make stable phase separated bilayers. We use an AFM to map the bilayer surface topology, and a synchronized home‐built confocal microscope to simultaneously map the distribution of the fluorescently labeled peptides on this bilayer. We find that small oligomers of hIAPP bind preferentially to the disordered (Ld) phase, compared to the ordered phase Lo, with a binding ratio of Ld:Lo = 9:1. Our studies thus quantify the membrane interaction of small n‐mers of hIAPP as a function of n, and also show that local membrane organization can play a dominant role in determining membrane interactions.

Support or Funding Information

Tata Institute of Fundamental Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Physicochemical Analysis of Outer Membrane Vesicles Isolated from Escherichia coli Harboring the Pks Genes
Authors: Morales‐Lozada Yermary , Torres‐Montañez Stephanie , Muñoz‐Santiago Ricardo , Díaz‐Cartagena Diana C. , Gómez‐Moreno Ramón , Baerga‐Ortiz Abel
Published: June 2021

Almost all gram‐negative bacteria secrete sphere‐like proteoliposomes named outer membrane vesicles (OMVs). These vesicles provide the bacteria a vehicle for the secretion of outer membrane molecules and periplasmic content. The pks island is a gene cluster present in some strains of bacteria in the human gut microbiota, which has been correlated with the development of colorectal cancer due to the promotion of tumor growth, DNA damage and toxicity in transiently infected eukaryotic cells. The pks island genes encode a sequence of enzymes for the biosynthesis of a compound that we hypothesize is transported from the bacteria to cells by OMVs. The main objective of this investigation is to analyze physically OMVs isolated from E. coli strains harboring and lacking the pks island genes. To achieve this objective, OMVs were analyzed by scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS) to assess any morphological differences between them. Results show that pks+ E. coli strains produce more vesicles than the pks− strain. As expected, vesicle diameters are around 50 – 280 nm. The OMVs from pks+ and pks− strains have similar diameter distribution between them, but markedly different compared against the lab strain DH10B. We conclude that the presence of pks island is associated with events in the production of OMVs but not with the structure and length of them.

Support or Funding Information

RISE Program Grant #5R25GM061151‐16

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Role of the BRCA2 C‐Terminus in Homologous Recombination
Authors: Chiang Alex , Ladino Natalie , Lee Kaya , Lu Ashley , Srikanth Nina , Stearns William , Wen Collin , Williams Samuel , Jensen Ryan B , D'Ausilio Morgan E
Published: June 2021

The tumor suppressor protein, BRCA2, acts as a mediator for DNA double stranded break repair via homologous recombination (HR). Mutations in BRCA2 lead to a higher risk of tumor formation, particularly in breast and ovarian cancers. The C‐terminus of BRCA2 plays an important role in the protein's overall ability to bind to DNA during HR. Of the identified tumor associated BRCA2 mutations, 27% are found in the C‐terminus, suggesting the significance of this region in tumor suppression. An important function of the C‐terminus is the ability to bind DSS1, a small, acidic protein found to be critical for DNA repair. DSS1 supports the stability of BRCA2 and is thought to act as a DNA mimic, dissociating replication protein A (RPA) from single stranded DNA (ssDNA). As RPA dissociates, the ssDNA becomes available for RAD51 binding and nucleoprotein filament formation leading to the repair of damaged DNA. When DSS1 is altered or absent, BRCA2 is unable to promote dissociation of RPA from ssDNA and a decrease in HR efficiency is observed. The Pingry School SMART Team, in conjunction with MSOE Center for BioMolecular Modeling, used 3D modeling and printing technology to examine the structure‐function relationship of BRCA2, DSS1, and ssDNA. While further research is necessary to more completely characterize the roles of DSS1 and the BRCA2 C‐terminus in HR, the Pingry School SMART Team model aims to describe the structural relationship between these proteins.

