bluuebunny/crossref_metadata_2025 huggingface dataset:
Alternative splicing of Cav1.3 in the mouse
Authors: Sabzanov Samuel , Mirkovic Jovan , Wang Ruizhi , Hossain Faruk , Ruggiu Matteo
Published: June 2021

L‐type voltage‐gated Ca2+ channel Cav1.3, which is encoded by CACNA1D, is one of four poreforming 1α‐subunits and it has been shown to undergo alternative splicing. Mutations of CACNA1D have been associated with neurological disorders such as Parkinson's Disease and Epilepsy. Previous studies have shown that a Calmodulin‐IQ domain present in some splicing isoforms of CACNA1D is critical for Cav1.3 binding affinity to the pharmaceutical inhibitor drug DHP. Our results further characterize the alternative splicing of Cav1.3 for the development of further potential clinical applications.

We were able to confirm four alternatively spliced regions, E8–E11, E12–E15, E32–36, and E46–E48 in Cacna1d. By semi‐quantitative RT‐PCR analysis, we found alternatively spliced variants of Cacna1d that were expressed at higher levels in brain‐derived tissues when compared to non‐brain derived tissues. Given how previously characterized alternatively spliced exons encode protein domains that are targeted by drugs that selectively inhibit Cav1.3 activity, the alternative splicing events described could also be important for drug discovery and therapeutic intervention.

Support or Funding Information

National Institute of General Medical Sciences Grant 1R15GM119099‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel AKT Activator SC79 Protects Airway Epithelial Cells Against Cadmium‐Induced Lung Injury
Authors: Gopallawa Indiwari , Lee Robert J
Published: June 2021

Study Objective

Activation of constitutively expressed nitric oxide synthase (NOS) isoforms has beneficial effects via increased ciliary beat frequency and antibacterial nitric oxide (NO) production in the airway and anti‐inflammatory effects in other tissues. It is known that activation of Ca2+/Calmodulin can activate endothelial (e) NOS and subsequent NO production. However, other signaling pathways (e.g., AKT, also known as protein kinase B) can activate e/neuronal (n) NOS. These signaling pathways have not been investigated in the airway cells. We hypothesized that activation of the AKT/eNOS pathway, which also activates nuclear factor erythroid 2–related factor 2 (NRF‐2) in many tissues, may have protective effects in lung cells during injury. We tested the responses of airway cells to SC79, a novel AKT activator. To model epithelial injury, we used cadmium (Cd2+), a highly reactive component of cigarette smoke that has been shown to disrupt epithelial barrier integrity and cause oxidative stress in some cell types.

Methods

To test if SC79 up‐regulates phospho (p)‐AKT, p‐eNOS, and/or NRF‐2 levels, A549 human type II alveolar epithelial cells were stimulated with SC79 for 2 hrs ± pre‐treatment with phosphoinositide‐3‐kinase (PI3K) inhibitor LY294002 for 1hr. Cell lysates were subjected for Western blotting. To determine the nuclear translocation of NRF‐2, cells were subjected to immunofluorescence (IF) with a specific NRF‐2 primary antibody after stimulation with SC79 for 2 hrs. To test if SC79 could protect against disruption of tight junction integrity during lung injury, 16HBE human bronchial epithelial cells were pretreated with Cd2+ for 1hr and then stimulated with SC79 for an additional 2hrs. Transepithelial electrical resistant (TEER) measurements were taken using an epithelial ohm meter.

Results

We observed an approximately 2‐fold induction of p‐AKT, p‐eNOS and NRF‐2 with SC79 treatment that lasted >2 hrs. Pre‐incubation with LY294002 blocked p‐AKT, p‐eNOS, and NRF‐2 induction. IF revealed a 2‐fold increase in nuclear‐localized NRF‐2 upon SC79 stimulation. Additionally, exposure of 16HBE cells to Cd2+ led to an almost complete reduction in TEER, which was blocked by SC79 for up to 24 hrs.

Conclusions

Our preliminary data suggest that SC79 upregulates pAKT, peNOS and NRF‐2 in lung cells through a mechanism involving PI3K. Furthermore, treatment with SC79 protects the epithelial integrity against cadmium‐induced injury. Future work will be directed toward testing the role(s) of eNOS and Nrf2 in the protective effects of SC79.

Support or Funding Information

R01DC016309, R21AI137484

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genesis of Antibiotic Resistance: XLI Herd Immunity Mitigates Waning Immunity
Authors: Martinez Thaliza , Soto Elsa , Escobedo Julio , Mendoza Jonathan , Barrientos April , Chavez Norma , Perez Ashley , Riojas Robert , Galindo Oscar , Kannan Subburaj
Published: June 2021

