bluuebunny/crossref_metadata_2025 huggingface dataset:
Location and Partners of Polysialic Acid in the Dorsal Horn and Trigeminal Nucleus of Adult Rat
Authors: Shahbazian Shila , Lee Albert , Everest‐Dass Arun , Bokiniec Phill , Packer Nicolle , Goodchild Ann
Published: November 2023

Polysialic acid (PSA) is a large cell‐surface glycan predominantly attached to the neural cell adhesion molecule (NCAM) in the mammalian brain. PSA has discrete expression patterns to specific areas of the CNS, postulated to have high levels of synaptic plasticity. The regulation, expression and interacting partners of PSA is complex and remains poorly understood. In this study, we aimed to examine the cellular distribution of PSA by immunohistochemistry in the dorsal horn and trigeminal nucleus of adult Sprague Dawley rats. Both regions receive significant sensory input thus are likely to have high levels of plasticity. We further investigated interacting partners of PSA in these regions using co‐immunoprecipitation (IP) followed by label‐free liquid chromatography tandem mass spectrometry (LC‐MS/MS) analysis.

We found that PSA was highly expressed in the superficial laminae of the dorsal horn, and within the trigeminal complex distribution was restricted to only the superficial layers of nucleus caudalis (TNc). Using LC‐MS/MS analysis, 14 proteins were identified in the dorsal horn as potential binding partners of PSA‐carrying NCAM. Three candidates (Receptor expression‐enhancing protein 5 (REEP5), Guanine nucleotide‐binding protein G(o) subunit alpha (GNOA1), and Clathrin heavy chain 1 (CLTC) were validated by IP and western blot analyses. Moreover, co‐IP of PSA from TNc preparations followed by western blot analyses of the three candidates confirmed their PSA status within TNc. This is the first study to demonstrate the detailed cellular distribution of PSA in the rat trigeminal nucleus and the identities of three potential interacting partners. These findings will improve our understanding of the mechanisms by which PSA affects synaptic plasticity and may reveal novel PSA functions.

Support or Funding Information

Anti‐polysialic acid antibody (mAb735) was kindly provided by prof. Rita Gerardy‐Schahn.
Axin Is Localized within Mitochondria and Regulates Energy Metabolism through Alterations of Oxidative Phosphorylation and Glycolysis
Authors: Shin Jee‐Hye , Kee Sun‐Ho
Published: November 2023

Axin is a scaffolding protein that antagonizes Wnt signaling and controls various cellular events, involving cell proliferation and death. Tankyrase inhibitors, such as XAV939 stabilizes the axin protein, provide a therapeutic strategy for cancers.

To study effects of axin expression on mitochondria, we established two axin‐expressing HeLa cells models: doxycycline‐inducible axin expression and enhanced axin expression by treatment with XAV939. Axin‐expressing cells showed localization of axin in mitochondria, especially the oxidative phosphorylation (OXPHOS) complex IV. Axin reduced complex IV subunits expression and altered supercomplexes formation. Moreover, axin led to functional alteration containing the reduction of cell proliferation, ATP level, complex IV activity, and basal respiration. Despite the respiratory effects, the glycolysis was increased and mitochondrial membrane potential was maintained, which even preserved in the presence of mitochondria‐affecting stimuli in axin‐expressing cells.

In conclusion, our results suggest that axin was expressed in complex IV of mitochondria, which modulated mitochondrial function and glycolysis.
MboII Restriction Fragment Length Analysis Of Estrogen Receptor 1 Gene
Authors: Saibu Morounke G , Ayorinde James , Adu Oluwatosin , Faduyile Francis , Wusu Dorcas , Iyapo Oluwadamilare , Kazeem Mutiu , Soyemi Sunday , Izuagie Sekinat
Published: November 2023

Introduction

Cervical cancer is the commonest genital tract malignant neoplasm that affect women worldwide. It is most notable in the lower‐resource countries of sub‐Saharan Africa. Understanding the restriction digestion pattern of Human estrogen receptor‐α gene (ESR‐1) would provide information on the possible mutations on the gene that can serve as potential diagnostic and therapeutic markers.

The present work is focused on determining the MboII restriction fragment length on esr1 gene of cervical cancer cells. The elucidation of this is vital in the diagnosis of the cervical cancer disease

Materials and Method

PCR technique was used to amplify the promoter region of exon1 of the esr1 gene for ten samples. MboII restriction enzyme specific for the promoter site was used to carry out restriction fragment analysis.

Result and Discussion

The result indicates that the ESR1 gene for cervical cancer has restriction patterns or sequences recognized by the restriction enzyme. It is important to note however, that there is need for similar work using large sample size in order to conclusively ascertain the presence of polymorphism in the ESR 1 promoter region and the restriction fragment length of the ESR1 gene. Seven out of the ten samples analyzed for restriction fragment length exhibited the presence of restriction site specific to the MboII restriction endonuclease. These restriction sites are vital for diagnostic purposes.

Conclusion

The result indicates that the ESR1 gene for cervical cancer has restriction patterns or sequences recognized by the restriction enzyme.

