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RalBP1 (Rlip) is a stress-activated protein that is believed to play a large role in aging and neurodegenerative diseases such as Alzheimer’s disease (AD) and other tauopathies. The purpose of our study was to understand the role of Rlip in mutant Tau-expressed immortalized hippocampal HT22 cells. In the current study, we used mutant Tau (mTau)-expressed HT22 neurons and HT22 cells transfected with Rlip-cDNA and/or silenced RNA, and studied the cell survival, mitochondrial respiration, mitochondrial function, immunoblotting, and immunofluorescence analysis of synaptic and mitophagy proteins and the colocalization of Rlip and mTau proteins. We found Rlip protein levels were reduced in mTau-HT22 cells, Rlip silenced HT22 cells, and mTau + Rlip RNA silenced HT22 cells; on the other hand, increased Rlip levels were observed in Rlip cDNA transfected HT22 cells. We found cell survival was decreased in mTau-HT22 cells and RNA-silenced HT22 cells. However, cell survival was increased in Rlip-overexpressed mTau-HT22 cells. A significantly reduced oxygen consumption rate (OCR) was found in mTau-HT22 cells and in RNA-silenced Rlip-HT22 cells, with an even greater reduction in mTau-HT22 + Rlip RNA-silenced HT22 cells. A significantly increased OCR was found in Rlip-overexpressed HT22 cells and in all groups of cells that overexpress Rlip cDNA. Mitochondrial function was defective in mTau-HT22 cells, RNA silenced Rlip in HT22 cells, and was further defective in mTau-HT22 + Rlip RNA-silenced HT22 cells; however, it was rescued in Rlip overexpressed in all groups of HT22 cells. Synaptic and mitophagy proteins were decreased in mTau-HT22 cells, and further reductions were found in RNA-silenced mTau-HT22 cells. However, these were increased in mTau + Rlip-overexpressed HT22 cells. An increased number of mitochondria and decreased mitochondrial length were found in mTau-HT22 cells. These were rescued in Rlip-overexpressed mTau-HT22 cells. These observations strongly suggest that Rlip deficiency causes oxidative stress/mitochondrial dysfunction and Rlip overexpression reverses these defects. Overall, our findings revealed that Rlip is a promising new target for aging, AD, and other tauopathies/neurological diseases.

Alzheimer’s disease (AD) is a devastating illness in elderly individuals, that currently has no known cure. Causal genetic factors only account for 1-2% of AD patients. However, other causal factors are still unknown for a majority of AD patients. Currently, multiple factors are implicated in late-onset AD, including unhealthy diet, physical inactivity, traumatic brain injury, chronic conditions, epigenetic factors, and environmental exposures. Although clinical symptoms of dementia are common to all races and ethnic groups, conditions that lead to dementia are different in terms of lifestyle, genetic profile, and socio-economic conditions. Increasing evidence also suggests that some elderly individuals age without cognitive impairments in their 60–90s as seen in rural West Texas, while some individuals progress with chronic conditions and cognitive impairments into their 60s. To understand these discriminations, we assessed current literature on demographic features of health in rural West Texas. This paper also outlines our initiated clinical study with a purpose of understanding the factors that allow some individuals to live without cognitive impairments at the age of 60–90 years, whereas others develop deficits in cognitive function around or above 60 years. Our ongoing study hopes to determine the factors that delay aging in some individuals by investigating various aspects including genetics, epigenetics, ethnicity, biology, culture, and lifestyle. This will be achieved by gathering information about participants’ ethnographic profiles, cognitive assessments, blood-profiles, brain scans, and blood-based genomic analyses in relation to lifestyle. The outcomes of our study will provide insights into healthy aging in rural West Texas.

TP53 (p53) functions as a stress-responsive, genome protective, tumor suppressor whose functions are lost or altered a majority of breast cancers. TP53 homozygous null mice uniformly die of spontaneous malignancy. The human RALBP1 gene (18p11.22) encodes is a stress-protective, anti-apoptotic mercapturic acid pathway transporter protein (RLIP76 or Rlip) regulated by the Ral/Rac/Rho pathways. In stark contrast to TP53 null mice, RALBP1-null mice are highly cancer resistant, even to the most potent known chemical carcinogens benzo[a]pyrene or dimethylbenzanthracene. We recently reported that pharmacologically or chemically induced haploinsufficiency of Rlip switches off spontaneous carcinogenesis in p53-null mice (Proc. Natl. Acad Sci USA, 2018; 115:3918), indicating an existential role of Rlip in p53-deficient cancer cells and suggesting the possibility that DNA-repair deficient cells could be particularly susceptible to Rlip depletion. Results of present studies demonstrated that RALBP1 depletion by antisense inhibited the growth of the MDA-MB231 cell line in-vitro and in xenografts. Remarkably, Rlip depletion with antisense or upon treatment with an orange-derived flavanone (2-hydroxyflavanone) depleted the expression of PALB2 as well as BRCA1 and BRCA2 in triple negative breast cancer cell lines, and synergistically increased the efficacy of PARP inhibition. These findings suggest that drugs that inhibit or deplete Rlip could be useful in sensitizing DNA-repair sufficient triple negative breast cancer to PARP inhibitors. Citation Format: Sanjay Awasthi, Chhanda Bose, Catherine Jones, Rakhshanda Rahman, Sharad S. Singhal, Sharda P. Singh. Partial RALBP1 loss depletes DNA repair pathway genes [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-18.

Background: Comparative genomic hybridization (CGH) microarrays are used forgenome-wide evaluation of copy number variations (CNV) of known prognostic significance;however, unannotated variants of uncertain significance (VUS) are frequently present. Toidentify potentially actionable targets, we retrospectively analyzed VUS loci using CGH datafrom 192 cases of cancer or genetic disorders treated at TTUHSC.Methods: DNA was hybridized onto CytoSure Constitution V3 arrays, scanned with theAgilent microarray D scanner, and analyzed by CytoSure Interpret Software.Results: We found 794 distinct CNVs, the most frequent being 14q32.22 (112rearrangements), 14q11.2 (100), 8p11.2 (98), 15q11.1-q11.2 (83), and 8p23.1 (77). In particular,8p11.22 alterations were found in many pediatric tumors, with gain/loss ratio of 4.7. Linkage ofTACC1, TM2D2, KAT6A and ADAM32 was indicated by a similar 5-year survival rate of 75.3%(n = 253), which was greater than in unaltered cases (62.2%, n = 15,809 cases) in The CancerGenome Atlas database.Conclusion: Knockdown of genes occurring at variants of uncertain significance (VUS)loci may help identify new therapy targets.

