The signal transducer and activator of transcription 3 (STAT3) protein is a major transcription factor involved in many cellular processes, such as cell growth and proliferation, differentiation, migration, and cell death or cell apoptosis. It is activated in response to a variety of extracellular stimuli including cytokines and growth factors. The aberrant activation of STAT3 contributes to several human diseases, particularly cancer. Consequently, STAT3-mediated signaling continues to be extensively studied in order to identify potential targets for the development of new and more effective clinical therapeutics. STAT3 activation can be regulated, either positively or negatively, by different posttranslational mechanisms including serine or tyrosine phosphorylation/dephosphorylation, acetylation, or demethylation. One of the major mechanisms that negatively regulates STAT3 activation is dephosphorylation of the tyrosine residue essential for its activation by protein tyrosine phosphatases (PTPs). There are seven PTPs that have been shown to dephosphorylate STAT3 and, thereby, regulate STAT3 signaling: PTP receptor-type D (PTPRD), PTP receptor-type T (PTPRT), PTP receptor-type K (PTPRK), Src homology region 2 (SH-2) domain-containing phosphatase 1(SHP1), SH-2 domain-containing phosphatase 2 (SHP2), MEG2/PTP non-receptor type 9 (PTPN9), and T-cell PTP (TC-PTP)/PTP non-receptor type 2 (PTPN2). These regulators have great potential as targets for the development of more effective therapies against human disease, including cancer. STAT3 gene) is the most prominent of all the STATs, because it has been established as a major oncogenic protein. In a majority of cancers, it is constitutively activated at tyrosine 705 (Y705), which disrupts mechanisms involved in cell proliferation, autophagy, differentiation, and/or cell survival, depending on the cell type . S,5. S4,5]iciently ,8,9.As previously mentioned, STAT3 is activated following phosphorylation at Y705 . STAT3 aThe rate and duration of phosphotyrosine-based signaling, such as the JAK-mediated activation of STAT3, is maintained by the activities of protein tyrosine phosphatases (PTPs). PTPs are a large and diverse family of enzymes, which catalyze the removal of the phosphate group from the tyrosine residue of phosphorylated proteins ,19,20,21PTPRD gene. It is classified as a member of the LAR subfamily of transmembrane PTPs, which includes LAR and PTPσ (PTPRS) anthracene (DMBA)-induced apoptosis. Furthermore, TC-PTP deficiency significantly increased epidermal thickness and hyperproliferation following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter. During two-stage skin carcinogenesis, the loss of TC-PTP promoted TPA-induced skin carcinogenesis via the upregulation of STAT3 and AKT signaling, whereas the inhibition of STAT3 or AKT recovered the effects of TC-PTP as a tumor suppressor in TC-PTP deficient cell lines (TC-PTP has several substrates such as EGF receptor, JAK, and STAT ,126. As he liver . Also, ihe liver ,129. Anohe liver . Studieshe liver ,136,137.he liver ,139, sughe liver . TC-PTP ll lines  141]. O. OPtpn2+PTPN2 have been associated with the chronic inflammatory bowel disease known as Crohn’s disease [TC-PTP is the only PTP known to regulate STAT1 other than SHP2. The interaction between STAT1 and TC-PTP may involve associated proteins, such as β-arrestin 1. For instance, during IFN-γ signaling, β-arrestin 1, acting as a scaffold, directly interacts with STAT1 and promotes STAT1 tyrosine dephosphorylation by recruiting TC45 . However disease .The STAT family of transcription factors are vital to the proper functioning of most cell types. They transduce the signals required for the activation of several key cellular processes. A STAT is activated primarily by tyrosine phosphorylation of its C-terminal tail. Tyrosine phosphorylation is a reversible post-translational modification that regulates proteins in multiple ways; however, during carcinogenesis, the rate and duration of tyrosine phosphorylation can become disrupted by genetic mutation or by the inactivation of important phosphotyrosine regulators. STAT3 is constitutively activated in many cancers; therefore, the mechanisms that regulate STAT3 have been, and continue to be, heavily investigated in order to identify newer and more effective targets for anti-cancer therapies. PTPs are the primary enzymes to dephosphorylate phosphotyrosine proteins. The current research has greatly increased our understanding of how STAT3 is regulated by PTP. To date, seven PTPs have been implicated in the regulation of STAT3: PTPRD, PTPRT, PTPRK, SHP1, SHP2, MEG2, and TC-PTP . However


We hypothesize that inflammatory mediators resulting from LMW PAH exposure in mouse lung epithelial cell lines are involved in the dysregulation of GJIC. We used mouse lung epithelial cell lines and an alveolar macrophage cell line in the presence of a binary PAH mixture . Parthenolide, a pan-inflammation inhibitor, reversed the PAH-induced inhibition of GJIC, the decreased CX43 expression, and the induction of KC and TNF. To further determine the direct role of a cytokine in regulating GJIC, recombinant TNF (rTNF) was used to inhibit GJIC and this response was further enhanced in the presence of the PAH mixture. Collectively, these findings support a role for inflammation in regulating GJIC and the potential to target these early stage cancer pathways for therapeutics.Polycyclic aromatic hydrocarbons (PAHs), prevalent contaminants in our environment, in many occupations, and in first and second-hand smoke, pose significant adverse health effects. Most research focused on the genotoxic high molecular weight PAHs (e.g., benzo[ Polycyclic aromatic hydrocarbons (PAH) are abundant toxicants in the environment , occupational settings, and in mainstream smoke, secondhand (sidestream) smoke (SHS), and thirdhand smoke exposures ,2,3,4. Ta]pyrene (B[a]P). B[a]P is a known group 1 carcinogen according to the IARC [a]P, in primary and SHS smoke from cigarettes and marijuana , in air pollution as components of particulate matter, as well as potential occupational hazards for those workers in industries such as coal, coke, and steel [a]P and benzo(e)pyrene P demonstrated increases in benzo[a]pyrene-7, 8-dihydrodiol-9, 10-oxide  resulting in DNA adducts P (a HMW PAH) to upregulate CYP1B1 in a rat alveolar type II cell line (RLE-6TN), potentially increasing the metabolism of procarcinogens and therefore playing a role in early tumor development [a]P in these RLE-6TN cells also increased reactivity with DNA as indicated by B[a]P-induced DNA adduct formation potentially leading to genotoxic events. Additionally, TNF increased mRNA production of inflammatory mediators, namely iNos, Cox2, Il6, and Il1β P in the C10 cells [a]P metabolite (BPDE) combined with the same two LMW PAHs in our mixture used for the present studies. In addition, we observed significant increases in GJIC dysregulation with the B[a]P and LMW PAH combination, as well as an increase in COX-2 mRNA expression, all supporting early stage tumor promotion associated phenotypes. However, in these studies we did not directly assess promotion per se because we applied the toxicants at the same time and thus concluded that at the least, these LMW PAHs should be considered co-carcinogens. However, currently, they are considered non-toxic, non-genotoxic, and non-carcinogenic in many cellular models [We previously published data that provided evidence that the LMW PAH mixture elicited a co-carcinogenic effect when combined with B[10 cells . In thesr models ,63,64. Dr models , and is Cx40−/− and Cx43−/−  KO mice had fibrosis like symptoms 8 wks after birth [Gap junction dysregulation and dysfunction is part of the evasion of growth suppression and the current hypothesis is that gap junctions can act as tumor suppressors, at least in early cancer development . Connexier birth . Howeverer birth . Intereser birth .Tlr4−/− mice inhibited gap junctions and CX43 expression compared to wildtype mice [Tumor promoting inflammation is also a hallmark of cancer  and our ype mice . Thus, iLastly, there are several future directions for our research. These studies did not investigate other lung expressed connexins, such as CX26 and CX32, the possibility of hemichannel involvement, nor tight junction protein involvement, all of which have potential roles in our LMW-PAH induced lung model . Cross tParthenolide  and ACHP (NFκB specific inhibitor) were purchased from TOCRIS . Indomethacin (COX1/2 inhibitor) was purchased from Sigma-Aldrich . CLI-095 (TLR4 inhibitor) was purchased from InvivoGen . Fluoranthene was obtained from AccuStandard  and 1-methylanthracene from Crescent Chemical . Glyburide  was purchased from Novus . All inhibitors and PAH were dissolved in DMSO , as done previously ,37,40. R50 for dysregulation of GJIC at 4 h (40 μM) or 24 h (15 μM) [Both the C10 and E10 cell lines, immortalized non-transformed alveolar type II cell lines, were obtained as a kind gift from Dr. Lori Nield . These cell lines were originally derived from BALB mice ,77 and a (15 μM) ,37,40. FMHS cells are an alveolar macrophage cell line originally derived from BALB/c mice . MHS celCytotoxicity was assessed in all cell lines using the CellTiter 96 Aqueous One Solution Cell Viability assay  following manufacturer’s instructions. Cells were grown in 96 well cell culture plates and serum deprived for 24 h prior to treatment with PAH, pharmaceutic inhibitors, rTNF, or their combinations.http://imagej.nih.gov/ij/). Treatment groups were compared to DMSO, the vehicle control, for final fraction of control (FOC) percentages. Three images were taken of each individual line, three cuts were made per dish, and there were three dishes per treatment, for a total of n = 9.GJIC activity was determined using the method described by Upham et al. (2011) . C10 celKC protein levels were evaluated using the mouse CXCL1/KC DuoSet ELISA  following manufacturer’s instructions. Cells were washed with saline before a 1 h treatment with inhibitor and a 4 h PAH treatments. At sample addition, the conditioned media retrieved was diluted 1:1 with reagent diluent (5% BSA in PBS) and incubated overnight in 96 well ELISA plate. The plate was read at 450 nm using the Tecan Infinite M200 Pro plate reader .Protein from C10 cells was extracted using 20% SDS containing Halt Protease & Phosphatase Single-Use Inhibitor Cocktail , similar to Osgood et al. . TweIl1b, Tlr4, Nalp3) can be found in Kc, Mcp1, Cox2, Il6, 18S) [One microgram of total RNA was reverse transcribed to cDNA  and ampll6, 18S) ,37,40.Our studies herein provide novel evidence that GJIC inhibition can be reversed in response to an anti-inflammatory inhibitor in lung epithelial cells. In addition, we show for the first time that a pro-inflammatory cytokine, such as TNF, can influence or potentially interact with an environmental toxicant, such as the PAHs, in lung epithelial cells to inhibit gap junction function as well as CX protein expression. Since we provide evidence that TNF originates from both epithelial cells or macrophages, this supports that both autocrine and paracrine mechanisms are involved. In the future, we will further investigate the mechanisms driving these observed responses, evaluate additional pathways for identification of biomarkers of exposure, and potential new therapeutic targets.


