Puya G. Yazdi

EVOLUTION OF LATE-LIFE MORTALITY IN DROSOPHILA MELANOGASTER

Abstract.— Aging appears to cease at late ages, when mortality rates roughly plateau in large‐scale demographic studies. This anomalous plateau in late‐life mortality has been explained theoretically in two ways: (1) as a strictly demographic result of heterogeneity in life‐long robustness between individuals within cohorts, and (2) as an evolutionary result of the plateau in the force of natural selection after the end of reproduction. Here we test the latter theory using cohorts of Drosophila melanogaster cultured with different ages of reproduction for many generations. We show in two independent comparisons that populations that evolve with early truncation of reproduction exhibit earlier onset of mortality‐rate plateaus, in conformity with evolutionary theory. In addition, we test two population genetic mechanisms that may be involved in the evolution of late‐life mortality: mutation accumulation and antagonistic pleiotropy. We test mutation accumulation by crossing genetically divergent, yet demographically identical, populations, testing for hybrid vigor between the hybrid and nonhybrid parental populations. We found no difference between the hybrid and nonhybrid populations in late‐life mortality rates, a result that does not support mutation accumulation as a genetic mechanism for late‐life mortality, assuming mutations act recessively. Finally, we test antagonistic pleiotropy by returning replicate populations to a much earlier age of last reproduction for a short evolutionary time, testing for a rapid indirect response of late‐life mortality rates. The positive results from this test support antagonistic pleiotropy as a genetic mechanism for the evolution of late‐life mortality. Together these experiments comprise the first corroborations of the evolutionary theory of late‐life mortality.

Rapamycin and Chloroquine: The In Vitro and In Vivo Effects of Autophagy-Modifying Drugs Show Promising Results in Valosin Containing Protein Multisystem Proteinopathy

Mutations in the valosin containing protein (VCP) gene cause hereditary Inclusion body myopathy (hIBM) associated with Paget disease of bone (PDB), frontotemporal dementia (FTD), more recently termed multisystem proteinopathy (MSP). Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43)-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem proteinopathies can now be included as disorders that can potentially be ameliorated by rapalogs.

Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact

Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer.

Nucleosome Organization in Human Embryonic Stem Cells

The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a “ground state” of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this “ground state” by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of nucleosome organization alone is sufficient to elucidate much of the circuitry of pluripotency. Our results, suggest that nucleosome organization is associated with numerous genomic and epigenomic processes and can be used to elucidate cellular identity.

A review of the biologic and pharmacologic role of docosapentaenoic acid n-3.

Fish oil contains a complex mixture of omega-3 fatty acids, of which eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) are the three predominant forms. There has been a plethora of previous research on the effects and associations of fish oil supplementation with various clinical manifestations. While the majority of this work was focused on EPA and DHA as the active compounds, emerging research has begun to elucidate the specific role that DPA plays in these physiological processes and its differences with the other omega-3 fatty acids. The purpose of this review is to focus on the new studies undertaken with DPA. This review summarizes the biochemical mechanisms involved in the biosynthesis and metabolism of DPA before focusing on its effects in cardiovascular disease, immune function, and psychiatric and cognitive health. The limited studies point toward a positive role that DPA supplementation can play in these processes and that is separate and distinct from traditional supplementation with DHA and EPA.

Effects of Arthrocen, an avocado/soy unsaponifiables agent, on inflammatory mediators and gene expression in human chondrocytes

Osteoarthritis (OA) is a chronic joint disease characterized by pain and stiffness. Recently, there has been great interest in the use of plant‐derived compounds and supplements in managing the symptoms of OA. Arthrocen is a plant‐based supplement consisting of avocado and soy unsaponifiable extracts in a 1 : 2 ratio. In an effort to unravel the potential mechanisms of its action on the cellular level, we utilized an in vitro assay to study its effects on cultured human chondrocytes. By pairing this assay with protein arrays on inflammatory markers, RNA‐Seq with downstream pathway analysis, and lipidomics on eicosanoids, we were able to further define its action at the molecular level. Specifically, we found a role for Arthrocen in attenuating the inflammatory response both at the protein and mRNA level. Furthermore, we discovered that Arthrocen diminished prostaglandin E2 (PGE2) levels in response to an inflammatory trigger. Additionally, unlike traditional COX‐2 inhibitors, this response rather specifically attenuated PGE2 levels in the presence of inflammation and without lowering levels of other eicosanoids. This implies that Arthrocen could potentially bring about the reduced pain produced by COX‐2 inhibitors without the known side effects of COX‐2 inhibition.

Hepatic proteomic analysis revealed altered metabolic pathways in insulin resistant Akt1+/−/Akt2−/− mice

OBJECTIVE The aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels. METHODS Two-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1(+/-)/AKT2(-/-) mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway Analysis was performed for the interpretation of the biological significance of the observed proteomic changes. RESULTS 11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1(+/-)/Akt2(-/-)) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways. CONCLUSION Our results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance.

In Vitro Effects of Arthrocen, an Avocado/Soy Unsaponifiables Agent, on Inflammation and Global Gene Expression in Human Monocytes

Osteoarthritis (OA) is the most common form of arthritis. Symptomatically characterized by stiffness and pain, OA is a chronic degenerative disease of joints. Of note, there is growing interest in the potential use of plant-based compounds for symptomatic treatment of OA. Arthrocen is a plant-derived agent consisting of a one to two ratio of avocado and soy unsaponifiable extracts. In order to decipher the potential mechanisms of Arthrocen’s action at the molecular level, we employed an in vitro assay using cultured human THP-1 cells (a model cell line for monocytes) to study its effects. By pairing protein arrays enriched for inflammatory markers, transcriptomic pathway analysis using RNA-Sequencing, and eicosanoid specific lipidomics, we have begun to unravel its potential mechanism of action. Specifically, we found that Arthrocen can attenuate the inflammatory response at the transcript level while inducing significant changes in numerous cytokines. Furthermore, we discovered that while Arthrocen alone did not increase IL-8 or MCP-1 levels, its presence had a synergistic effect on the observed increase in response to LPS stimulation. Additionally, this synergistic effect of Arthrocen on LPS stimulation of IL-8 and MCP-1 protein levels was also observed at the mRNA level and suggests a regulatory mechanism at the transcriptional level. Interestingly, Arthrocen induced no changes in any of the eicosanoids studied. This multi-omics approach implies that Arthrocen functions at the level of gene transcription to dampen inflammation mediated by monocytes in OA.

Novel remodeling of the mouse heart mitochondrial proteome in response to acute insulin stimulation

BACKGROUND AND AIM Mitochondrial dysfunction contributes to the pathophysiology of diabetic cardiomyopathy. The aim of this study was to investigate the acute changes in the mitochondrial proteome in response to insulin stimulation. METHODS AND RESULTS Cardiac mitochondria from C57BL/6 mice after insulin stimulation were analyzed using two-dimensional fluorescence difference gel electrophoresis. MALDI-TOF MS/MS was utilized to identify differences. Two enzymes involved in metabolism and four structural proteins were identified. Succinyl-CoA ligase [ADP forming] subunit beta was identified as one of the differentially regulated proteins. Upon insulin stimulation, a relatively more acidic isoform of this protein was increased by 53% and its functional activity was decreased by ∼32%. CONCLUSIONS This proteomic remodeling in response to insulin stimulation may play an important role in the normal and diabetic heart.