Simple Summary Epidemiological evidence suggests that breast cancer risk is lowered by Ω-3 and increased by Ω-6 polyunsaturated fatty acids (PUFAs). Paradoxically, the Ω-6 PUFA metabolite 4-hydroxynonenal (4-HNE) inhibits cancer cell growth. This duality prompted us to study whether arachidonic acid (AA) would enhance doxorubicin (dox) cytotoxicity towards breast cancer cells. We found that supplementing AA or inhibiting 4-HNE metabolism potentiated doxorubicin (dox) toxicity toward Her2-dependent breast cancer but spared myocardial cells. Our results suggest that Ω-6 PUFAs could improve outcomes of dox chemotherapy in Her2-overexpressing breast cancer. Abstract Her2-amplified breast cancers resistant to available Her2-targeted therapeutics continue to be a challenge in breast cancer therapy. Dox is the mainstay of chemotherapy of all types of breast cancer, but its usefulness is limited by cumulative cardiotoxicity. Because oxidative stress caused by dox generates the pro-apoptotic Ω-6 PUFA metabolite 4-hydroxynonenal (4-HNE), we surmised that Ω-6 PUFAs would increase the effectiveness of dox chemotherapy. Since the mercapturic acid pathway enzyme RALBP1 (also known as RLIP76 or Rlip) that limits cellular accumulation of 4-HNE also mediates dox resistance, the combination of Ω-6 PUFAs and Rlip depletion could synergistically improve the efficacy of dox. Thus, we studied the effects of the Ω-6 PUFA arachidonic acid (AA) and Rlip knockdown on the antineoplastic activity of dox towards Her2-amplified breast cancer cell lines SK-BR-3, which is sensitive to Her2 inhibitors, and AU565, which is resistant. AA increased lipid peroxidation, 4-HNE generation, apoptosis, cellular dox concentration and dox cytotoxicity in both cell lines while sparing cultured immortalized cardiomyocyte cells. The known functions of Rlip including clathrin-dependent endocytosis and dox efflux were inhibited by AA. Our results support a model in which 4-HNE generated by AA overwhelms the capacity of Rlip to defend against apoptosis caused by dox or 4-HNE. We propose that Ω-6 PUFA supplementation could improve the efficacy of dox or Rlip inhibitors for treating Her2-amplified breast cancer.

MicroRNAs (miRNAs) are found in nerve terminals, synaptic vesicles, and synaptosomes, but it is unclear whether synaptic and cytosolic miRNA populations differ in Alzheimer’s disease (AD) or if synaptosomal miRNAs affect AD synapse activity. To address these questions, we generated synaptosomes and cytosolic fractions from postmortem brains of AD and unaffected control (UC) samples and analyzed them using a global Affymetrix miRNAs microarray platform. A group of miRNAs significantly differed (P 200-fold) in their expressions in different comparisons: (1) UC synaptosome vs UC cytosol, (2) AD synaptosomes vs AD cytosol, (3) AD cytosol vs UC cytosol, and (4) AD synaptosomes vs UC synaptosomes. MiRNAs data analysis revealed that some potential miRNAs were consistently different across sample groups. These differentially expressed miRNAs were further validated using AD postmortem brains, brains of APP transgenic (Tg2576), Tau transgenic (P301L), and wild-type mice. The miR-501-3p, miR-502-3p, and miR-877-5p were identified as potential synaptosomal miRNAs upregulated with disease progression based on AD Braak stages. Gene Ontology Enrichment and Ingenuity Pathway Analysis of synaptosomal miRNAs showed the involvement of miRNAs in nervous system development, cell junction organization, synapse assembly formation, and function of GABAergic synapse. This is the first description of synaptic versus cytosolic miRNAs in AD and their significance in synapse function.

Rlip76 (Rlip) is a multifunctional membrane protein that facilitates the high metabolic rates of cancer cells through the efflux of toxic metabolites and other functions. Rlip inhibition or depletion results in broad-spectrum anti-cancer effects in vitro and in vivo. Rlip depletion effectively suppresses malignancy and causes global reversion of characteristic CpG island methylomic and transcriptomic aberrations in the p53-null mouse model of spontaneous carcinogenesis through incompletely defined signaling and transcriptomic mechanisms. The methylome and transcriptome are normally regulated by the concerted actions of several mechanisms that include chromatin remodeling, promoter methylation, transcription factor interactions, and miRNAs. The present studies investigated the interaction of Rlip depletion or inhibition with the promoter methylation and transcription of selected cancer-related genes identified as being affected by Rlip depletion in our previous studies. We constructed novel promoter CpG island/luciferase reporter plasmids that respond only to CpG methylation and transcription factors. We found that Rlip depletion regulated expression by a transcription factor-based mechanism that functioned independently of promoter CpG methylation, lipid peroxidation, and p53 status.

The purpose of our study is to determine DDQ (diethyl (3,4-dihydroxyphenethylamino) (quinolin-4-yl) methylphosphonate)—a newly discovered molecule that has been shown to protect against phosphorylated tau (p-tau) in Alzheimer’s disease (AD) pathogenesis. We used a well-studied tau (P301L) transgenic mouse model to achieve our goal. We administered DDQ into 12-month-old Tau mice, at 20 mg/kg body weight intraperitoneally two times per week for 2 months. We also assessed DDQ levels in the blood, skeletal muscle and brain using biochemical and molecular techniques. We investigated the mRNA and protein levels of mitochondrial dynamics, biogenesis, synaptic, p-tau and longevity genes sirtuins in DDQ-treated tau mice using real-time quantitative PCR (q-RT-PCR), immunoblotting and immunofluorescence techniques. Our extensive pharmacodynamics investigations revealed that skeletal muscle had the greatest peak levels of DDQ, followed by serum and brain. Interestingly, DDQ-treated tau mice had higher levels of mitochondrial fusion, biogenesis, synaptic genes and sirtuins than DDQ-untreated tau mice. In addition, DDQ-treated tau mice had lower levels of mitochondrial fission and p-tau than untreated tau mice. The current findings, combined with our prior findings, firmly show that DDQ possesses anti-aging, anti-amyloid-beta and anti-p-tau properties, making it a promising molecule for reducing age-related, amyloid-beta and p-tau-induced synaptic and mitochondrial toxicities in AD.