Support or Funding Information

No conflict of Interest Disclosure
The Bioactive Properties of Acetone Extract of Cola Acuminata in Breast Cancer Cells
Authors: McGovern Janachia , Gray Wesley
Published: November 2023

Breast cancer (BC) is the leading cause of death in women in the United States. Currently there are many treatment options, however the side effects associated with these treatments usually result in a decrease in the standard of life. Previously we reported the identification of a bioactive fraction from the natural product Cola Acuminata (Biz‐3) which is capable of inhibiting cancer cells. In this study we tested the hypothesis that bioactive substances in the Cola Acuminata (Biz‐3) extract contains compounds that may provide a natural treatment option for BC. Thus, our objective was to isolate the bioactive compound from the Biz‐3 and reveal which ones are responsible for inhibiting the MCF 7 breast cancer cell growth. The Biz‐3 extract was isolated using SPE reverse phase chromatography and the resulting analytes of interest were characterized using a combination of thin layer chromatography and UV‐Vis chromatography and a MTT cell inhibitory assay. Elution of the SPE column with solvents of increasing polarity resulted in three distinct ethanol peaks with the majority of the analytes appearing in the 70% ethanol fraction. UV‐Vis spectrum reveals that Biz‐3 contains at least five different compounds absorbing between 210 and 265nn. To determine in which of the SPE peaks the cell inhibitory activity co‐eluted, MCF‐7 cells were induces with different concentrations of the SPE peaks for 1, 3 and 5 days. Our preliminary data suggests that the growth inhibitory activity co‐elutes with the 30% SPE ethanol peak. However, we observed a stimulation of the MCF‐7 cell growth in the presence of the 70% and 100% SPE ethanol peaks. Our overall results demonstrate that the bioactivity present in Biz‐3 can be isolated using SPE and it's likely that two different bioactive compounds are present in Cola Acuminate.

Support or Funding Information

NIH grant, Grant No. NIH R15AT006821
Avocado Oil Improves Renal Vasorelaxation in Hypertensive Rats; Probable Role of Decreased Mitochondrial ROS Generation and Enhanced Antioxidant Capacity
Authors: de la Paz Jose Lucio Hernandez , Marquez‐Ramirez Cristian , Aguilar‐Toral Roxana , Moeno‐Jimenez Mario , Raya‐‐Farias Andres , Salgado‐Garciglia Rafael , Godinez‐Hernandez Daniel , Saavedra‐Molina Alfredo , Cortes‐Rojo Christian
Published: November 2023

Kidney disease is a major complication of hypertension. Both mitochondrial dysfunction and increased ROS generation are involved in the exacerbated vasoconstriction observed in rodent models of hypertension in response to the increased activity of angiotensin II. Thus, the prevention of mitochondrial dysfunction along with decreased ROS generation or increased activity of antioxidant systems may improve the response to vasorelaxant and/or vasoconstrictor agents, leading to attenuation of hypertensive kidney damage. An increasing body of evidence indicates that avocado oil (AO) intake improves the mitochondrial function and its redox state in kidney, liver and brain of diabetic rats, which has been attributed to the wide array of lipophilic antioxidants contained in AO. We have observed that avocado oil consumption protects from kidney mitochondrial dysfunction elicited by L‐NAME‐induced hypertension. However, it is unknown whether this might lead to attenuation of oxidative stress because decreased ROS generation and improvement of kidney vasodilator responses. Thus, we aimed to test the effect of AO intake in rats treated with L‐NAME on ROS generation, GSH/GSSG ratio (i.e a marker of oxidative stress) in kidney mitochondria and vasodilator responses to a cholinergic agonist in isolated perfused kidney. ROS generation stimulated by complex I substrates increased twice in L‐NAME treated rats while the addition of antimycin A, a complex III inhibitor, exacerbated this effect, suggesting a role for complex III in ROS overproduction. Despite this, L‐NAME did not modified the GSH/GSSG ratio. L‐NAME rats also exhibited a decline in kidney vasorelaxation induced by carbachol. AO supplementation by 90 days (1mL/250g body weight) inhibited ROS generation by below of the values observed in control rats, increased twice the GSH/GSSG ratio and normalized the vasorelaxation stimulated by carbachol. These results suggest that AO improves renal vasorelaxation in association with a decrease in mitochondrial ROS generation and enhancement of antioxidant capacity.

Support or Funding Information

Supported by Programa de Investigación 2016 de la Coordinación de la Investigación Científica de la UMSNH (CIC‐UMSNH) (CCR).
Effects of uric acid and melatonin exposure on mitochondrial complex‐I respiration, triglyceride accumulation and aconitase activity in skeletal muscle cells
Authors: Maarman Gerald Jerome , Andrew Brittany Morgan , Blackhurst Dee , Ojuka Edward
Published: November 2023

Background

Hyperuricemia is associated with gout, hepatic steatosis and type 2 diabetes. It induces oxidative stress, lipogenesis and mitochondrial dysfunction in the liver. However, the skeletal muscle is the greatest surface area exposed to hyperuricemia and yet no studies have investigated its effects on skeletal muscle.

Aims

To establish whether hyperuricemia modulates mitochondrial function, oxidative stress and triglyceride accumulation in myotubes and whether antioxidant treatment with melatonin can counteract these effects.

Methods

Differentiated C2C12 myotubes were exposed to uric acid (750μM, 72 hrs.) or uric acid + melatonin (10nM melatonin, 72 hrs). Mitochondrial function was assessed (high‐resolution respirometry), spectrophotometry was used to assess oxidative damage to aconitase and triglyceride content. All values were compared to respective control groups (without uric acid and without melatonin).

Results

Uric acid (1) reduced OXPHOS (60%) in the presence of pyruvate, glutamate and malate, (2) reduced aconitase activity by 37% and (3) increased triglyceride accumulation (52%) in C2C12 myotubes. Melatonin treatment restored OXPHOS (40%) and reduced triglyceride content (47%).

Conclusion

Hyperuricemia dysregulates complex‐1 mediated mitochondrial respiration, induces damage to aconitase and triglyceride accumulation. Melatonin restored complex‐1 mediated mitochondrial function and normalized triglyceride accumulation. Melatonin could therefore be considered a therapy against hyperuricemia.

