Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.

Investigating the role of apoptosis regulator EndoG on exogenous DNA uptake, stability, replication and recombination

Endonuclease G (EndoG) is a well conserved mitochondrial nuclease with dual lethal and vital roles in the cell. It non-specifically cleaves endogenous DNA following apoptosis induction, but is also active in non-apoptotic cells for mitochondrial DNA (mtDNA) replication and may also be important for replication, repair and recombination of genomic DNA. The aim of our study was to examine whether EndoG exerts similar activities on exogenous DNA substrates such as plasmid DNA (pDNA) and viral DNA vectors, considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA and nuclease activity in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four day time-course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus (a cationic liposome transfection reagent), targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances it may non-specifically cleave intracellular DNA regardless of its origin. To investigate possible effects of EndoG on viral DNA vectors, we constructed and evaluated AdsiEndoG, a first generation adenovirus (Ad5 ΔE1) vector encoding a shRNA directed against EndoG mRNA, along with appropriate Ad5 ΔE1 controls. Infection of HeLa cells with AdsiEndoG at a multiplicity of infection (MOI) of 10 p.f.u./cell resulted in an early cell proliferation defect, absent from cells infected at equivalent MOI with control Ad5 ΔE1 vectors. Replication of Ad5 ΔE1 DNA was detected for all vectors, but AdsiEndoG DNA accumulated to levels that were 50 fold higher than initially, four days after infection, compared to 14 fold for the next highest control Ad5 ΔE1 vector. Deregulation of the cell cycle by EndoG depletion, which is characterized by an accumulation of cells in the G2/M transition, is the most likely reason for the observed cell proliferation defect. The enhanced replication of AdsiEndoG is consistent with this conclusion, as Ad5 ΔE1 DNA replication is intimately related to cell cycling and prolongation or delay in G2/M greatly enhances this process. Furthermore, infection of HeLa with AdsiEndoG at MOI of 50 p.f.u./cell resulted in an almost complete disappearance of viable, adherent tumour cells from culture, whereas almost a third of the cells were still adherent after infection with control Ad5 ΔE1 vectors, relative to the non-infected control. Therefore, targeting of EndoG by RNAi is a viable strategy for improving the oncolytic properties of first generation adenovirus vectors. In addition, AdsiEndoG-mediated knockdown of EndoG reduced homologous recombination between pDNA substrates in HeLa cells. The effect was modest but, nevertheless demonstrated that the proposed role of EndoG in homologous recombination of cellular DNA also extends to exogenous DNA substrates.

A comparative study of chitin synthase activity in two Mortierella species

An in vitro investigation of some important factors controlling the activity of chitin synthase in cell-free extracts of two Mortierella species has been carried out. Mixed membrane fractions from mycelial homogenates of Mortierella candelabrum and Mortierella pusilla were found to catalyse the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine into an insoluble product characterized as chitin by its insolubility in weak acid and alkali, and the release of glucosamine and diacetylchitobiose on hydrolysis with a strong acid and chitinase, respectively. Apparent Km values for UDP-GlcNAc were 1.8 mM and 2.0 mM for M. pusilla and ~ candelabrum, respectively. Polyoxin D was found to be a very potent competitive inhibitor with values of the constant of inhibition, Ki' for both species about three orders of magnitude lower than theKm for UDP-GlcNAc. A divalent cation, Mg+2 , Mn+2 or Co+2 , was required for activity. N-acetylglucosamine, the monomer of chitin, stimulated the activity of the enzyme. The crude enzyme preparation of ~ candelabrum, unlike that of ~ pusilla, showed an absolute requirement for both Mg+2 and N-acetylglucosamine. Large differences in response to exogenous proteases were noted in the ratio of active to inactive chitin synthase of the two species. A fifteen fold or greater increase was obtained after treatment with acid protease (from Aspergillussaitoi) as compared to a two- to four-fold activation of the M. pusilla membrane preparation treated similarly. During storage at 4°C over 48 hours, an endogenous activation of chitin synthase of ~ pus ilIa was achieved, comparable to that obtained by exogenous protease treatment. The high speed supernatant of both species inhibited the chitin synthase activity of the mixed membrane fractions. The inhibitor of ~ pus ilIa was effective against the pre-activated enzyme whereas that of M. candelabrum inhibited the activated enzyme. Several possibilities are discussed as to the role of the different factors regulating the enzyme activity. The suggestion is made from the properties of chitin synthase in the two species that in vivo a delicate balance exists between the activation and inactivation of the enzyme which is responsible for the pattern of wall growth of each fungus.

