Paralysis after a spinal cord injury (SCI) induces physiological adaptations that compromise the musculoskeletal and metabolic systems. Unlike non-SCI individuals, people with spinal cord injury experience minimal muscle activity which compromises optimal glucose utilization and metabolic control. Acute or chronic muscle activity, induced through electrical stimulation, may regulate key genes that enhance oxidative metabolism in paralyzed muscle. We investigated the short and long term effects of electrically induced exercise on mRNA expression of human paralyzed muscle. We developed an exercise dose that activated the muscle for only 0.6% of the day. The short term effects were assessed 3 hours after a single dose of exercise, while the long term effects were assessed after training 5 days per week for at least one year (adherence 81%). We found a single dose of exercise regulated 117 biological pathways as compared to 35 pathways after one year of training. A single dose of electrical stimulation increased the mRNA expression of transcriptional, translational, and enzyme regulators of metabolism important to shift muscle toward an oxidative phenotype . However, chronic training increased the mRNA expression of specific metabolic pathway genes , mitochondrial fission and fusion genes , and slow muscle fiber genes . These findings support that a dose of electrical stimulation (∼10 minutes/day) regulates metabolic gene signaling pathways in human paralyzed muscle. Regulating these pathways early after SCI may contribute to reducing diabetes in people with longstanding paralysis from SCI. Muscle paralysis after a spinal cord injury (SCI) triggers a cascade of events that disrupts the metabolic homeostasis of paralyzed muscle. Healthy skeletal muscle is involved with over 70% of daily glucose utilization Gene expression profiling is one method to survey the genome for mRNA transcripts common to a specific phenotype Key muscle transcription factors and co-activators are known to be responsive to metabolic and mechanical stress induced through muscle contractions in healthy people The purpose of this study is to determine the effects of a single dose and a chronic dose of electrical stimulation on human paralyzed muscle. We expect a single dose of muscle activity will result in an increased expression of metabolic transcription factors associated with the transformation of fibers from fast to slow. In contrast, we expect the chronically trained muscle will show the long term stable expression of genes associated with the maintenance of oxidative metabolic pathways, despite the minimal daily activity performed (10 minutes/day). Overall, these findings will support the hypothesis that a minimal dose of muscle activity, performed regularly, is an important and powerful metabolic regulator of skeletal muscle health, even in people with paralysis, who cannot exercise throughout the day.Five human subjects (30.40±4.39 years of age) with complete paraplegia were analyzed in this study. The protocol was approved by the University of Iowa Human Subjects Review Board, and all subjects provided written informed consent before participating. Three subjects received a single dose of electric muscle stimulation unilaterally 3 hours before a bilateral soleus muscle biopsy; whereas two subjects were muscle biopsied twice after training for >1 year . All subjects had complete paraplegia (ASIA-A) at or below T4 and had been paralyzed for over 1.5 years. None of the subjects used electrical muscle stimulation of the soleus prior to the start of this study. The training subjects sustained over 81% compliance with the training protocol. The training subjects discontinued training 5 days prior to the soleus muscle biopsy procedure to capture the homeostatic effect of long term training and minimize the influence an acute dose of activity has on gene regulation. All biopsies were normalized to the same person's limb that did not receive training. The control limb has previously been shown to remain physiologically identical when the opposite limb is being trained The acutely stimulated subjects had a single session of unilateral muscle stimulation of the soleus muscle. None of these individuals had received prior electrical stimulation. Subjects sat in their wheelchair while the experimenter positioned one leg into the testing apparatus in order to elicit an isometric contraction of the soleus muscle Electrical stimulation was delivered to the plantar flexor muscles using reusable adhesive carbon electrodes. Stimulation was provided by a constant current electrical stimulator with a 0 to 400 milliamp range at 400 volts . The stimulator was controlled by digital pulses from a data-acquisition board  housed in a microcomputer under custom software control. Single pulses were given at increasing intensity until maximal twitch torque was observed via an oscilloscope. Stimulation intensity was increased an additional 50% and remained at this level for the remainder of the experiment to insure supra maximal activation. Subjects received 5 warm-up contractions  to potentiate the plantar flexor muscles and minimize the risk of muscle strain. After the warm-up contractions, the plantar flexor muscles were activated with 10 Hz stimulation trains (7 pulses) every 2100 ms for 120 isometric contractions. After a 5 minute rest, subjects received a second bout of 120 contractions at the same intensity and frequency. After the second bout, an experimenter used an indelible ink pen to mark the trained leg with the time that stimulation ended in preparation for the 3 hr post-activation bilateral soleus biopsy. The biopsy sampled fibers that had received the acute dose of electrical stimulation.The right soleus muscle was trained for at least one year then discontinued training 5 days prior to obtaining bilateral muscle biopsies. The training protocol consisted of 4 bouts of 125 contractions  per day with a 5 minute rest period between each bout 3. An MR was only obtained from subject 2, as subject 1 had metal fragments retained within his body from his initial injury ©  script, which utilized image registration and segmentation algorithms implemented in the MATITK toolkit 3, providing an estimate to the total area for each tissue type within the limb. The trained and untrained limbs were separated into two regional sites  each consisting of 30 image slices. The proximal calf maximized the inclusion of the soleus muscle, while the distal thigh was used as a control to illustrate the specificity of training to the soleus muscle. The total muscle, fat, and background volumes were compared between the trained and untrained side. The total muscle and fat tissue content within each region was totaled and a ratio of muscle to fat volume was calculated.Due to the known association between metabolic risk and the fat to muscle ratio, we decided to illustrate the differences in phenotype between the trained and untrained limbs by using an axial-plane spin-echo, T1-weighted magnetic resonance (MR) image from the trained muscle and the untrained muscles of the calf and thigh (1.5 T Siemens Avanto Scanner). A three-dimensional gradient-echo-based sequence was used for high-resolution imaging. The acquisition parameters included repetition time (TR) of 15.0 ms and echo-time (TE) of 6.7 ms with a 512×256 matrix covering a field of view of 46 cm×18 cm and a 2.5-mm slice distance, creating a voxel volume of about 1.58 mmThe biopsy procedure has been previously described The Affymetrix Human Exon 1.0 ST arrays were normalized using a Robust Multi-array Average (RMA) and transformed into a log2 hybridization signal, reflecting the mean signal intensity of all exon probes specific for a particular mRNA transcript using Partek Genomic Suites . The mRNA log2 hybridization signals were further analyzed using two techniques: a pathway analysis system and expression level profiling.The pathway analysis was performed using the gene set enrichment analysis (GSEA) algorithm implemented in Partek Genomic Suites The exon arrays were analyzed using an expression profiling technique, which compared the mRNA hybridization signals between the acute and chronic groups relative to each participant's control leg. MRNA with a log2 hybridization signal less than 2 standard deviations away from the mean hybridization signal were discarded from the dataset, restricting the analysis to mRNA transcripts with high signal intensity relative to background intensities. A fold-change (FC) was calculated for each gene transcript in the acute and chronic groups relative to the control limb. A gene transcript was considered to be differentially expressed, if 1) the FC was greater than 2.0 or less than 0.5 (indicating a 2.0 fold decrease in expression) and 2) the p-value for the gene was below 0.05 for an independent paired t-test comparing the stimulated and control. Due to the small sample size within each group, two muscles samples were independently extracted and hybridized for each subject, providing a technical replicate to minimize systematic errors.