Support or Funding Information

National Research Foundation (South Africa) and the University of Cape Town.
Insulin‐dependent glucose uptake is regulated by mitochondrial Ca2+ handling in skeletal muscle fibers
Authors: Contreras‐Ferrat Ariel Eduardo , Díaz‐Vegas Alexis , Campos Cristian , Utreras Yildy , Valladares Denisse , Rosales Giovanni , Llanos Paola , Arias Manuel , Jaimovich Enrique
Published: November 2023

Introduction

The mitochondria functions rapidly to respond to high energy food supply but the role of mitochondrial Ca2+ levels in the context of insulin signaling has not been explored.

Material and Methods

Mice were fed with normal chow diet (NCD) or high fat diet (HFD) for 1 (St‐HFD) or 8 (Lt‐HFD) weeks, (short‐ and long‐term) respectively. Changes in calcium levels, mitochondria membrane potential and glucose uptake were evaluated in living fibers from Flexor digitorum brevis muscle using fluorescent dyes.

Results

Insulin induced an increase in cytoplasmic and mitochondrial Ca2+ in adult fibers that was significantly smaller in fibers from short‐term HFD fed mice and completely inhibited in Lt‐HFD. In fibers from NCD fed mice, insulin‐dependent mitochondrial Ca2+ uptake was blunted by both inositol‐1,4,5‐trisphosphate receptor inhibition and knockdown. The mitochondrial potential was larger in fibers from short‐term HFD fed mice v/s NCD derived fibers and the recruited of active mitochondria was increased. The glucose analogue (2‐NBDG) uptake and the redistribution of GLUT4myc‐eGFP chimera induced by insulin were decreased in the absence of mitochondrial Ca2+ uptake in conditions of type 1inositol 1,4,5‐trisphosphate receptor knockdown.

Discussion

These effects suggest a role for mitochondrial Ca2+ uptake in a retrograde regulation of insulin signaling by mitochondria.

Support or Funding Information

Financed by FONDECYT 11130267, 1151293, PIA‐ACT1111
Caveolin‐1 Regulates Mitochondrial Dynamics by Suppressing Mfn‐2 and Drp‐1 recruitment
Authors: Jiang Ying , Toth Peter , Oliveira Suellen , Liu Yiyao , Hart Peter , Rehman Jalees , Bonini Marcelo , Minshall Richard D
Published: November 2023

Oxygen disposition by mitochondria determines whether electrons from NADH are used for energy (ATP) or H2O2 production. Both ATP and H2O2 have widely recognized (though different) roles in cell signaling. By switching the electron flow in order to produce either metabolite in different proportions mitochondria broadly affect the cellular metabolism, cell cycle and phenotypic fate decisions. The balance between H2O2 and ATP is determined in large part by the reorganization of the mitochondrial network with fission favoring H2O2 and mitochondrial fusion favoring ATP. Fusion and fission are regulated by mitofusin 2 (Mfn‐2) and dynamin‐related protein 1 (Drp‐1), respectively. Here, we report our recent findings that caveolin‐1 (Cav‐1), a protein normally associated with signaling on the plasma membrane regulates mitochondrial fission/fusion dynamics, oxygen consumption, and H2O2 production in MDA‐MB‐231 breast cancer cells expressing relatively high levels of Cav‐1. In 231 cells, Cav‐1 siRNA simultaneously increased both Mfn‐2 and Drp‐1 expression in the mitochondrial fraction. However, less fragmented mitochondria in Cav‐1 knockdown cells suggested that enhanced fusion via Mfn‐2 overcame Drp‐1 mediated fission. Furthermore, co‐IP and fluorescence resonance energy transfer (FRET) studies showed that Cav‐1 directly interacts with both Mfn‐2 and Drp‐1 in plasma membrane fractions. Also, fluorescence recovery after photobleaching (FRAP) of mitochondrial matrix targeted DS‐Red increased after Cav‐1 knockdown indicating that the mitochondrial network may be more plastic in absence of Cav‐1 due to increase in mitochondria‐associated Mfn‐2 and Drp‐1. We observed reduced Oxygen consumption and H2O2 production in cells treated with Cav‐1 siRNA. ATP biosensor showed higher ATP/ADP ratio after 2‐deoxy‐glucose (2‐DG) treatment in Cav‐1 knockdown cells demonstrating that mitochondrial ATP generation was blocked by Cav‐1. These results suggest that Cav‐1 plays an important role in the regulation of mitochondrial fission/fusion dynamics and function by binding to Mfn‐2 and Drp‐1 at the plasma membrane, thus limiting their translocation to mitochondria where they are known to regulate fission/fusion dynamics.

Support or Funding Information

Financial support provided by the National Natural Science Foundation of China (Nos.11272083, 31470906) (YJ, YL) and NIH P01 HL60678 (RM, MGB).
Combined BH3 and Metabolic Profiling as a Method to Define Therapeutic Response and Resistance in Grade IV Astrocytomas
Authors: Silva Monica Rodriguez , Gonzalez‐Arias Sergio , Chambers Jeremy W.
Published: November 2023

Background and Significance

Grade IV astrocytomas, formerly known as glioblastoma multiforme (GBM), are the most common primary brain tumors and have the highest mortality. The therapeutic standard for managing this malignancy remains a combination of surgery, chemotherapy, and radiotherapy; however, there is no cure, nor has there been any significant advancement in the clinical approach to GBM since this protocol was established in 2005. This study aims to better understand the molecular and metabolic characteristics of GBM‐derived cell lines to better define treatment groups and potentially identify new avenues for therapy. This study utilized continuous, commercially‐available GBM cell lines U87, U118, A172, and H4, and GBM cell lines from the NCI60 bank (SF268, SF295, SF539, SNB19, SNB75 and U251) and examined the concentrations of Bcl‐2 family proteins on mitochondria and metabolic activity for each of the cell lines. The measures were correlated to IC50 values for temozolomide (TMZ).