Based on the observations from the livestock show in the Maverick County, Texas, (MCJLS) (January 2006 to January 2017), and data analysis from the Texas Department of State Health Services, Health and Human Services (TDSHS‐HHS): Pertussis Emerging and Acute Infectious Disease Infectious Disease Control Unit (IDCU) and CDC, a prototype for “Transmission Model” conferring the herd protection (Herd Immunity) in combination with WASH interventions is presented here. Pertussis, a respiratory illness commonly known as whooping cough, is a contagious disease caused by Bordetella pertussis. The respiratory infection is spread via aerosol (in bacteria‐contaminated air droplets) from an infected person's cough or sneeze. Transmission often occurs from older family members to infants or children. Five doses of the DTaP vaccine, is recommended while protection gained from pertussis vaccines decline over time referred as “waning immunity.” To counter the decrease in immunity, a TDaP vaccine, a booster vaccine for tetanus and diphtheria with limited protection for pertussis. Here we present a hypothesis that immunity acquired through “community living inclusive of herd of livestock” (Herd) by the agrarian community mitigate the waning immunity there by confer the protection from the pertussis despite the observed antibiotic resistance observed across the nation and also emerging trend across the globe. Annually the MCJLS in Eagle Pass, Texas with over 300 youth exhibitors, presenting steers, goats, lambs, Hogs, and also chickens. Our observations and information gathered during the past ten years of the stock show could be best summarized as the proximity and duration of the exposure to the livestock animals and community (model) living represent the “WASH” interventions suggested in the transmission model for “Herd Immunity” (Am J Trop Med Hyg. 2016; 95(5):1201). Based on the Texas ‐ Pertussis Incidence Rates Maps by Year, Maverick County has relatively lowest pertussis incidence. Hypothetically, the scheduled DTap/TDap vaccinations conferred immunity weakens over the period of time would make the HSR8 susceptible for the pertussis which is contrary to the incidence report of TDSHS‐HHS‐IDCU. Furthermore, community living in HSR8 exemplifies the community model conferring cross protection among the agrarian community members, in addition to the protection from the scheduled pertussis immunization in combination with WASH interventions serve as a prototype for “Transmission Model” for “Herd Immunity”

Support or Funding Information

Supported by the Professional Development Funds by SWTJC to Subburaj Kannan

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Whole egg consumption increases plasma choline and betaine without affecting TMAO levels and gut microbiome in overweight postmenopausal woman
Authors: Zhu Chenghao , Sawrey‐Kubicek Lisa , Bardagjy Allison S. , Houts Hannah , Zhang Ruihan , Xu Weiqing , Rhodes Chris H. , Sacchi Romina , Randolph Jody M. , Steinberg Francene M. , Zivkovic Angela M.
Published: June 2021

As a crucial part of the symbiotic system, the gut microbiome has been shown to be a metabolic organ that presents important connections to many diseases and conditions, including cardiovascular diseases (CVD), the world wide leading cause of death. Trimethylamine N‐oxide (TMAO) is a plasma metabolite that is positively correlated with CVD. Trimethylamine (TMA) is produced by gut bacteria from dietary choline, betaine, or L‐carnitine, and is then converted in the liver to TMAO, which in turn affects hepatic and intestinal lipid and cholesterol metabolism. Because eggs are rich in choline, it has been speculated that their consumption may increase plasma TMAO. The objective of this study was to examine the effect of two eggs per day on plasma TMAO level and how it is related with gut microbiome composition in mildly hypercholesterolemic postmenopausal women. In this randomized, cross‐over study, 20 overweight human subjects were given two whole eggs and the equivalent amount of yolk‐free substitute as breakfast for four weeks, in randomized order, with a four‐week washout in between. Fasting blood draws and stool were collected at the beginning and of each treatment period. Plasma TMAO, choline, betaine and other metabolites were analyzed using LC/MS, while gut microbiome composition was analyzed using 16S amplicon sequencing. Plasma choline and betaine were significantly increased after whole egg but not yolk‐free substitute, however TMAO level was not significantly affected by treatments. Gut microbiome composition showed large inter‐individual variability at baseline and in response to the treatments. Parabacteroides, Ruminococcus, and Faecalibacterium were slightly increased after whole egg but not yolk‐free substitute for the majority of the subjects, while Bilophila slightly increased after yolk‐free substitute but not whole egg. The consumption of two eggs per day in overweight, postmenopausal mildly hypercholesterolemic women significantly increased plasma choline and betaine, but not TMAO, with a subtle effect on gut microbiome composition.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Influence of Gut Microbiome on Induction of Obesity
Authors: Hanafi Ihab Sheikh , Gupta Akash , Ayar Mirat , Doke Saloni
Published: June 2021

Obesity is considered as a multifactorial disease which depends on the complex interaction of host genetics, diet and other environmental factors. Specific alterations in gut microbiota can lead to obesity. There are a few organisms in the gut which have a great influence by the diet we eat. Organisms such as Actinobacteria, Firmicutes and Bacteriodetes are some of the common ones. The role of the gut microbiota in understanding the onset and development of obesity is gaining importance. This review covers the latest available data regarding the influence of different factors on gut microbiota in relation to obesity. Potential Therapeutic Targets and Probiotic Treatment –The gut bacteria model attributes obesity to the variable makeup of gut microbiota and its ability to modulate host energy homeostasis, adiposity and inflammation. To counteract these Student effects, strains of probiotics have been developed to manipulate gut microbiomes and improve health. This highlights recent human study data assessing the effects of probiotic supplementation on the prevention and treatment of obesity.