The establishment of two novel bovine cell lines by transfection with the putative transforming region of bovine adenovirus type 3

Human adenoviruses (Ads), members of the family adenoviridae, are medium-sized DNA viruses which have been used as valuable research tools for the study of RNA processing, oncogenic transformation, and for the development of viral vectors for use in gene delivery and immunization technology. The left 12% of the linear Ad genollle codes for products which are necessary for the efficient replication of the virus, as well as being responsible for the forlllation of tumors in animallllodels. The establishlllent of the 293 cell line, by immortalization of human embryonic kidney cells with th~ E1 region of Ad type S (AdS), has facilitated extensive manipulation of the Ads and the development of recombinant Ad vectors. The study of bovine adenoviruses (BAVs), which cause mild respiratory and gastrointestinal infections in cattle has, on the other hand, been limited primarily to that of infectivity, immunology and clinicallllanifestations. As a result, any potential as gene delivery vehicles has not yet been realized. Continued research into the molecular biolo~gy of BAVs and the development of recolllbinant vectors would benefit from the development of a cell line analogous to that of the 293 cells. In an attelllpt to establish such a cell line, the recombinant plaslllid pKC-neo was constructed, containing the left 0-19.7% of the BAV type 3 (BAV3) genome, and the selectable marker for resistance to the aminoglycoside G418, a neomycin derivative. The plasmid construct was then used to transfect both the Madin-Darby bovine kidney (MDBK) -iicell line and primary bovine lung cells, after which G418-resistant foci were selected for analysis. Two cell lines, E61 (MDBK) and E24 (primary lung), were subsequently selected and analysed for DNA content, revealing the presence of the pKC-neo sequences in their respective genomes. In addition, BAV3 RNA transcripts were detected in the E61 cells. Although the presence of E1 products has yet to be confirmed in both cell lines, the E24 cells exhibit a phenotype characteristic of partial transformation by E1. The apparent immortalization of the primary lung cells will permit exploitation of their ability to take up exogenous DNA at high efficiency.

CCK, dietary variety, and food intake in rats /

Among the environmental factors that can affect food intake is the extent of dietary variety available in the environment. Numerous studies have demonstrated that variety in a meal can increase the amount of food consumed in humans, rats, and other species. A physiological mechanism that has been demonstrated to affect food intake is the gut peptide cholecystokinin (CCK) which is released from the upper small intestine during the ingestion of food. Peripherally administered CCK has a robust inhibitory effect on the intake of a single-food meal. Thus, dietary variety and CCK both affect meal size, with dietary variety increasing intake and CCK decreasing intake. This raises the question ofhow dietary variety and CCK might interact to affect meal size. Previous studies of CCK's effects have focused on situations in which only one food was available for consumption. However, in an animal's natural environment it would frequently occur that the animal would come across a number of foods either simultaneously or in quick succession, thus providing the animal access to a variety of foods during a meal. Accordingly, the effect ofCCK on food intake in single-food and multiple-food meals was examined. It was found that food intake was greater in multiple-food than in single-food meals provided that foods in the multiplefood meal were presented either simultaneously or in increasing order of preference. When foods in the multiple-food meal were presented in decreasing order of preference, intake was similar to that observed in single-food meals. In addition, it was found that CCK inhibited food intake in a dose-dependent manner, and that its effects on food intake were similar regardless of meal type. Therefore, the inhibitory effects ofCCK were not diminished when a variety of foods were available for consumption. Furthermore, the finding that CCK did not differentially affect the intake of the two types of meals does not provide support for the recent-foods hypothesis which postulates that CCK decreases food intake by reducing the palatability of only recently consumed foods. However, it is consistent with the all-foods hypothesis, which predicts that CCK reduces food intake by decreasing the palatability of all foods. The 600 ng/kg dose of the CCK^-antagonist lorglumide significantly antagonized the inhibitory effect of exogenous CCK on food intake, and the magnitude of this effect was similar for both types of meal. These results suggest that exogenous CCK inhibits food intake through the activation ofCCK^ receptors. However, when administered by itself, the 600^ig/kg dose of lorglumide did not increase food intake in either single-food or multiple-food meals, suggesting that peripheral endogenous CCK may not play a major role in the control of food intake.