Results

Western blot analysis of pro‐survival and pro‐apoptotic Bcl‐2 proteins revealed that U118, U87 and SNB19 expressed high levels of Bcl‐2, while A172 had increased Bcl‐xL expression. Interestingly, Bcl‐2 and Bcl‐xL were not detected in H4 cells. Incidentally, pro‐apoptotic BH3‐only protein (Bid, Bim, Puma, etc.) levels were increased in H4 and A172 when compared to the U118 and U87 cell lines. Metabolic analysis of the cell lines revealed that U118 and SF295 cells were glutaminolytic, while U87, A172, and H4 were classified as glycolytic. We assessed cellular viability in the presence of increasing doses of TMZ for each cell line. We found that U118 and SNB19 cells were the most resistant followed by U87, SF295, U251, A172, SF539, SF268, SNB75 and H4 respectively. Interestingly, MGMT levels did not influence chemo‐responsiveness in these cell lines.

Conclusion

We found that Bcl‐2 protein profiling was a useful means to determine therapeutic response and resistance. This was further enhanced by metabolic stratification, wherein glycolytic cells were shown to be more sensitive TMZ than glutaminolytic cells that may possess more stable mitochondria. In the immediate future, we will increase the cellular profiling to include mitochondrial dynamics and quality control as well. Using these approaches, we will produce a method to define GBM responses and outcomes based on their mitochondrial and metabolic context.

Support or Funding Information

M.R.S. was supported by NIH/NIGMS R25 GM061347. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Effects of Turnera diffusa Will. over blood glucose and oxidative stress in kidney mitochondria diabetic‐STZ rats
Authors: Esquivel‐Gutiérrez Edgar R. , Saavedra‐Molina Alfredo , Alcaraz‐Meléndez Lilia
Published: November 2023

Turnera diffusa Will (damiana) is used in Mexican traditional medicine for treatment of various disorders including diabetes mellitus (DM). Not with standing, there is not a study that proves this effect on DM‐STZ for a long period. Also, there are not reports about the effects of damiana over the oxidative stress; a marker of damage in DM, in kidney mitochondria. By this reason, we set a main aim in this work for evaluate the antioxidant ability of this important medicinal plant in a worldwide pathology DM. To achieve this purpose we use STZ‐DM rats (65mg/kg body weight). We administered aqueous extract (150mg/kg body weight) from aerial parts of damiana. The blood glucose and animal weight was recorded every five days during 3 and 5 weeks of treatment. At the end of the treatments rats were sacrificed and kidney removed for mitochondria isolation by differential centrifugation. Lipid peroxidation, nitric oxide (NO) and nitrosylation profile in mitochondrial proteins were evaluated. Our results shown that T. diffusa in this investigation has no capacity to reduce the blood glucose at any time after 3 or 5 weeks of treatment, but induce to DM‐STZ animals to maintain the body weight, an important sign in DM. Our investigation also reveals the protection versus lipid peroxidation at the end of 5 weeks of treatment in control and DM groups (from 7.60 ± 0.38 to 6.48 ± 0.87 and 7.802 ± 0.70 to 3.358 ± 0.04 nmoles/mg prot, respectively), decreases the NO levels in both 3 and 5 weeks of treatment (from 0.703 ± 0.03 to 0.248 ± 0.05 and 0.754 ± 0.07 to 0.433 ± 0.02 μMole/mg prot, respectively). Nonetheless, nitrosylation of mitochondrial proteins remains unchanged compared with the control groups in all treatments. Finally, either control groups with or without treatments do not modifies the parameters measured in any case. In conclusion, aqueous extract from aerial parts of damiana has the capacity to mitigate the oxidative stress present in rat kidney mitochondria in DM‐STZ rats and the protector effects in this pathology is due to antioxidant properties more than lower the blood glucose.

Support or Funding Information

EREG is Postdoctoral Fellow CONACYT.
Hypoxic Adaptation by a Unique Mechanism of Reprogramming Mitochondrial Metabolism
Authors: Tessman Robert T , Chavan Hementkumar , Du Gouqing , Sun Daxi , Schuetz John , Krishnamurthy Partha
Published: November 2023

Hypoxia plays critical roles in the pathobiology of heart disease, cancer, stroke, and chronic lung disease, which are responsible for 60% of deaths in the United States. Evidence suggests that hypoxic survival often requires reprogramming of mitochondrial basal metabolism. However, our understanding of the fundamental mechanisms that regulate mitochondrial metabolism are still incomplete. During our studies evaluating the physiological significance of the mitochondrial transporter ABCB6, we uncovered a novel pathway in which loss of ABCB6 expression shifts the balance between mitochondrial respiration and glycolysis because ABCB6 expression decreased mitochondrial respiratory rate and increased glycolysis rate. More importantly, we found that the major transcriptional sensor of oxygen hypoxia‐inducible factor (HIF) was involved in the activation of Abcb6 expression under hypoxic conditions. Further, ABCB6 promoter contains hypoxia response (HRE) elements. These HREs are functional because the promoter is activated by the hypoxia mimetic, desferoxamine as well as directly by co‐transfected HIF1‐alpha. Moreover, in cells with a defective HIF, ABCB6 expression is not upregulated by hypoxia. Finally, ectopic overexpression of ABCB6 reveals that ABCB6 protects cells from the cytotoxicity induced by hypoxia. These studies reveal that ABCB6, a mitochondrial ABC transporter, is regulated by hypoxia and the major hypoxia transcription factor HIF1‐alpha. This upregulation by hypoxia is compatible with ABCB6 overexpression providing a survival advantage under hypoxic conditions.