Support or Funding Information

Dr. Rajeev Pandey ‐ Department of Biochemistry, Spartan Health Sciences University, St. Lucia, West Indies

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Designer Biosynthetic Pathways for Photosynthetic Biofuels and Bioproducts: Opportunities and Challenges
Authors: Lee James Weifu
Published: June 2021

With synthetic biology, genetically engineered (GE) cyanobacteria are now a potential photosynthetic platform for bioproducts, in that genetic transformation with a series of transgenes can encode for biosynthetic pathways to enable the conversion of CO2, water and sunlight to biofuels (e.g. butanol). This presentation will disclose designer biosynthetic pathways for photosynthetic production of bioproducts such as butanol and/or related higher alcohols from carbon dioxide and water. The designer photosynthetic organisms are created such that the endogenous photobiological regulation mechanism is tamed, and the reducing power (NADPH) and energy (ATP) acquired from the photosynthetic process are used for synthesis of butanol and/or related higher alcohols from carbon dioxide and water. This photosynthetic biofuels‐production method eliminates the problem of recalcitrant lignocellulosics by bypassing it. Furthermore, this approach enables the use of seawater and/or groundwater for photobiological production of higher alcohols (such as 1‐butanol) without requiring freshwater or agricultural soil, since the designer photosynthetic organisms can be created from certain marine algae and/or cyanobacteria that can use seawater and/or certain groundwater. However, there are still significant challenges. For example, it is also important to understand the bio‐risk of such organisms before the synthetic biology approach could be used for commercial application. Recently, using plasmid‐transformed cyanobacteria, we experimentally demonstrated that the horizontal gene transfer from GE cyanobacteria to E. coli could occur as soon as 1–3 days of liquid co‐cultures. On the other hand, there was no detectable transfer event for the integrated transgenes so far. These results suggested that even though GE cyanobacteria carrying integrated transgenes possess a more permanent change in the genomic DNA, the bio‐risk of these GE organisms in the context of gene sharing is less severe than plasmids‐carrying GE cyanobacteria. In any case, biosafety is still a potential challenge that must also be addressed.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does Urbanization Drive Adaptive Evolution in White Clover (Trifolium repens)?
Authors: Akinwole Philips , Sanders Anya , Golde Spencer
Published: June 2021

Urban areas are functioning ecosystems with profound effects on the ecology and evolution of organisms. In fact, urban ecosystems with myriad deviations from natural systems provides potent evolutionary stimuli, however, the mechanisms through which urbanization drives natural selection on populations are poorly understood. To facilitate our understanding of the selective factors driving adaptation in cities, we examined adaptive phenotypic changes by quantifying the relative frequency of cyanogenesis (hydrogen cyanide production after tissue damage) in 110 white clover (Trifolium repens) populations along urbanization gradients in three Midwest (USA) cities. White clover is naturally polymorphic for cyanogenesis; a chemical defense against herbivores. We found that the frequency of cyanogenic plants within populations increased towards the urban center in two of three cities, but no consistent pattern in genetic diversity in one city. The area of a city strongly predicted the average cyanogenesis frequency within a city. Population density did not account for significant variations in the clines along urbanization gradient. Our study suggests that plant populations are adapting to urbanization gradients, but more studies are needed to assess whether the capacity of this species to adapt to urban ecosystems is a general phenomenon.

Support or Funding Information

We thank Biology Department, DePauw University, Greencastle, IN for funding.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional characterization of microsomal fatty acid desaturation pathway genes in Buglossoides arvensis: A stearidonic acid rich oilseed plant
Authors: Prasad P , Sreedhar RV
Published: June 2021

Buglossoides arvensis seed oil contains a significant amount of stearidonic acid (SDA, 18:4 n−3), an unusual omega‐3‐fatty acid with nutraceutical potential superior to α‐linoleic acid (18:3 n−3). Stearidonic acid biosynthesis occurs by sequential desaturation of oleic acid, linoleic acid and α‐linolenic acid by Delta‐12‐desaturase (FAD2), Delta‐15‐desaturase (FAD3) and Delta‐6‐desaturase (D6D), respectively. Molecular characterization of these enzymes was essential to exploit them as potential target genes for transgenic production of SDA in traditional oilseed crops. In the present study, putative full‐length coding sequence of BaFAD2, BaFAD3 and BaD6D genes were identified from B. arvensis developing seed transcriptome data (NCBI SRA ID: SRX2191688). Absolute quantification of gene expression levels by quantitative RT‐PCR showed that BaFAD2, BaFAD3 and BaD6D genes were ubiquitously expressed in both vegetative and reproductive tissues but with different levels of transcript abundance. BaFAD2, BaFAD3 and BaD6D were cloned in yeast expression vector pYES/NTC and were functionally characterized by heterologous expression in Saccharomyces cerevisiae strain INVSC1. Overexpression followed fatty acid feeding studies in INVSC1 strain revealed that the encoded enzyme activities of BaFAD2 and BaFAD3 efficiently converted oleic acid and linoleic acid into linoleic acid and α‐linolenic acid, respectively. BaD6D efficiently converted both linolenic and α‐linolenic acid substrates in to γ‐linolenic acid and stearidonic acid, respectively. In conclusion, the identification and characterization of these new fatty acid desaturase genes not only provide an insight into the biosynthesis of SDA in B. arvensis but also expand the repository of fatty acid desaturase targets available for genetic engineering of omega‐3 fatty acid traits in traditional oilseed crops.