The purpose of our study is to understand the role of the Ralbp1 gene in oxidative stress (OS), mitochondrial dysfunction and cognition in Alzheimer’s disease (AD) pathogenesis. The Ralbp1 gene encodes the 76 kDa protein Rlip (aka RLIP76). Previous studies have revealed its role in OS-related cancer. However, Rlip is transcriptionally regulated by EP300, a CREB-binding protein that is important for synaptic plasticity in the brain. Rlip functions as a stress-responsive/protective transporter of glutathione conjugates (GS-E) and xenobiotic toxins. OS causes rapid cellular accumulation of Rlip and its translocation from a tubulin-bound complex to the plasma membrane, mitochondria and nucleus. Therefore, Rlip may play an important role in maintaining cognitive function in the face of OS-related injury. This study is aimed to determine whether Rlip deficiency in mice is associated with AD-like cognitive and mitochondrial dysfunction. Brain tissue obtained from cohorts of wildtype and Rlip+/- mice were analyzed for OS markers, expression of genes that regulate mitochondrial fission/fusion, and synaptic integrity. We also examined mitochondrial ultrastructure in mouse brains obtained from these mice and further analyzed the impact of Rlip deficiency on gene networks of AD, aging, inhibition of stress-activated gene expression, mitochondrial function, and CREB signaling. Our studies revealed a significant increase in the levels of OS markers and alterations in the expression of genes and proteins involved in mitochondrial biogenesis, dynamics and synapses in brain tissues of these mice. Furthermore, we compared the cognitive function of wildtype and Rlip+/- mice. Behavioral, basic motor and sensory function tests in Rlip+/- mice revealed cognitive decline, similar to AD. Gene network analysis indicated dysregulation of stress-activated gene expression, mitochondrial function, and CREB signaling genes in the Rlip+/- mouse liver. Our results suggest that the Rlip deficiency-associated increase in OS and mitochondrial dysfunction could contribute to the development of OS-related AD processes. Therefore, the restoration of Rlip activity and endogenous cytoprotective mechanisms by pharmacological interventions is a novel approach to protect against AD.

Simple Summary Rlip knockout has been reported to prevent cancer in highly cancer-susceptible mice lacking p53, and Rlip knockdown kills many types of cancer cells. In humans, breast cancer shows diverse characteristics, including HER2-driven subtypes and viral-driven subtypes. HER2 can be targeted; however, escape of the cancer from targeted therapies remains a problem. In this work we evaluated the capacity of Rlip knockout to prevent breast cancer in genetically engineered mouse models of HER2-driven breast cancer (Erbb2 model) and polyomavirus-driven breast cancer (PyVT model). We found that in Erbb2 mice, Rlip knockout significantly delayed oncogenesis and reduced the expression of genes associated with poor prognosis in patients. In PyVT mice, Rlip knockout did not delay oncogenesis or tumor growth, but Rlip knockdown reduced tumor metastasis to the lung. We conclude that Rlip inhibitors may significantly improve survival in HER2-positive patients, but are unlikely to offer benefits to patients with polyomavirus-associated tumors. Abstract We recently reported that loss of one or both alleles of Ralbp1, which encodes the stress-protective protein RLIP76 (Rlip), exerts a strong dominant negative effect on both the inherent cancer susceptibility and the chemically inducible cancer susceptibility of mice lacking one or both alleles of the tumor suppressor p53. In this paper, we examined whether congenital Rlip deficiency could prevent genetically-driven breast cancer in two transgenic mouse models: the MMTV-PyVT model, which expresses the polyomavirus middle T antigen (PyVT) under control of the mouse mammary tumor virus promoter (MMTV) and the MMTV-Erbb2 model which expresses MMTV-driven erythroblastic leukemia viral oncogene homolog 2 (Erbb2, HER2/Neu) and frequently acquires p53 mutations. We found that loss of either one or two Rlip alleles had a suppressive effect on carcinogenesis in Erbb2 over-expressing mice. Interestingly, Rlip deficiency did not affect tumor growth but significantly reduced the lung metastatic burden of breast cancer in the viral PyVT model, which does not depend on either Ras or loss of p53. Furthermore, spontaneous tumors of MMTV-PyVT/Rlip+/+ mice showed no regression following Rlip knockdown. Finally, mice lacking one or both Rlip alleles differentially expressed markers for apoptotic signaling, proliferation, angiogenesis, and cell cycling in PyVT and Erbb2 breast tumors. Our results support the efficacy of Rlip depletion in suppressing p53 inactivated cancers, and our findings may yield novel methods for prevention or treatment of cancer in patients with HER2 mutations or tumor HER2 expression.

The purpose of our study is to determine the protective effects of the newly discovered molecule DDQ (diethyl (3,4-dihydroxyphenethylamino)(quinolin-4-yl) methylphosphonate) against aging in an in vitro, mouse primary hippocampal neurons, HT22 cells, and in vivo, 24-month-old C57BL6/J mice. Using biochemical and molecular methods, we studied the half-life period in the blood and brain, optimized the dose, determined dose-response (using 1, 5, 10, 20, and 50 mg/kg body weight), and measured the levels of blood, skeletal muscle, and brain. Using Morris water maze (cognitive behavior), q-RT-PCR (mRNA and protein levels of longevity genes SIRTUINS), transmission electron microscopy (mitochondrial number and length), and Golgi-Cox staining (dendritic spine number and length) were assessed in 24-month-old C57BL6/J mice. Out of 5 different doses of DDQ, the 20 mg/kg body weight dose showed the strongest protective effects against aging in C57BL6/J mice. The half-life time of DDQ is 20 h in the serum and 12 h in the brain. Our extensive pharmacodynamics analysis revealed high peak levels of DDQ in the skeletal muscle, followed by serum and brain. Using mouse primary hippocampal (HT22) neurons and 24-month-old C57BL6/J mice, we tested the protective effects of DDQ. Interestingly, longevity genes SIRTUINS were upregulated in DDQ-treated HT22 cells, and DDQ-treated aged wild-type mice relative to DDQ-untreated cells and untreated aged control mice. Dendritic spines and the quality of mitochondria were significantly increased in DDQ-treated aged mice. Current study findings, together with our previous study observations, strongly suggest that DDQ has anti-aging effects and warrants further investigations of anti-inflammatory, anti-DNA damage, and telomerase activity studies.