Support or Funding Information

P20RR021940‐7

T32ES007079
Effect of aqueous leaf extract of Pennisetum purpurem in alloxan‐induced diabetic wistar rats
Authors: Omeodu Stephen Ike , Onyegeme‐Okerenta Blessing Minaopunye
Published: November 2023

Some biochemical indices (aspartate aminotransferase [AST], alanine aminotransferase [ALT], total protein, amylase and glucose) were evaluated in alloxan‐induced diabetic wistar rats treated with aqueous leaf extract of Pennisetum purpurem. Sixteen wistar albino rats were grouped into four. Group 1 served as the control while Groups 2 – 4 were administered with 120 mg/kg body weight alloxan – a toxic glucose analogue that resulted in a hyperglycemic state. Group 2 did not receive any treatment and served as the negative control. Groups 3 and 4 were treated with 1200 and 1500 mg/kg body weight aqueous extract of P. purpurem respectively for seven days and thereafter, the rats were sacrificed and the effects of the plant extract on the liver investigated. Results showed that there was significant reduction (p<0.05) in AST, ALT and glucose levels when groups treated with 1200 and 1500 mg/kg body weight aqueous extract of P. purpurem were compared with the control group. On the other hand, there was no significant reduction in amylase and Total protein concentration (p>0.05) when the group treated with 1200 mg/kg body weight extract of P. purpurem was compared with the control group. However, there was significant reduction (p<0.05) in amylase and Total protein concentration when the group treated with 1500 mg/kg body weight aqueous extract of P. purpurem was compared with the control group. This study shows that the aqueous extract of P. purpurem caused a significant reduction in serum glucose, AST, ALT, amylase and total protein in alloxan‐induced diabetic rats.

Support or Funding Information

Research was funded by the authors.
Quantification of Ethanolic Dandelion Root Extract on the Growth Inhibition of Saccharomyces cerevisiae
Authors: Samuelson Hanna Mary , Villari Taylor , Dunkley Rachel , Mann Francis M.
Published: November 2023

Taraxacum officinale, more commonly known as dandelion, has anti‐carcinogenic properties that have been demonstrated to induce apoptosis in human leukemia cells. While some active components, such as taraxinic acid, have been characterized, the complex effect of dandelion root extract on eukaryotic cell growth warrants further analysis. In this study, Sacchromyces cerevisae (yeast) was used as the eukaryotic sample because of their definite cell cycle. Concentrations varying between 1%‐100% of ethanolic dandelion root extract were applied and the effect on growth was measured over time. The information collected showed a decreasing viability of yeast growth as the concentration of dandelion extract decreased, which is supported by previous research. This effect is rescuable and does not result in increased cell death at low volumes of dandelion extract. However, increasing volumes of dandelion extract resulted in improved growth, which lead to the indication that the inhibitory effect could be overcome by increasing concentration of another molecule in the dandelion extract. This contradictory effect indicates dandelion extract contains activating as well as inhibitory molecules. Further fractionation and characterization of these molecules was attempted to better understand the overall effect of dandelion extract.
Mapping Epitopes of Pan‐Ebolavirus Antibodies From Human Survivors
Authors: David Joshua A , Murin Charles D , Flyak Andrew , Turner Hannah L , Fusco Marnie L , Saphire Erica Ollmann , Crowe James E , Ward Andrew B
Published: November 2023

Members of the Ebolavirus genus including Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV) and Budibugyo ebolavirus (BDBV) cause viral hemorrhagic fever and have high case fatality rates. The current outbreak in Western Africa is the largest on record and has infected over 28,000 individuals, yet no currently approved vaccines or therapeutics exist. Previous reports have revealed that ZMapp™, a cocktail of three antibodies that target the glycoprotein (GP) of EBOV, can revert an active infection in nonhuman primates. A group of recently identified antibodies from human survivors of BDBV and EBOV infections were shown to neutralize their respective virus. Some of the BDBV antibodies were also shown to be cross‐reactive against SUDV and EBOV; however, the basis of cross‐reactivity remains unknown. Here we report single‐particle negative stain EM reconstructions of each antibody in complex with GP. The cross‐reactive antibodies have overlapping epitopes and bind at the top of GP in a subdomain termed the glycan cap which contains multiple N‐linked glycosylation sites. Interestingly, many BDBV‐specific and EBOV‐specific antibodies bind in very close proximity. Therefore, the glycan cap contains structural motifs that influence species cross‐reactivity and neutralization potential of targeting antibodies. Higher resolution structures of these antibodies will be valuable for understanding the details of this important site of vulnerability.