Support or Funding Information

This research is financially supported by the Council of Scientific and Industrial Research (CSIR), New Delhi, under the 12th five‐year plan project LIPIC (BSC0401). Prasad P is grateful to UGC, New Delhi, India for providing senior research fellowship and United States‐India Educational Foundation, India for providing Fulbright Fellowship.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Topical Application of Wogonin Provides a Novel Treatment of Knee Osteoarthritis
Authors: Smith Jacob F , Starr Evan G , Goodman Michael A , Hanson Romney B , Palmer Trent A , Woolstenhulme Jonathan B , Weyand Jeffery A , Sterling Matthew T , Marchant Andrew D , Rose Brandon J , Porter James P , Eggett Dennis L , Kooyman David L
Published: June 2021

Osteoarthritis (OA) is a degenerative joint disease characterized by joint pain, decreased functional mobility, and deformation of articular cartilage and consequentially diminishing quality of life. Wogonin (WG), a compound extracted from the Skullcap Baicalensis plant, has been shown to have anti‐inflammatory effects on the Toll‐like Receptor 4 (TLR‐4) inflammatory pathway and antioxidant properties via Nrf2. We have shown that TLR‐4 is a major receptor for pain in OA. In our study, we examined the pain reducing, anti‐inflammatory, and chondroprotective effects of WG when applied as a topical cream (TC). After destabilizing the menisci of mice to induce OA, we examined the severity and progression of OA with and without the topical application of WG. Using a running wheel to track mice activity, we found that mice with WG treatment were statistically more active than mice without WG treatment. OA progression analyzed using Modified Mankin and OARSI scoring showed a significant attenuation of OA severity among mice treated with WG. Immunohistochemistry revealed a significant decrease in protein expression of TGF‐β1, but no significant differences were found among the biomarkers HTRA1, MMP‐13, or BMP‐2, suggesting an alternative pathway of cartilage rescue.

Support or Funding Information

BYU Office of Research and Creative Works

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prospective Changes in Fasting Glucose Inversely Affects Spexin Levels in Females with Prediabetes
Authors: Al‐Daghri Nasser , Wani Kaiser , Yakout Sobhy , Al‐Hazmi Hazim , Amer Osama , Hussain Syed Danish , Sabico Shaun , Almusharaf Sara , Alenad Amal , Alokail Majed , Clerici Mario
Published: June 2021

Background

Spexin (SPX) is a novel peptide thought to have a role in various metabolic regulations. Given its presumed body‐weight regulatory function, we aimed to determine whether lifestyle intervention programs focusing on weight loss and improvement in fasting glucose (FG) among people with impaired glucose regulation also induces changes in circulating levels of SPX.

Materials and Methods

A total of 160 Saudi males and females with prediabetes were randomly selected from a larger cohort of 294 who underwent a 6‐month lifestyle modification and/or metformin treatment to improve their glycemic status. Participants were split in two groups based on differences in glucose levels post‐intervention, with the first 50% (improved group) having the most significant reduction in FG. SPX was measured in both time‐points.

Results

Changes in SPX was significant only in this group [baseline median (Q1–Q3) 164pg/ml (136–227) vs follow‐up 176pg/ml (146–285); p<0.01]. When stratified by sex, the significant increase was observed only in females [159pg/ml (127–252) vs 182.5 (152,369.1); p<0.01], not in males. Furthermore, SPX levels showed a significant inverse association with FG (β=−0.22, p=0.003) only in females after adjustment for age and BMI.

Conclusions

The study demonstrated that prospective changes in FG inversely modulates SPX levels in a sexually dimorphic manner.

Support or Funding Information

Deanship of Scientific Research, KSU and the Chair for Biomarkers of Chronic Diseases (CBCD), Biochemistry Department, College of Science, King Saud University

Association of spexin with glycemic indexes. Fasting glucose (3A), insulin (3B) and HOMA‐IR (3C) on X‐axis and spexin on Y‐axis. Significant inverse correlations between SPX and glycemic indices were seen only in females. “r” depicts pearson correlation coefficient and “p” is the associated p‐value. P‐value <0.05 is considered significant

image

Association of spexin with glycemic indexes. Fasting glucose (3A), insulin (3B) and HOMA‐IR (3C) on X‐axis and spexin on Y‐axis. Significant inverse correlations between SPX and glycemic indices were seen only in females. “r” depicts pearson correlation coefficient and “p” is the associated p‐value. P‐value <0.05 is considered significant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Knock‐on effects of gestational diabetes in offspring learning and anxiety without influence of oxidative stress: Effects dependent on sex and age
Authors: Huerta‐Cervantes Maribel , Peña‐Montes Donovan , López‐Vázquez Miguel Angel , Olvera‐Cortés María Esther , Cortés‐Rojo Christian , Manzo‐Avalos Salvador , Saavedra‐Molina Alfredo
Published: June 2021