Τ method described by Schmittgen and Livak Muscle samples were homogenized in lysis buffer using a tissue homogenizer and hard tissue grinding tip (Omni). A column-based RNA extraction was subsequently performed using the RNEasy Fibrous Tissue Kit (Qiagen). DNAse was included in the protocol to ensure absence of genomic DNA in final samples. RNA samples were eluted in water and quantified via nanodrop method. In addition, the quality of each sample was assayed using the Agilent 2100 Bioanalyzer. High quality RNA samples were reverse transcribed using iScript supermix (Bio-Rad). Input quantity of RNA was standardized across all reactions (500 ng each). All cDNA samples were stored at −86°C. At the time of qPCR plate preparation, cDNA samples were diluted five-fold in water and analyzed via SYBR green technology using a custom PrimePCR plate (Bio-Rad). All samples were analyzed in duplicate at a final concentration of .5 ng/µL cDNA per 10 µL reaction using an ABI 7900 machine. Expression levels were converted to a relative fold change (FC) of the acutely stimulated or chronically trained limb to the control limb using the comparative CThe chronically paralyzed muscle phenotype is highly fatigable, slows its contractile speed during repetitive activation, and potentiates early during electrical stimulation, findings that are consistent with fast fatigable glycolytic muscle . Long teWe measured over 16,000 mRNA transcripts from each muscle sample obtained. Using a conservative false discovery rate (FDR) of 10% and the gene ontology (GO) biological process database, we identified 117 and 35 pathways that were significantly up regulated after an acute dose and chronic dose of electrical stimulation, respectively . Both thsingle session of electrical muscle stimulation; however, after chronic stimulation training there were 66 genes with a 2 fold increase in expression, and 20 genes had a 2 fold decrease in expression. The top 10 genes with the highest relative fold change . PGC-1α, NR4A3, IFRD1, FOS, and ABRA trigger cellular processes needed to shift the muscle toward an oxidative phenotype, while CYR61 aids in initiating angiogenesis in muscle. PGC-1α, NR4A3, EGR1, IFRD1, FOS, and ABRA were over 2 fold higher following an acute bout of electrical stimulation in paralyzed muscle. In contrast, these genes were minimally altered in chronically trained paralyzed muscle. In the chronic group, 5 out of the 10 high intensity transcripts function as metabolic enzymes, protein transporters, and oxidative muscle proteins . MYH6, MYL3, and MYH7 were at least 3 fold higher in chronically trained muscle relative to acutely stimulated paralyzed soleus muscle. Importantly, chronic training decreased genes associated with fast twitch muscle  and atrophy (MSTN) . An acutAfter a preliminary analysis, we looked at the influence of acute and chronic soleus stimulation on a subset of genes linked to skeletal muscle phenotypes or metabolic capacity, specifically targeting genes linked to skeletal muscle transcription factors, fast-twitch muscle proteins, slow-twitch muscle proteins, glycolysis enzymes, fatty acid oxidation enzymes, tricarboxylic acid cycle enzymes, oxidative phosphorylation enzymes, and mitochondrial fission and fusion proteins. PGC-1α, NR4A3, and ABRA are early response genes that were up regulated relative to the non-stimulated limb 3 hours after a single bout of electrical muscle stimulation.  However,Glycolysis and fatty acid oxidation are the 2 primary pathways used to metabolize macromolecules in skeletal muscle. PDK4 was increased relative to the control limb 3 hours after a single session of electrical muscle stimulation training , but was not increased with chronic muscle training  . PDHA11. ACADL wThe common substrate, acetyl CoA, following aerobic glycolysis or fatty acid oxidation is further oxidized within mitochondria via the tricarboxylic acid cycle and oxidative phosphorylation pathways. Genes responsible for monocarboxylic acid transport into the mitochondria  and BRP44L ) were increased after chronic electrical muscle stimulation training and unchanged 3 hours after a single session of electrical muscle stimulation . A subseWe verified the expression levels of five genes  using qPCR. Similar to the exon microarray results, ABRA, EGR1, and NR4A3 expression was increased following an acute dose of electrical stimulation but was decreased or unchanged in the chronically trained muscle . ChronicWe examined the mRNA expression response after a single session of exercise with electrical stimulation in paralyzed soleus muscle and compared that to the mRNA expression profile of the chronically trained paralyzed soleus muscle. The objective of this study was to advance our understanding of the influence of acute or chronic exercise on regulating gene expression important for metabolic health of skeletal muscle in humans with paralysis. The major findings of this study are 1) acute stimulation of paralyzed muscle regulates over 100 biological pathways as compared to less than 30 in chronically trained muscle; 2) acute electrically induced exercise up regulates transcriptional, translational, and enzyme regulators of metabolic pathways that shift muscle toward an oxidative phenotype ; 3) long term electrically induced exercise increased the chronic expression of glycolysis , fatty acid oxidation , tricarboxylic acid , and oxidative genes ; 4) long term electrically induced exercise increased oxidative muscle fiber  and mitochondrial fission/fusion  genes, but repressed glycolytic muscle  and muscle atrophy (MSTN) genes. Taken together, these findings support that the minimal dose (0.6% of muscle activity per day) of electrically induced exercise used in this study was sufficient to initiate and maintain several metabolic pathways in human paralyzed muscle. Whether preserving the metabolic capacity of skeletal muscle in humans after SCI improves systemic metabolic health remains unknown.In 1995, we reported that the acutely paralyzed soleus muscle in humans is fatigue resistant and oxidative, but transforms into a highly fatigable, glycolytic muscle after 1 year of paralysis This study demonstrated that a single dose of electrically induced exercise stressed muscle and triggered the transcription of genes important for controlling metabolic pathways. Further, chronic training of paralyzed muscle resulted in a consistent increase of genes and proteins important for an oxidative, slow-twitch phenotype. It is noteworthy that skeletal muscle adaptation requires a repetitive and long term (over a 1 year time) delivery of a stressor in order to up regulate the full complement of oxidative enzymes necessary to improve muscle endurance Two factors likely contributed to the magnitude of gene signaling based on the design of this protocol. First, the frequency of stimulation was set to create an unfused tetany, which generated ∼50% of the maximal muscle force generating capacity, under isometric conditions. We chose this level of stimulation because we believed 50% of maximal force mechanically strains tissues that modulate muscle signaling and we wished to limit the total force of the muscle to a level that would not induce injury to the underlying skeletal and soft-tissues. Second, we supra maximally activated the muscle, which assured all fibers of the soleus muscle were activated. This level of muscle activity is unique and specific to electrical stimulation induced exercise. For example, this dose of exercise cannot be duplicated in humans under volitional control, as motor unit recruitment, via the size principle The unique method of stimulating paralyzed muscle induced the peroxisome-proliferator-activated-receptor-gamma-coactivator-alpha (PGC-1α) gene. Increased PGC-1α expression results in muscle hypertrophy, oxidative, and slow-twitch protein expression, while decreased levels lead to the conversion to glycolytic proteins and a transformation to a faster-twitch, less oxidative muscle fiber The nuclear orphan receptor-1  assists in regulating muscle metabolic phenotypes, with increased expression resulting in a more oxidative muscle fiber Actin-binding Rho-activating protein (ABRA) has also been shown to be instrumental in triggering the transformation of muscle to a more oxidative muscle fiber phenotype through the activation of the PGC-1α pathway We limited our analysis to genes with a large differential expression (>2 FC) compared to the untrained limb. However, limiting to a>2 FC eliminates transcripts that are differentially expressed but to a lesser magnitude. We explored genes that did not meet the 2 FC thresholds, but have important roles in regulating oxidative metabolism. BRP44 and BRP44L are two genes that control the transport of pyruvate from the cytosol to the mitochondria for aerobic oxidation The mitochondrion is a critical organelle for skeletal muscle metabolism, generating over 90% of a cell's energy The findings support that exercise, through electrical stimulation, is a powerful method to stress skeletal muscle. Exercised skeletal muscle has a high oxidative capacity, and can more rigorously respond to insulin and metabolize glucose Future studies are underway to deliver interventions shortly after the injury that efficiently regulates skeletal muscle with the goal to optimize the health of people with paralysis. The optimal electrical stimulation dose  to most efficiently/effectively elicit long term cellular adaptations of paralyzed muscle are not known. By targeting specific gene signaling pathways regulated by exercise, future studies may develop “scientifically grounded” electrical stimulation interventions before initiating expensive and long-term training programs to restore the metabolic capacity of paralyzed skeletal muscle. Future studies examining various doses of electrical stimulation may selectively regulate quite different gene signaling pathways and be prescribed based on the needs of the individual with SCI .The results of this study provide a “snapshot” into the effects that a defined dose of muscle activity has on gene regulation in human paralyzed muscle. We used an intervention to sufficiently “stress” the paralyzed muscle while also not injuring the underlying muscle or skeletal structures. In addition, we used a biopsy technique that minimized the amount of tissue harvested in order to reduce risk of secondary complications associated with venous stasis in humans with paralysis. We required nearly all of the harvested tissues to sufficiently extract enough RNA to examine cell signaling pathways, which was the primary objective of this study. However, this precluded us from carrying out comprehensive proteomic studies and microscopy studies. Indeed, we do not know how long gene expression was altered after 3 hours or the dose needed to induce an actual change in protein. We do know that chronic training of ∼10 minutes of activity/day caused a significant change in the functioning phenotype  and a stable up regulation of several metabolic signaling pathways. While the number of participants in this study was not large, each subject had a control limb serving as a within subject comparison, constituting the identical genotype to contrast the effects of the electrical stimulation intervention. In addition, the long term trained subjects, while limited in number, represent a novel example of the capacity for muscle tissue to adapt when exposed to a regular (>1 year) but a modest dose of electrically induced exercise (0.6%/day).A modest dose of acute or chronic electrical stimulation of paralyzed muscle regulated distinct biological pathways. Taken together, these findings support that acute and chronic electrical stimulation intervention (∼10 minutes/day) regulates key metabolic gene signaling pathways. Early regulation of these pathways after SCI may assist in preventing diabetes in people with SCI.