Support or Funding Information

This work was supported by NIAID Center for Excellence in Translational Research Grant U19AI109762 (to E.O.S., and A.B.W.). J.A.D. was supported by The Scripps Research Institute Summer Undergraduate Research Fellowship Program.
The Polyamine Pathway of Leishmania donovani as a Potential Therapeutic Target
Authors: Roberts Sigrid , Paradis Dustin , Perdeh Jasmine , Harrelson John , Jan Boitz , Yates Phil , Ullman Buddy
Published: November 2023

Parasites of the genus Leishmania cause a variety of diseases, including fatal visceralizing infections, and affect an estimated 12 million people worldwide. Due to the absence of effective vaccines, therapeutic treatment has offered the only avenue of defense. Unfortunately, the current arsenal of drugs used to treat this neglected disease is far from ideal and the need for new therapeutic targets and a better understanding of host‐parasite interactions is urgent. Polyamines are ubiquitous, essential cations that are especially important for rapidly growing cells and the synthesis pathway for polyamines has been validated as a potential target in the related parasite, Trypanosoma brucei. In Leishmania, the polyamine biosynthetic pathway consists of four enzymes: arginase (ARG), ornithine decarboxylase (ODC), spermidine synthase (SPD), and S‐adenosylmethionine decarboxylase (ADOMETDC). These enzymes sequentially generate ornithine, putrescine and spermidine. We have generated a complete set of gene deletion mutants in Leishmania donovani (LdΔarg, LdΔodc, LdΔspd, and LdΔadometdc), in order to evaluate the polyamine biosynthetic enzymes as therapeutic targets. In vitro studies showed that the gene deletion mutants depend on the provision of downstream metabolites to survive. An evaluation of cell growth between the different cell lines uncovered that the only vital role of ornithine is as precursor for polyamine synthesis. However, putrescine, which has previously been postulated to be merely a substrate for spermidine formation, has additional essential functions and may even be more critical for cellular proliferation than spermidine. In vivo infectivity studies revealed that Ldodc exhibits profoundly reduced infectivity compared to wild type parasites, while Ldspd and Ldadometdc show a less pronounced, albeit significant, reductions in infectivity. In contrast, the Ldarg gene deletion has no effect on virulence. The observed discrepancies in infectivity phenotypes suggest a model in which the polyamine precursor ornithine is readily salvaged by intracellular parasites, whereas the polyamines putrescine and spermidine are severely limited. Our long‐term goal is to exploit the polyamine pathway for therapeutic development against leishmaniasis.

Support or Funding Information

School of Pharmacy Research Incentive Grant

NIH R01 AI041622
A role for underlying glycan structure in influenza binding: extending the species specificity paradigm
Authors: Woods Robert J , Grant Oliver C , Hadden Jodi A , Smith Hannah M.K. , Peng Wenjie , De Vries Robert , McBride Ryan , Paulson James C.
Published: November 2023

Recently, a new source for carbohydrate specificity data has emerged through the extensive development of glycan microarrays. Glycan array screening provides rapid insight into binding specificities, limited only by the number of elements in the array, the largest of which currently contain on the order of 600 members. Although rich in information, array data do not provide direct insight into the 3D structural properties of the glycan‐protein complexes responsible for the observed specificities.

Here we employ a new technology [1,2], Computational Carbohydrate Grafting (CCG), to generate 3D models of glycan‐hemagglutinin complexes that provide a structural rationalization for the sometimes perplexing data from glycan array screening. Given a structure for the protein in complex with a minimal binding determinant, CCG can be employed to generate a 3D model of any related glycan bound to the complex, by splicing the additional branches of the glycan into the bound fragment. This virtual approach overcomes many of the experimental challenges associated with generating structures for these complexes. CCG is a high‐throughput screening method that can be readily validated by comparison with specificity data from glycan array screening.

We show that CCG predictions explain differences in specificity for a range of sialylated N‐linked glycans interacting with human and avian influenza hemagglutinins. The analysis suggests that glycans with poly‐LacNAc extensions can bind in a bidentate fashion to the trimeric HA, consistent with affinity enhancements seen for some hemagglutinins in glycan array data. CGG analysis may prove valuable in elucidating the preferred receptors for influenza adhesion.

Support or Funding Information

Supported by the National Institutes of Health GM094919 (RJW), and AI050143 & AI099141 (JCP), and the Science Foundation of Ireland 08/IN.1/B2070 (RJW).
Investigating the use of Chk1 Inhibitors for Triple‐Negative Breast Cancer
Authors: Bindeman Wendy , Redwood Abena , Piwnica‐Worms Helen
Published: November 2023

Triple‐negative breast cancer (TNBC) accounts for 15–20% of all diagnosed breast cancers. It is characterized by lack of expression of estrogen receptor and progesterone receptor and an absence of HER2 amplification. TNBC is aggressive, highly metastatic, and ~50% of patients do not respond to first line chemotherapy. TNBC frequently harbors mutations in TP53, suggesting that therapies that exploit this vulnerability may be an effective treatment option. In particular, p53‐deficient cells are hypothesized to be highly sensitive to inhibitors of checkpoint kinase 1 (Chk1) due to checkpoint abrogation. We evaluated the efficacy of two clinically relevant Chk1 inhibitors, LY2606368 (LY) and SCH900776 (SCH), to elucidate the reasons for differing cellular response to the two drugs and evaluate the potential for synergy of each drug in combination with different DNA damaging agents. We hypothesized that both agents would synergize with gemcitabine regardless of p53 status, but would only synergize in combination with cisplatin in p53‐deficient cells. We tested the effects of LY and SCH alone and in combination with cisplatin or gemcitabine on cell viability, cell proliferation, and relevant signaling pathways in a TNBC cell line. Our data suggest that the differential cellular response to the two drugs is mainly due to a difference in potency. LY, but not SCH, preferentially sensitizes p53‐deficient cells to cisplatin. Notably, both LY and SCH synergize with gemcitabine regardless of p53 status, suggesting that Chk1 inhibitors + gemcitabine might be effective in both p53‐proficient and p53‐deficient backgrounds. Further studies are necessary to more fully evaluate the synergistic potential between cisplatin and LY or SCH, to characterize the sensitivity of cells carrying mutant p53, and to verify these results in vivo.