Gestational diabetes (DG) is an impairment of glucose metabolism during pregnancy, an increment of blood glucose in the intrauterine milieu results in abnormal fetal development and an increase in the risk of developing metabolic disorders as type 2 diabetes mellitus, neurodegenerative diseases and behavioral abnormalities in the offspring. In addition, increased oxidative stress has been reported in the offspring of rats under experimental GD in several brain regions at early ages. However, the insult in the offspring behavior in young and adult stages and oxidative stress in cerebral regions as hippocampus and cortex remains unclear. The aim of this study was to evaluate the effect of gestational diabetes in female and male offspring on behavior and oxidative stress in juvenile and adult stages. Twelve Wistar female rats were place mate with a control male. Gestation pregnant rats (day 7) were divided randomly into two groups: Gestation control rats (GC) and gestational diabetic rats (GD). GD rats were injected with a single intraperitoneal injection of a low dose of streptozotocin (STZ), only rats exhibiting blood glucose levels 140–300 mg/dL were considered for the study. Two‐months old rats (juvenile) and six‐months old rats (adult) male and female offspring from GC and GD were employed in this work. We evaluate emotionality and anxiety in the elevated plus maze, and open field, the Morris water maze was employed to evaluate spatial learning, and the radial maze for short‐term working memory. Oxidative stress was evaluated measuring the oxidative stress biomarkers (lipid‐peroxidation and GSH:GSSG ratio) and reactive oxygen species production (ROS) in homogenate of hippocampus and cortex. We found an impairment in spatial learning of offspring DG female rats, and a low anxiety level in offspring DG male on juvenile stage, this disturbs did not remain at adult stage. On the other hand, we have not found changes in lipid‐peroxidation and GSH:GSSG ratio and ROS production. In conclusion, these results suggest that GD affects learning and anxiety behavior without the involvement of oxidative stress in the juvenile stage of gestational diabetes offspring.

Support or Funding Information

Financial support grant from CONACYT (MHC), and Coordinación de Investigación Científica (UMSNH) (2.16 to ASM).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Uremic Toxins Decrease Skeletal Muscle Mitochondrial Energy Transfer Through Disruption of the Electron Transport System
Authors: Thome Trace , Berru Fabian N , Kumar Ravi A , Hahn Dongwoo , Ferreira Leonardo F , Ryan Terence E
Published: June 2021

Chronic kidney disease (CKD) impacts more than 25 million Americans and is associated with higher risk of all‐cause and cardiovascular mortality. Impaired kidney function leads to retention of metabolic waste products, termed uremic toxins, that negatively impact skeletal muscle resulting in increased fatigue, weakness, and muscle atrophy. Previous evidence has implicated mitochondria within the skeletal muscle as a primary mediator of muscle dysfunction in CKD, yet the underlying mechanisms are unknown. Therefore, the purpose of this study was to define the impact of uremic toxins on mitochondrial energetics. Skeletal muscle mitochondria were isolated from healthy C57BL/6N mice and exposed to vehicle (DMSO) or varying doses of the following uremic toxins: indoxyl sulfate, indole‐3‐acetic‐acid, L‐kynurenine, kynurenic acid, and methylguanidine. We employed a comprehensive mitochondrial phenotyping platform that included assessments of mitochondrial OXPHOS conductance across several levels of energy demand, hydrogen peroxide production (JH2O2), and dehydrogenase flux (using NADH autofluorescence). Exposure to uremic toxins resulted in a dose‐dependent decrease in OXPHOS conductance for all toxins, with 100nM exposure resulting in an average decrease of ~22% supported by pyruvate/malate (all P<0.05, n= 5–6/group). Uremic toxins did not decrease pyruvate dehydrogenase activity even at millimolar concentrations (all P>0.64), suggesting the decreased OXPHOS conductance occurs downstream of matrix dehydrogenases. Consistent with decreased OXPHOS conductance, uremic toxins dose‐dependently increased JH2O2 by 2–5‐fold (all P<0.01, n=4/group). These findings provide direct evidence that uremic toxins negatively impact skeletal muscle mitochondrial energetics, resulting in decreased energy transfer. Impaired mitochondrial energetics appears to be mediated downstream of matrix dehydrogenases, through either direct interaction within the electron transport system or ATP synthase.

Support or Funding Information

Partially supported by AHA Grant 18CDA34110044 to TER

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sensitizing Prostate Cancer Cells to IL‐27 Immunotherapy by Chemotherapy Induced Immunogenic Modulation
Authors: Kumar Shreya , Figueiredo Marxa
Published: June 2021