Support or Funding Information

This work was supported by The Pingry School.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hissing cockroaches may save water by closing their spiracles.
Authors: Vrtar Andrew , Keene Blake , Contreras Heidy Lorena
Published: June 2021

We examined the effects of humidity on the metabolic rates and respiratory patterns of Gromphadorhina portentosa to determine whether insects transition from continuous, cyclical and discontinuous (DGC) respiration in response to water conservation. Eight male G.portentosa were placed under 5 different humidity treatments (0, 23, 40, 60, 80% RH). Using flow through respirometry we: 1) determined the effect of humidity on metabolic rate; 2) observed if changes in metabolic rate were correlated with changes in closed/flutter (CF) or the open (O) phase of DGC; and 3) determined whether increased spiracular closure was correlated with an increase in water retention. Although G. portentosa had similar rates of CO2 release when placed under 0, 40, 60 and 80% RH, cockroaches placed at 23% RH had a significantly higher metabolic rate. There was no effect of humidity on the duration of the CF phase of the DGC. However, the O phase of DGC was significantly longer when G. portentosa was placed at humidity levels above 23% RH. Interestingly we saw that the average rate of mass lost to the environment did not change when cockroaches were placed at humidity levels ranging from 0–80% RH. This suggests that modulation of the spiracles allows G. portentosa to regulate the amount of water lost to the environment.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Acute Aerobic Exercise on Transcriptomics in Skeletal Muscle of Lean vs Overweight/Obese Men
Authors: Devarshi Prasad P , Jones Aarin D , Taylor Erin M , Henagan Tara M
Published: June 2021

Aerobic exercise improves skeletal muscle mitochondrial function and improved insulin sensitivity in lean and overweight and obese (Ov/Ob) individuals; however, the genes important in determining the beneficial effects of exercise in both lean and Ov/Ob individuals are still largely unknown. Here, we aimed to identify genes similarly expressed in lean and Ov/Ob skeletal muscle following an acute bout of exercise. 30 men, aged 19–30 years, were recruited and divided into two groups: lean (BMI<25, 18.5–24.1 kg/m2, n=15) and Ov/Ob (BMI≥25, 25.5–36.9 kg/m2, n=15). Four hours after eating a standardized high‐carbohydrate breakfast (7 kcal/kg; 60% carbohydrate, 25% fat, 15% protein), participants performed an acute bout of cycling exercise (50% VO2max, spending ~650 kcal). Vastus lateralis muscle biopsy samples were collected prior to (Pre) and immediately following (Post) the acute exercise bout. In skeletal muscle, gene expression by RNA‐seq was determined using the Illumina platform. For analysis, TopHat was used for sequence alignment and Cuffdiff for differential gene expression analysis with FDR set at 0.05. ~97% of all sequenced reads from RNA‐seq aligned to the genome. Gene Ontology analysis was performed using DAVID. Analysis showed that 297 genes were differentially regulated between lean Pre and Ov/Ob Pre, 1230 genes between lean Pre and lean Post, 1123 genes between Ov/Ob Pre and Ov/Ob Post and 161 between lean Post and Ov/Ob Post. In both lean and Ov/Ob, genes upregulated following exercise were enriched for GO terms related to lipid metabolism, mitochondrial biogenesis/function and insulin signaling. Pathway analysis revealed that the AMP‐activated protein kinase (AMPK) signaling pathway was one of the pathways enriched in both lean Post and Ov/Ob Post, with 11 genes in the pathway upregulated in lean Post and 9 in Ov/Ob Post. In conclusion, it is possible that similar expression of genes following exercise may account for the increased responsiveness in mitochondrial function and insulin signaling in both lean and Ov/Ob.

Support or Funding Information

This work was supported by Ralph W. and Grace M. Showalter Research Trust grant and Indiana CTSI funds to TMH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heat Shock Protein Response During a Prolonged Ultraendurance Cycling Event in Hot and Humid Conditions
Authors: Lopez Virgilio , Colburn Abigail T , Wright Skylar S , Dadzie Ekow C , Armstrong Lawrence , Vingren Jakob , Boyett James , McDermott Brendon , Butts Cory , Melford Colin , Munoz Colleen , Kunces Laura , Seal Adam , Lee Elaine C
Published: June 2021