Support or Funding Information

Komen Foundation; Cancer Prevention & Research Institute of Texas
ProKinO: A unified resource for mining the cancer kinome
Authors: Kannan Natarajan
Published: November 2023

The massive and exponential growth of genomic, structural, biochemical and clinical data on the human kinome poses major challenges in using existing data for cancer and clinical research. Here we describe an ontological framework for integrating and conceptualizing diverse forms of protein kinase data in a machine readable, human understandable form. We demonstrate the utility of this framework in mining the cancer kinome and in generating testable hypotheses for experimental studies. Through the iterative process of aggregate ontology querying, hypothesis generation and experimental validation, we identify novel mutational hotspots in key regulatory regions of the kinase domain, and demonstrate the impact of identified mutations on kinase activity and drug sensitivity. We find that mutations mapping to the regulatory spine activate the kinase domain through distinct mechanisms and contribute to drug resistance. User‐friendly tools to mine cancer mutations in the context of naturally occurring variants, pathways, and drug sensitivity profiles will be discussed.

Support or Funding Information

NIH RO1 GM 11409‐01
USP24‐inhibited c‐Myc expression represses lung cancer formation
Authors: HUNG UA
Published: November 2023

Lung cancer is a malignant lung cancer characterized by high incidence and motility. c‐Myc is a transcription factor that plays an important role in oncogenic activation to influence cellular metabolism and proliferation. Ubiquitin‐specific peptidase 24 (USP24) is one of the member of USPs family to regulate protein ubiquitination. However, it is lack of evidence to uncover the role of USP24 in cancer formation. In this study, we found that cell proliferation was significantly increased by USP24 knockdown in A549 cells as accompanied by the increase of c‐Myc protein. In particular, the RNA level and protein stability of c‐Myc were not affected by USP24 knockdown, suggesting that USP24 affects c‐Myc through regulating the translational pathway. Furthermore, c‐Myc participated in USP24‐knocked down‐induced proliferation, not migration. To further clarify the mechanism underlying the regulation of c‐Myc by USP24 knockdown. Herein rapamycin, the mTOR inhibitor, prevented c‐Myc up regulation caused by USP24 knockdown, suggesting that USP24 affect the protein level of c‐Myc by regulating mTOR‐mediated translation. We found that the downstream signaling of mTOR is activated by USP24 knockdown, including S6K and RPS6 phosphorylation. In addition, USP24 knockdown induced the binding of activated RPS6 to 5′‐UTR of c‐Myc, indicating that USP24 regulates c‐Myc through mTOR‐mediated translation. Understanding the role of USP24 in regulating mTOR pathway will contribute to develop strategies to fight lung cancer.
Ceramide is A Key Factor That Regulates The Crosstalk Between TGF‐β and Sonic Hedgehog Signaling at The Basal Cilia To Control Cell Migration and Tumor Metastasis
Authors: Gencer Salih , Oleinik Natalia , Dany Mohammed , Ogretmen Besim
Published: November 2023

Bioactive sphingolipid ceramide is a second messenger in cell membrane in response to inflammation and stress. Recent studies indicate that ceramide species with different fatty‐acid chain lengths play diverse biological functions in various cellular processes, highlighting the importance of ceramide synthases (CerS) in these processes. Migration and cell mobility, a part of these processes, also are effected by ceramide metabolism. However, the molecular mechanism of CerS/ceramide involved is unknown. Here, we investigated the effect of CerS/and ceramide on migration and its related signal pathways in situ and in vivo model. Interestingly, our data show that among CerS only CerS4/ceramide is involved to cell migration and tumor metastasis. Here, we also have generated CerS4−/− mice for in vivo studies. Interestingly, we observed that genetically loss of CerS4 resulted in severe irreversible alopecia, which was associated with hyper‐proliferation and migration of keratinocytes. Mechanistically, we show here that genetic loss or shRNA‐mediated knockdown of CerS4 enhances cell migration by which ligand‐independent signaling of TGF‐beta receptors I and II in various cell types, including keratinocytes, mouse embryonic fibroblasts and cancer cells. Moreover, we found that ceramide directly interact with Smad7 and this interaction was decreased by shRNA‐mediated knockdown of CerS4. Thus, ceramide‐Smad7 binding modulates plasma membrane association of TGF‐βR1 at primary basal cilia, and inhibits its signaling through Sonic‐Hedgehog (Shh) for migration. Furthermore, Ceramide accumulation at the primary basal cilia was decreased by knockdown of CerS4, and this was associated with direct interaction of TGF‐βR1 and SMO receptors in cilia. In fact, inhibition of TGF‐βR /Shh signaling or cilia formation using molecular or pharmacologic inhibitors almost completely prevented cell migration in response to CerS4 knockdown. These data revealed that CerS4/ceramide signaling plays key roles in the regulation of cell migration and metastasis via controlling the TGF‐βR and Shh axis at primary basal cilia.