The theme of the 2018 Barshop Symposium on Aging at the Mayan Ranch in Bandera, Texas, USA was ‘Exercise regulation of biological aging’. Drs. Nicolas Musi and Darpan Patel were the conference organizers. The keynote address was delivered by Dr. Charlotte Peterson, Professor and Director of the Center for Muscle Biology at the University of Kentucky, USA, followed by scientific sessions on 1) Effects of exercise on mitochondrial biology; 2) Effects of exercise on inflammation and senescence; 3) Aging, exercise and function; 4) Muscle as a secretory organ; 5) Exercise and metabolism; 6) Exercise and cancer. These platform presentations were complimented by interactive poster presentations, which are represented by respective abstracts for this publication.1,2, Christopher S. Fry1, Rosario Maroto1, Blake B. Rasmussen1,*Ted G. Graber1Department of Nutrition and Metabolism, 2Division of Rehabilitation Science University of Texas Medical Branch at Galveston (UTMB), TX, USAIn older adults, sarcopenia and frailty produce a downward trajectory of decreased ability to perform activities of daily living, increased incidence of falls, eventual loss of independence, and increased mortality rate. To study the basic mechanisms underlying these diseases, and to assess potential therapies such as exercise, we need to comprehensively evaluate physical ability and exercise capacity in mice as they age. We hypothesized that older C57BL/6 male mice  would have reduced functional/exercise capacity compared to adult mice . We tested this hypothesis with grip test (forelimb strength), rotarod , inverted cling , treadmill (cardiovascular endurance), and voluntary wheel running . We then developed a composite scoring system comprised of the individual functional tests. In subsets from both age groups we determined muscle function using in vivo and in vitro contractile physiology. After adjusting for body mass with univariate ANCOVAs, older mice had reduced capacity in inverted cling , grip test , rotarod , and voluntary wheel running . Additionally, maximum dorsiflexion torque , and soleus  and EDL  peak force were lower in older mice. The composite scoring system determined reduced physical ability in older mice (p < 0.001) overall. However, older mice performing in the top 25% maintained function, and were indistinguishable from average adult mice (p = 0.335). In conclusion, our composite test is a powerful, non-invasive, and repeatable system to measure exercise capacity and functional aptitude in in future preclinical aging research.blrasmus@utmb.edu*Correspondence to: Blake B. Rasmussen, UTMB, TX. Email: NIA P30 AG024832 – Pepper Center Pilot Project (TGG)1, F. Bello2, V. Mulgrave3, A. Alsayegh3, T. Ajisebutu2, T. Falegan2, D. Lee1, J.S. Allard1,*A. Jaldi1Department of Physiology & Biophysics, Howard University College of Medicine, Washington, D.C., USA 2Department of Biology, Howard University, Washington, D.C., USA 3Department of Nutritional Sciences, Howard University, Washington, D.C., USAOur previous study showed that long-term high-dose treatment with the insulin-sensitizing drug metformin decreased brain-derived neurotrophin (BDNF) levels in mouse brain. However, several studies have shown metformin to be beneficial in the brain, as well as peripherally. In order to attenuate any possible deleterious effects of metformin, we combined a lower-dose metformin treatment with voluntary exercise. The overall goal of this study is to understand the impact of metformin combined with voluntary exercise on amyloid-beta plaque deposition, levels of brain BDNF and pro-inflammatory cytokines using an adult, female APP+/PS1+ mouse model. In cage, voluntary running-wheel activity was monitored and metformin was given at a dose of 70 mg/kg body weight for one year. Preliminary results reveal a significant increase in voluntary running activity of mice treated with metformin. Amyloid-β 42/40 ratio in metformin treated sedentary mice was significantly lower than that of non-treated sedentary mice. The combination of metformin and exercise, however, did not affect amyloid-beta plaque load, levels of mature BDNF or pro-inflammatory cytokines in the hippocampus. These results provide new information on the impact of long-term treatment with metformin in combination with exercise on AD-like pathology.joanne.allard@howard.edu*Correspondence to: Joanne Allard, Howard University College of Medicine at Washington, D.C. Email: 1, Rojina Ranjit1, Holly Van Remmen1,*Bumsoo Ahn1Department of Aging and Metabolism, Oklahoma Medical Research Foundation at Oklahoma City (OMRF), OK, USASarcopenia, the age-related loss of muscle mass and contractile dysfunction, universally affects the elderly and is closely associated with frailty and reduced healthspan. Despite the inevitable consequences of aging, no pharmacological therapies are available to mitigate sarcopenia. Ghrelin is a gut-released hormone known to increase appetite and body weight through acylation-dependent  receptor activation in pituitary and hypothalamus. Literature shows a direct impact of unacylated ghrelin (UnAG) on skeletal muscle in acute myopathies (e.g. denervation), independent of its receptor activation. However, the impact of UnAG on a progressive loss of muscle mass in sarcopenia has not been tested. Thus, we are testing the hypothesis that UnAG can ameliorate sarcopenia. To test the hypothesis, we used a mouse model of accelerated aging, mice lacking CuZnSOD (Sod1KO). UnAG was continuously administered to two-month old Sod1KO mice for 3 weeks. Serum UnAG level trended to decrease in Sod1KO mice compared with WT, but UnAG increased circulating UnAG level by 2–3 folds. Unlike AG, UnAG did not affect food consumption or body weight. Importantly, the UnAG treatment prevented loss of skeletal muscle mass and contractile dysfunction observed in Sod1KO mice. Our data indicate a direct role of UnAG on sarcopenia independent from food consumption and body weight. These findings implicate UnAG as a potential therapeutic strategy and warrant future aging studies.Holly-VanRemmen@omrf.org*Correspondence to: Holly Van Remmen, OMRF, TX, USA. Email: 1,*, Shane Sprague1, Pamela Reed1, Naomi L. Sayre1Sadiya Ahmad1University of Texas Health Science Center at San Antonio (UTHSCSA), TX, USAStroke is the leading cause of death and the primary cause of long-term disability in the U.S. Expression of Apolipoprotein E4 predisposes stroke patients to poor long-term outcome. This study aims to test one possible mechanism by which ApoE4 contributes to cognitive decline after stroke. Here, we examine the effect of a major ApoE4 receptor, low density lipoprotein receptor related protein 1 (LRP1) on sensitivity to stress in astrocytes. LRP1 binds and moves extracellular ligands and plasma membrane proteins into the endocytic system. Others have shown that LRP1 regulates cell-surface TNF receptor (TNFR1) in non-astrocytic cells. We propose LRP1 similarly regulates TNFR1 in astrocytes to attenuate inflammatory response after stroke. Studies have shown ApoE4 slows the recycling of endocytic LDL receptors. We hypothesize that ApoE4 inhibits the ability of LRP1 to remove TNFR1 from the plasma membrane. This is expected to increase cytokine sensitivity, resulting in worse outcome after stroke. We investigated the effect of LRP1 on astrocyte TNFα signaling and response in immortalized ApoE null mouse astrocytes subjected to lentiviral-mediated knockdown of LRP1. We found that LRP1 deficient cells have increased TNFR1 activity and significant loss of viability after prolonged TNFα stimulation. Our results indicate that loss of LRP1 renders astrocytes more sensitive to TNFα. These studies will elucidate how astrocyte-LRP1 contributes to outcome after stroke, and help explain one potential way ApoE4 exerts pathological effects. A better understanding of the long-term processes after stroke will allow identification of therapies which will improve the morbidity and mortality associated with stroke.ahmedsn@uthscsa.edu*Correspondence to: Sadiya Ahmad, UTHSCSA, TX, USA. Email: 1,*, Kennedy Mdaki1, and Yuguang Shi1John-Paul Andersen1Barshop Institute for Longevity and Aging Studies, Department of Pharmacology, University of Texas Health Science Center at San Antonio (UTHSCSA), TX, USAAster-B also known as GRAMD1B has been characterized as a non-vesicular Plasma Membrane (PM) to Endoplasmic Reticulum (ER) cholesterol transporter. However, little is known as to its fate after it becomes localized in the ER. Here we identified a potential role for Aster-B as a regulator of cholesteryl ester trafficking to the mitochondria as well as a novel function of COPI vesicles in its associated pathway.andersenj@uthscsa.edu*Correspondence to: John-Paul Andersen, UTHSCSA, TX, USA. Email: 1,*, Hanyu Liang1,2, Nicolas Musi1,2Eric Baeuerle1Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio (UTHSCSA), TX, USA. 2Geriatric Research Education and Clinical Center, Audie L. Murphy VA Medical Center, San Antonio, TX, USASkeletal muscle insulin resistance is an early-stage dysfunction observed in the pathogenesis of type 2 diabetes (T2DM) in humans. Development of insulin resistance is a multifactorial process between organ systems, and recently, the gut and its microbial contents (the microbiome) have been hypothesized to contribute a larger-than-anticipated role to the development of obesity and type 2 diabetes. These include alteration of the bacterial profile such as reduced Bifidobacterium and Bacteroidetes in the gut, increased intestinal permeability, and production of bacterial lipopolysaccharide (LPS) leading to low-grade systemic inflammation termed endotoxemia. This endotoxemia effect may be primarily mediated through the toll-like receptor TLR4-NF-κB signaling pathway, which is increased in insulin-resistant diseases such as type 2 diabetes. To date, the contributions of the human gut microbiome to endotoxemia and skeletal muscle insulin resistance in obesity and type 2 diabetes have not yet been evaluated. This study will investigate (i) the human gut microbiome profile in lean, obese, and diabetic patients; (ii) endotoxemia modulation in human subjects and its effects on insulin sensitivity and intestinal permeability; and (iii) the effects of endotoxemia and associated inflammation on skeletal muscle insulin resistance in vitro. I hypothesize that alterations in gut microbiome in obese and type 2 diabetic patients result in increased metabolic endotoxemia and systemic inflammation through the TLR4-NF-κB pathway, which contributes to insulin resistance in skeletal muscle tissue. I also hypothesize that the endotoxemia effect can be lessened by promotion of a more favorable microbiome population through synbiotic supplementation or by LPS