The heat shock protein (HSP) response is critical to the cellular stress response. Circulating, extracellular stress‐inducible HSP72 is known to respond to exercise‐heat stress, but little is known about other, constitutively expressed and fundamental members of the HSP family. The aim of this study was to investigate the extracellular HSP27, HSP60, and HSP90α response during a prolonged cycling event in hot and humid conditions. Seventy‐six subjects (mean±SD: age, 51.64±10.47 years; height, 176.44±6.85 cm; mass, 86.78±13.93 kg) were recruited at the Hotter'n Hell 100 mile cycling event in Wichita Falls, Texas. Blood and urine samples, as well as data on body weight and gastrointestinal temperature (TGI) were recorded before (pre) and after (post) the event. Plasma was isolated from blood samples, stored at −80°C, and later analyzed with StressMarq Biosciences ELISAs for HSP27, HSP60, and HSP90α concentrations. Student's t‐test was used to test for significant differences of means. TGI significantly increased from pre to post (pre: 37.14±0.55 °C; post: 38.12±0.79 °C; p<0.05). HSP27 and HSP90α were both significantly elevated post race (pre27: 2.73±0.51 ng × mL−1; post27: 3.46±0.73 ng × mL−1; pre90α: 18.03±3.27 ng × mL−1; post90α: 21.53±4.84 ng × mL−1; p < 0.05). However, there was no significant difference in HSP60 plasma concentration from pre to post (pre: 44.05±7.38; post: 43.65±6.81; p=0.77). In conclusion, significant increases in circulating HSP27 and HSP90α suggest that chaperones required for thermotolerance and protein degradation or folding during heat stress increase in extracellular compartments post exercise‐heat stress. However, our data suggest that chaperones specifically involved in mitochondrial protein assembly or molecular danger signals like HSP60 are not elevated in blood fractions. Ongoing work will characterize cellular expression of these proteins in circulating peripheral blood mononuclear cells.

Support or Funding Information

New investigator start‐up, McNair Scholar Program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Photobiomodulation And Exercise Training Contribute To Protect The Cardiovascular System In Experimental Monoarthritis
Authors: Zamuner Stella Regina , Zamuner Luis Fernando , de Paulo Alves Silva Marcelo , de Angelis Katia , Chavantes Maria Cristina
Published: June 2021

It has been shown that the inflammatory process causes autonomic changes in arthritis. This study examined the effect of non‐invasive therapies involving photobiomodulation therapy (PBMT) and exercise training (ET) on cardiovascular changes in experimental monoarthritis. Male Wistar rats (n=40) were randomly divided into five groups: sedentary control (C); arthritis (A); arthritis plus PBMT (AP); arthritis plus exercise training (AT); AP plus AT (APT). Monoarthritis was induced by intra‐articular injection of zymosan (1 mg in 50 μl of a sterile saline solution) into one rear knee joint. Moderate‐intensity training was performed on a treadmill (8 weeks/5 days a week, 40–60% maximum exercise test, 50 min/day) and PBMT was applied at 660 nm (power output 5 mW, power density 0.1 W/cm2, energy density 2.5 J/cm2, spot size 0.04 cm2 and 20 sec irradiation time), twice a week. Evaluations of cardiac function, cardiovascular autonomic modulation, leukocyte recruitment as well as articular structure of the knee joint were measured. Ethics Committee: AN009/2014. Trained rats presented lower body weight, an increase in the maximum speed of running and lower heart rate compared to sedentary groups. Hemodynamic data showed no statistical difference among groups. In frequency domain analysis, low frequency (LF) power, indicating mainly sympathetic activity, was increased in A and AP groups, ET caused a decreased in LF power in these groups. High frequency (HF) power, indicating parasympathetic activity, was decreased in A and AP groups and ET caused an increase of HF power. The association treatment induced a reduction of LF/HF, which is associated with resting bradycardia. In addition, a better tissue organization fibers and less inflammatory cell were observed in both AP, AT and APT group, as compared to A group. In conclusion, PBMT and ET (associated or not) exert beneficial effects in arthritic rats. These benefits were related to the autonomic cardiovascular balance and the improvement of the inflammatory process in the knee joints.