Bone‐metastasis occurs in most patients suffering from advanced prostate cancer. Treatment options for such patients are currently limited and immunogenic modulation (IMO) is emerging as a possible therapeutic option. IMO involves changes in cancer cell‐phenotype in response to treatment with sub‐clinical doses of chemotherapy which makes cells more susceptible to an immune attack. By utilizing the immune system to eliminate tumors, one can effectively reduce chemotherapy toxicity and the associated side‐effects. Moreover, an IMO approach may enable one to treat tumors unresponsive or resistant to chemotherapy. Our goal for this project is to characterize and optimize the response of prostate cancer cells to sub‐lethal doses of chemotherapy drugs. We examined gene expression changes in cancer cells in vitro following exposure to varying sub‐lethal concentrations of chemotherapeutics and optimized cellular response with respect to the duration of treatment, drug concentrations, and different cell types by using quantitative Polymerase Chain Reaction (qPCR), viability assays, and flow cytometry approaches. Expression levels of five markers of IMO were investigated in two prostate cancer cell lines. For example, the carcinoembryonic (CEA) gene, which is a tumor antigen and previously associated with IMO, was significantly upregulated in response to sublethal doses of chemotherapy and thus was selected for subsequent characterization. Of the five drugs that we tested across two cell lines, Cabozantinib and Docetaxel showed the most promising response in upregulating CEA. PC3 cells (a human prostate cancer adenocarcinoma cell line) showed a ~15‐fold change in CEA expression 48 h post‐docetaxel treatment. Similarly, Cabozantinib promoted a ~11‐fold CEA upregulation after 72 h of treatment. These gene expression changes were confirmed using flow cytometry. These results suggest that sub‐lethal doses of chemotherapy might induce tumor cell sensitivity to CEA‐specific T‐cell mediated killing. Further studies in in vivo models to profile tumor‐infiltrating immune effectors will help characterize the associated immune response and examine whether chemotherapy and immunotherapy (IL‐27) can act in synergy to enhance the anti‐tumor immune response. We anticipate that such combinational therapy will open new avenues for treating bone‐metastatic prostate cancer while reducing chemotherapy‐associated toxicity.

Support or Funding Information

NIH NCI R01CA196947

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Experience‐dependent axon targeting in the olfactory system
Authors: Belser Nathalee , Gong Naihua , Matsunami Hiroaki
Published: June 2021

Olfactory sensory neurons that express the same olfactory receptors are scattered in the olfactory epithelium (OE) and their axons coalesce into a few bundles of axons or glomeruli on each olfactory bulb. A single odorant particle binds to specific olfactory receptors (OR) and activates a pattern of glomeruli, facilitating the ability to smell. The mechanism wherein these OSN coalesce is still unclear. As a step toward understanding glomeruli map formation, we determined the effect of postnatal odor experience on OSN axon targeting of two related ORs, muscarinic type 71 (M71) and type 72 (M72) using a cognate ligand, 2‐hydroxyacetophenone. M71 and M72 differ in only 11 amino acid sequences and therefore respond to an overlapping set of odorants.

We obtained mice that express both M71 and M72 receptors and exposed them to 1/1000 of 2‐hydroxyacetophone for 16 hours intervals daily from birth to day 21. Mice were sacrificed at day 21 and dissected to view their olfactory bulb under fluorescence microscope that highlighted M71 glomeruli with green signal and M72 glomeruli with red signal. M71 and M72 glomeruli were categorized as intermingling, adjacent, or separate based on their proximity on the olfactory bulb.

Mice stimulated with 1/1000 2‐hydroxyacetophone had M71 and M72 glomeruli at closer distances in the olfactory bulb than control. The distance was analyzed according to minimum distances between each glomerulus, which was done by collecting the pixel coordinates of each object and determining the pair of coordinates between the two glomeruli with a minimum distance. We found that odor experience affected M71 and M72 olfactory sensory neurons (OSNs) that their axons coalesced onto closer glomeruli in the olfactory bulb compared to control mice.

Support or Funding Information

Funding Acknowledgements: Research Supplement to Promote Diversity in Health‐Related Research, National Institute of Deafness and other Communication Disorders (NIDCD).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synergy between Resolvins and Immune Checkpoint Blockade in a Novel Transplantable FANCC−/− Murine Head and Neck Tumor Model
Authors: Gartung Allison , Gilligan Molly M. , Bielenberg Diane R. , Fishbein Anna , Wells Susanne , Huang Sui , Kieran Mark W. , Serhan Charles N. , Panigrahy Dipak
Published: June 2021

Fanconi anemia (FA) patients have increased incidence of malignancies including aggressive head and neck cancer. Traditional cancer therapies, including chemotherapy and radiation, in head and neck cancer patients with mutations in the FA pathway (e.g. FANCC−/−) have failed and the development of novel therapeutics has been hindered by a lack of relevant pre‐clinical models. While FA knockout mice recapitulate tumorigenic properties and the stromal microenvironment, these genetic models exhibit extreme variability in time to develop invasive disease (e.g. 80–400 days), presenting a tremendous challenge to evaluate novel FA cancer therapies. Transplantable tumor systems are optimal to assess the anti‐tumor activity of new therapeutic approaches, however, few to no such models for FA have been characterized to date. Controlling inflammation is now recognized as a critical component of successful cancer treatment. While FA patients exhibit excessive inflammatory cytokines, a new direction has emerged with the discovery of endogenous omega‐3 fatty acid‐derived lipid mediators, such as resolvins, which have potent pro‐resolving activity without being immunosuppressive. These lipid autacoids act through promoting the clearance of cellular debris by macrophage phagocytosis, resulting in reduced localized pro‐inflammatory cytokines. As immunotherapy response can be predicted by DNA repair deficiencies that occur in FA, we hypothesize that stimulation of resolution of inflammation and checkpoint blockade would inhibit FANCC−/− tumor growth. Utilizing FANCC−/− and wild type (WT) murine head and neck squamous cell carcinoma cells, we have developed a robust transplantable tumor model in immunocompetent mice. FANCC−/− tumors exhibited accelerated growth and histologic findings demonstrated numerous FA tumor cells with dysplastic nuclei and Ki‐67‐positive proliferating cells compared to WT tumors. FANCC−/− tumors also expressed the immune checkpoint proteins PD1, PD‐L1, and CTLA4. Immune checkpoint blockade (e.g. anti‐PD1) and resolvins alone or in combination suppressed FANCC−/− subcutaneous and orthotopic (e.g. tongue) tumor growth and prolonged survival. Therefore, we have established a transplantable FANCC−/− murine head and neck tumor model whereby various novel therapies can be efficiently evaluated prior to clinical translation. The synergy between resolvins and immune checkpoint inhibitors to suppress FANCC−/− head and neck tumor growth provides a rationale for targeting immunotherapy and the resolution of inflammation for the treatment of FA cancers.