removal.baeuerle@livemail.uthscsa.edu*Correspondence to: Eric Baeuerle, UTHSCSA, TX, USA. Email: 1,*, Habil Zare2, Bess Frost3Adrian Beckmann1Institute of Biotechnology, Barshop Institute of Longevity and Aging, UTHSCSA, TX, USA. 2Department of Computer Science, Texas State University at San Antonio, TX, USA. 3Department of Cell Systems and Anatomy, Barshop Institute of Longevity and Aging, University of Texas Health Science Center at San Antonio, TX, USATauopathies are a class of neurodegenerative disorders associated with deposits of insoluble tau protein within the brain. At over 5 million cases currently diagnosed among Americans, Alzheimer’s disease (AD) is the most common type of tauopathy. As one of two hallmark pathologies of AD, pathogenic tau has emerged as a promising target for therapeutic targeting. Mutations in the tau gene are associated with dominantly inherited familial tauopathies termed frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), demonstrating that tau dysfunction is sufficient to drive neurodegeneration. An active cellular program maintains the terminally differentiated, postmitotic state of neurons. When this program is perturbed, post-mitotic neurons can re- activate the cell cycle, which is known to drive neuronal death. In my preliminary analysis, I have identified prospero, a protein that orchestrate the expression and silencing of genes that maintain terminal neuronal differentiation, among the top ten significantly downregulated genes in brains of a Drosophila model of tauopathy. In addition, I identified 51 genes that are differentially expressed in tau transgenic Drosophila that are known to be regulated by prospero, suggesting that pathogenic tau may disrupt the cellular program that maintains terminal neuronal differentiation. Therefore, I hypothesize that pathological tau causes neuronal death by dysregulating prospero and staufen, thereby disrupting the cellular program that maintains terminal differentiation in neurons. Using genetic manipulations of tau transgenic and control Drosophila, I have identified filamentous actin to be a key driver in the depletion of prospero and discovered that targets of prospero affect tau-induced neurotoxicity.beckmann@livemail.uthscsa.edu*Correspondence to: Adrian Beckmann, Institute of Biotechnology, Barshop Institute, UTHSCSA, TX, USA. Email: 1, Adam Salmon1,*Michael Bene1University of Texas Health Science Center at San Antonio, TX, USA4-Phenylbutyrate (PBA), an FDA approved drug for urea cycle disorders, has been extensively researched for its potential use in many age-related diseases including: Alzheimer’s, Parkinson’s, type II diabetes, heart disease, and various cancers. One gene whose expression has been reported to increase in response to PBA is the Parkinson’s related oxidative stress response gene DJ-1/PARK7. DJ-1 promotes cell survival pathways and protects mitochondrial function. Most research looking at PBA’s effects on DJ1 have concentrated on neurons, therefore PBA’s effects on DJ-1 in other cell types are largely unknown. To address this question dose response experiments were performed in vitro with mouse liver cells, C2C12 muscle cells, and tail fibroblasts. DJ-1 protein levels were increased in response to PBA in both liver cells and tail fibroblasts, but not C2C12 cells. Notably, tail fibroblasts from a male mouse had significantly different DJ-1 levels compared to fibroblasts taken from a female mouse. Additionally, due to DJ-1’s ability to complex with PINK1/Parkin and mitochondrial complex I, it was expected that PBA may be altering mitochondrial function through DJ-1. Interestingly, it was found that despite no alteration to DJ-1 expression, PBA produced changes to mitochondrial respiration in C2C12 cells, reducing basal respiration and relative ATP production, while leaving relative maximum respiration unchanged. PBA has been approved by the NIA Intervention Testing Program to determine possible effects on mammalian lifespan and future in vivo studies may further elucidate the role of PBA in altering physiological function and determine possible effectiveness in a broader range of patients.salmona@uthscsa.edu*Correspondence to: Adam Salmon, Barshop Institute, UTHSCSA, TX, USA. Email: 1, Bumsoo Ahn1, Gavin Pharaoh1, Ashwini Poopal1, Rojina Ranjit1, Holly Van Remmen1,*Jacob L. Brown1Aging and Metabolism Research Program, OMRF, Oklahoma City, OK, USAOxidative stress is associated with sarcopenia; however, the effect of oxidative stress on myogenesis is unknown. Therefore, the purpose of this study was to examine myogenic factors in inducible MnSOD muscle specific knockout (Sod2 mKO) mice. Methods: Skeletal muscle Sod2 was deleted with Cre recombinase containing mutated estrogen receptor ligand-binding domains driven by α-actin promoter. Wildtype (WT) or Sod2 floxed mice were injected with tamoxifen at ~6 months old to delete the floxed gene and were sacrificed at 11–13 months old. Sod2 protein and activity level were measured to confirm deletion of the transgene. Muscle wet weights, contractility, respiration and reactive oxygen species (ROS) were also measured. Hematoxylin and eosin staining was used to assess muscle size and morphology. Myogenic markers were measured by rt-qPCR and/or western blot. Data were analyzed by t-test, and α was set at 0.05. Results: Sod2 was not detectable in Sod2 mKO muscle. Muscle size, respiration, ROS and contractility were not different between groups; however, there was a mean increase in ROS. Centrally located nuclei were ~10 fold higher in Sod2 mKO mice, so myogenic response was assessed. MyoD mRNA content and Myogenin protein content were measured but were found to be not different between groups; however, eMHC mRNA content was ~50% lower in Sod2 mKO mice. Conclusions: Centrally located nuclei is associated with regenerating fibers; however, myogenic response was lacking. This may indicate satellite cell dysfunction. Satellite cell response to muscle damage will be assessed in future studies.Holly-VanRemmen@omrf.org*Correspondence to: Holly Van Remmen, OMRF, TX, USA. Email: 1,*, Qianqian Liu1, James F. Nelson1, Jonathan A.L. Gelfond1Catherine J. Cheng1University of Texas Health Science Center at San Antonio (UTHSCSA), TX, USAThe female survival advantage is one of the most robust characteristics of human longevity, but the underlying biological mechanisms are unknown. The National Institute on Aging’s Interventions Testing Program (ITP) was designed to overcome the limitations of inbred strains by evaluating lifespan-extending compounds in genetically heterogeneous UMHET3 mice. In this combined dataset of 12,223 UM-HET3 mice from the ITP, we report that the female survival advantage parallels that of human populations: greatest in early adulthood and diminishing progressively thereafter. This finding is observed at 3 sites in 6 cohort years. We further report that, out of 13 lifespan-extending interventions tested, those with a male-specific effect primarily decrease male mortality before median lifespan, whereas interventions that were effective in both sexes are more likely to reduce mortality at later ages (>30 months).ChengCJ@livemail.uthscsa.edu*Correspondence to: Catherine J Cheng, UTHSCSA, TX, USA. Email: 1,2,3,*, Karl A. Rodriguez1,4, Alfred L. Fisher1,2,3,5Meghna N. Chinchankar1Barshop Institute for Longevity and Aging Studies, 2Center for Healthy Aging, 3Division of Geriatrics, Gerontology, and Palliative Medicine, Department of Medicine, 4Department of Cell Systems and Anatomy, UTHSCSA, San Antonio, TX, USA. 5Division of Geriatrics, Gerontology, and Palliative Medicine, Department of Medicine, University of Nebraska Medical Center, Omaha, NE, USAProtein degradation is essential for the preservation of the proteome. Its reduced efficiency leads to protein aggregation which potentiates several proteotoxic disorders. Paradoxically, our lab reported that the Caenorhabditis elegans rpn-10(ok1865) proteasome mutant exhibits enhanced proteostasis, elevated stress resistance and extended lifespan. RPN-10 is an ubiquitin receptor on the 19S regulatory particle that targets polyubiquitinated substrates to its 20S proteasomal core for degradation. The proteasome dysfunction of the rpn-10 mutant is characterized by reduced ubiquitin fusion degradation. We ascertained that the compensatory upregulation of autophagy and SKN-1/Nrf-mediated responses partially contribute to the robust rpn-10 mutant phenotype. To elucidate this protective mechanism, our RNA-sequencing data analysis revealed that several ERQC genes are transcriptionally upregulated in the rpn-10 mutant. We hypothesized that the rpn-10 mutant exhibits enhanced ER proteostasis which mediates its stress resistance. Accordingly, we found that cytosolic proteostasis and longevity depends on the ER master chaperone hsp-3/-4 (BiP) and ER ATPase cdc-48.2 (p97/VCP) in the rpn-10 mutant. Moreover, it exhibits altered ER homeostasis compared to the wild-type. Complementarily, the attenuated expression of the aggregation-prone mutant α-1 antitrypsin (ATZ) reporter proves that ER proteostasis is ameliorated in the rpn-10 mutant. Via a suppressor screen of the ATZ phenotype in the rpn-10 mutant, we identified H04D03.3 (putative homolog of the proteasome-associated protein ECM29) is critical, thus signifying an unexpected role for the rpn-10 mutant proteasome in maintaining ER proteostasis. Altogether, it appears that mild proteasomal dysfunction induces an ERQC adaptation that underlies the improved proteostasis and increased longevity of the rpn-10 mutant.Chinchankar@livemail.uthscsa.edu*Correspondence to: Meghna N. Chinchankar, Barshop Institute, UTHSCSA, TX, USA. Email: 1, Ning Zhang1, Nicolas Musi1,2, Andrew M. Pickering1,3,*E. Sandra Chocron1Barshop institute for Longevity and Aging Studies, UT Health San Antonio, TX, USA. 2Geriatric Research, Education, and Clinical Center (GRECC), South Texas Veterans Health Care System (STVHCS) at San Antonio, TX, USA. 3Department of Molecular Medicine, School of Medicine, UT Health San Antonio, TX, USAMitochondrial Thioredoxin Reductase (Txnrd2) is a rate limiting enzyme in the mitochondrial thioredoxin system which serves as one of the major mitochondrial ROS scavenging pathways. Txnrd2 is also a repressor of the ASK-1 oxidative stress induced apoptotic pathway. Our group previously identified a correlation with the expression of this protein and long-lived species and its overexpression prolonged lifespan in Drosophila. We have generated a Txnrd2 transgenic (T-tg) mouse which has ubiquitously heighted (two-fold) Txnrd2 expression. We have found that overexpression of Txnrd2 leads to increased Oxygen Consumption, enhanced mitochondrial membrane potential and increased resistance to mitochondrial