Mechanisms underlying Retinoic acid-induced chemoattraction in molluscan neurons

Retinoic acid, a derivative of vitamin A, is known to play diverse roles in development and regeneration. Previous research in the mollusc Lymnaea stagnalis has shown that a gradient of all-trans retinoic acid attracts the growth cones of cultured neurons. The present study investigates the sub-cellular mechanisms within the growth cones of Lymnaea pedal A neurons which mediate the attractive response to a gradient of alltrans retinoic acid. In this study, the mechanism of growth cone turning is shown to be local, as neurites mechanically isolated from their cell body retain the capacity to turn towards an exogenous gradient of all-trans retinoic acid. The turning response is dependent on the initiation of protein synthesis and calcium influx, but does not appear to involve signaling through protein kinase C (PKC). The retinoid X receptor (RXR), which classically functions as a transcription factor, was also shown to be involved in the turning response, functioning locally through a non-genomic pathway. These data show, for the first time in any species, that all-trans retinoic acid's chemotropic action involves a local mechanism involving non-genomic signaling through the RXR. As retinoic acid is known to playa role in regeneration, understanding the mechanisms underlying retinoic acid signaling may lead to further advances in regenerative neuroscience.

Impact of different irrigation strategies on grapes and wine quality of four grapevine cultivars (Vitis sp.) in cool climate conditions. An investigation into the relationships among ABA, water status, grape cultivar and wine quality

Niagara Peninsula of Ontario is the largest viticultural area in Canada. Although it is considered to be a cool and wet region, in the last decade many water stress events occurred during the growing seasons with negative effects on grape and wine quality. This study was initiated to understand and develop the best strategies for water management in vineyards and those that might contribute to grape maturity advancement. The irrigation trials investigated the impact of time of initiation (fruit set, lag phase and veraison), water replacement level based on theoretical loss through crop evapotranspiration (ETc; 100,50 and 25%) and different irrigation strategies [partial root zone drying (PRD) versus regulated deficit irrigation (RD!)] on grape composition and wine sensory profiles. The irrigation experiments were conducted in a commercial vineyard (Lambert Vineyards Inc.) located in Niagara-on-the-Lake, Ontario, from 2005 through 2009. The two experiments that tested the combination of different water regimes and irrigation time initiation were set up in a randomized block design as follows: Baco noir - three replicates x 10 treatments [(25%, 50% and 100% of ETc) x (initiation at fruit set, lag phase and veraison) + control]; Chardonnay - three replicates x seven treatments [(25%, 50% and 100% of ETc) x (initiation at fruit set and veraison) + control]. The experiments that tested different irrigation strategies were set up on two cultivars as follows: Sauvignon blanc - four replicates x four treatments [control, fully irrigated (100% ETc), PRD (100% ETc) and RDI (25% ETc)]; Cabemet Sauvignon - four replicates x five treatments [control, fully irrigated (100% ETc), PRD (100% ETc), RDI (50% ETc) and RDI (25% ETc)]. The controls in each experiment were nonirrigated. The irrigation treatments were compared for many variables related to soil water status, vine physiology, berry composition, wine sensory profile, and hormone composition [(abscisic acid (ABA) and its catabolites]. Soil moisture profile was mostly affected by irrigation treatments between 20 and 60 em depth depending on the grapevine cultivar and the regime of water applied. Overall soil moisture was consistently higher throughout the season in 100 and 50% ETc compare to the control. Transpiration rates and leaf temperature as well as shoot growth rate were the most sensitive variables to soil water status. Drip irrigation associated with RDI treatments (50% ETc and 25% ETc) had the most beneficial effects on vine physiology, fruit composition and wine varietal typicity, mainly by maintaining a balance between vegetative and reproductive parts of the vine. Neither the control nor the 100 ETc had overall a positive effect on grape composition and wine sensory typicity. The time of irrigation initiation affected the vine physiology and grape quality, the most positive effect was found in treatments initiated at lag phase and veraison. RDI treatments were overall more consistent in their positive effect on grape composition and wine varietal typicity comparing to PRD treatment. The greatest difference between non-irrigated and irrigated vines in most of the variables studied was found in 2007, the driest and hottest season of the experimental period. Soil water status had a greater and more consistent effect on red grapevine cultivars rather than on white winegrape cultivars. To understand the relationships among soil and plant water status, plant physiology and the hormonal profiles associated with it, abscisic acid (ABA) and its catabolites [phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy-ABA (TOH-ABA), 8' -hydroxy-ABA, neophaseic acid and abscisic acid glucose ester (ABA-GE)] were analyzed in leaves and berries from the Baco noir and Chardonnay irrigation trials over two growing seasons. ABA and some of its catabolites accurately described the water status in the vines. Endogenous ABA and some of its catabolites were strongly affected in Baco noir and Chardonnay by both the water regime (i.e. ET level) and timing of irrigation initiation. Chardonnay grapevines produced less ABA in both leaves and berries compared to Baco noir, which indicated that ABA synthesis is also cultivar dependant. ABA-GE was the main catabolite in treatments with high water deficits, while PA and DPA were higher in treatments with high water status, suggesting that the vine produced more ABA-GE under water deficits to maintain rapid control of the stomata. These differences between irrigation treatments with respect to ABA and catabolites were particularly noticeable in the dry 2007 season. Two trials using exogenous ABA investigated the effect of different concentrations of ABA and organs targeted for spraying, on grape maturation and berry composition of Cabemet Sauvignon grapevines, in two cool and wet seasons (2008-2009). The fIrst experiment consisted of three replicates x three treatments [(150 and 300 mg/L, both applications only on clusters) + untreated control] while the second experiment consisted in three replicates x four treatments [(full canopy, only clusters, and only leaves sprayed with 300 ppm ABA) + untreated control]. Exogenous ABA was effective in hastening veraison, and improving the composition of Cabemet Sauvignon. Ability of ABA to control the timing of grape berry maturation was dependant on both solution concentration and the target organ. ABA affected not only fruit composition but also yield components. Berries treated with ABA had lower weight and higher skin dry mass, which constitutes qualitative aspects desired in the wine grapes. Temporal advancement of ripening through hormonal control can lead to earlier fruit maturation, which is a distinct advantage in cooler areas or areas with a high risk of early frost occurrence. Exogenous ABA could provide considerable benefits to wine industry in terms of grape composition, wine style and schedule activities in the winery, particularly in wet and cool years. These trials provide the ftrst comprehensive data in eastern North America on the response of important hybrid and Vitis vinifera winegrape cultivars to irrigation management. Results from this study additionally might be a forward step in understanding the ABA metabolism, and its relationship with water status. Future research should be focused on ftnding the ABA threshold required to trigger the ripening process, and how this process could be controlled in cool climates.