The purpose of our study is to determine the protective effects of the newly discovered molecule DDQ (diethyl (3,4-dihydroxyphenethylamino)(quinolin-4-yl) methylphosphonate) against mutant APP and amyloid-beta (Aβ) in Alzheimer's disease (AD). To achieve our objective, we used a well characterized amyloid-beta precursor protein (APP) transgenic mouse model (Tg2576 strain). We administered DDQ, a 20 mg/kg body weight (previously determined in our laboratory) intra-peritoneally 3-times per week for 2 months, starting at the beginning of the 12th month, until the end of the 14th month. Further, using biochemical and molecular methods, we measured the levels of DDQ in the blood, skeletal muscle, and brain. Using Morris Water Maze, Y-maze, open field, and rotarod tests, we assessed cognitive behavior after DDQ treatment. Using q-RT-PCR, immunoblotting, transmission electron microscopy, and Golgi-cox staining methods, we studied mRNA and protein levels of longevity genes SIRTUINS, mitochondrial number & length, and dendritic spine number and length in DDQ-treated APP mice. Our extensive pharmacodynamics analysis revealed high peak levels of DDQ in the skeletal muscle, followed by serum and brain. Our behavioral analysis of rotarod, open field, Y-maze, and Morris Water Maze tests revealed that DDQ ameliorated cognitive decline (Morris Water Maze), improved working memory (Y-Maze), exploratory behavior (open field), and motor coordination (rotarod) in DDQ-treated APP mice. Interestingly, longevity genes SIRTUINS, mitochondrial biogenesis, fusion, mitophagy, autophagy and synaptic genes were upregulated in DDQ-treated APP mice relative to untreated APP mice. Dendritic spines and the quality mitochondria were significantly increased in DDQ treated APP mice. Current study findings, together with our previous study observations, strongly suggest that DDQ has anti-aging, and anti-amyloid-beta effects and a promising molecule to reduce age-and amyloid-beta-induced toxicities in AD.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and is the most common cause of dementia in older people. AD is associated with the loss of synapses, oxidative stress, mitochondrial structural and functional abnormalities, microRNA deregulation, inflammatory responses, neuronal loss, accumulation of amyloid-beta (Aβ) and phosphorylated tau (p-tau). AD occurs in two forms: early onset, familial AD and late-onset, sporadic AD. Causal factors are still unknown for a vast majority of AD patients. Genetic polymorphisms are proposed to contribute to late-onset AD via age-dependent increases in oxidative stress and mitochondrial abnormalities. Recent research from our lab revealed that reduced levels of Rlip76 induce oxidative stress, mitochondrial dysfunction and synaptic damage, leading to molecular and behavioral phenotypes resembling late-onset AD. Rlip76 is a multifunctional 76 kDa protein encoded by the RALBP1 gene, located on chromosome 18. Rlip is a stress-protective ATPase of the mercapturic acid pathway that couples clathrin-dependent endocytosis with the efflux of glutathione–electrophile conjugates. Rlip is evolutionarily highly conserved across species and is ubiquitously expressed in all tissues, including AD-affected brain regions, the cerebral cortex and hippocampus, where highly active neuronal metabolisms render the cells highly susceptible to intracellular oxidative damage. In the current article, we summarize molecular and cellular features of Rlip and how depleted Rlip may exacerbate oxidative stress, mitochondrial dysfunction and synaptic damage in AD. We also discuss the possible role of Rlip in aspects of learning and memory via axonal growth, dendritic remodeling, and receptor regulation. We conclude with a discussion of the potential for the contribution of genetic polymorphisms in Rlip to AD progression and the potential for Rlip-based therapies.

Background Dietary factors may modulate many complex interactions between the microbiome, metabolome, and immune system and can have an impact on the functional status of older adults. Sulforaphane (SFN), a natural compound and Nrf2-related activator of cytoprotective genes, provides a wide range of biological effects from cancer prevention to reducing insulin resistance. We have shown that SFN increased survival and improved cardiac and skeletal muscle function in a mouse model of aging. This study aims to investigate the anti-aging effects of SFN on the gut microbiome and metabolome.Results Young (6-8 weeks of age) and old (21-22 months of age) male C57BL/6J mice were provided regular rodent chow or chow containing SFN for 2 months. Fecal samples were collected right before and at the completion of SFN administration. We profiled the gut microbiome and applied global metabolomic profiling to fecal samples. Multi-omics datasets were analyzed individually and integrated to investigate the relationship between SFN diet, the microbiome, and metabolome. Microbial diversity, composition and functional capacity varied substantially across different age groups. On a global level, in old mice we observed that the SFN diet restored the gut microbiome to mimic that in young mice. In old mice, the SFN diet enriched bacteria associated with an improved intestinal barrier function and the production of anti-inflammatory compounds. In addition, the tricarboxylic acid cycle, central in cellular respiration, was decreased and amino acid metabolism-related pathways were increased. SFN diet induced metabolite biomarkers in old mice that are associated majorly with the genera, Oscillospira , Ruminococcus , and Allobaculum.Conclusion In old mice, SFN directed the metabolic potential to that of young animals. Integrated microbiome and metabolome analyses revealed metabolite biomarkers that could be modulated by bacteria and contribute to the anti-aging effects of SFN. Collectively, our results provide evidence in support of a novel hypothesis that SFN diet exerts anti-aging effects by influencing the gut microbiome and metabolome. Although further investigations are needed to identify precise mechanisms, modulating the gut microbiome by SFN may have the potential to promote healthier aging.

RLIP76 (RAL-binding protein-1, Rlip) is a stress-protective mercapturic-acid-pathway transporter protein that also plays a key role in regulating clathrin-dependent endocytosis as a Ral effector. Targeted inhibition or depletion of Rlip causes regression of xenografts of many cancers and is capable of abrogating tumor formation in p53-null mice. This is associated with the reversion of the abnormal methylomic profile of p53-null mice to wild-type. In a query of The Cancer Genome Atlas (TCGA) databases, we found that Rlip expression was associated with poor survival and with significant differences in the frequencies of PIK3CA mutation, MYC amplification, and CDKN2A/B deletion, which were the most commonly mutated, amplified, and deleted genes, respectively, among TCGA breast cancer patients. We conducted the present study to further examine the effects of Rlip inhibition and to evaluate the in vitro and in vivo efficacy in breast cancer. Using immunogold electron microscopy, we found that plasma-membrane Rlip was accessible to cell-surface antibodies in the MCF7 (ER+) breast cancer cell line. Rlip depletion resulted in decreased survival of MCF7 and MDA-MB-231 cells and increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity and DNA laddering, indicating apoptotic cell death. Additionally, in vitro knockdown of Rlip inhibited EGF endocytosis and WNT/MAPK signaling. Xenograft studies in nude mice showed regression of breast cancer via antisense-mediated depletion of Rlip mRNA as well as by anti-Rlip antibody. Finally, knockdown of Rlip by antisense locked nucleic acid oligonucleotides increased markers for apoptotic signaling and decreased markers for proliferation, angiogenesis, and cell cycling in MCF7 and MDA-MB-231luc xenografts. Our findings validate Rlip as an attractive target in breast cancer.