Support or Funding Information

National Cancer Institute grant RO1 01CA170549‐02; ROCA148633‐01A4; Credit Unions Kids at Heart Team.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Translocation and Targeting of PGK1 to HTATSF1 Regulate Cancer Cell Metastatic Ability
Authors: Tseng Yu‐Wen , Chang Yu‐Chan , Hsiao Michael
Published: June 2021

Phosphoglycerate kinase (PGK) is involved in glycolysis and metabolic pathways. Dysfunction of PGK may induce metabolic reprogramming and the Warburg effect. In this study, we have identified PGK1, but not PGK2, may play a key role in tumorigenesis and is associated with metastasis ability. We observed an inverse correlation between PGK1 and survival rate in several clinical cohorts through bioinformatics statistics and immunohistochemistry staining analyses. Surprisingly, we found that PGK1 was significantly increased in adenocarcinoma type compared with other subtypes. For this purpose, we established PGK1‐base proteomics datasets by pull‐down assay. We further investigated HIV‐ Tat Specific Factor 1 (HTATSF1), a potential binding partner through protein‐protein interactions. Therefore, we confirmed PGK1 indeed bind to HTATSF1 by two‐way immunoprecipitation experiments. In addition, we generated several mutant clones of PGK1 through side‐directed mutagenesis includes N‐terminal region, enzyme catalytic domain and C‐terminal region. Our data showed that PGK1 binds to HTATSF1 with or without enzyme activity and substrate recruitment. Moreover, our immunofluorescence staining also facilitates PGK1 has been translocated from cytoplasm to nucleus and colocalization with HTATSF1. Combined with all evidences, we proposed a novel model that the binding of PGK1 with HTATSF1 may exert functioning control of cancer metastasis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FAS receptor regulates HNSCC cancer stemness ability and pulmonary metastasis through Notch pathway
Authors: Chang Wei‐Min , Hsiao Michael
Published: June 2021

Up to 80% of head and neck squamous cell carcinoma (HNSCC) distance metastasis is occurring in lung through seeding of circulating cancer stem cells. It is crucial to understand the genes that are involved in HNSCC pulmonary colonization which may provide an important therapy niche in treating HNSCC patients successfully. Here, we found the FAS receptor knockout SAS cells could diminished the in vitro stemness ability and completely abolished the HNSCC pulmonary colonization in the immunodeficiency NSG mice. Even pretreated with single dose of FAS neutralize antibody could also significantly reduce the lung colonies forming numbers in vivo. FAS knockout could also prevent the in vitro cell migration and invasion abilities in the Boyden chamber assay. Using the reporter assays of common oncogenic signaling pathways, our results showed that Notch, HIF1A, RhoA, and STAT1 were suppressed in the FAS knockout cells. Moreover, the microarray analysis revealed that FAS knockout could also decrease stemness gene expressions, such as XIST, WDR66, HMGA2, and the ligand of Notch, Jag1 expressions. These genes and pathways reversal and complementation assays were performed and confirmed that indeed Notch pathway is one of the major downstream pathways of FAS‐mediated HNSCC stemness signaling to promote lung cancer metastasis. Taken together, our study found the novel FAS‐XIST axis might be mediated through Jag1/Notch pathway that may be contributing to HNSCC patient's lung metastasis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distribution of Mast Cells (MC), Toll‐Like Receptor 2 (TLR2) and Receptor for Advanced Glycation End Products (RAGE) May Reflect the Nature of Tumor Neovascularization in Human Medulloblastoma
Authors: Szukiewicz Dariusz , Laure‐Kamionowska Milena , Maslinska Danuta
Published: June 2021