oxidative damage in MEFs. We have also found that female T-tg mice showed a leaner trend and reduced food consumption, with improved glucose tolerance but no difference in insulin sensitivity. These mice showed a lower Oxygen consumption and CO2 production with lower energy expenditure in individual metabolic cages. We further tested their exercise capacity where the T-tg mice had a similar performance to control mice. These results suggest that Txnrd2 overexpression can lead to metabolic changes that need to be further understood, but seem to be beneficial for glucose handling and homeostasis in female mice.PickeringAM@uthscsa.edu*Correspondence to: Andrew M. Pickering, Barshop Institute, UTHCSA, TX, USA. Email: Voelcker foundation.1,*, Liu Y1, Thyne KM1, Salmon AB1Dorigatti JD1The Sam and Ann Barshop Institute for Longevity and Aging Studies, UTHCSA, San Antonio, TX, USAβ-Guanidinopropionic acid (β-GPA) is a naturally occurring creatine analog reported to activate AMP Activated Protein Kinase (AMPK) signaling. β-GPA has been reported to improve glucose metabolism, promote mitochondrial biogenesis, alter mitochondrial fuel preference, and enhance muscle exercise resistance in young animals. However, there is a knowledge gap regarding whether β-GPA may improve or prevent age-related declines in such physiological measures when delivered later in life. We consequently hypothesize that β-GPA will prevent age-related physiological declines in function by altering mitochondrial energetics to preserve cellular function. We test this hypothesis using young and old genetically heterogeneous mice (HET3) fed 1% β-GPA or control chow for 4 months. In contrast to reports of decreased body mass in young rodents, we report that aged males supplemented with β-GPA are protected from late-life declines in body mass. Further, while young rodents are reported to exhibit improved exercise tolerance and better glucose tolerance with β-GPA, aged mice did not see improvements in these measures, though HbA1c levels were decreased in males. Unexpectedly, content of mitochondrial oxidative phosphorylation complexes was generally unchanged by β-GPA, while Complex 1 was reduced in β-GPA treated males. Additionally, Acetyl Coenzyme A Carboxylase (ACC) phosphorylation was increased in females supplemented with β-GPA but no corresponding increase in fatty acid oxidation was observed in permeabilized soleus. These results raise an important point regarding the timing of interventions to improve healthy aging; while some interventions may have rejuvenating effects in the old, others may require earlier intervention to provide benefit.dorigatti@uthscsa.edu*Correspondence to: Jonathan Dorigatti, Barshop Institute, UTHSCSA, TX, USA. Email: 1,2, Nancy S. Gonzalez2, Teppei Fujikawa2,3,4,*Takuya Yoshida1Department of Clinical Nutrition, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan. 2Department of Cellular and Integrative Physiology, Long School of Medicine, 3Center for Biomedical Neuroscience, 4Mouse Genome Engineering and Transgenic Facility, Long School of Medicine, UTHSCSA, TX, USAExercise training induces metabolic adaptations such as strengthening skeletal muscle function, leading to beneficial effects of exercise on metabolism. However, the precise mechanism underlying metabolic adaptations to exercise, in particular, the contribution of the central nervous system (CNS), is largely unknown. Our previous study demonstrates that steroidogenic factor-1 (SF-1) in the ventromedial hypothalamic nucleus (VMH) is key to the metabolic adaptions to exercise. Here we investigate that the role of neuronal activities of SF-1-expressing neurons (SF-1 neurons) in the regulation of metabolic adaptations. We utilized optogenetics which allows us to specifically activate SF-1 neurons. After SF-1 neuronal activation by optogenetics, we measured mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), which is the master regulator of skeletal muscle function as well as one of readouts of exercise effects to the skeletal muscle. We found that activation of SF-1 neurons is sufficient to increase PGC-1α in the skeletal muscle. We further found that blocking sympathetic nervous system (SNS) blunted increased PGC1α in the skeletal muscle by the activation of SF-1 neurons. Overall, our data suggest that the activation of SF-1 neurons-SNS axis is sufficient to recapitulate exerciseinduced PGC-1α in the skeletal muscle.fujikawa@uthscsa.edu*Correspondence to: Teppei Fujikawa, UTHSCSA, TX, USA. Email: This study was supported by UT System Rising STARs Program (to T.F.) and San Antonio Area Foundation Research Grant (to T.F.)1, Cang Chen1, Anindita Bhattachcharjee1, Xiaogang Wang1, Guo Xiaoqian1, Guo Ge1, Senlin Li1Michael Guderyon1Department of Medicine, Biology of Aging, UTHSCSA, TX, USAHematopoietic stem cell transplantation (HSCT) offers an effective treatment modality for patients diagnosed with a number of diseases of the blood and immune systems. HSCT uses mobilization factors to collect donor cells before patients undergo toxic conditioning regimens using chemotherapy and/or radiation. Here, we employ a novel non-cytotoxic conditioning regime using FDA-approved mobilization factors in concert with multiple cycles of transplantation to examine health-associated benefits of transplanting youthful donor HSCs into old recipients. We report that mobilization of endogenous hematopoietic stem cells (HSCs) using G-CSF and AMD3100 sufficiently conditions recipients to accept donor cells, achieving over 70% long-term engraftment in C57BL6/NIA mice. In ongoing studies, we define health-associated benefits of replacing old endogenous HSCs with young donor cells by examining healthspan markers such as food intake, body weight, median and overall longevity, and the accumulation of health-associated deficits. This approach is particularly attractive because, in addition to eliminating the need for irradiation or chemotherapy, each of the essential reagents have previously been approved by the FDA for human use. Thus, these studies have the potential to improve current transplantation regimes by replacing traditional HSCT methods with this novel approach and may lead to the development of the first cell-based anti-aging therapy.Guderyon@uthscsa.edu*Correspondence to: Michael Guderyon, University of Texas Health Science Center at San Antonio. Email: 1,*Jason Hadley1Biology of Aging, UTHSCSA, TX, USAProtein palmitoylation is a reversible post-translational modification. It has gained a rapid attention recently as its ability to spatially and temporally modulate a diverse range of signaling pathways in cells. Depalmitoylases are a class of enzymes that catalyze the removal of this lipid modification and subsequently alters the subcellular localization of the target protein. Recently, we identified LF8 as a potential novel depalmitoylase and found its positive association with insulin sensitivity in mouse liver. In addition, we have generated LF8 knockout mice, and our preliminary data indicate that deletion of LF8 gene led to exacerbate high fat diet-induced obesity phenotype. These results reveal an important role of depalmitoylation in regulating energy homeostasis and identify a novel target molecule for enhancing insulin sensitivity and treating type 2 diabetes.hadleyj@livemail.uthscsa.edu*Correspondence to: Hadley Jason, University of Texas Health Science Center at San Antonio. Email: 1,*, Bess Frost2Simon A. Levy1Neuroscience discipline, IBMS PhD program, Graduate School of Biomedical Sciences, UTHSCSA, TX, USA. 2Department of Cell Systems and Anatomy, UTHSCSA, TX, USATauopathies are neurodegenerative diseases characterized by fibrillar tau protein aggregates. In Alzheimer’s disease these aggregates form neurofibrillary tangles, one of the disease’s pathological hallmarks. Tau pathology propagates through the brain hierarchically over time and along circuitry, suggesting a prion-like cell-to-cell mode of spreading. Tau is now known to spread between cells and form stably transmitted strains in vitro and in vivo, but the underlying mechanisms have not been well characterized. We are creating a Drosophila model to study prion-like tau propagation in a shortlived animal model amenable to screening approaches. We aim to model two key features of prionlike tau transmission: 1) cell-to-cell spread and 2) a capacity to recruit soluble tau into aggregates. Native gel electrophoresis indicates that tauP301L forms oligomers in Drosophila neurons in vivo, suggesting a capacity for aggregation. Based on this finding, tauP301L was expressed in retinal neurons and its spread was assessed by fluorescence in situ hybridization to detect tau mRNA and immunofluorescence to detect tau protein. Tau protein was found in cells lacking tau mRNA, indicating cell-to-cell tau spread. To detect aggregation seeding of soluble tau by spreading tau, seedcompetent tauP301L fused to N-terminal luciferase (LucN), will be expressed in one set of neurons, while wild-type tau fused to C-terminal luciferase (LucC) will be expressed in another. LucN-labeled tau recruiting soluble LucC-tagged tau into aggregates will produce luminescence. This model will allow investigations into the mechanisms controlling tau spread in adult brains, with the goal of developing treatment strategies targeting prion-like pathogenic tau spread.levys@livemail.uthscsa.edu*Correspondence to: Simon A. Levy, UTHSCSA, TX. Email: 1,2,3, Vanessa Martinez2, Elizabeth Fernandez1,2,4, Randy Strong1,2,3,4Paul Anthony Martinez1Department of Pharmacology, 2Barshop Institute for Longevity and Aging Studies, 3Center for Biomedical Neuroscience, UTHSCSA, TX, USA. 4Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care Network (STVHCS) at San Antonio, TX, USAParkinson’s disease (PD) is second most common neurodegenerative disease affecting ~1–2% of individuals over the age of 65 years. Neurodegeneration of dopaminergic neurons in the nigrostriatal pathway results in a reduction of striatal dopamine (DA) that is causally related to impaired motor function. A pathological hallmark of PD, is the presence of alpha-synuclein (αSyn)-rich Lewy bodies in surviving DA neurons. While it remains unclear what mechanism(s) underlies the selective vulnerability of DA neurons, a growing body of evidence implicate the dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde (DOPAL). DOPAL is the first product of dopamine catabolism by the enzyme monoamine oxidase. We previously reported that deletion of the two aldehyde dehydrogenase (ALDH) enzymes (Aldh1a1 and Aldh2) that are responsible for the detoxification of DOPAL, results in elevated levels of DOPAL, degeneration of tyrosine hydroxylaseimmunoreactive neurons and deficits in motor performance. Additionally, mutations