Scheme depicts hypothesized decreases of heart and SKM function during aging via ROS and partial reversal by SFN activation of Nrf2 that results in significant restoration of function of both types of muscle. A decline in heart and skeletal muscle function was observed in aged mice, with altered mitochondrial structure and gene expression, accompnied by decreases in mitochondrial complex activity, Nrf2 binding to antioxidant‐responsive DNA elements and physical endurance. The addition of sulforaphane (SFN) to the diet improved these age‐related changes in older mice to levels observed in younger ones. We demonstrated in this paper that SFN alleviates age‐associated oxidative damage and improves mitochondrial and cardiac function as well as physical endurance in old mice., Age‐associated mitochondrial dysfunction and oxidative damage are primary causes for multiple health problems including sarcopenia and cardiovascular disease (CVD). Though the role of Nrf2, a transcription factor that regulates cytoprotective gene expression, in myopathy remains poorly defined, it has shown beneficial properties in both sarcopenia and CVD. Sulforaphane (SFN), a natural compound Nrf2‐related activator of cytoprotective genes, provides protection in several disease states including CVD and is in various stages of clinical trials, from cancer prevention to reducing insulin resistance. This study aimed to determine whether SFN may prevent age‐related loss of function in the heart and skeletal muscle. Cohorts of 2‐month‐old and 21‐ to 22‐month‐old mice were administered regular rodent diet or diet supplemented with SFN for 12 weeks. At the completion of the study, skeletal muscle and heart function, mitochondrial function, and Nrf2 activity were measured. Our studies revealed a significant drop in Nrf2 activity and mitochondrial functions, together with a loss of skeletal muscle and cardiac function in the old control mice compared to the younger age group. In the old mice, SFN restored Nrf2 activity, mitochondrial function, cardiac function, exercise capacity, glucose tolerance, and activation/differentiation of skeletal muscle satellite cells. Our results suggest that the age‐associated decline in Nrf2 signaling activity and the associated mitochondrial dysfunction might be implicated in the development of age‐related disease processes. Therefore, the restoration of Nrf2 activity and endogenous cytoprotective mechanisms by SFN may be a safe and effective strategy to protect against muscle and heart dysfunction due to aging.

The purpose of our study is to understand the role of the RALBP1 gene in oxidative stress (OS), mitochondrial dysfunction and cognition in Alzheimer’s disease (AD) pathogenesis. The RALPB1 gene encodes the 76 kDa protein RLIP76 (Rlip). Rlip functions as a stress-responsive/protective transporter of glutathione conjugates (GS-E) and xenobiotic toxins. We hypothesized that Rlip may play an important role in maintaining cognitive function. The aim of this study is to determine whether Rlip deficiency in mice is associated with AD-like cognitive and mitochondrial dysfunction. Brain tissue obtained from cohorts of wildtype (WT) and Rlip+/− mice were analyzed for OS markers, expression of genes that regulate mitochondrial fission/fusion, and synaptic integrity. We also examined mitochondrial ultrastructure in brains obtained from these mice and further analyzed the impact of Rlip deficiency on gene networks of AD, aging, stress response, mitochondrial function, and CREB signaling. Our studies revealed a significant increase in the levels of OS markers and alterations in the expression of genes and proteins involved in mitochondrial biogenesis, dynamics and synapses in brain tissues from these mice. Furthermore, we compared the cognitive function of WT and Rlip+/− mice. Behavioral, basic motor and sensory function tests in Rlip+/− mice revealed cognitive decline, similar to AD. Gene network analysis indicated dysregulation of stress-activated gene expression, mitochondrial function and CREB signaling genes in the Rlip+/− mouse brain. Our results suggest that Rlip deficiency-associated increases in OS and mitochondrial dysfunction could contribute to the development or progression of OS-related AD processes.

Small molecules containing carboxylic acid functional groups are ubiquitous throughout biology, playing vital roles in biological chemistry ranging from energy metabolism to cellular signaling. This paper describes a new derivatization reagent, 4-bromo-N-methylbenzylamine, which was selected for its potential to derivatize mono-, di- and tri-carboxylic acids, such as the intermediates of the tricarboxylic acid (TCA) cycle. This derivatization procedure facilitated the use of positive electrospray ionization (ESI) tandem mass spectrometry (MS/MS) detection of derivatized species allowing for clear identification thanks to the easily recognizable isotope pattern of the incorporated bromine. A liquid chromatography (LC)-MS/MS method was developed which provided limits of detection between 0.2 and 44 μg L−1 in under 6 min, depending on the analyte and total analysis time. This method was successfully applied in both in vitro and in vivo models. Electronic supplementary material The online version of this article (doi:10.1007/s10337-017-3421-0) contains supplementary material, which is available to authorized users.

Background Neuroinflammation, typified by elevated levels of interleukin-1 (IL-1) α and β, and deficits in proteostasis, characterized by accumulation of polyubiquitinated proteins and other aggregates, are associated with neurodegenerative disease independently and through interactions of the two phenomena. We investigated the influence of IL-1β on ubiquitination via its impact on activation of the E3 ligase parkin by either phosphorylated ubiquitin (P-Ub) or NEDD8. Methods Immunohistochemistry and Proximity Ligation Assay were used to assess colocalization of parkin with P-tau or NEDD8 in hippocampus from Alzheimer patients (AD) and controls. IL-1β effects on PINK1, P-Ub, parkin, P-parkin, and GSK3β—as well as phosphorylation of parkin by GSK3β—were assessed in cell cultures by western immunoblot, using two inhibitors and siRNA knockdown to suppress GSK3β. Computer modeling characterized the binding and the effects of P-Ub and NEDD8 on parkin. IL-1α, IL-1β, and parkin gene expression was assessed by RT-PCR in brains of 2- and 17-month-old PD-APP mice and wild-type littermates. Results IL-1α, IL-1β, and parkin mRNA levels were higher in PD-APP mice compared with wild-type littermates, and IL-1α-laden glia surrounded parkin- and P-tau-laden neurons in human AD. Such neurons showed a nuclear-to-cytoplasmic translocation of NEDD8 that was mimicked in IL-1β-treated primary neuronal cultures. These cultures also showed higher parkin levels and GSK3β-induced parkin phosphorylation; PINK1 levels were suppressed. In silico simulation predicted that binding of either P-Ub or NEDD8 at a singular position on parkin opens the UBL domain, exposing Ser65 for parkin activation. Conclusions The promotion of parkin- and NEDD8-mediated ubiquitination by IL-1β is consistent with an acute neuroprotective role. However, accumulations of P-tau and P-Ub and other elements of proteostasis, such as translocated NEDD8, in AD and in response to IL-1β suggest either over-stimulation or a proteostatic failure that may result from chronic IL-1β elevation, easily envisioned considering its early induction in Down’s syndrome and mild cognitive impairment. The findings further link autophagy and neuroinflammation, two important aspects of AD pathogenesis, which have previously been only loosely related.