Iridovirid infection is associated with the catastrophic loss in aquaculture industry and the population decline of wild amphibians and reptiles, but none of the iridovirid life cycles have been well explored. Here, we report the detailed visualization of the life cycle of Singapore grouper iridovirus (SGIV) in grouper cells by cryo-electron microscopy (cryoEM) and tomography (ET). EM imaging revealed that SGIV viral particles have an outer capsid layer, and the interaction of this layer with cellular plasma membrane initiates viral entry. Subsequent viral replication leads to formation of a viral assembly site (VAS), where membranous structures emerge as precursors to recruit capsid proteins to form an intermediate, double-shell, crescent-shaped structure, which curves to form icosahedral capsids. Knockdown of the major capsid protein eliminates the formation of viral capsids. As capsid formation progresses, electron-dense materials known to be involved in DNA encapsidation accumulate within the capsid until it is fully occupied. Besides the well-known budding mechanism through the cell periphery, we demonstrate a novel budding process in which viral particles bud into a tubular-like structure within vacuoles. This budding process may denote a new strategy used by SGIV to disseminate viral particles into neighbor cells while evading host immune response. Iridovirids are large dsDNA icosahedral viruses composed of more than 45 isolates that infect a diverse array of hosts13457Chilo iridescent virus (CIV) in genus Iridovirus9ChloriridovirusLymphocystivirus1112MegalocytivirusRanavirus151617Iridovirid replication has been extensively studied in members from all 5 genera of the family, including invertebrate iridescent virus 6 (IIV6) and In brief, iridovirids enter a cell via endocytosis and the viral DNA is transported to the nucleus, where the first stage of viral DNA replication takes place. This is followed by the second stage of DNA replication that occurs in the cytoplasm. The infection subsequently leads to the formation of a morphologically distinct area within the cytoplasm, the viral assembly site (VAS), where the viral proteins and DNA are concentrated for virion assembly. Mature virions accumulate in large paracrystalline arrays or bud from the plasma membrane12Recently, electron microscopy technology has enabled scientists to make major advances in determining the molecular structures of various macromolecular complexes, including virus particles. The combination of cryofixation and high resolution electron microscopy/tomography (CryoEM/ET) enables analysis of the complex 3D architecture of viral particles and the viral assembly site. Cryofixation involves ultra-rapid cooling of small samples to the temperature of liquid nitrogen, which provides markedly improved structural and biochemical preservation of cells compared to traditional chemical fixation protocols222320Ranavirus of Iridoviridae family. It was first isolated in Singapore and has caused catastrophic economic loss in grouper fish farms2829Singapore grouper iridovirus (SGIV) is a member of the genus This study examined the SGIV life cycle in grouper cells by transmission electron microscopy including cryoEM/ET. We show the detailed process of viral morphogenesis starting from SGIV entry into the cell, viral assembly, and finally egress of viral particles. Knockdown of the expression of SGIV major capsid protein (MCP) significantly impedes the formation of viral intermediates and infectious particles. A novel budding process involving the formation of budding vacuoles is described, which hints at a new immune evasion mechanism used by SGIV.In order to study the mechanism of iridovirid infection in their natural host, we used high pressure freezing (HPF) and freeze substitution (FS) to capture the main events of SGIV infection in grouper embryonic cells (GECs). Electron microscopy imaging of the resin-embedded cryofixed specimen revealed various important steps in the SGIV life cycle including entry , assemblAt 4–12 hpi, the VAS was completely formed. Various viral intermediates and mature virions could be observed inside the VAS , 12 hpi.At 8 hpi, the VAS expanded in size as mature viral particles accumulate, and this pushed the nucleus to the side of the cell. Interestingly, a novel egression strategy was observed during this stage, in which membrane tubules containing mature virions emerged inside a large cytoplasmic vacuole, forming intracellular tubular networks , 8 hpi. At 24–48 hpi, the VAS has greatly expanded in size and now occupies the majority of the cytoplasm. This led to further compression of the nucleus at the cell membrane, and it now starts to adopt a deformed morphology. Vast amounts of mature viral particles densely accumulated inside the cell, forming paracrystalline arrays at the boundary of the VAS , 48 hpi.To capture the early stages of the capsid assembly, SGIV-infected GECs were maintained at 4 °C to slow the viral replication process. GECs at 1 or 2 hpi were preserved by HPF-FS and subsequently observed via TEM and tomography . VariousDuring the early stage of capsid formation, crescent shaped intermediates were detected. These intermediates had a double-shell morphology, with each end curving towards the concave side to form the crescent shape . These eoC or 27 oC.Following our EM/ET imaging of these early intermediates, we carried out quantitative analysis on two different batches of infected GECs to determine the distribution of the viral intermediates. At 1 hpi, the viral intermediates observed were all early stage intermediates, predominantly circle or crescent shaped structures, with some membranous sheets and headphone shaped structures also present. However, by 2 hpi, the percentage of partial hexagonal shaped structures had increased to about 40–50% of the total population , supportAlthough the capsid opening was larger and easily noticeable in some partially formed viral particles, in many 2D images visualization of the capsid opening was highly ambiguous and orientation-dependent, due to the asymmetrical nature of the viral intermediates. Therefore, we used 3D electron tomographic reconstruction to further verify the existence of the capsid opening in the partially formed virus . Figure To further investigate whether the electron dense materials were associated with the viral genome, we immuno-labelled viral DNA and a core-associated protein ORF075R with antibody-conjugated gold markers of 15 nm and 25 nm respectively using the Tokuyasu method. The gold particles of 15 nm diameter were abundantly detected in partially-filled capsids but were less abundant in fully filled capsids . This fe072 were infected with SGIV and harvested at 48 hpi. The efficiency of asMO072 knockdown on reduction of MCP was assessed by MALDI-TOF/TOF MS/MS. The yield of infectious particles was examined by TCID50, and the VAS was visualized using TEM.To analyze the role of SGIV MCP (ORF072) on the viral capsid assembly, GECs that had been transfected with asMO072 knockdown, the expression level of MCP was reduced compared to controls  were detected . Followicontrols . The knoed cells . The majed cells  or no paed cells . In all  virions .Tilt series of high pressure freezing, freeze substitution (HPF-FS) specimen were taken to investigate a novel viral egress process in the infected GECs. It is characterized by the formation of a large intracellular vacuole, appears in the regions where the mature virus accumulates, usually very close to the VAS. Notably, some mature viral particles were found to attach and bud into those vacuoles. LymphocystivirusDuring its life cycle, iridovirid produces enveloped and unenveloped viral particles, both of which are infectious. Using the high pressure freezing (HPF) method, which preserves the virus structure much better than conventional chemical fixationViral assembly often takes place in specific intracellular compartments where viral components concentrate, which increases the efficiency of the processAt the early stages of SGIV assembly, we observed a variety of membranous structures which were either linear (membranous sheets) or circular in shape  , and therana grylio virus (RGV) envelope protein 2L (a homolog of SGIV ORF019)36SGIV lipid-binding proteins, such as ORF026, 075, 089, 090, and 101, may interact with the ER-derived membranes and serve as precursors to recruit capsid proteins to form viral intermediates, crescent-shaped structures with two shells, the inner lipid envelope (black arrow head) and the outer capsid (white arrow head) . EnvelopThe progressive curvature change from crescent shape to hexagonal capsid occurs concurrently with the buildup of electron dense materials underneath the inner lipid envelope . We assuThe electron dense materials underneath the inner envelope shell may be involved in viral DNA encapsidation. As the immature particle becomes mature, there is a corresponding increase in density . Using g43Most of the viral intermediates were observed in the center of the VAS at 1 or 2 hpi, yet mature virions moved to the VAS periphery, where they either bud out (frequently observed around 6–8 hpi when infected with a MOI of 5–10) or form paracrystalline arrays (often observed after 48 hpi when infected with a MOI of 3 or lower). The budding from the cell periphery has been reported previouslyIn the first step of this new budding process, vacuoles were formed around the VAS. It is unknown whether those vacuoles were newly generated or transformed from organelles such as mitochondria. We observed that mitochondria surrounding the VAS were enlarged and lost their internal structures . This ha485051In summary, our study presents the first clear visualization of the outermost layer of unenveloped virions, and we show that this layer is the frontier for viral interaction with the cell membrane for viral entry. Using cryoEM/ET imaging, structural information of viral morphogenesis from early precursors to various intermediates and finally mature particles is also shown for the first time, and formation of mature viral particles requires the major capsid protein ORF072. Taken together, we unravel a detailed life cycle and a novel budding process used by SGIV to disseminate viral particles. The schematic in Epinephelus tauvina3. Freshly confluent monolayers of GECs were infected with SGIV at a multiplicity of infection (MOI) of approximately 1 at RT. When approximately 70% of cells were detached, the medium containing SGIV was harvested and the titers were determined by a TCID50 testGrouper embryonic cells (GECs) from the brown-spotted grouper GECs were infected with SGIV at different MOI values and temperatures. Infection with high MOIs (more than 10) at 4 °C was carried out to examine events at early stages of the viral life cycle , while infection at low MOIs at 27 °C allowed the capture of events at late stages of the viral