or multiplication of the SNCA gene which encodes for the principal component of Lewy bodies, αSyn, implicates αSyn in PD. Evidence suggests αSyn undergoes aggregation to form neurotoxic oligomers. DOPAL promotes and stabilize αSyn oligomers. The selective generation of DOPAL in DAergic neurons and its ability to stabilize neurotoxic αSyn oligomers, led us to hypothesize that, αSyn is mechanistically related to the neurochemical and behavioral manifestations of PD that result from elevated DOPAL. To test this hypothesis, we generated mice deficient in Aldh1a1 and Aldh2 crossed them to mice that overexpress the human wildtype αSyn. We found that overexpression of αSyn in the presence of elevated levels of DOPAL was associated with exacerbation of deficits in motor performance. The results of neurochemical assays will be discussed.martinezp3@uthscsa.edu*Correspondence to: Anthony Martinez, Barshop Institute, UTHSCSA, TX, USA. Email: VA/BLR&D (I01 BX001641) and NIH/NIA-Nathan Shock Center of Excellence in Basic Biology of Aging (P30-AG013319)1, Abdulrahman Alsayegh1, Aida Jaldi1, Joanne Allard1,*Verona Mulgrave1Department of Physiology & Biophysics, Howard University College of Medicine, Washington, D.C, USA.Possession of the APOE e4 allele has been associated with a higher risk for developing Alzheimer’s disease. Recent studies have suggested that ApoEε4 (ε4) down-regulates expression of brain-derived neurotrophic factor (BDNF) a critical promoter of synaptic plasticity and neuron survival. Upregulation of BDNF via aerobic activity is well documented and aerobic exercise has been shown to improve cognitive function. Our recent pilot study on elderly African Americans showed a disparity in exercise-induced BDNF upregulation between ε4 and ε3 carriers. Through this study we strive to assess the impact of APOE variance on exercise capacity and exercise induced regulation of biomarkers of brain health and function. C57Bl6, APOE ε3 add APOEε4 mice were placed in exercise and sedentary groups. Baseline locomotor function was measured using rotarod training and in-cage running-wheel activity. Mice in exercise groups were subjected to 30 minutes of treadmill running, 5days a week for 8 weeks. Voluntary activity in C57Bl6 mice was significantly greater than ε3 and ε4 mice. In turn, ε3 mice were significantly more active than ε4 mice. Despite activity level differences, hippocampal levels of mature BDNF were similar. Various blood cytokine and lipid levels varied between genotype and activity groups. Exercise activity conferred variable effects on activity and blood biochemistry in APOE variant mice.joanne.allard@howard.edu*Correspondence to: Joanne Allard, Howard University College of Medicine at Washington, D.C. Email: 1,*, Laura Quintanilla1, Andrew M. Pickering1Erin Munkácsy1Barshop institute of Aging Studies and Longevity, UTHSCSA, TX, USAThe proteasome has key roles in neuronal proteostasis, including protein turnover, removal of misfolded or oxidized proteins, presynaptic protein turnover, synaptic efficacy, and synaptic plasticity. Proteasome dysfunction occurs in the brain under Alzheimer’s disease (AD) and related dementias and there is strong evidence that β-amyloid (Aβ) inhibits proteasome function. We show in Drosophila models of AD that proteasome depletion accelerates, while proteasome augmentation delays, AD-like pathology and cognitive deficits. Surprisingly, Aβ accumulation was not reduced by proteasome upregulation, suggesting that proteasome dysfunction triggered by Aβ inhibition drives downstream neurodegeneration rather than altering Aβ accumulation.Munkacsy@uthscsa.edu*Correspondence to: Erin Munkácsy, Barshop Institute, UTHSCSA, TX, USA. Email: 1,2, Stacy Hussong1,2,3,*, Rakez Kayed4, Veronica Galvan1,2,3Angela Olson1Department of Cellular & Integrative Physiology, 2Barshop Institute for Longevity and Aging Studies, UTHSCSA, TX, USA. 3STVHCS at San Antonio, TX, USA. 4Department of Neurology, UTMB, TX, USAThe accumulation of molecular damage in somatic cells can trigger cellular senescence, an irreversible state of cell cycle arrest accompanied by the expression of proinflammatory mediators known collectively as the ‘senescent-associated secretory phenotype’ (SASP). During the pathogenesis of Alzheimer’s disease (AD) and other tauopathies, the microtubule-stabilizing factor tau is phosphorylated, becomes misfolded, and detaches from microtubules, destabilizing the microtubule cytoskeleton. Misfolded tau forms soluble aggregates that are released extracellularly and are transmitted transneuronally, promoting native tau phosphorylation and aggregation in target cells. We recently showed that, in addition to neurons, soluble extracellular tau aggregates propagate to brain microvascular endothelial cells, where microtubule destabilization triggers senescence/SASP. Neuronally-originated extracellular tau can also reach nearby astrocytes, which express abundant tau. Markers of cellular senescence and inflammation are increased in AD brain, including in astrocytes, and we had previously found that AD tauopathy models also display markers of senescence. In the present study, we tested the central hypothesis that soluble aggregated tau propagates to astrocytes and induces astrocyte senescence/SASP. We found that soluble aggregated tau propagates into primary human astrocytes, and that tau transmission to astrocytes triggered endogenous tau phosphorylation and microtubule destabilization, followed by upregulation of markers of permanent cell cycle arrest and the acquisition of SASP. These data provide the first evidence that soluble aggregated tau propagates to astrocytes and indicate that tau aggregate propagation triggers astrocyte senescence/SASP. We further found that primary neurons cocultured with astrocytes undergoing tau-induced senescence show reduced dendritic arborization and dendritic spine density, suggesting that astrocyte senescence may contribute to synaptic dysfunction in AD. Drugs that eliminate senescent cells are FDA-approved and antibody-based approaches to remove tau from brain are already in clinical trials. Our studies may thus advance the implementation of these interventions for the treatment of AD and other tauopathies.hussong@uthscsa.edu*Correspondence to: Stacy Hussong, Barshop Institute, UTHSCSA, TX, USA. Email: NIA T32 AG021890; NIA R01 AG057964-011, Joseph M. Valentine2, Kathryn R. Sickora2, Ashley Zapata2, Eric Baeuerle1,2, Qiang Shen3, Miranda E. Orr1,2,4,*Alec Peterson1San Antonio Geriatric Research, Education and Clinical Center, STVHCS at San Antonio, TX, USA. 2Barshop Institute for Longevity and Aging Studies, UTHSCSA, TX, USA. 3Research Imaging Institute, University of Texas Health Science Center San Antonio at San Antonio, TX, USA. 4Glenn Biggs Institute for Alzheimer’s & Neurodegenerative Diseases at San Antonio, TX, USASenescent cell accumulation is associated with tissue pathology, functional decline and decreased lifespan. In the brain, post-mitotic neurons containing aggregated tau in the form of neurofibrillary tangles (NFTs) are a source of cellular senescence. We are investigating the effects of NFT-associated cellular senescence on brain structure and function. Target pathways of interest were derived from studies on postmortem human brain tissue from patients with Alzheimer’s disease and progressive supranuclear palsy. Cellular and molecular pathways disrupted by neuronal senescence were identified by comparing laser capture micro-dissected neurons with NFTs to neurons without NFTs. Transgenic tau mouse models were utilized to investigate specific disease mechanisms responsible for observations in human tissue. Pharmacological interventions were tested in transgenic mice to investigate therapeutic potential of disrupting candidate pathways. Treatment efficacy was evaluated by measuring translationally relevant outcomes . Neurons with NFTs differentially regulate genes encoding for extracellular matrix and growth factor proteins that alter glial cell biology. Transgenic mouse models acquire pathologies involving all glial cell types, including oligodendrocytes. Analyses indicate tau-associated aberrant myelin gene and protein expression, and NFT-associated white matter hyperintensity (WMH) pathology. Pharmacological removal of senescent cells rescues myelin-associated pathologies in transgenic mice with advanced disease. Tau-induced cellular senescence contributes to WMH pathology and oligodendroglia dysfunction. Aberrant myelin protein expression and WMH pathology is sensitive to senolytic treatment. White matter pathologies, specifically WMH, are associated with cognitive decline and occur in 89% of patients with Alzheimer’s disease; treatment with senolytics may provide benefit to these patients.orrm3@uthscsa.edu*Correspondence to: Miranda Orr, Barshop Institute, UTHSCSA, TX, USA. Email: 1, Paolo Piatti1, Wei Guo1, Jessica Valadez1, Jae-Hyun Yang2, Joe Ollar1, Yap Chew1, David Sinclair2,*, Xiaojing Yang1Anand Gopalan1Zymo Research Corp at Irvine, CA, USA. 2Department of Genetics, Harvard Medical School at Boston, MA, USAAge is a driving factor of many major diseases including cancer, cardiovascular disease, arthritis, osteoporosis and type 2 diabetes. Recent studies have shown that epigenetic changes, especially DNA methylation, are aging related processes. Notably, we have developed a Human DNAge™ test based on Steve Horvath’s epigenetic clock. DNAge™ has been recognized as a robust and precise tool for the determination of biological age, and the study of the mechanisms underlying agerelated disease. Considering the invaluable power of mice as a primary research model, we developed Mouse DNAge™, a murine DNA methylation panel targeting over 2000 epigenomics loci. Mouse DNAge™ takes advantage of our proven targeted bisulfite sequencing approach, called SWARM™ (Simplified Whole-panel Amplification Reaction Method), to accurately quantify and track the epigenetic aging of mice. Epigenetic profiles have been shown to be tissue-specific and therefore, we further built tissue-specific epigenetic clocks for murine whole blood, muscle, liver and brain. To this end, we used the elastic net regression of DNA methylation levels of the targeted loci and the chronological age of over 200 tissue samples collected from mice between 9 and 129 weeks old. Using our Mouse DNAge™ blood clock, we demonstrated the relationship between DNAge™ rate of acceleration and life span amongst different mice strains. Interestingly, we also discovered that only 15% of aging-associated loci overlap between Mouse DNAge™ blood and brain clocks. This demonstrates the importance of tissue-specific clocks for the study of aging mechanisms and intervention strategies targeting specific mice tissues and organs.david_sinclair@hms.harvard.edu*Correspondence to: David Sinclair, Department of Genetics, Harvard Medical School at Boston, MA, USA. Email: 1, Sija He1, Jingjing Zhang2, Fen Liu2, Juli Bai1,2, Lily Dong3, Feng Liu1,2George Plasko1Department of Pharmacology, UTHSCSA, TX, USA. 2Department of Metabolism and Endocrinology, Metabolic Syndrome Research Center, Second Xiangya Hospital, Central South University, Changsha, Hunan, China. 