Medulloblastoma is a neuroepithelial rapidly growing tumor originating from primitive neuroectodermal tissue. It accounts for approximately 12–25% of brain tumors in pediatric population, being the most common primary solid brain tumor in children, with a strong male preponderance. Vascular proliferation may be present but is not a constant feature of this lesion, but as in other tumors neovascularization is an important mechanism underlying medulloblastoma progression and poor prognosis of the disease. Recently, the set of angiogenic mediators derived from fragments of detroyed host necrotic cells has gained scientific attention. These so‐called damage‐associated molecular pattern (DAMP) molecules activate target cells via multiple surface receptors including TLR2 and RAGE. In addition, numerous MC in the surrounding of solid tumors are a source of many compounds that are potentially involved in tumor angiogenesis. The aim of the study was to examine the expression of TLR2 and RAGE receptors and the distribution of MC associated with the tumor neovascularization in medulloblastoma of the human brain. Immunohistochemical stainings of TLR2, RAGE and MC were performed using specific primary antibodies and immunoreactions were visualized using biotinylated secondary antibodies and ABComplex/HRP or an alkaline phosphatase‐avidin‐biotin conjugate. Then, sections were lightly counterstained with Mayer's hematoxylin and the mean expression of TLR2 and RAGE as well as the distribution of MC were examined in the regions of interest (ROIs) using quantitative immunohistochemistry. Striking reduction of the MC numer by 88.9% was observed in the necrotic areas of the medulloblastoma. MC were scattered confusedly in the necrotic areas while in necrotic‐free regions, MC were numerous only in the perivascular space of large brain and meningeal vessels at the border of the tumor. The RAGE receptor was highly expressed in the endothelium of all capillaries within necrosis‐free regions of medulloblastoma. Immunohistochemistry revealed that in the tumor necrotic area, TLR acccompanies vascular network formation. However, the capillary network in the necrosis‐free regions was TLR2 immunonegative. It can thus be concluded that TLR2 plays an significant role in the recognition of ligands released from necrotic medulloblastoma cells and participates in tumor neovascularization. The RAGE receptor and MC operate almost exclusively in necrotic‐free regions of medulloblastoma influencing different parts of the tumor vascular network.

Support or Funding Information

WUM grant: 2M2‐W1‐16/2M2‐W2‐17

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changes in Hepatic Stellate Cell MicroRNA Regulation Leads to Altered Activation States Through Modulation of TGF‐β Signaling Pathway Activity
Authors: Srivastava Ankita , Parrish Austin , Hoek Jan , Vadigepalli Rajanikanth
Published: June 2021

microRNAs (miRNAs) are a class of small, non‐coding RNAs ~22 nucleotides in length that are involved in post‐transcriptional regulation of a wide variety of processes critical for normal cell function, such as development, cell differentiation, cell proliferation, and cell death. Differential expression of specific miRNAs in the context of disease states has been previously demonstrated and linked to changes in disease phenotypes, including alcoholic liver disease. Studies from our lab and others have identified several miRNAs that may be key regulators of liver regeneration, such as miR‐21, whose role in liver regeneration and liver disease is well‐characterized, as well as several other miRNAs that show differential expression in response to changes in miR‐21 levels. Dysregulation of miR‐21 expression has been implicated in the alteration of hepatic stellate cells (HSCs) as a potential mechanism for decreased liver regeneration, in particular through their activation and resultant fibrogenic effects mediated by TGF‐β/SMAD/SMURF signaling. In this study, we have characterized the response by HSCs to changes in miRNA levels as well as induction by TGF‐β. Our findings show that manipulation of the levels of miR‐21 led to altered expression of signaling components involved in the TGF‐β signaling pathway. Furthermore, additional perturbations with TGF‐β alongside these miRNA changes demonstrated alterations in signatures of HSC activation. Taken together, our results show that changes in miRNA levels are important in the regulation of hepatic stellate cell activation.

Support or Funding Information

Research was supported by the following NIH grants from NIAAA R01 AA018873, F31 AA024969, NIBIB U01 EB023224.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FGF19 and Met co‐activation in murine liver induces HCC: Biological and clinical relevance
Authors: Tao Junyan , Yang Hong , Preziosi Morgan , Singh Sucha , Liu Silvia , Bell Aaron , Chen Xin , Monga Satdarshan Pal
Published: June 2021

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer‐related deaths worldwide. While several tyrosine kinase inhibitors (TKIs) have been approved as first or second line therapy for HCC, the overall efficacy of these agents is limited. Thus, developing useful animal models may shed light on the biology of HCC tumor and provide a platform to evaluate the potential therapeutic agents. In the current study, we found about 37% HCC patients have c‐Met and FGF19 up‐regulation together in about 110 HCC patient samples from China. Concomitant expression of FGF19 and c‐Met worked synergistically to induce tumor after 13 weeks post co‐delivery of the sleeping beauty plasmids and transposase via hydrodynamic tail vein injection. H&E staining showed the tumor histology to be reminiscent of HCC. Immunochemistry staining showed that these tumors had upregulation of glycogen synthetase and cell cycle protein cyclin‐D1. Ki‐67 staining was also increased in the tumor nodule. pERK and pmTOR were also activated in the tumor areas by IHC staining. The HCC tissue was assessed by RNA‐seq and compared to normal FVB mice. Such analysis revealed alterations in about 15 KEGG pathways including MAPK, RAS, RAP1, PI3K/AKT and others. Thus, we have generated a highly relevant animal model of a subset of human HCC, which can be used to address molecular mechanisms and test targeted therapy.

Support or Funding Information

1R01DK62277, 1R01DK100287,1R01CA204586 and Endowed Chair for Experimental Pathology (S.P.M.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.