life cycle . The infected cells were harvested by centrifugation at different time points. Infected GECs were then subjected to high pressure freezing , freeze substitution (FS), ultramicrotomy, and staining, before observation under a FEI Tecnai T12 electron microscope.2O in acetone for freeze substitution (FS), which was carried out using a Leica FS machine, followed by epoxy infiltration as per the previously described protocolCryofixation of SGIV-infected cells was performed using an HPF Compact 01 system. In brief, about 3 μl of sample was inserted in a hexadicine coated aluminum platelet, quickly frozen by the HPF machine, and then transferred to vials containing 1% osmium tetroxide (OsO4), 0.5% uranyl acetate, and 5% HThe specimens were visualized on the FEI Tecnai T12 electron microscope operated at 120kv. The tilt series were recorded using the FEI tomography software from –60° to +60° with 2° intervals at a defocus of 6 μm on a 4k x 4k CCD camera (Gatan), with magnification was set to 5,000–25,000x depending on the size of the viral assembly intermediates. The tilt series were aligned using IMOD software packageGEC cells infected with SGIV were maintained at 4 °C and 27 °C after infection and collected at 1 and 2 hpi. Based on the classification of capsid intermediates , the num2. Images were recorded using a Gatan Ultrascan 4k × 4k CCD detector with magnification of 63,290×.Cryofixation of SGIV particles was done by a plunge freezer (Vitrobot Mark IV). Approximately 3μl of purified virus particles was applied onto a glow discharged quantifoil grid with a hole size of 2 μm. The grid was plunge-frozen in liquid ethane, blotted for 1 second at 100% humidity. The specimen was visualized using a Titan Krios electron microscope with the defocus ranging from −1.5 to −3μm, at a dose of 16 to 20e/Å072) and control (asMOcontrol) are 5′-CGCCAGCACCCGTTGTACAAGTCAT-3′ and 5′-CCTCTTACCTCAGTTACAATTTATA-3′, respectively. These sequences were designed to bind the target mRNA around the start codon region and block translation. The asMO was transfected into GECs by electroporation via a nucleofector machine as previously described072 and asMOcontrol, incubated for 40 h, followed by inoculation with SGIV at an MOI of 3, and incubated for another 48 h before harvesting for subsequent analysis.The asMO sequences of SGIV major capsid protein ORF072 . The peak areas of peptides corresponding to MCP were calculated using Data Explorer TM Software (Applied Biosystems), with a large peak area representing a high amount of the protein. The relative amount of MCP was shown as the percentage of the peak area in the knockdown samples divided by the corresponding peak area in the control samples. The lower the percentage, the more efficient the knockdown.Reduction of MCP (ORF072): The efficiency of knockdown in the reduction of MCP expression was assessed by label-free quantitative proteomics5950g for 1 min, the supernatant was collected and underwent serial dilution for final solutions of 102 to 1010 for TCID50 test in GECs.Formation of viral particles: Following transfection, harvested cells were processed for cryoEM to visualize the effect of MCP knockdown on virus assembly. The knockdown effects on the yield of infectious particles were quantitatively assessed using TCIDGECs infected with SGIV at 27 °C for 24 h were processed using Tokuyasu method61For immunolabeling, ultrathin sections were first incubated in 0.12% Glycerin for 15 min, followed by blocking with 1% BSA for 30 mins and then washing three times with PBS. The primary antibody of anti-SGIV core-associated protein ORF075 was generated as a gift from the National Institute of Diagnostics and Vaccine Development for Infectious Diseases in Xiamen University, Xiamen, China, following the approved institutional protocol. Mouse anti-DNA antibody was purchased from Millipore (MAB1293). The two antibodies were added at a dilution of 1:50 and incubated for 1 h, washed three times with PBS, followed by conjugation with goat anti-rabbit gold particles  and the goat anti-mouse gold particles  at a dilution of 1:40 for 1h. Grids were fixed with 1% glutaraldehyde for 5 min, washed four times with water, and incubated with a mixture of 1 part of 1% uranyl acetate: 9 parts of 2% methylcellulose for 5 min. Grids were dragged on a filter paper to remove the excess liquid and air dried before viewing using a FEI Tecnai T12 microscope.How to cite this article: Liu, Y. et al. Visualization of Assembly Intermediates and Budding Vacuoles of Singapore Grouper Iridovirus in Grouper Embryonic Cells. Sci. Rep.6, 18696; doi: 10.1038/srep18696 (2016).


We generated isogenic human cancer cell lines of different origin with wild-type p53 or p53 knockdown and we demonstrated that proteasome inhibitors induce p53-independent apoptosis in these cells. Using RNA-interference or proteasome inhibitors to inhibit FOXM1 we found that suppression of FOXM1 sensitized human cancer cells to apoptosis induced by DNA-damaging agents or oxidative stress. We encapsulated thiostrepton into micelle-nanoparticles and after injection we detected accumulation of nanoparticles in tumors and in the livers of treated mice. This treatment led to inhibition of human xenograft tumor growth in nude mice. Our data indicate that targeting FOXM1 increases apoptosis and inhibits tumor growth.The oncogenic transcription factor FOXM1 is one of the key regulators of tumorigenesis. We found that FOXM1 upregulates its own transcription and its protein stability depends on its interaction with the chaperone nucleophosmin. We also determined that FOXM1 is negatively regulated by the tumor suppressor p53. We identified the thiazole antibiotics Siomycin A and thiostrepton as inhibitors of transcriptional activity and FOXM1 expression via proteasome inhibition. In addition, we found that all tested proteasome inhibitors target FOXM1. We showed synergy between thiostrepton and bortezomib in different human cancer cell lines and  Recentlin vitro . Particuin vitro . In thisWhen we compared levels of FOXM1 in normal human fibroblasts, fibroblasts with p53 knockdown, and corresponding immortal/oncogenic cell lines with inactivated p53, we found that partial deletion or inactivation of p53 leads to upregulation of FOXM1 expression . SimilarWe found that FOXM1 binds and activates its own proximal promoter (unpublished data) and is involved in a positive autoregulatory loop  that is Using mass spectrometric analysis and immunoprecipitation/Western blot experiments, we showed that the chaperone nucleophosmin (NPM) forms a complex with FOXM1 . SeveralWe suggested that FOXM1 might be “the Achilles' heel of cancer”  and an aWe demonstrated that the anticancer activities of thiostrepton and Siomycin A are linked to their activity as proteasome inhibitors in human tumor cells . We testWe also found that all tested proteasome inhibitors such as bortezomib, MG132, and others inhibit FOXM1 transcriptional activity and FOXM1 expression, suggesting that negative regulation of FOXM1 by proteasome inhibitors is a general feature of these drugs that contributes to their anticancer properties . Since aNAC (N-acetyl-L-cysteine) is used to inhibit ROS activity and to test the activity of ROS (reactive oxygen species) inducers. However, we identified NAC as a novel inhibitor of proteasome inhibitors that was able to prevent cellular effects linked to proteasome inhibition, such as suppression of FOXM1, stabilization of multiple proteins, apoptosis, and accumulation of ubiquitin conjugates . Most imPreviously, we showed that proteasome inhibitors suppress FOXM1 and induce apoptosis in human cancer cell lines of different origin. We decided to examine the role of p53 in proteasome inhibitor-induced apoptosis using isogenic human colon, breast, and liver cancer cell lines that differ only in their p53 status, and we found that the proteasome inhibitors induce p53-independent apoptosis . ApoptosCombination of anticancer drugs is a viable strategy for the treatment of cancer. Using combinations of drugs at nontoxic concentrations may reduce the probability of cancer cells becoming drug-resistant. We tested the combination of the thiazole antibiotic/proteasome inhibitor thiostrepton and the proteasome inhibitor bortezomib (Velcade) in various human tumor cell lines , 26. We γ-irradiation and bortezomib/thiostrepton and they underwent stronger cell death compared to treatments with single agents. We tested the effect of combinatorial treatment on long-term survival by performing clonogenic assay. Human cancer cells were treated with γ-irradiation along with thiostrepton/bortezomib and the colony forming ability was assessed 10 days later. Combinatorial treatment drastically inhibited colony formation compared to control cells [The role of the transcription factor FOXM1 in apoptosis is not understood well. We decided to examine functions of FOXM1 in apoptosis by developing human cancer cell lines with stable FOXM1 knockdown and analyzing their responses to different anticancer agents. First, we treated MIA PaCa-2 (pancreatic cancer) and MDA-MB-231 (breast cancer) vector controls and FOXM1 knockdown (KD) human cell lines with increasing concentrations of the DNA-damaging agent, doxorubicin. We showed that the FOXM1-KD cell lines were more sensitive to programmed cell death induced by doxorubicin . Next, wol cells . Using aol cells . These dProteasome inhibitors suppress FOXM1, but paradoxically knockdown of FOXM1 additionally sensitizes human cancer cells to apoptosis induced by proteasome inhibitors . Control in vitro and in vivo. We found that the combinations of ROS inducers with FOXM1/proteasome inhibitors stimulated robust apoptosis in human cancer cell lines. The proteasome inhibitor bortezomib in combination with the ROS inducer β-phenylethyl isothiocyanate suppressed the growth of human breast tumor xenografts in nude mice [It has been shown that FOXM1, which is overexpressed in a majority of human tumors, protects cancer cells from oxidative stress. We proposed that the combination of FOXM1 suppression and induction of ROS by specific drugs would specifically kill human cancer cells. We demonstrated that FOXM1 suppression by RNAi elevates intracellular ROS levels and overexpression of the scavenger MnSOD that is target of FOXM1 partially reversed this effect . These dude mice . Since bude mice . in vivo suppression of oncogenic FOXM1 by the proteasome inhibitor thiostrepton correlated with increased apoptosis in drug-treated tumors [ in vivo and their accumulation in the liver suggest that this nanoparticle-thiostrepton formulation may be potentially useful for treatment of liver cancer [We demonstrated earlier that Siomycin A and thiostrepton suppressed tumor growth in a human breast cancer xenograft model . Howeverd tumors . The strr cancer . in vivo [We showed proapoptotic synergy between bortezomib and thiostrepton in human cancer cell lines . It has  in vivo . To dete in vivo . Xenogra in vivo . Using d in vivo  that thi in vivo . Inducti in vivo . Animals in vivo . The bod in vivo . in vivo through the administration of FOXM1-specific siRNA [ in vivo delivery agent for FOXM1-siRNA by direct intratumoral injection at a dose of 10 μg siRNA/tumor, 3 times per week [We decided to examine the possibility of suppressing FOXM1 in xenograft tumorsic siRNA . We usedper week . First, per week . After 1per week . Suppres