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1R01 DE016572
Sodium Hydrogen Exchanger 1 and Calcineurin Homologous Protein: A Critical for Survival Mechanism in Non‐Small Cell Lung Cancer
Authors: Cottle Wayne Taylor , Wallert Mark , Provost Joseph John
Published: November 2023

Calcineurin Homologous Protein 1 and 2 (CHP1 and CHP2) are competing cofactors which regulate the exchange activity of the Sodium Hydrogen Exchanger Isoform 1 (NHE1), a transmembrane proton pump vital in maintaining pH homeostasis. Early in tumor development, cancer cells experience an acidic microenvironment and serum deprivation as a result of anaerobic metabolism and poor diffusion of nutrients. To survive, cells must engage regulatory mechanisms to maintain intracellular pH (pHi). NHE1 regulation by CHP1 is required for basal activity, and CHP2 is thought to compete for, and augment this role by an unknown mechanism. CHP2 is also uniquely expressed in malignant tissues but the function of this is unclear. To characterize the roles of NHE1 and CHP2 on pHi in serum starvation, NHE1 and CHP2 RNAi knock down (KD) in H1299 cell lines were generated and serum deprived. NHE1 KD cells showed a decrease in pHi of 0.19 (s=0.02, n=12) and CHP2 KD showed a decrease of 0.23 (s=0.02, n=12). CHP2 KD cells also exhibit an overall lower resting pHi than NHE1 and H1299 wild‐type cell both in serum and serum deprivation. These data suggest that both NHE1 and CHP2 play a significant role in pHi regulation and that the cells are more susceptible to acidification without CHP2 expression. Western blot analysis of cultured cells and human tissues reveals an increase in CHP2 expression in nearly each lung tumor cell line and tissue examined. Real‐time PCR (RT‐PCR) in serum‐deprived conditions measured RNA expression of NHE1, CHP1, and CHP2. Serum deprivation in H1299 lung cells show no significant change in gene expression of either NHE1 (n=3) and CHP1 (n=3) after serum starvation. This supports existing western blot data suggesting that transcriptional regulation of NHE1 and CHP1 does not change under serum deprivation. CHP2 expression in paired diseased and adjacent normal tissue is also examined. By characterizing the mechanism for tumor survival in serum deprivation we can develop new modalities of treatment for Non‐Small Cell Lung Cancer patients.
ER‐Dependent Ca++‐Mediated Production of Cytosolic ROS as an Effector for Induction of Mitochondrial Apoptotic Signal and ATM in Gallic Acid‐treated Human Oral Cancer Cell
Authors: Lu Yao‐Cheng , Lin Meng‐Liang , Su Hong‐Lin , Chen Shih‐Shun
Published: November 2023

Release of calcium (Ca++) from the endoplasmic reticulum (ER) has been proposed to be involved in induction of apoptosis by oxidative stress. Using ER Ca++ release inhibitor dantrolene and mitochondrial Ca++ uptake inhibitor Ru‐360, we demonstrated that Ca++ release from the ER was associated with loss of mitochondrial membrane potential, increase in reactive oxygen species (ROS), and apoptosis of human oral cancer (OC) cells induced by gallic acid (GA). Chemical chaperone 4‐phenylbutyrate inhibits tunicamycin‐induced induction of 78 kDa glucose‐regulated protein, C/EBP homologous protein and pro‐caspase‐12 cleavage, cytosolic Ca++ increase, and apoptosis, but did not attenuate the increase in cytosolic Ca++ level and apoptosis induced by GA. Ataxia telangiectasia mutated (ATM)‐mediated c‐Jun N‐terminal kinase (JNK) phosphorylation and apoptosis in response to GA was blocked by dantrolene. The specificity of ROS‐mediated ATM activation was confirmed by the treatment of N‐acetylcysteine, an ROS scavenger. Blockade of ATM activation by a specific inhibitor KU55933, short hairpin RNA, or kinase‐dead ATM overexpression suppressed JNK phosphorylation but did not completely inhibit cytosolic ROS production, mitochondrial cytochrome c release, pro‐caspase‐3 cleavage, and apoptosis induced by GA. When taken together, these results indicate that GA induces OC cell apoptosis by inducing the activation of mitochondrial apoptotic signal and ATM, likely through ER Ca++‐mediated ROS production.

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Effect of O‐GlcNAcylation in tumor microenvironment on B16 melanoma progression
Authors: Moriwaki Kazumasa , Takeshige Yoshiko , Segawa Naoko , Asahi Michio
Published: November 2023

O‐GlcNAcylation, a protein modification which transfers O‐linked N‐acetylglucosamine (O‐GlcNAc) to serine or threonine residues, plays crucial roles in regulating cellular signaling through competition with phosphorylation on the same residues of intracellular molecules. Recent works indicate that increased O‐GlcNAcylation is a general feature of cancer cells and contributes to various cancer phenotypes, including cell proliferation, survival, invasion and metastasis, energy metabolism, and epigenetics. Additionally, it is reported O‐GlcNAcylation is increased in diabetes mellitus and may contribute to tumor progression. However, the role of O‐GlcNAcylation in tumor microenvironment is not fully elucidated. Here, we generated O‐GlcNAc transferase (OGT), an enzyme catalyzing the O‐GlcNAcylation, transgenic (OGT‐TG) mice (C57BL/6J) and examined the effect of O‐GlcNAcylation in tumor microenvironment on tumor progression using a subcutaneous transplantation model of B16 melanoma. We first confirmed that OGT‐TG mice showed increased O‐GlcNAcylation in various tissues. In our transplantation model, B16 tumor growth was significantly increased in OGT‐TG mice compared with wild type mice. In the tumor mass grown in OGT‐TG mice, NF‐κB activity and proinflammatory cytokine production were reduced. In the tumor cells, p38 MAPK activity was downregulated and, on the other hand, the Erk1/2 signaling pathway was upregulated. P38 MAPK inhibitor, SB230580, induced activation of the Erk1/2 signaling pathway in B16 melanoma cells. Taken together, these data suggest that O‐GlcNAcylation in tumor microenvironment can promote cancer progression through downregulation of p38 MAPK and subsequent upregulation of the Erk1/2 signaling pathway. Increased O‐GlcNAcylation in tumor microenvironment may be one of the mechanisms by which diabetes mellitus elevates the capability of promoting cancer progression.