3Department of Cell Systems & Anatomy, UTHSCSA, TX, USAObesity has an established association with insulin resistance, type 2 diabetes mellitus, dyslipidemia, and atherosclerosis, with a heavy burden on the healthcare system. Preventing the progression from obesity to insulin resistance requires an understanding of the regulatory mechanisms involved in the loss of insulin sensitivity. Adipose tissue is well known to function as an endocrine organ that produces many kinds of adipokines, such as leptin, adiponectin, resistin, and retinol-binding protein 4 etc. In the current study, we report the identification and initial characterization of a novel adipokine tetranectin. Tetranectin, which is coded by the C-type lectin domain family 3 member B (CLEC3B) gene, is ubiquitously expressed in various mouse tissues, whereas it is highly enriched in white adipose tissue. We found that the serum level of tetranectin was much higher in both obese and diabetic patients. Knocking out the tetranectin gene in mice protected against high fat diet-induced insulin resistance and glucose intolerance without effects on food intake and body weight. Mechanistically, tetranectin targets liver tissues and its deficiency improves insulin-stimulated AKT phosphorylation at serine 473. Taken together, we have identified a novel adipokine which mediates metabolic crosstalk among tissues to maintain systemic insulin sensitivity. Further investigation of tetranectin’s function could yield a new target for the development of effective therapeutic treatment for obesity and its associated metabolic diseases.liuf@uthscsa.edu*Correspondence to: Feng Liu, UTHSCSA, TX, USA. Email: 1, Shylesh Bhaskaran1, Rojina Ranjit1, Kavithalakshmi Sataranatarajan1, Pavithra Premkumar1, Kendra Huseman1, Holly Van Remmen1,2,*Rizwan Qaisar1Aging and Metabolism Research Program, OMRF, TX, USA. 2Oklahoma City VA Medical Center, Oklahoma City, OK, USAMolecular targets to reduce muscle weakness and atrophy due to oxidative stress have been elusive. Here we show that activation of Sarcoplasmic Reticulum (SR) Ca2+ ATPase (SERCA) with CDN1163, a novel small molecule allosteric SERCA activator, ameliorates the muscle impairment in the CuZnSOD deficient (Sod1-/-) mouse model of oxidative stress. Sod1-/- mice are characterized by reduced SERCA activity, muscle weakness and atrophy, increased oxidative stress and mitochondrial dysfunction. 7 weeks of CDN1163 treatment completely restored SERCA activity and reversed the 23% reduction in gastrocnemius mass and 22% reduction in specific force in untreated Sod1-/- versus wild type mice. These changes were accompanied by restoration of autophagy protein markers to the levels in wild-type mice. CDN1163 also reversed the increase in mitochondrial ROS generation and oxidative damage in muscle tissue from Sod1-/- mice. Taken together our findings suggest that the pharmacological restoration of SERCA is a promising therapeutic approach to counter oxidative stress associated muscle impairment.Holly-VanRemmen@omrf.org*Correspondence to: Holly Van Remmen, OMRF, TX, USA. Email: 1,*, Liu Y1, Salmon AB1Thyne KM1The Sam and Ann Barshop Institute for Aging and Longevity Studies Barshop Institute, UTHSCSA, TX, USACaloric restriction (CR) is perhaps the most well-studied and robust method for improving lifespan in a variety of model organisms. However, restriction of the essential amino acid methionine can produce similar physiological effects to CR without reducing caloric intake. Like CR, methionine restriction (MR) extends longevity, reduces oxidative stress, and improves metabolism. While CR and MR share physiological similarities, there is growing evidence of disparate molecular mechanisms between the two interventions. The side chain of methionine contains sulfur suggesting a potential importance of sulfur redox in MR. This sulfur is easily oxidized to form methionine sulfoxide which is potentially detrimental to protein function. Eukaryotic cells have evolved methionine sulfoxide reductase A (MsrA) as an enzymatic means to repair this oxidation. Here, we questioned whether methionine oxidation, mediated by MsrA, may play an important role in the physiological outcomes of MR by testing this intervention in mice lacking MsrA (MsrA KO). Under MR, MsrA KO mice experience reduced body weight and fat as expected, and also experience improved glucose tolerance as a functional outcome. However, other metabolic markers including O2 consumption and CO2 production in MsrA KO mice were unaffected by MR which contrasts the increase observed in wild type MR mice. Moreover, we noted significant differences between MsrA KO and wild type mice under MR in other markers of healthspan such as activity. These results suggest that methionine oxidation and/or MsrA play important roles in at least some of the mechanistic outcomes mediated by MR.thyne@livemail.uthscsa.edu*Correspondence to: Kevin Thyne, Barshop Institute, UTHSCSA, TX. Email: 1,*, Stacy A. Hussong1,2, Stephen F. Hernandez1, and Veronica Galvan1,2Candice E. Van Skike1Barshop Institute for Aging and Longevity Studies and the Department of Cellular and Integrative Physiology, UTHSCSA, TX, USA. 2Department of Veterans Affairs, STVHCS at San Antonio, TX, USAOur lab has recently reported the accumulation of tau oligomers in the cerebral microvasculature of human patients with tauopathies, including Alzheimer’s disease (AD), dementia with Lewy bodies, and progressive supranuclear palsy, as well as in the Tg2576 (hAPPswe) mouse model of AD. However, the functional consequences of cerebrovascular tau accumulation have yet to be determined. We assessed the coupling between increased neural activity and increased cerebral blood flow, which is a highly regulated processes called neurovascular coupling (NVC). Therefore, the aim of the present study was to establish cerebrovascular dysfunction in several different models of tauopathy. Laser Doppler flowmetery was used to measure the increase in cerebral blood flow in response to whisker stimulation, an indicator of NVC, in the following mouse models: JNPL3 (P301L), PS19 (P301S), and hTau mice compared to appropriate WT controls for each strain. At seven months, all transgenic mice exhibited neurovascular coupling deficits compared to WT controls. We also assessed endothelium-dependent vasodilation in vivo in the PS19 (P301S) and hTau mice. The tau transgenic mice showed reduced evoked CBF in response to the endothelium-dependent vasodilator, acetylcholine compared to WT controls. Both NVC and endothelium-dependent vasodilation are dependent on nitric oxide synthase activity. In order to assess the impact of tau oligomers on nitric oxide synthase function, we treated neurons or endothelial cells with oligomeric tau and measured the relative phosphorylated and total protein concentration of nNOS and eNOS from neurons and endothelial cells, respectively. Our results indicate that oligomeric tau inhibits the activation of both nNOS and eNOS, which may underlie the cerebrovascular dysfunction observed in these models of tauopathy. These data suggest a link between cerebrovascular function and tauopathy. It is not yet clear whether impaired cerebrovascular function is a cause or consequence of tauopathy, particularly with regards to the accumulation of soluble tau aggregates within the cerebrovasculature.vanskike@uthscsa.edu*Correspondence to: Candice E. Van Skike, Barshop Institute, UTHCSA, TX, USA. Email: CVS: Alzheimer’s Association AARF-17–504,221; SFH: NIH R25NS080684; VG: 1R01AG057964-01, 1-I01-BX002211-01 VA Merit Review Award, Owens Foundation, Bailey Family Alzheimer’s Fund.1,*, Charles F. Hodgman1, Joshua S. Godwin1, R. Andrew Shanely1Kevin A. Zwetsloot1Department of Health & Exercise Science; Integrated Muscle Physiology Laboratory, Appalachian State University at Boone, NC, USAPhytoecdysteroids are natural plant steroids synthesized by a variety of hardy plants. Previous work by our group has shown that administration of phytoecdysteroids, such as 20hydroxyecdysone (20E), in old mice can lead to an increase in protein synthesis signaling and skeletal muscle fiber size. To investigate whether phytoecdysteroids enhance skeletal muscle recovery from eccentric contraction-induced damage, young (6.1 ± 0.4 mo) and old (26.5 ± 0.5 mo) mice were subjected to injurious eccentric contractions (EC), followed by 7 days of 20E supplementation or placebo (PLA). Mice were anesthetized with isoflurane and then in vivo isometric contractions were performed  to obtain torque-frequency relationships (TF) of the anterior crural muscle group (PRE), followed by 150 EC. Following recovery from anesthesia, the mice received either 20E (50 mg•kg-1 BW) or PLA  by oral gavage. Mice were gavaged daily for 6 days and on day 7, TF was re-assessed (7-day). Significant decreases in TF in young and old mice were measured immediately after EC (both p < 0.001) and PLA groups remained depressed at day 7 . However, 20E supplementation completely recovered TF after 7 days in both young and old mice . These findings suggest that 20E may exert anabolic or ergogenic effects in skeletal muscle of male mice that accelerate recovery of in vivo isometric skeletal muscle function following EC-induced injury.vanskike@uthscsa.edu*Correspondence to: Kevin A. Department of Health & Exercise Science; Integrated Muscle Physiology Laboratory, Appalachian State University at Boone, NC, USA. Email:


Dual-specificity phosphatases (DUSPs) are important regulators of neuronal cell growth and differentiation by targeting proteins essential to neuronal survival in signaling pathways, among which the MAP kinases (MAPKs) stand out. DUSPs include the MAPK phosphatases (MKPs), a family of enzymes that directly dephosphorylate MAPKs, as well as the small-size atypical DUSPs, a group of low molecular-weight enzymes which display more heterogeneous substrate specificity. Neuroblastoma (NB) is a malignancy intimately associated with the course of neuronal and neuroendocrine cell differentiation, and constitutes the source of more common extracranial solid pediatric tumors. Here, we review the current knowledge on the involvement of MKPs and small-size atypical DUSPs in NB cell growth and differentiation, and discuss the potential of DUSPs as predictive biomarkers and therapeutic targets in human NB. MYCN  and ALK (encoding an oncogenic receptor tyrosine kinase (RTK); about 3% of cases) genes amplification . Children with MYCN amplification regardless of age, those older than 12 months with disseminated tumors, or those older than 18 months with unfavorable histology, are considered to be part of the high-risk NB group (about 40% of cases). Very low-, low-, and intermediate-risk NB patients show a 5-year survival of 90–95%, whereas high-risk NB patients show a 5-year survival of 40–50% [Neuroblastoma (NB) is the most common malignancy diagnosed in the first year of life, with an average age at diagnosis of 18 months, and constitutes the most frequent extracranial solid tumor in infants, accounting for about 7% of total pediatric tumors. Although novel treatments have improved the survival of NB patients , the high-risk forms of NB are the major cause of pediatric cancer death, rendering about 15% of pediatric cancer mortality . NB is af 40–50% ,12,13,14ALK RTK gene, which is mainly expressed in the developing nervous system [PHOX2B, encoding a transcription factor essential in neuronal differentiation and development of the autonomic nervous system, which is mutated in patients with congenital central hypoventilation syndrome (CCHS) [DUSP12 gene, which encodes a large-size atypical DUSP proposed to target MAPKs, were associated with low-risk NB [Familial NB accounts for 1–2% of NB cases, with two major genes showing germline mutations in association with the disease: the s system ,16; and e (CCHS) . Comprehe (CCHS) ,19,20,21e (CCHS) ,23,24. I-risk NB ,26. In tMYCN amplification, the major hallmark of high-risk NB, associates with poorly differentiated NB tumors [MYCN amplification and ALK hyperactivation [NB can be considered a neural crest-related developmental tissue disease in which alterations in neuronal differentiation, driven by the unbalanced action of pro-proliferative and pro-differentiation factors on the maturation and migration of neural crest cells and neuroblasts, play a fundamental etiologic role. Highly differentiated NB tumors have a favorable clinical outcome, and spontaneous regression linked to neuroblast apoptosis is frequent even in metastatic cases ,27,28,29B tumors ,31,32, aB tumors ,34. In aB tumors ,36,37. Stivation ,39. NB ctivation ,41. Humativation ,43,44  in relapsed NB samples obtained after chemotherapy [Genes related to the RAS-ERK1/2 MAPK pathway display somatic alterations in about 5% of sporadic primary NB tumors, with a much higher percentage of alterations  family. They have in common the presence of a single catalytic PTP domain, which in the members of several DUSP subfamilies, has the dual capability to dephosphorylate both Ser/Thr- and Tyr-phosphorylated residues  in proteins. Enzymes from other DUSP subgroups, however, have RNA, lipids, or other biomolecules as their major substrates ,94,95,96The functional role of MKPs and small-size atypical DUSPs as physiologic MAPK inactivators advocates for their high potential as important players in NB cell growth and differentiation . In Figu2O2 treatment of SH-SY5Y cells triggered apoptosis which was accompanied by early reduction and late induction of DUSP1 protein content, in inverse correlation with pERK1/2 levels, and siRNA suppression of DUSP1 expression attenuated H2O2-induced cell death [2O2 treatment. In this regard, treatment of SH-SY5Y cells with silver nanoparticles (AgNP) inducing neuronal differentiation increased reactive oxygen species (ROS) generation, which was accompanied by decreased mRNA levels in several DUSPs, including DUSP1, and increased pAKT and pERK1/2 levels [DUSP1 (MKP1), DUSP4 (MKP2), and DUSP5 constitute a group of highly transcriptionally inducible MKPs with nuclear localization and distinct MAPK substrate specificity. DUSP1 mainly dephosphorylates JNKs and p38s, whereas DUSP4 and DUSP5 are more specific for ERK1/2 as a substrate ,115,116.ll death . It woul2 levels ,123. In 2 levels , whereas2 levels . Interes2 levels . Treatme2 levels , and P192 levels . On the 2 levels . These fDUSP4 has been regarded as a potential tumor suppressor, as well as a promoter of chemotherapy resistance, in several human cancers ,127,128.DUSP5, together with DUSP6, is an ERK1/2-specific MKP whose transcription is induced in an ERK1/2-dependent manner ,131,132.2O2 displayed reduced DUSP6 protein levels, which was associated with a higher pERK1/2 content and cell death [DUSP6 transcription can be repressed by MYCN in BE(2)-C and LAN-1 NB cells [DUSP6 gene transcription and DUSP6 protein stability. They prompt further investigations of the potential of DUSP6 as a modulator of NB malignancy.DUSP6 (MKP3), DUSP7 (MKPX), and DUSP9 (MKP4) are cytoplasmic MKPs with specificity towards ERK1/2 dephosphorylation. Similar to DUSP5, DUSP6 is inducible by growth and differentiating agents that activate the ERK1/2 pathway ,134,135,ll death . This efll death . Besidesll death . Differell death . As mentll death , and DUSNB cells . TogetheExpression of both DUSP7 and DUSP9 protein has been detected in NB tumor samples, although, in the case of DUSP9, with a low frequency  Figure . Human SDUSP16 gene loss caused perinatal lethality in mice, associated with hydrocephalus, brain overgrowth by expansion of neural progenitors, and increased midbrain pp38 content [DUSP8, DUSP10 (MKP5), and DUSP16 (MKP7) are MKPs with substrate specificity towards JNKs and p38s MAPKs. These three MKPs are larger than the rest of the MKPs, with N- or C-terminal extensions in their amino acid sequences. DUSP10 and DUSP16 have been involved in autoimmunity and inflammation processes, and little is known about the physiological function of DUSP8. Interestingly,  content . RA trea content ,78.DUSP3 (VHR) is the prototype of small-size atypical DUSPs, and displays substrate specificity towards ERK1/2 and JNKs. However, some other proteins, including STAT5, FAK, growth factor receptors, and nuclear proteins regulating DNA damage repair, have been shown to be tyrosine-dephosphorylated by DUSP3 and have been proposed as DUSP3 physiological substrates ,137. As DUSP13A and DUSP13B are two small-size atypical DUSPs encoded in the same gene and generated by the use of alternative open reading frames, whose physiologic substrate specificity is poorly known . DUSP13ADUSP23 gene in NB tumors revealed higher methylation in MYCN-amplified tumors. In addition, DUSP23 mRNA was at lower levels in NB patients with poor outcomes, when compared to patients free of disease [DUSP23 (VHZ) has been reported to affect MAPKs activation both by phosphatase-dependent and -independent mechanisms ,140, as  disease . This suDUSP26 (MKP8) was originally identified as a DUSP mainly expressed in embryonal cancers and displaying substrate specificity towards p38 in cells . SubsequDUSPs have been proposed as cancer biomarkers in several human cancers, and recent advances in the understanding of the biology of DUSPs have made these enzymes potentially targetable molecules in cancer therapy: (1) DUSPs regulate cell growth, differentiation, and apoptosis through dephosphorylation of key effector proteins from major intracellular signaling pathways; (2) DUSPs present a well-defined catalytic mechanism towards well-defined substrates, especially those in the MAPK family; and (3) DUSPs activities can be efficiently repressed by small molecule inhibitors ,109,146.


In 2017, the Joint United Nations Programme on HIV/AIDS (UNAIDS) estimated that worldwide, 36.9 million persons were living with human immunodeficiency virus (HIV) infection, the virus infection that causes acquired immunodeficiency syndrome (AIDS). Among persons with HIV infection, approximately 75% were aware of their HIV status, leaving 9.4 million persons with undiagnosed infection . The primary HIV testing indicator is the number of persons who have received HIV testing services, categorized by HIV result, age group, sex, and testing service delivery approach. Age group categories are classified as <1 year, 1–9 years, 10–14 years, 15–19 years, 20–24 years, 25–49 years, and ≥50 years. Sex is not reported for children aged <10 years. Delivery approaches for HIV testing services include 1) community-based testing in mobile clinics; 2) voluntary drop-in centers; 3) facility-based provider-initiated testing in tuberculosis, sexually transmitted infection, outpatient, and antenatal clinics; 4) testing in hospital emergency and inpatient departments; and 5) since October 1, 2016, index testing.This report includes the six most recent fiscal quarters for which index testing data were available . Among 33 countries reporting index testing data during this period, seven countries  that reported <1,000 persons tested using index testing and four countries  that reported <500 tests during October 1, 2017–March 31, 2018 were excluded from this analysis. Also excluded were Nigeria and Ukraine in response to requests from the country offices. The number of HIV tests reported using index testing and the percentage of positive tests among different demographic groups in CDC-supported PEPFAR programs in 20 countriesFrom October 1, 2016, to March 31, 2018, CDC-supported implementing partners reported a total of 1,700,998 persons tested for HIV using index testing among the 20 countries evaluated, including 889,599 (52%) persons aged ≥15 years and 799,976 persons aged <15 years . OverallDuring the six fiscal quarters covered by this report, the number of persons tested for HIV using index testing among the 20 countries increased from 166,108 to 356,573 from the first to sixth quarter . During Among the 865,126 persons aged ≥15 years tested for whom sex was reported, 55% were females and 45% were males . HIV indFrom October 1, 2016, to March 31, 2018, CDC-supported PEPFAR partners in 20 countries reported administering a total of 66 million HIV tests over all approaches, 70% of which occurred in facility settings and 30% in community-based settings. The HIV positivity rate using all testing approaches was 1.0% among persons aged <15 years and 4.1% among persons aged ≥15 years  infection and offers these contacts HIV testing services.From October 2016 to March 2018, both the number of persons tested for HIV and the number who received a diagnosis of HIV infection using index testing increased in 20 CDC-supported countries. With an HIV positive rate that is more than twice that of all HIV testing approaches combined, index testing was found to be a more efficient approach to HIV case finding.Expanding index testing services could help increase the number of HIV-positive persons who know their HIV infection status, increase the number who receive antiretroviral treatment, and, as a result, reduce the number of persons who can transmit the virus.