Critically ill patients experience hypermetabolism increas ing substrate utilization, especially glucose oxidation. Glycogen is the main source of glucose in the body, being 85% and 15% stored in skeletal muscle and liver respectively. Since glycogen stores are limited we evaluated the hypothesis that critical illness could be associated with glycogen depletion leading to skeletal muscle catabolism for gluconeogenesis and eventually resulting in cachexia, an important determinant of future ICU survival and ICU-acquired weakness.n = 3), hemorrhagic shock/abdominal hypertension (n = 1), hypovolemic shock/post major oncologic surgery (n = 1), trauma (n = 3), and burn injury (n = 1).Nine critically ill patients (58.75 ± 25 to 75 years old) with an ICU stay from 1 day to 5 weeks were evaluated for skeletal muscle glycogen content using a rapid, non-invasive high-frequency ultrasound methodology . Scans were obtained from the rectus femoris and vastus lateralis muscles. Glycogen content was measured with a score from 0 to 100 according to the MuscleSound® scale. Patients had a variety of primary diagnoses including septic shock (Six out of nine patients had no glycogen present in the muscle (score = 0). The other three patients had glycogen scores between 5 and 15 which are well below scores of healthy individuals (reference 50 to 70). As a comparison we collected post-competition levels in competitive athletes, which decrease their glycogen stores (score 15 to 25) but are well above those of most critically ill patients we have studied.This is the first time that muscle glycogen stores have been evaluated in critical illness. Our data show severe glycogen depletion in ICU patients which probably leads to muscle catabolism necessary for gluconeogenesis, eventually resulting in cachexia. This finding poses severe metabolic challenges for ICU patients in which interfering with recovery can contribute to poor survival. In light of our findings, re-evaluation of nutritional protocols and potential anabolic/anticatabolic therapy to decrease muscle catabolism may improve survival. Different therapeutics that may prevent hypermetabolism (such as beta-blockers) should be re-evaluated along with anabolic agents  which could counteract the severe catabolic response in critical illness. Monitoring of muscle glycogen repletion could signal the transition from the catabolic to anabolic phase.


Thawing of permafrost soils is expected to stimulate microbial decomposition and respiration of sequestered carbon. This could, in turn, increase atmospheric concentrations of greenhouse gasses, such as carbon dioxide and methane, and create a positive feedback to climate warming. Recent metagenomic studies suggest that permafrost has a large metabolic potential for carbon processing, including pathways for fermentation and methanogenesis. Here, we performed a pilot study using ultrahigh throughput Illumina HiSeq sequencing of reverse transcribed messenger RNA to obtain a detailed overview of active metabolic pathways and responsible organisms in up to 70 cm deep permafrost soils at a moist acidic tundra location in Arctic Alaska. The transcriptional response of the permafrost microbial community was compared before and after 11 days of thaw. In general, the transcriptional profile under frozen conditions suggests a dominance of stress responses, survival strategies, and maintenance processes, whereas upon thaw a rapid enzymatic response to decomposing soil organic matter (SOM) was observed. Bacteroidetes, Firmicutes, ascomycete fungi, and methanogens were responsible for largest transcriptional response upon thaw. Transcripts indicative of heterotrophic methanogenic pathways utilizing acetate, methanol, and methylamine were found predominantly in the permafrost table after thaw. Furthermore, transcripts involved in acetogenesis were expressed exclusively after thaw suggesting that acetogenic bacteria are a potential source of acetate for acetoclastic methanogenesis in freshly thawed permafrost. Metatranscriptomics is shown here to be a useful approach for inferring the activity of permafrost microbes that has potential to improve our understanding of permafrost SOM bioavailability and biogeochemical mechanisms contributing to greenhouse gas emissions as a result of permafrost thaw. The northern permafrost region contains ~1672 gigatonnes (Gt) of organic carbon, nearly twice the amount of atmospheric carbon. Roughly 1470 Gt or 88% of this carbon occurs in perennially frozen soils and deposits -producers (methanogens) growing on hydrogen (H2) and carbon dioxide (CO2) with some species also growing on formate, methanol, or methylamine  genes   may bias phylogenetic representation in culture . We sought to provide the first transcriptional data supporting the hypothesis that microbial degradation of SOM biopolymers leads to increased CO2 production and methanogenesis, and to elucidate the biochemical mechanisms underlying these processes. Parallel geochemical analysis of soil age, carbon and nitrogen content, and lipid biomarkers provided additional information on the sources and composition of permafrost SOM.Here, we performed a pilot study using ultrahigh throughput Illumina HiSeq sequencing of reverse transcribed mRNA  aluminum foil and kept frozen inside coolers with deeply frozen blue ice packs. At the Toolik Field Station, the melted active layer was kept inside the cooler and the frozen core sections were kept in a freezer at −5°C for up to 2 h until subsampling was completed: within 2 h after coring an uncontaminated subsampling surface area was created by removing the outer 1 cm of frozen soil with a sterile knife. Cores were split laterally at the desired depths using a sterilized hammer and a chisel, and subsamples were obtained only from the central part of each interval with sterilized scalpel and tweezers. Thus, the distance between the core liner and subsampled sediment was at least 2 cm to avoid cross contamination.Using a SIPRE style auger system , a 130-cm-long (8 cm diameter) core was recovered on July 27, 2008 from moist acidic tundra (MAT) near the Kuparuk River at the Toolik Long Term Ecological Research (LTER) Field Station, Alaska (68°38′41.455″N:149°24′09.682″W) , and five depth intervals from the underlying permafrost . For lipid analysis, subsamples (~2 g) were stored frozen at −20°C at the Woods Hole Oceanographic Institution (WHOI) until subsequent lipid extraction. For the subsequent extraction of RNA, ~2 g aliquots of pristine soils were transferred to 5 ml cryovials and immediately flash frozen in liquid nitrogen. In addition, 20 ml sterile serum flasks were completely filled with soil leaving no headspace, capped airtight, and incubated in the dark at 4°C. As samples were capped airtight with no headspace, they remained anoxic for the duration of the incubation. Subsamples for RNA extraction were also obtained on the last day of fieldwork . All samples for RNA work were stored in liquid nitrogen during airfreight transport to WHOI and RNA was extracted immediately upon arrival as detailed below.13C, δ15N) from the exact same intervals were provided previously  before being extracted 3 times with methyl tert-butyl ether. Subsequently, elemental sulfur was removed by filtering extracts through acid-washed copper. Samples were resuspended in hexane prior to separation of compound classes by solid phase extraction. Discovery DSC-NH2 stationary phase  (1 g) was packed into glass columns and samples were separated into five fractions: F1 5 ml hexane; F2 8 ml of 4:1 hexane/methylene chloride; F3 10 ml of 9:1 methylene chloride/acetone; F4 15 ml of 2% formic acid in methylene chloride  we calculated the carbon preference index (CPI), Paq index, and Pwax index. CPIalkane reflects relative inputs of higher plants vs. algae and bacteria since the former have a strong odd/even predominance. Consequently, high CPIalkane values (>5) reflect mainly contributions from higher plants  using a microwave-accelerated reaction system (MARS6)  in which samples were heated at 100°C for 15 min with continuous stirring  indicate inputs from submerged or floating macrophytes and may reflect wet conditions.The Pwax index value (>0.7) reflects waxy inputs from higher plants and may indicate drier conditions  following the instructions of the manufacturer. The homogenization step started while the soil was still frozen to minimize degradation of RNA during thawing of the flash frozen soil samples. Standard procedures to further minimize degradation of RNA during the extraction process involved the use of filter tips and certified RNAse free disposables and reagents. Surfaces were cleaned with RNase AWAY™ , and all extraction procedures were performed in a HEPA-filtered horizontal laminar flow hood  inside a HEPA-filtered and positive-pressured ancient DNA dedicated lab at WHOI to eliminate aerosol contamination by bacterial and fungal cells/spores. DNA was removed using the Turbo DNA-free® kit (Life Technologies), increasing the incubation time to 1 h to ensure rigorous DNA removal. Extraction blanks were performed (adding sterile water instead of sample) to ensure that aerosolized contaminants did not enter sample and reagent tubes during the extraction process. Short RNA fragments (mostly produced during the extraction protocol) and residual inhibitors  were removed from the extracted RNA using the MEGA-Clear® RNA Purification Kit (Life Technologies). We followed the protocol all the way through the optional precipitation/concentration step, resuspending the RNA pellet in 10 μl of certified RNAse free sterile water (Life Technologies). Prior to cDNA amplification, the removal of contaminating DNA in RNA extracts was confirmed by the absence of visible amplification of SSU rRNA genes after 35 cycles of PCR using the RNA extracts as template, and the quality of the RNA was verified by agarose gel electrophoresis. All downstream analyses  described briefly in the following sections followed the protocols developed and reported recently  are expected to thaw in the Alaskan Arctic as a result of continuing Arctic warming  of the purified RNA: Five nanograms of purified high quality RNA as determined fluorometrically with Quant-iT™ RiboGreen® RNA reagent (Life Technologies) was used as template for selective mRNA amplification using the Ovation RNA-Seq v2 System® . We followed the manufacturers instructions for cDNA amplification, and the resulting quantity of cDNA was checked fluorometrically using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies). Quality of the amplified cDNA was checked on a Bioanalyzer  prior to Illumina® sequencing. cDNA of the triplicate extracts were pooled and Illumina® library preparation and paired-end sequencing was performed at the University of Delaware Sequencing and Genotyping Center . We acknowledge that because the original RNA extracts were stored ~5 years at −80°C prior to metatranscriptome construction, it is possible that some RNA could have been lost or partially degraded.