2&prime, hydroxyflavanone (2HF) is a dietary flavonoid with anticancer activity towardsmultiple cancers. Here, we report that topically applied 2HF inhibits the growth of intradermalimplants of melanoma in immunocompetent mice. 2HF induced apoptosis and inhibited the growthof the human SK-MEL-24 as well as murine B16-F0 and B16-F10 melanoma cell lines in vitro.Apoptosis was associated with depletion of caspase-3, caspase-9, and PARP1 in B16-F0 and SKMEL-24 cells. Caspase-9 and MEKK-15 were undetected even in untreated B16-F10 cells. Signalingproteins TNF&alpha, and phospho-PDGFR-&beta, were depleted in all three cell lines, MEKK-15 was depletedby 2HF in SK-MEL-24 cells. 2HF enhanced sunitinib (an MEK and PDGFR-&beta, inhibitor) and AZD2461 (a PARP1 inhibitor) cytotoxicity. 2HF also depleted the Ral-regulated, stress-responsive,antiapoptotic endocytic protein RLIP76 (RALBP1), the inhibition of which has previously beenshown to inhibit B16-F0 melanoma growth in vivo. Functional inhibition of RLIP76 was evidentfrom inhibition of epidermal growth factor (EGF) endocytosis by 2HF. We found that topicallyapplied 2HF&ndash, Pluronic Lecithin Organogel (PLO) gel inhibited B16-F0 and B16-F10 tumorsimplanted in mice and caused no overt toxicity despite significant systemic absorption. 2HFtreatment reduced phospho-AKT, vimentin, fibronectin, CDK4, cyclinB1, and BCL2, whereas itincreased BIM and phospho-AMPK in excised tumors. Several cancer signals are controlled byendocytosis, a process strongly inhibited by RLIP76 depletion. We conclude that 2HF&ndash, PLO gel maybe useful for topical therapy of cutaneous metastases of melanoma and could enhance theantineoplastic effects of sunitinib and PARP1 inhibitors. The mechanism of action of 2HF inmelanoma overlaps with RLI76 inhibitors.

While fluorescence in situ hybridization (FISH) is traditionally used to detect copy number variations (CNV) at known pathogenic chromosomal loci, the comparative genomic hybridization (CGH) microarray is a platform capable of simultaneous detection of CNVs at thousands of loci. CGH microarrays are typically used to assess loci of known prognostic significance; however loci variants of uncertain significance (VUS) are frequently present. To identify potentially actionable targets, we retrospectively analyzed VUS loci from 192 cases of cancer or genetic disorders using CGH data generated for treatment guidance at the Texas Tech University Health Sciences Center Cytogenetics Laboratory, a CLIA-certified Children's Oncology Group reference lab serving the West Texas population. The majority of cases were of pediatric malignancies, though some adult cancers and benign hematological diseases were included. Patient DNA was hybridized onto CytoSure Constitution V3 arrays (60k loci), scanned with the Agilent microarray D scanner, and analyzed by CytoSure Interpret Software. TACC1, TM2D2, KAT6A and ADAM32 were knocked down in H358 lung cancer (p53-null), MCF7 (ER+) and MDA-MB-231 (triple-negative) breast cancer, and B16-F0 murine melanoma cell lines using siRNA, followed by evaluation of growth inhibition by MTT assay. Colcemid block was used to evaluate metaphase reduction and chromosomal abnormalities following TACC1 knockdown. We found 794 distinct CNVs, the most frequent being 14q32.22 (112 cases), 14q11.2 (100 cases), 8p11.22 (98 cases), 15q11.1-15q11.2 (83 cases), and 8p23.1 (77 cases). In particular, the 8p11.22 locus was amplified in many pediatric cancers, including acute lymphoblastic leukemia (18 gains/2 losses), lymphoma (6/1), neuroblastoma (5/0), pediatric breast tumors (5/0), and Wilm's tumor (4/0). Linkage of TACC1, TM2D2, KAT6A and ADAM32, all genes residing at the 8p11.22 locus, was indicated by a similar average 5-year overall survival rate (75.3 ± 1.7%, n=253) among patients harboring alterations in these genes in The Cancer Genome Atlas (TCGA) database. Knockdown of TACC1, TM2D2, and KAT6A reduced growth of H358, MCF7, MDA-MB-231, and B16-F0 cells (P < 0.05). ADAM32 depletion inhibited H358, MCF7, and MDA-MB-231 cells (P < 0.05), but not B16-F0 cells. Because TACC1 function is linked to mitosis through the aurora kinases, we subsequently studied metaphase of B16-F0 cells after TACC1 depletion, found an 80% reduction metaphase cells, and increased morphological abnormalities. In summary, we report a high frequency of 8p11.2 gains in our pediatric oncology patients and demonstrate that evaluation of growth inhibition of cancer cells following knockdown of genes at VUS loci may help to identify new cancer therapy targets. These results further suggest that TACC1 could be a target for treating triple negative breast cancer, melanoma, and p53-null lung cancer. Citation Format: Aditya Rajan, Sharda P. Singh, Santhosh Chavali, Chhanda Bose, Vijay Tonk, Sanjay Awasthi. Tacc1 is amplified in a wide range of pediatric malignancy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5461.

A need exists for developing new therapies to improve cardiovascular outcomes in end-stage kidney disease. Three new areas that address novel pathophysiological mechanisms and/or therapeutic approaches toward cardiovascular events in chronic kidney disease patients include the use of an anti-inflammatory agent, the role of catalytic iron, and protein carbamylation. In preliminary studies, hydroxychloroquine, which has multiple anti-inflammatory properties, preserved vascular compliance for the aorta and major vessels, as well as reduced the extent of severity of atherosclerosis in ApoE-/- mice. The ability of iron to rapidly and reversibly cycle between 2 oxidation states makes iron potentially hazardous by enabling it to participate in the generation of powerful oxidant species. We have shown that high catalytic iron in the general population is associated with a 4-fold increase in prevalent cardiovascular disease (CVD), even after accounting for traditional risk factors. In addition, the highest levels of catalytic iron are present in dialysis patients and, more specifically, patients with prevalent CVD have several-fold higher catalytic iron levels compared with controls without CVD. These data suggest the utility of iron chelators for preventing and treating CVD in patients with chronic kidney disease and should be further investigated. Carbamylation of proteins results from nonenzymatic chemical modification by isocyanic acid derived from urea and an alternative route, the myeloperoxidase-catalyzed oxidation of thiocyanate. We have shown carbamylated low-density lipoprotein to have all the major biological effects relevant to atherosclerosis including endothelial cell injury, increased expression of cell adhesion molecules, and vascular smooth muscle cell proliferation. In 2 separate clinical studies, plasma levels of carbamylated protein independently predicted an increased risk of CVD and death.

Breast cancer is the most common malignancy in women of the Western world. Doxorubicin (DOX) continues to be used extensively to treat early-stage or node-positive breast cancer, human epidermal growth factor receptor-2 (HER2)-positive breast cancer, and metastatic disease. We have previously demonstrated in a mouse model that sulforaphane (SFN), an isothiocyanate isolated from cruciferous vegetables, protects the heart from DOX-induced toxicity and damage. However, the effects of SFN on the chemotherapeutic efficacy of DOX in breast cancer are not known. Present studies were designed to investigate whether SFN alters the effects of DOX on breast cancer regression while also acting as a cardioprotective agent. Studies on rat neonatal cardiomyocytes and multiple rat and human breast cancer cell lines revealed that SFN protects cardiac cells but not cancer cells from DOX toxicity. Results of studies in a rat orthotopic breast cancer model indicated that SFN enhanced the efficacy of DOX in regression of tumor growth, and that the DOX dosage required to treat the tumor could be reduced when SFN was administered concomitantly. Additionally, SFN enhanced mitochondrial respiration in the hearts of DOX-treated rats and reduced cardiac oxidative stress caused by DOX, as evidenced by the inhibition of lipid peroxidation, the activation of NF-E2-related factor 2 (Nrf2) and associated antioxidant enzymes. These studies indicate that SFN not only acts synergistically with DOX in cancer regression, but also protects the heart from DOX toxicity through Nrf2 activation and protection of mitochondrial integrity and functions.