Danio rerio) embryo toxicological model, to characterize metabolic profiles associated with exposure to AFB1. Exposure to AFB1 was associated with dose-dependent acute toxicity  and developmental deformities at micromolar (≤ 2 µM) concentrations. Toxicity of AFB1 was stage-dependent and specifically consistent, in this regard, with a role of the liver and phase I enzyme  bioactivation. Metabolic profiles of intact zebrafish embryos exposed to AFB1 were, furthermore, largely consistent with hepatotoxicity previously reported in mammalian systems including metabolites associated with cytotoxicity , glutathione-based detoxification, and multiple pathways associated with the liver including amino acid, lipid, and carbohydrate  metabolism. Taken together, these metabolic alterations enabled the proposal of an integrated model of the hepatotoxicity of AFB1 in the zebrafish embryo system. Interestingly, changes in amino acid neurotransmitters , as a key modulator of neural development, supports a role in recently-reported neurobehavioral and neurodevelopmental effects of AFB1 in the zebrafish embryo model. The present study reinforces not only toxicological pathways of AFB1 , but also multiple metabolites as potential biomarkers of exposure and toxicity. More generally, this underscores the capacity of NMR-based approaches, when coupled to animal models, as a powerful toxicometabolomics tool.Aflatoxin B1 (AFB1) is a widespread contaminant of grains and other agricultural crops and is globally associated with both acute toxicity and carcinogenicity. In the present study, we utilized nuclear magnetic resonance (NMR), and specifically high-resolution magic angle spin (HRMAS) NMR, coupled to the zebrafish ( Aspergillus, which is endemic to soil organic matter, but also among the most common fungal contaminants of food and feed crops including, in particular, grains , nuts/seeds, legumes, and various other staples  and post-harvest . Aflatoxins are consequently detected in a wide range of plant and animal-based  agricultural products. In fact, aflatoxins were first discovered in the 1960s following an outbreak of so-called “turkey X disease”, which resulted in a massive  number of deaths among domestic turkeys linked, in turn, to contamination of feed grains by A. flavus, as well as particularly high sensitivity of poultry to aflatoxins [Aflatoxins are potent mycotoxins specifically produced by the fungal genus iew, see ). Contamlatoxins ,3.Aspergillus contamination of agricultural crops primarily occur within the tropical and sub-tropical climates , where economically-developing nations are disproportionately located. Accordingly, it has been estimated that as many as 4.5 billion people worldwide are chronically exposed to aflatoxins [Given the recognized adverse effects of aflatoxins on human health, regulatory limits have been adopted by many nations. However, similar guidelines are often either not uniformly established or effectively enforced in many developing countries due, in particular, to widespread subsistence farming, which makes monitoring and regulation exceedingly difficult . ExacerbToxicologically, aflatoxins are associated with both acute and sub-acute adverse health effects. With respect to acute intoxication, aflatoxicosis following exposure to high concentrations of aflatoxins, which primarily target hepatocytes, leads to hemorrhagic necrosis, edema, and other symptoms associated with liver damage  and can frequently (>30% of cases) be fatal, particularly among children ,6,7. DiaOf the more than 20 variants produced, aflatoxin B1 (AFB1) is generally considered the most toxic variant. In addition to acute toxicity, AFB1 is classified by the International Agency for Research on Cancer (IARC) as a Group 1 carcinogen . The carcinogenic potential of AFB1 is specifically linked to hepatocellular carcinoma (HCC), which accounts for as much as 70% of human cancer deaths in areas where aflatoxicosis is endemic ,9, and iDanio rerio) embryo model, to investigate metabolic changes associated with AFB1 exposure. Embryonic and other early life  stages of the zebrafish are well established as a toxicological model, in general, and have been specifically shown in several previous studies [In the present study, we utilized techniques based on nuclear magnetic resonance (NMR), specifically applied to the zebrafish  NMR techniques, which has been recently shown to enable both highly quantitative and qualitative  analyses of major metabolites in the developing zebrafish embryo ,17,18,1950 (after 24 h exposure) of 2.1, 1.8, 1.1, and 0.5 µM at 24, 48, 72, and 96 hpf, respectively. Acute toxicity of AFB1, based on embryo lethality, was evaluated over a range of concentrations (up to 2 µM) in zebrafish embryos, specifically following 24 h of exposure at representative developmental stages . Dose-dependent toxicity was observed  below 2 µM; moreover, toxicity was clearly stage-dependent and specifically increased with age of embryos . AFB1 wa50), AFB1 impaired development, resulting in embryo deformity. Deformities were generally observed among approximately 20–30% of surviving embryos at or below lethal concentrations. At 72 hpf, for example, embryos exposed to concentrations below the approximate LC50  were consistently characterized by malformation of the head and bending of the tail and upper body  enabled unambiguous identification, and subsequent quantitation, of metabolites  following 24-hour exposure to AFB1 at 72 hpf (p < 0.01), tryptophan , and tyrosine , as well as isoleucine , glutamate , glutamine , and glycine  was observed, whereas a highly significant (p < 0.0001) decrease in cysteine (Cys) was measured. Chemical shifts used to identify amino acids are specific for amino acids that are not incorporated into protein, and quantification, therefore, reflects the concentration of the “free” amino acid pools for each. Notably, the non-proteinogenic amino acid neurotransmitter, γ-aminobutyric acid (GABA), also significant increased (p < 0.05). Numerous metabolites associated with carbohydrate metabolism, and cellular energetics, were significantly altered by AFB1 treatment including: (1) decreases in glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P), as well as glucose  itself; (2) highly significant increases in lactate  as the product of lactate dehydrogenase and/or anaerobic glycolysis; and (3) increases in several metabolites associated with cellular energetics including ATP, NADH, and NAD+ (p < 0.05). Significant increases in fatty acids  and cholesterol  were observed, alongside a concomitant increase in acetate as both an intermediate of lipid metabolism and anaplerotic catabolism  into the Krebs cycle (as acetyl CoA). Alongside changes in lipids, significant increases in the polar headgroups, choline (Cho) and myo-inositol (m-Ins), of phospholipids’ characteristic cellular membranes were observed. Finally, a significant decrease (p < 0.05) in glutathione (GSH) as a phase II detoxification mechanism was measured following AFB1 exposure (compared to controls). Of these, a total of 19 metabolites were shown to increase or decrease significantly  and carcinogenicity, although a complete picture of the pathways of toxicity remains to be fully clarified. To elucidate pathways and potential exposure biomarkers, we utilized HRMAS NMR to characterize alterations in the metabolic profiles of intact zebrafish embryos following exposure to AFB1. Solution-state NMR techniques have been previously applied ex vivo [Contamination of crop plants by aflatoxinogenic  ex vivo ,21,22,23 ex vivo ,19. Simi50 = 2.3 µM, versus 1.1 µM in the present study; see 50 of AFB1 .Aligned with previous studies ,12,13, aGiven the reported role of hepatocytes and, moreover, cytochrome P450 enzymes  in the metabolic bioactivation AFB1 to reactive epoxides , it is proposed that observed stage-dependence of toxicity in the zebrafish embryo is related to the development of the liver and associated expression of these phase I detoxification enzymes. Although initial differentiation  of the liver in zebrafish embryos begins at 24 hpf, complete development (i.e. “outgrowth and expansion”) does not occur until approximately 72–96 hpf ,26. ReleMetabolic profiling of zebrafish embryos by HRMAS NMR  in the pAlongside these general indicators of hepatotoxicity, in our study, the measured alterations of three major metabolic pathways—namely amino acid, lipid, and carbohydrate  metabolism —which arCentral to these metabolic changes is a significant alteration of the levels of several relevant amino acids following exposure of zebrafish embryos to AFB1. Exposure of 72 hpf embryos to AFB1 altered levels of several amino acids including significant increases of Phe, Tyr, Trp, Ile, Glu, Gln, and Gly and a significant decrease of Cys . AlteredIn addition, levels of the non-essential amino acids Glu, Gln, and Gly were, likewise, elevated in AFB1-exposed embryos. Elevated Glu is noteworthy, in this regard, as conversion of Glu to α-ketoglutarate (αKG) via either transamination or glutamate dehydrogenase represents a key alternative (to pyruvate/acetyl CoA) as an entry point for the Krebs cycle to meet cellular energy demands . Furtherp > 0.05; mALT and mAST) of Glu to αKG, to supply the Krebs cycle, for which these two amino acids are the primary products. Specifically, previously-demonstrated disruption of mitochondrial function including Krebs cycle and coupling of oxidative phosphorylation  is, in fact, widely recognized as an indicator of cytotoxicity, in general, and of AFB1 hepatocytotoxicity specifically . Releaseobserved . That saIt is proposed that stage-dependent (and presumably hepatocyte-dependent) toxicity of AFB1, and alterations of metabolic pathways observed here, result from impairment of energy metabolism and associated anaplerotic reactions  during embryo development . The effAlongside carbohydrate/energy metabolism, hepatocytes are the primary location of lipid metabolism including both biosynthetic and catabolic functions. Indeed, hepatic damage is clinically associated with lipid accumulation  or so-called “fatty liver.” Hepatotoxicity of AFB1 would, therefore, also closely correlate with loss of lipid metabolic function and, thus, observed increases in FA and Chol . Indeed,Finally, in addition to recognized hepatotoxicity, AFB1 has been very recently found to impair locomotor function and disrupt neural development in zebrafish embryos and larvae . AlthougOf these, Glu and GABA are particularly notable given their shared pathways of “recycling”  via the glutamate/GABA-glutamine cycle and synchronized association with the Krebs cycle . With reCoupling of NMR-based techniques to early life stages of the zebrafish was demonstrated in the present study , unless otherwise mentionedDanio rerio) were maintained in recirculating aquarium systems according to established rearing procedures [Adult wild-type zebrafish . Percentage survival of the embryos was evaluated and scored for lethal or teratogenic effects, using a Zeiss CKX41 inverted microscope with phase contrast optics, amounted time-lapse recorder, and the analysis software . Lethal or teratogenic effects were recorded according to Weigt et al. [50) were calculated by probit analysis. To evaluate acute toxicity and developmental defects caused by AFB1, zebrafish embryos at representative stages  were treated with varying concentrations of AFB1  for 24 h in 35 mm-diameter polystyrene dishes  and replicates (N = 3) for quantitative NMR analyses. Accordingly, ~100 embryos were collected (after 24 h) from both controls (N = 3) and pooled (N > 3) AFB1 exposures. Following washing (3-times with MilliQ water) to remove residual AFB1, embryos were transferred to 4-mm zirconium oxide rotors  for replicate (N = 3) measurements by NMR. As a reference , deuterated phosphate buffer  containing 0.1% (w/v) 3-trimetylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP) was added, and the rotor was transferred immediately to the NMR spectrometer. All HRMAS NMR experiments were done on a Bruker DMX 600-MHz NMR magnet with a proton resonance frequency of 600 MHz, which was equipped with a 4-mm HRMAS dual 1H/13C inverse probe with a magic angle gradient and spinning rate of 6 kHz. Measurements were carried out at a temperature of 277 K using a Bruker BVT3000 control unit. Acquisition and processing of date were done with Bruker TOPSPIN software . Metabolic profiling by HRMAS NMR was performed as adapted from previous studies ,19. Embr1H HR-MAS NMR spectra. Each one-dimensional spectrum was acquired applying a spectral width of 8000 Hz, domain data points of 16k, a number of averages of 512 with 8 dummy scans, a constant receiver gain of 2048, an acquisition time of 2 s, and a relaxation delay of 2 s. The relaxation delay was set to a small value to remove short 2T components due to the presence of lipids in intact embryo samples. All spectra were processed by an exponential window function corresponding to a line broadening of 1 Hz and zero-filled before Fourier transformation. NMR spectra were phased manually and automatically baseline corrected using TOPSPIN 2.1 . The total analysis time  of 1H-HRMAS NMR spectroscopy for each sample was approximately 20 min.A rotor synchronized Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence with water suppression was used for one-dimensional 2t domain over the spectral width of 4k, and 512 1t increments were collected with 16 transients, a relaxation delay of 2 s, an acquisition time of 116 ms, and a pre-saturated water resonance during relaxation delay. Data were zero-filled with 2048 data points and weighted with a sine bell window function in both dimensions (prior to Fourier transformation). To preclude the possibility of sample degradation during COSY experiments, the 1D 1H HRMAS spectra were measured before and after 1H-1H COSY measurements  homo-nuclear correlation spectroscopy (¹H-¹H COSY), specifically in magnitude mode, was performed using a standard pulse program library. For COSY, 2048 data points were collected in the urements . F-values larger than 2.8 (p < 0.05) were considered significant.All of the spectra were referenced, baseline- and phase-corrected, and analyzed by using MestReNova v.8.0 . Quantification of metabolites was performed by Chenomx NMR Suite 8.2 . This enabled qualitative and quantitative analysis of an NMR spectrum by fitting spectral signatures from an HMDB database to the spectrum. The concentrations of metabolites were subsequently calculated based on a ratio relative to tCr, since the external reference may lead to the misleading results, and Cr resonance has been previously shown to be a reliable internal reference in a wide range of animal studies. Statistical analysis of NMR quantification was done by one-way analysis of variance (ANOVA) using OriginPro v. 8 , and calculated For multivariate analysis, AMIX  was used to generate bucket tables from the one-dimensional spectra of control and AFB1treated embryos, excluding the region between 4.20 and 6.00 ppm to remove the larger water signal. The one-dimensional CPMG spectra were normalized to the total intensity and binned into buckets of 0.04 ppm. The data were mean-centered and scaled using the Pareto method in the SIMCA software package . Unsupervised principle component analysis (PCA) was performed on the data using the SIMCA software as described earlier .