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), with a quality score cutoff of 28. Approximately 375 million paired-end reads that passed quality control were imported into CLC Genomics Workbench 6.0®  and assembled using the paired-end Illumina assembler. Contigs were assembled over a range of kmer sizes  with a minimum contig size cutoff of 300 nucleotides. The kmer size of 50 resulted in the highest number of contigs and thus these contigs were chosen for use in downstream bioinformatics analyses. To reduce the formation of chimeric assemblies, we used a 2 × 105 paired-end sequencing approach on the Illumina HiSeq platform and performed assemblies without scaffolding. Reads were mapped onto the contigs using the read mapping option in CLC Genomics Workbench to retain information on relative abundance of contigs.Quality control of the dataset was performed using FastQC  and assigned to clusters of orthologous gene (COG) families using the Rapid Analysis of Multiple Metagenomes with a Clustering and Annotation Pipeline (RAMMCAP) . For identification of fungal ORFs, additional RAMMCAP analyses were performed using the standard genetic code for eukaryotes and the alternative yeast genetic code (−t 1 and −t 12 in advanced options). cDNA contigs were also submitted to MG-RAST  Li,  using thContigs were assigned to high-level taxonomic groups (Class level and above) using a Naïve Baysien Classifier (NBC) that compares against a database containing all DNA sequences in NCBI that classified as either Bacteria, Archaea, Fungi, or viral  searches using the translated amino acid sequences of M. barkeri genes as a reference. Expressed genes with homology M. barkeri proteins were subjected to metabolic pathway mapping in KEGG (http://www.genome.jp/kegg/pathway.html). Expressed genes with homology to the M. barkeri genome involved in methanogenesis pathways were visualized using Adobe Illustrator .The bioinformatics pipeline for metatranscriptomic genome recruitment followed that described recently , with the MG-RAST matR library (metagenomics.anl.gov). To maintain abundance information, assembled contig sequences from each sample were uploaded to MG RAST with the read mapping abundance added to the fasta headers as specified on the MG RAST website. Differences in overexpression between frozen and thawed permafrost samples were determined by a One-Way ANOVA test with a P-value cutoff of 0.025. The relatively low number of rRNA reads that were present in the dataset despite the selective amplification of mRNA using the Ovation RNA-Seq v2 System® were removed prior to comparison. Data were normalized in MG RAST with a log-based transformation , where Xs,i represents an abundance measure (i) in sample (s). Log transformed counts from each sample were then standardized (data centering) according to the equation , where Zs,i is the standardized abundance of an individual measure Ys,i (log transformed from previous equation). From each log transformed measure of (i) in sample (s), the mean of all transformed values (Ys) is subtracted and the difference is divided by the standard deviation (σs) of all log-transformed values for the given sample. After log transformation and standardization, the values for the functional categories within each sample were scaled from 0  to 1 , which is a uniform scaling that does not affect the relative differences of values within or between samples. The top 100 processes which differed most substantially in the level of gene expression between frozen and thawed samples and which passed the One-Way ANOVA test were used as input for heatmap presentation, hypothetical proteins and proteins of unknown function were removed from the list of top 100 processes after creating the heatmap.Analyses of gene overexpression in frozen vs. thawed samples was performed using the R statistical package  and lipid biomarkers , reflected mainly higher plant sources ] ]   Figure  and cond13:0-C17:0)]  were assembled de-novo from each Illumina-sequenced metatranscriptome, with 87% (SD = 3%) of original reads mapping to contigs per sample  with an average length of 616 -response occurs in the frozen soils  Figure . This suGenes encoding for all major classes of hydrolases involved in the cleavage of complex biopolymers into C1 and C2 substrates were expressed in both frozen and thawed soils Figure . The relThe diversity of taxa expressing hydrolase encoding transcripts in the deeper permafrost decreased markedly relative to 33.5 cm, with Firmicutes-derived hydrolase encoding transcripts increasing in relative representation after thaw Figure . A similUpon thawed conditions in the permafrost table, relative expression of fungal, γ-Proteobacterial, and Firmicutes genes encoding hydrolases acting on recalcitrant ether bonds increased Figure . Ether bRelative expression of genes with homology to cyanobacterial peptidases and hydrolases acting on non-peptide C-N bonds and anhydrides was highest in the deepest analyzed thawed permafrost interval Figure . The actmcrA), which catalyzes the last step in methanogenesis and is present in all methanogens , which is indicative of heterotrophic methanogenic metabolism, was also detected after thaw, suggesting C1 and C2 substrates as sources of biogenic methane. Further metabolic mapping of expressed genes to the M. barkeri genome indicated an increase in active biochemical pathways utilizing acetate, trimethylamine, and methanol as methanogenic substrates  (a protein necessary completing the pathway from methanogenesis from CO2), was not detected, suggesting a predominance of heterotrophic methanogenesis after thaw. A marked increase in acetoclastic methanogensis was recently also reported from thawed permafrost soils in northern Sweden  increased humification rates, (ii) DOM shifted toward lower molecular weight compounds with lower aromaticity, (iii) lower organic oxygen content, and (iv) more abundant microbially produced compounds  genes (not detected in frozen permafrost), which can serve as a proxy for acetogenesis (Lever, fhs in the upper permafrost were assigned predominantly to Acetobacterium carbinolicum, whereas various Clostridium species are likely involved in acetate production in deeper permafrost (Figure M. barkeri (Figure In all thawed permafrost samples, an increase in s Lever,  (Figure s Lever, . Transcrt Figure . The gent Figure  after thi Figure . Acetogei Figure .Our pilot study shows that the analysis of metatranscriptomes is a sensitive approach capable of identifying a wide spectrum of important metabolic processes in frozen and thawed permafrost. In general, the transcriptional profile under frozen conditions suggests a dominance of stress responses, survival strategies, and maintenance processes whereas upon thaw a rapid enzymatic response to decomposing SOM was observed. Furthermore, our analysis indicates that acetoclastic methanogenesis may be a dominant form of methanogenesis in thawing permafrost soils fueled, in part, by acetate production from acetogenic bacteria. Due to large heterogeneity in permafrost SOM and microbial composition, future metatranscriptome studies should be performed from a wider variety of permafrost soil types and locations, and in comparison with the active layer. Future studies comparing metatranscriptomes from thawing permafrost should also contain biological replicates to provide robust statistical power for hypothesis testing. Furthermore, paired geochemical analysis of isotopically labeled substrates and metabolites during thaw incubation experiments as well as the analysis of the stable isotopic composition and concentration of dissolved methane should be performed to validate mRNA-based inferences of metabolic pathways. Measurements of soil pH would further be useful to contextualize microbial responses to permafrost thaw at the transcriptional level. Metatranscriptomics is shown here to be a useful approach for inferring the activity of permafrost microbes, that has potential to improve our understanding of permafrost SOM bioavailability and biogeochemical mechanisms contributing to greenhouse gas emissions as a result of warming induced permafrost thaw.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mate preference behavior is an essential first step in sexual selection and is a critical determinant in evolutionary biology. Previously an environmental compound (the fungicide vinclozolin) was found to promote the epigenetic transgenerational inheritance of an altered sperm epigenome and modified mate preference characteristics for three generations after exposure of a gestating female.The current study investigated gene networks involved in various regions of the brain that correlated with the altered mate preference behavior in the male and female. Statistically significant correlations of gene clusters and modules were identified to associate with specific mate preference behaviors. This novel systems biology approach identified gene networks (bionetworks) involved in sex-specific mate preference behavior. Observations demonstrate the ability of environmental factors to promote the epigenetic transgenerational inheritance of this altered evolutionary biology determinant.Combined observations elucidate the potential molecular control of mate preference behavior and suggests environmental epigenetics can have a role in evolutionary biology.The online version of this article (doi:10.1186/1471-2164-15-377) contains supplementary material, which is available to authorized users. The current molecular paradigm for neo-Darwinian evolutionary biology is that random DNA sequence mutations, gene flow, and genetic drift promote phenotype variation that allows an adaptation event to facilitate natural selection . AlthougPreviously we demonstrated that exposure of a gestating female rat to an environmental compound during fetal gonadal sex determination promoted epigenetic reprogramming of the male germline –15. ThesInvestigation of the epigenetic transgenerational inheritance of altered brain genome activity and behaviors previously demonstrated anxiety-like behavior increased in females and decreased in males, which correlate to alterations in specific brain region transcriptomes . AlteredSystems biology analysis has allowed biological phenomena such