Human Endogenous Retrovirus (HERV) Insertional Polymorphisms

Human endogenous retroviruses (HERVs) are the result of ancient germ cell infections of human germ cells by exogenous retroviruses. HERVs belong to the long terminal repeat (LTR) group of retrotransposons that comprise ~8% of the human genome. The majority of the HERVs documented have been truncated and/or incurred lethal mutations and no longer encode functional genes; however a very small number of HERVs seem to maintain functional in making new copies by retrotranspositon as suggested by the identification of a handful of polymorphic HERV insertions in human populations. The objectives of this study were to identify novel insertion of HERVs via analysis of personal genomic data and survey the polymorphism levels of new and known HERV insertions in the human genome. Specifically, this study involves the experimental validation of polymorphic HERV insertion candidates predicted by personal genome-based computation prediction and survey the polymorphism level within the human population based on a set of 30 diverse human DNA samples. Based on computational analysis of a limited number of personal genome sequences, PCR genotyping aided in the identification of 15 dimorphic, 2 trimorphic and 5 fixed full-length HERV-K insertions not previously investigated. These results suggest that the proliferation rate of HERVKs, perhaps also other ERVs, in the human genome may be much higher than we previously appreciated and the recently inserted HERVs exhibit a high level of instability. Throughout this study we have observed the frequent presence of additional forms of genotypes for these HERV insertions, and we propose for the first time the establishment of new genotype reporting nomenclature to reflect all possible combinations of the pre-integration site, solo-LTR and full-length HERV alleles.