Support or Funding Information

Grant fund: CNPq and UNINOVE

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Terminal sequences in the M protein of bovine parainfluenza virus type 3 is important for virus egress
Authors: Ueda Haruna
Published: June 2021

Bovine parainfluenza virus type 3 (BPIV3), which is one of the most important respiratory pathogens in cattle, impairs the global cattle industry. To gain insights into the virus particle formation of BPIV3, we focused on the membrane (M) protein, which is thought to interact with the host cell membrane and virus ribonucleoprotein (RNP) complex. First, we generated M‐gene‐deficient BPIV3 (ΔM) that can propagate in cells transfected with M protein‐expressing plasmids. With ΔM and modified M gene, we found that 10‐ amino‐acid or 4‐amino‐acid deletion from its N terminal or C terminal completely abrogated virus infectivity. Furthermore, vero cells transfected with M genes produced and released virus‐like particle (VLP) even without virus infection. VLP production was also induced with an M gene in which the terminal sequence was deleted for a few amino acids. These results suggest that the terminal structures of M protein interact with RNP, which affects virus infectivity. Though the actual three‐dimensional structures of both terminals are still unclear, these findings demonstrate the importance of a few amino acids in the terminal of M protein for virus infection. These results can contribute to further research on virus M protein function and antiviral development.

Support or Funding Information

Ohkura T, Minakuchi M, Sagai M, Kokuho T, Konishi M, Kameyama K, Takeuchi K. Infection of the upper respiratory tract of hamsters by the bovine parainfluenza virus type 3 BN‐1 strain expressing enhanced green fluorescent protein. Virology. 476:134–40; 2015

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic fluid and electrolyte imbalance contributes to brain inflammation and the development of cardiovascular complications in streptozotocin‐treated diabetic rats
Authors: Alzahrani Omar Awad , Habeeballah Hamza Habeeb , Alahmadi Emad Awad , Badoer Emilio , Stebbing Martin
Published: June 2021

We previously showed specific microglial activation within cardiovascular control centers, in the brains of streptozotocin (STZ) diabetic rats. Microglial activation within the PVN of STZ diabetic rats was not evident until the 6th week after STZ treatment, and was preceded by neuronal activation in the PVN and associated with chronic dehydration and electrolyte imbalance (1). Furthermore, giving STZ diabetic rats 1% saline to drink after the induction of diabetes increased blood pressure (BP) (2). Therefore, we sought to investigate the complex relationships among PVN microglial activation, the development of diabetic cardiovascular complications and the effect of saline intake. Male Sprague‐Dawley rats were given a single injection of STZ via the tail vein to induce diabetes. Throughout the experiment fluid intake and BP parameters were monitored in control rats and diabetic rats given tap water or saline to drink. Echocardiography was used to measure cardiac function and tail cuff was used to record BP. Microglial morphology was quantified using immunohistochemistry for CD11b (1). Here we show that, two weeks after the induction of diabetes in STZ diabetic rats drinking 1% saline, BP was elevated and microglial activation was prominent in the PVN in comparison to diabetic rats drinking tap water. However, by week 6, STZ diabetic rats given tap water to drink showed elevated polydipsia, blood parameters consistent with dehydration, activation of PVN microglia, and functional signs of cardiomyopathy, whereas STZ diabetic rats drinking 1% saline did not, suggesting a delicate balance between electrolyte intake and electrolyte loss in these animals. In a separate set of experiments STZ diabetic rats were given 0.9% saline,+/‐20 mM KCl or 1 mM MgCl to drink. At the second week after the induction of diabetes, rats drinking 0.9% saline showed no elevations in blood pressure; whereas STZ diabetic rats drinking saline + 20 mM KCl or 1 mM MgCl showed BP elevations. All STZ diabetic rats were given low level insulin supplementation to prevent dehydration and showed no sign of cardiomyopathy even after 10 weeks of diabetes. We conclude that neuroinflammation in the PVN and other cardiovascular control centers, secondary to chronic fluid and electrolyte imbalances may contribute to the development of diabetic complications and preventing dehydration may be therapeutic

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.