as mate preference to be considered from the molecular to physiological level. The gene bionetwork  analysisObservations demonstrate an environmental compound exposure can induce an epigenetic reprogramming of the germline that promotes epigenetic transgenerational inheritance of altered mate preference behavior. Although no direct epigenetic modifications in the brain were examined, the environmentally induced epigenetic transgenerational model used indicates epigenetics can be involved in the induction of the altered behavioral phenotypes. Sex-specific effects were observed in both the male and female brain transcriptome and behavior correlations. The gene networks in specific brain regions that statistically correlate with various mate preference behaviors provides insight into this environmentally modified transgenerational behavior. This systems biology approach has elucidated novel mechanisms to be considered in mate preference biology.The experimental design involved the development of transgenerational control and vinclozolin lineage animals for a mate preference behavioral analysis . SubsequFemales of both control and vinclozolin lineages preferred control lineage males over vinclozolin lineage males . The matFor the microarray analysis each F3 generation control and vinclozolin lineage male and female animal had six different brain regions analyzed which totaled 134 different microarrays. The microarray data were pre-processed and demonstrated two abnormal arrays that were omitted for further analysis  Figure . This reIn considering the mate preference behaviors, the female is the discriminatory sex to choose a mate, while the male is non-discriminatory and has phenotypes and behaviors to be selected , 39. TheThe six male brain regions and associated gene modules had a number of statistically significant correlations with the mate preference parameters Table . TherefoThe direct connection gene sub-networks for the critical male amygdala (M-Amy) and cingulate cortex (M-CngCtx) turquoise modules are shown in Figure The epigenetic transgenerational inheritance of the altered mate preference behavior requires the transmission of an altered epigenome in the germline (sperm) , 13, 15.A systems biology analysis of environmentally induced epigenetic transgenerational inheritance of altered mate preference behavior was performed to suggest a potential role for epigenetics in evolutionary biology. Previous research has demonstrated that environmental toxicants such as the fungicide vinclozolin can promote a reprogramming of the germline epigenome during fetal gonadal sex determination that then transmits altered phenotypes and adult onset disease states transgenerationally in the absence of future environmental exposure . This isThe vinclozolin induced epigenetic transgenerational phenotypes previously identified included adult onset rat disease after 12 months of age of male infertility, mammary tumors, prostate disease, kidney disease and immune abnormalities . TherefoObservations from the current study need to consider the effects on the female brain as potentially altering female discrimination and preference. The effects on the male brain are presumed to be associated with the characteristics (e.g. auditory cues and pheromone production) being selected. This altered mate preference behavior suggests the existence of an environmentally altered epigenetic transgenerational inheritance of mate preference behavior . The curA novel gene bionetwork analysis was developed to identify gene networks correlated to disease . The appAnalysis of significant pathways and cellular processes potentially influenced by the differentially expressed gene sets and networks did not identify predominant or over represented pathways. One pathway identified that previously has been shown to correlate with sexual selection is the olfactory transduction pathway –52. HoweIn contrast, analysis of gene networks identified unique gene sub-networks and gene modules associated with each brain region differentially expressed gene sets. The specific inter-connected genes were unique and overall networks of connected genes distinct. Therefore, the different functions associated with each brain region and associated with the altered mate preference behavior were identified. A statistical correlation of the gene modules for each brain region with the different mate preference parameters measured identified a number of statistically significant correlations. All but one female brain region (Hipp) had statistically significant correlations with the Plexiglas behavior parameter. The female behavior directly associates with the discrimination and mate preference choice. Interestingly, all the male brain regions had some modules with statistically significant correlations with the Wire Mesh behavior parameter. The male behavior and/or phenotype  associates with the selected behaviors and characteristics of the non-discriminant sex. Therefore, direct correlations with the gene modules, specific brain regions and mate preference behavior parameters were identified. The specific gene modules and behavioral parameters statistically correlated were distinct between the sexes and brain regions, but strong correlations of the gene networks to the mate preference behavior was established. Interestingly, both the Plexiglas and Wire Mesh are indicators of interest and assessment of the stimulus animal.The gene bionetwork analysis and statistical correlation with the mate preference behavior provides insight into the molecular basis of how various male and female brain regions correlate and in part control the various behavioral parameters. Observations provide one of the first genomic and systems biology analysis of mate preference behavior Figures  and 7. TDarwin  considerMate preference, in its simplest form, states that males compete for females and females choose between them. Although most research has focused on how females choose males, male choice of females is also important –58. ThisEvolution favors reproductive success, and it is in the individual's interest to focus on selecting the best mate and to avoid mating with the wrong species , 60. MakThere are a number of sexual selection hypotheses, all of which emphasize that females choosing optimally will produce young whose viability and survivorship are enhanced by the female’s choice of mate . The mosThe ability of an environmental factor to alter mate preference behavior suggests a critical role of environment in evolutionary biology. This is distinct from the generally accepted role of the environment in natural selection where environment is the active factor in the selection of an adaptive phenotype, but alternately here it involves the induction of phenotypes that can be acted on by natural selection. Since the majority of environmental factors can not alter DNA sequence or promote mutagenesis , an addiMale and female rats of the F3 generation of Vinclozolin (Vinclozolin-Lineage) and DMSO Control (Control-Lineage) Lineages were selected out of litters from untreated F2 generation mothers in Dr. Michael Skinner’s laboratory at Washington State University according to established protocols . Brieflyad libitum access to tap water and standard rat chow (Purina rodent chow #5LL2 Prolab RMH 1800 diet). The animal room was on a 14:10 light/dark schedule. For environmental enrichment, a 7 cm diameter PVC pipe was placed in each cage.Each dyad of animals was randomly placed in a six-wide, five-high metal housing rack in standard translucent polycarbonate rat cages (46 × 24 × 20.5 cm) with All experimental protocols for the procedures with rats were pre-approved by the Washington State University Animal Care and Use Committee  and by the University of Texas at Austin Animal Care and Use Committee .After habituation to the testing arena, each individual was tested individually  or in pairs  with all individuals; the order of the testing was rotated during the course of both the male and female trials. All tests were conducted during the dark phase of the light cycle, beginning at 1200 h, 4 h after the progesterone injection, in a room illuminated with low levels of red light. Before trials, to confirm that females were receptive, each female was placed with a sexually experienced but otherwise experimentally naïve male; all females exhibited robust lordosis (arched back and lifted head posture) in response to mounting by the male.Partner preference tests consisted of placing an individual  in the center of a large three-chamber glass-testing arena (122 × 46 × 54 cm). At either end was a small compartment (28 × 28 × 12.5 cm) containing the stimulus rats separated by a Wire-mesh barrier to allow exchange of olfactory, visual, and tactile cues. The area directly in front of the stimulus cage was marked by tape. Tests were conducted 2 h after the onset of the dark cycle under red-light illumination and lasted 10 min; all tests were videotaped for further review and analysis. At the end of each test, all animals were removed, and the entire testing arena was washed with a household cleaner and then wiped down with 70% ethanol to remove scent marks and residual odors. All males were tested with both types of females as stimulus animals , and all females were tested with both types of males as stimulus animals  (Movie S1).http://www.jwatcher.ucla.edu) computer software to quantify the behavior of each experimental animal. Time spent with a stimulus animal was recorded as soon as all four paws of the experimental animal crossed over the line of tape marking the boundary of that stimulus animal’s compartment. As soon as one paw crossed over the tape back into the center compartment, the time recorded with the experimental animal was stopped. Preference behaviors were defined as those directed to the stimulus animal and included time spent in contact with the Wire Mesh separating the experimental and stimulus animal (Wire Mesh), during which the animals often touched noses through the Wire Mesh , and contacted the Plexiglas surface surrounding the front of the stimulus cage; the cumulative total time in these preference behaviors toward each stimulus animal was also calculated . Other activity measured included undirected walking and sniffing , standing still with minimal head movement (still). Videos demonstrating the test can be viewed as supporting information (Movie S1).The videotaped trials were analyzed by using JWatcher v1.0  within 3–5 min. This procedure was done on iced tissue. The dissected brain areas were placed in chilled Trizol (150 l) in 1.5 ml Eppendorf tubes according to manufacturers specifications in each tube. No tissue fragment was more than 3 mm but in those instances, but where there were multiple fragments the amount of Trizol was doubled (approximately). After all animals were dissected, the Eppendorf tubes were vortexed (15 sec) and then frozen