Gadolinium-based contrast agents are linked to nephrogenic systemic fibrosis in patients with renal insufficiency. The pathology of nephrogenic systemic fibrosis is characterized by abnormal tissue repair: fibrosis and ectopic ossification. The mechanisms by which gadolinium could induce fibrosis and ossification are not known. We examined in vitro the effect of a gadolinium-based contrast agent on human peripheral blood mononuclear cells for phenotype and function relevant to the pathology of nephrogenic systemic fibrosis using immunofluorescence, flow cytometry, real-time PCR, and osteogenic assays. We also examined tissues from patients with nephrogenic systemic fibrosis, using IHC to identify the presence of cells with phenotype induced by gadolinium. Gadolinium contrast induced differentiation of human peripheral blood mononuclear cells into a unique cellular phenotype—CD163 + cells expressing proteins involved in fibrosis and bone formation. These cells express fibroblast growth factor (FGF)23, osteoblast transcription factors Runt-related transcription factor 2, and osterix, and show an osteogenic phenotype in in vitro assays. We show in vivo the presence of CD163 + /procollagen-1 + /osteocalcin + cells in the fibrotic and calcified tissues of nephrogenic systemic fibrosis patients. Gadolinium contrast–induced CD163 + /ferroportin + /FGF23 + cells with osteogenic potential may play a role in systemic fibrosis and ectopic ossification in nephrogenic systemic fibrosis.

The alpha class glutathione s-transferase (GST) isozyme GSTA4-4 (EC2.5.1.18) exhibits high catalytic efficiency towards 4-hydroxynon-2-enal (4-HNE), a major end product of oxidative stress induced lipid peroxidation. Exposure of cells and tissues to heat, radiation, and chemicals has been shown to induce oxidative stress resulting in elevated concentrations of 4-HNE that can be detrimental to cell survival. Alternatively, at physiological levels 4-HNE acts as a signaling molecule conveying the occurrence of oxidative events initiating the activation of adaptive pathways. To examine the impact of oxidative/electrophilic stress in a model with impaired 4-HNE metabolizing capability, we disrupted the Gsta4 gene that encodes GSTA4-4 inmice. The effect of electrophile and oxidants on embryonic fibroblasts (MEF) isolated from wild type (WT) and Gsta4 null mice were examined. Results indicate that in the absence of GSTA4-4, oxidant-induced toxicity is potentiated and correlates with elevated accumulation of 4-HNE adducts and DNA damage. Treatment of Gsta4 null MEF with 1,1,4-tris(acetyloxy)-2(E)-nonene [4-HNE(Ac)3], a pro-drug form of 4-HNE, resulted in the activation and phosphorylation of the c-jun-N-terminal kinase (JNK), extracellular-signal-regulated kinases (ERK 1/2) and p38 mitogen activated protein kinases (p38 MAPK) accompanied by enhanced cleavage of caspase-3. Interestingly, when recombinant mammalian or invertebrate GSTs were delivered to Gsta4 null MEF, activation of stress-related kinases in 4-HNE(Ac)3 treated Gsta4 null MEF were inversely correlated with the catalytic efficiency of delivered GSTs towards 4-HNE. Our data suggest that GSTA4-4 plays a major role in protecting cells from the toxic effects of oxidant chemicals by attenuating the accumulation of 4-HNE.

Patients with chronic kidney disease (CKD) have high risk of cardiovascular complications. Plasma levels of carbamylated proteins produced by urea-derived isocyanate or thiocyanate are elevated in CKD patients and that they are significant predictors of cardiovascular events and all-cause mortality. Carbamylated LDL (cLDL) has pro-atherogenic properties and is known to affect major biological processes relevant to atherosclerosis including endothelial cell injury. The underlying mechanisms of cLDL-induced endothelial cell injury are not well understood. Although autophagy has been implicated in atherosclerosis, cLDL-mediated induction of autophagy and its role in endothelial cell injury is unknown. Our studies demonstrate that human coronary artery endothelial cells (HCAECs) respond to cLDL by specific induction of key autophagy proteins including LC3-I, beclin-1, Atg5, formation of lipid-conjugated LC3-II protein, and formation of punctate dots of autophagosome-associated LC3-II. We demonstrated that autophagy induction is an immediate response to cLDL and occurred in a dose and time-dependent manner. Inhibition of cLDL-induced autophagy by a specific siRNA to LC3 as well as by an autophagy inhibitor provided protection from cLDL-induced cell death and DNA fragmentation. Our studies demonstrate that autophagy plays an important role in cLDL-mediated endothelial cell injury and may provide one of the underlying mechanisms for the pathogenesis of cLDL-induced atherosclerosis in CKD patients.

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor EPOR. Recent studies, however, have shown that the EPOR is additionally present in various cancer cells and EPO induces the proliferation of these cells, suggesting a different function for EPO other than erythropoiesis. Therefore, the purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation and signaling cascades involved in this process, using the rat pancreatic tumor cell line AR42J. Our results showed that AR42J cells expressed EPOR, and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated cells indicated an increased percentage of cells in the S phase, whereas cell numbers in G0/G1 phase were significantly reduced. Phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH2terminal kinase 1/2 (JNK1/2) was rapidly stimulated and sustained after EPO addition. Treatment of cells with mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 or JNK inhibitor SP600125 significantly inhibited EPO-enhanced proliferation and also increased the fraction of cells in G0/G1 phase. Furthermore, the inhibition of JNK using small interference RNA (siRNA) suppressed EPO-enhanced proliferation of AR42J cells. Taken together, our results indicate that AR42J cells express EPOR and that the activation of both ERK1/2 and JNK1/2 by EPO is essential in regulating proliferation and the cell cycle. Thus both appear to play a key role in EPO-enhanced proliferation and suggest that the presence of both is required for EPO-mediated proliferation of AR42J cells.

The aim of this study was to evaluate changes in the regulation of lipid metabolism and mitogen-activated protein kinases (MAPK) in the liver of C57BL/6 mice as they age. This was done by assessing the status of total cholesterol content and its enzyme, acyl-CoA: cholesterol acyltransferase (ACAT), in liver microsomal preparations and the low-density lipoprotein receptor (LDLr) mRNA expression in the livers of 4-24-month-old C57B/6 mice, without exogenous cholesterol feeding. With aging, there was an increase in cholesterol content and ACAT activity in liver microsomes. Northern blot analysis and real-time quantitative polymerase chain reaction data showed that ACAT-2 mRNA increased with age as well. LDLr expression decreased significantly in an age-dependent manner. In addition, we studied the basal and activated forms of MAPK, e.g. extracellular regulatory kinase (ERK-1/2), c-jun NH2-terminal kinase (JNK-1/2) and p38 MAPK. During aging, there was a considerable decrease in phosphorylated ERK-1/2 level while JNK-1/2 and p38 MAPK levels increased with age. Our studies showed an altered LDLr expression and altered phosphorylated MAPK in the liver of C57BL/6 mice during aging. These alterations might contribute to the development of atherosclerosis, hypercholesterolemia and other cholesterol-related conditions.