METHOXYPYRAZINES AND LADYBUG TAINT IN WINES

Methoxypyrazines are aroma active compounds found in many wine varietals. These compounds can be of either grape-derived nature or can be introduced into wines via Coccinellidae beetles. Regardless of their origin, methoxypyrazines can have either a beneficial role for wine quality, contributing to the specificity of certain wine varietals (Cabernet sauvignon, Cabernet franc, Sauvignon blanc) or a detrimental role, particularly at higher concentrations, resulting in overpowering green, unripe and herbaceous notes. When methoxypyrazines of exogenous nature are responsible for these unpleasant characteristics, wines are considered to be affected by what is generally known as Ladybug taint (LBT). This is work is a collection of studies seeking to create a sensitive analytical method for the detection and quantification of methoxypyrazines in wines; to investigate the role of different Coccinellidae species in the tainting of wines with LBT and identify the main compounds in ladybug tainted wines responsible for the typical green herbaceous characteristics; to determine the human detection threshold of 2,5-dimethyl-3-methoxypyrazine in wines as well as investigate its contribution to the aroma of wines; and finally to survey methoxypyrazine concentrations in a large set of wines from around the world. In the first study, an analytical method for the detection and quantitation of methoxypyrazines in wines was created and validated. The method employs multidimensional Gas Chromatography coupled with Mass Spectrometry to detect four different methoxypyrazines (2,5-dimethyl-3-methoxypyrazine, isobutyl methoxypyrazine, secbutyl methoxypyrazine and isopropyl methoxypyrazines) in wine. The low limits of detection for the compounds of interest, improved separation and isolation capabilities, good validation data, as well as the ease of use recommend this method as a good alternative to the existing analytical methods for methoxypyrazine detection in wine. In the second study the capacity of two Coccinellidae species, found in many wine regions – Harmonia axyridis and Coccinella septempunctata - to taint wines is evaluated. Coccinella septempunctata is shown to be as capable as causing LBT in wines as Harmonia axyridis. Dimethyl methoxypyrazine, previously thought to be of exogenous nature only (from Coccinellidae haemolymph), is also detected in control (untainted) wines. The main odor active compounds in LBT wines are investigated through Aroma Extract Dilution Assay. These compounds are identified as isopropyl methoxypyrazine, sec- and iso- butyl methoxypyrazine. In the third study, the human detection threshold for dimethyl methoxypyrazine in wine is established to be 31 ng/L in the orthonasal modality and 70 ng/L retronasally. After wines spiked with various amounts of dimethyl methoxypyrazine are evaluated sensorally, dimethyl methoxypyrazine causes significant detrimental effects to wine aroma at a concentration of 120 ng/L. The final study examines methoxypyrazine (dimethyl methoxypyrazine, isopropyl methoxypyrazine, secbutyl methoxypyrazine and isobutyl methoxypyrazine) concentrations in 187 wines from around the world. Dimethyl methoxypyrazine is detected in the majority of the red wines tested. Data are interpreted through statistical analyses. A new measure for predicting greenness/herbaceousness in wines - methoxypyrazine “total impact factor” is proposed.

Mode of action of a Drosophila FMRFamide in inducing muscle contraction

Drosophila melanogaster is a model system for examining the mechanisms of action of neuropeptides. DPKQDFMRFamide was previously shown to induce contractions in Drosophila body wall muscle fibres in a Ca(2+)-dependent manner. The present study examined the possible involvement of a G-protein-coupled receptor and second messengers in mediating this myotropic effect after removal of the central nervous system. DPKQDFMRFamide-induced contractions were reduced by 70% and 90%, respectively, in larvae with reduced expression of the Drosophila Fmrf receptor (FR) either ubiquitously or specifically in muscle tissue, compared with the response in control larvae in which expression was not manipulated. No such effect occurred in larvae with reduced expression of this gene only in neurons. The myogenic effects of DPKQDFMRFamide do not appear to be mediated through either of the two Drosophila myosuppressin receptors (DmsR-1 and DmsR-2). DPKQDFMRFamide-induced contractions were not reduced in Ala1 transgenic flies lacking activity of calcium/calmodulin-dependent protein kinase (CamKII), and were not affected by the CaMKII inhibitor KN-93. Peptide-induced contractions in the mutants of the phospholipase C-β (PLCβ) gene (norpA larvae) and in IP3 receptor mutants were similar to contractions elicited in control larvae. The peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. Peptide-induced contractions were not potentiated by 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and were not antagonized by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. Additionally, exogenous application of arachidonic acid failed to induce myogenic contractions. Thus, DPKQDFMRFamide induces contractions via a G-protein coupled FMRFamide receptor in muscle cells but does not appear to act via cAMP, cGMP, IP3, PLC, CaMKII or arachidonic acid.

Action of octopamine and tyramine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10−5 and 10−4 M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10−5 M OA increased synaptically driven contractions by ∼1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

The action of octopamine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10(-5) and 10(-4) M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10(-5) M OA increased synaptically driven contractions by ≈ 1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.