We have shown previously that the chemoprotective activity of diallyl disulfide (DADS), a naturally occurring anticancer agent in garlic, against benzo[a]pyrene (BP)-induced forestomach carcinogenesis in mice correlates strongly with its inductive effects on the expression of Pi class glutathione (GSH) transferase mGSTP1-1. The present structure-activity relationship studies were designed to define the role of allyl groups and the disulfide chain in mGSTP1-inducing activity of DADS. Hepatic mGSTP1 mRNA levels rose rapidly upon treatment of mice with DADS, reached a maximum between 12 and 24 h (< or =5.7-fold induction) and fell to control levels by 48 h after DADS treatment. Induction of mGSTP1 mRNA in the forestomach was maximal between 6 and 12 h after DADS treatment (< or =4.7-fold induction). The mGSTP1 mRNA expression was either unaltered (liver) or moderately increased (forestomach) upon treatment of mice with dipropyl disulfide (DPDS), which is a naturally occurring saturated analog of DADS. These results indicated that the allyl groups are critical for the mGSTP1-inducing activity of DADS. A statistically significant increase in the expression of mGSTP1 mRNA was also observed in the liver and forestomach of mice treated with diallyl monosulfide (DAMS), albeit to a much lesser extent compared with DADS. These results indicated that the oligosulfide chain length in garlic organosulfides (OSCs) is equally important for their mGSTP1-inducing activity. The role of the disulfide chain in DADS-mediated induction of mGSTP1 was further investigated by testing a pair of alkadienes (1,7-octadiene and 1,8-nonadiene) having structural similarity to DADS. Both DADS and the alkadienes carry allyl groups at both ends of a linear molecule and the distance between the allylic carbon atoms is similar in both compounds, but the central disulfide chain of DADS is replaced with an alkyl chain in the alkadienes. The alkadienes were either ineffective or moderately active in increasing mGSTP1 expression. In conclusion, the results of the present study clearly indicate that the presence of terminal allyl groups as well as the central disulfide chain is required for maximum induction of mGSTP1 in vivo by garlic-derived OSCs.

The syntheses of 5-amino (4–12,15) and 5,8-diamino(16–17) isoquinoline derivatives. their antifilarial activity and their effect on metabolic activities of filariids are delineated. Some of the screened compounds have shown promising filaricidal response against Acanthocheilonema viteae in rodents.

Tachyzoites are considered to be the most important stage of Toxoplasma gondii which causes toxoplasmosis. T. gondii is, an obligate intracellular parasite which infects a wide range of cells. The present study was designed to develop a method for an early detection of T. gondii tachyzoites. The method comprised of a binding assay which was analyzed using principal component and cluster analysis. Our data showed that glycoconjugates GC1, GC2, GC3 and GC10 exhibit a significantly higher binding affinity for T. gondii tachyzoites as compared to controls (T. gondii only, PAA only, GC 1, 2, 3, and 10 only).

Objectives: The aim of the current study was to investigate whether nicotine treatment would induce the proliferation of isolated rat primary pancreatic acinar cells in culture by activating mitogen‐activated protein kinase (MAPK) signalling and exocrine secretion. Materials and Methods: A nicotine dose‐ and time‐response curve was initially developed to determine the optimal dose and time used for all subsequent studies. Proliferation studies were conducted by cell counting and confirmed further by bromodeoxyuridine (BrdU) incorporation and flow cytometry assays. MAPK signalling studies were conducted by Western blot analysis. Localization of ERK1/2 signals, with or without nicotine and the MAPK inhibitor, was visualized by immunofluorescence. Results: Nicotine treatment caused dose‐dependent activation of extracellular signal‐regulated kinases (ERK1/2), the maxima occurring at 100 µm and at 3 min after treatment; the response was suppressed by the ERK1/2 inhibitor. Maximal nicotine‐induced cell proliferation occurred at 24 h, and UO126‐treatment significantly reduced this response. Exposure of cells to 100 µm nicotine for 6 min significantly enhanced both baseline and cholecystokinin‐stimulated cell function, and these effects were not affected by treatment with the inhibitor of ERK1/2 but were suppressed by mecamylamine, a nicotinic receptor antagonist. Conclusions: Our results suggest that nicotine treatment induced cell proliferation of isolated pancreatic acinar cells and that this is coupled with the activation of MAPK signalling with no effect on its function. Hence, in primary cells, the mechanism of induction and regulation of these two processes, cell proliferation and cell function, by nicotine treatment are independent of each other.

Cardiovascular disease is the largest cause of morbidity and mortality among patients with chronic kidney disease (CKD) and end-stage kidney disease, with nearly half of all deaths attributed to cardiovascular disease. Hydroxychloroquine (HCQ), an anti-inflammatory drug, has been shown to have multiple pleiotropic actions relevant to atherosclerosis. We conducted a proof-of-efficacy study to evaluate the effects of hydroxychloroquine in an animal model of atherosclerosis in ApoE knockout mice with and without chronic kidney disease. Forty male, 6-week-old mice were divided into four groups in a 2 x 2 design: sham placebo group; sham treatment group; CKD placebo group; and CKD treatment group. CKD was induced by a two-step surgical procedure. All mice received a high-fat diet through the study duration and were sacrificed after 16 weeks of therapy. Mice were monitored with ante-mortem ultrasonic echography (AUE) for atherosclerosis and vascular stiffness and with post-mortem histology studies for atherosclerosis. Therapy with HCQ significantly reduced the severity of atherosclerosis in CKD mice and sham treated mice. HCQ reduced the area of aortic atherosclerosis on en face examination by approximately 60% in HCQ treated groups compared to the non-treated groups. Additionally, therapy with HCQ resulted in significant reduction in vascular endothelial dysfunction with improvement in vascular elasticity and flow patterns and better-preserved vascular wall thickness across multiple vascular beds. More importantly, we found that presence of CKD had no mitigating effect on HCQ's anti-atherosclerotic and vasculoprotective effects. These beneficial effects were not due to any significant effect of HCQ on inflammation, renal function, or lipid profile at the end of 16 weeks of therapy. This study, which demonstrates structural and functional protection against atherosclerosis by HCQ, provides a rationale to evaluate its use in CKD patients. Further studies are needed to define the exact mechanisms through which HCQ confers these benefits.