on dry ice. The brain regions collected were according to Paxinos & Watson : olfactoBrain area samples from individual rats were homogenized in 150 μl Trizol and then 600 μl Trizol was added to final volume of 750 ml. Samples were stored at −80 or −20°C until RNA extraction. For microarray analysis, from 4 to 6 biological replicas  were prepared as above for each brain area Control or Vinclozolin group depending on samples availability . Hybridized chips were scanned on Affymetrix Scanner 3000. CEL files containing raw data were then pre-processed and analyzed with Partek Genomic Suite 6.5 software  using an RMA, GC-content adjusted algorithm. Raw data pre-processing was performed in 12 groups, one for each male or female brain area. Comparison of array sample histogram graphs for each group showed if data for all chips were similar and appropriate for further analysis  arrayed for each transcript and many genes are represented on the chip by several transcripts. The hybridization to each probe must be consistent to allow a statistically significant quantitative measure of resulting gene expression signal. Therefore, the microarray provides an unbiased and highly stringent quantitative procedure compared to other protocols . In conthttp://www.skinner.wsu.edu. For gene annotation, Affymetrix annotation file RaGene1_0stv1.na31.rn4.transcript.csv was used.All microarray CEL files from this study have been deposited with the NCBI gene expression and hybridization array data repository GEO (GEO series accession number: GSE33830) and can be also accessed through T test p-value ≤ 0.05. The union of the differentially expressed genes from the different treatments resulted in 1,693 genes for males and 1833 for females being identified and used for constructing a weighted gene co-expression network [f(x) = xβ. The parameter β of the power function is determined in such a way that the resulting adjacency matrix  is approximately scale-free. To measure how well a network satisfies a scale-free topology, we use the fitting index proposed by Zhang & Horvath [R2 of the linear model that regresses log(p(k)) on log(k) where k is connectivity and p(k) is the frequency distribution of connectivity). The fitting index of a perfect scale-free network is 1.The network analysis was restricted to genes differentially expressed between the control and the treatment groups based on previously established criteria: (1) fold change of group means ≥ 1.2 or ≤ 0.83; (2)  network , 72. Unl Horvath   is defined as the sum of the power-function transformed correlations between the gene g and all the other genes in the whole network while the TO-based connectivity  is defined as the sum of the topological overlaps between the gene g and all the other genes. By default, connectivity used throughout the paper refers to TO-based connectivity to.all.To distinguish between modules, each module was assigned a unique color identifier, with the remaining, poorly connected genes colored grey. The hierarchical clustering over the topological overlap matrix (TOM) and the identified modules is shown Figure . In thisGene Co-expression Network Analysis Clarification: Gene networks provide a convenient framework for exploring the context within which single genes operate. Networks are simply graphical models comprised of nodes and edges. For gene co-expression networks, an edge between two genes may indicate that the corresponding expression traits are correlated in a given population of interest. Depending on whether the interaction strength of two genes is considered, there are two different approaches for analyzing gene co-expression networks: 1) an unweighted network analysis that involves setting hard thresholds on the significance of the interactions, and 2) a weighted approach that avoids hard thresholds. Weighted gene co-expression networks preserve the continuous nature of gene-gene interactions at the transcriptional level and are robust to parameter selection. An important end product from the gene co-expression network analysis is a set of gene modules in which member genes are more highly correlated with each other than with genes outside a module. Most gene co-expression modules are enriched for GO functional annotations and are informative for identifying the functional components of the network that are associated with disease .This gene co-expression network analysis (GCENA) has been increasingly used to identify gene sub-networks for prioritizing gene targets associated with a variety of common human diseases such as cancer and obesity , 76–79. http://vortex.cs.wayne.edu) [http://www.genome.jp/kegg/tool/search_pathway.html). Global literature analysis of various gene lists was performed using Pathway Studio 8.0 software .Resulting lists of differentially expressed genes for each male or female brain area as well as for each module generated in the combined network and some generated in separate networks analysis were analyzed for KEGG  pathway enrichment using Pathway-Express, a web-based tool freely available as part of the Onto-Tools (yne.edu)  as well Additional file 1: Movie S1: The first 19 sec of a 10-min mate-preference trial is shown. The trial is conducted under dim red light during the nocturnal (active) phase of the rats' light cycle. At the beginning of the video, the male is in the center of the chamber. The chamber is demarcated into thirds by tape on its floor. A stimulus female can be seen at the far end of the apparatus; the other stimulus female is not visible due to the position of camera. The stimulus females are free-moving in their chambers, but they are separated from the male by a wire mesh that is bounded by Plexiglas barrier. This enables the animals to communicate by olfactory, pheromonal, or behavioral cues, but physical interaction is limited to touching across the wire mesh. The trial begins with the removal of a holding box that confines the male. The male can be seen moving into the zone in front of one stimulus female and then moving across the central portion of the cage to the other stimulus female (out of sight). Several behaviors of the male can be seen on the video such as sniffing, facial investigation, walking, and standing of the female. The male is also seen investigating the various parts of the chamber, including the wire mesh, surrounding Plexiglas partition, and the glass walls of the chamber. Behaviors were scored for each male toward each pair of opposite lineage (Control- or Vinclozolin-Lineage) stimulus females. (AVI 4 MB)Additional file 2: Table S1: Behavior and Sample Information. (PDF 60 KB)Figure S1B. Samples Histograms After Pre-Processing . Figure S1. Sample histograms and box plots for male (S1A) female (S1B) microarray signal values after pre-processing with RMA, GCcontent adjusted algorithm. Plots for F3 generation control (red) and F3 generation vinclozolin (blue) chips for female amygdala (A), cingulate cortex (B), enterorhinal cortex (C), hippocampus (D), olfactory bulbs (E), and preoptic areaanterior hypothalamus (F). (PDF 4 MB)Additional file 3: Figure S1A: Samples Histograms After Pre-processing . Additional file 4: Table S2: Genes Differentially Expressed in F3 Generation Vinclozolin Versus Control Lineage Male and Female Rat Brain Regions. (PDF 618 KB)Top cellular pathways affected by signature gene lists and chosen modules from separate networks. (PDF 80 KB)Additional file 5: Additional file 6: Table S3: Pathways Affected Male and Female Brain Region Signature Gene Lists and Chosen Modules from Separate Networks. (PDF 54 KB)Additional file 7: Table S4: Correlation between combined network modules and behavior trait for F3-Vinclozolin rat brain regions. (PDF 2 MB)Additional file 8: Figure S2: (Color) Brain Region Specific Signature List Direct Connection Gene Sub-Networks. Legend: Figure S2. Direct connection sub-networks for signature lists:female amygdala (A), female preoptic area-anterior hypothalamus(B), female hippocampus (C), female enterorhinal cortex (D), female cingulate cortex(E), female olfactory bulbs (F), male amygdala (G), male hippocampus (H), male cingulate cortex(I), male enterorhinal cortex (J), male olfactory bulbs (K) obtained by global literature analysis using Pathway Studio 8.0 software . Numbers in brackets on figures subtitles indicate number of genes in the list. Only directly connected genes are shown. Some sub-networks  show gene location in the cell . Node shapes and color code: oval and circle – protein; diamond – ligand; circle/oval on tripod platform – transcription factor; ice cream cone – receptor; crescent – kinase or protein kinase; irregular polygon – phosphatase; red color indicates up-regulated genes, blue – down-regulated. Arrows with plus sign show positive regulation/activation, arrows with minus sign – negative regulation/inhibition; grey arrows represent regulation, lilac - expression, purple – binding, green – promoter binding, and yellow – protein modification. (PDF 66 KB)Additional file 9: Table S5: Correlation between separate network modules and behavior trait for F3-Vinlozolin rat brain regions. (PDF 66 KB)


Lrp+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis.Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the contribution of the LDL receptor-related protein 1 (LRP1) to this process is not known. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR)-deficient background (macLRP1-/-). After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from  Key events during the development of atherosclerosis include the accumulation of lipoprotein particles in the subendothelial arterial intima, where they are taken up by recruited monocytes/macrophages . This prin vivo. Early studies confirmed that native forms of LDL particles are not taken up by macrophages and therefore do not contribute to excessive cholesterol uptake in these cells -cholesterol (PerkinElmer) in MGM for 24 hours at 37°C. Following incubation, the cells were washed twice with PBS, incubated in low serum growth medium , and treated with or without 8-Br-cyclic AMP (Sigma) for 16–18 hours. To induce efflux, cells were washed twice with PBS and incubated in DMEM containing 50 μg/mL HDL (Intracel). At the indicated time points, the medium was collected and centrifuged at maximum speed for 10 minutes and cells were harvested by addition of NP-40 lysis buffer. Radioactivity in the medium and cell lysate was quantified by liquid scintillation counting, and percent cholesterol efflux was calculated as follows: (cpmMedia/(cpmMedia + cpmCell)) x 100 after subtracting background efflux (efflux in the absence of HDL).Cholesterol efflux assays were carried out essentially as described . ThioglyAfter a 4 hour fasting period, mice  were injected with 500 mg/kg body weight Triton WR1339  in 0.9% NaCl. At the indicated timepoints following injection, blood samples were obtained by tail snipping and the serum was assayed for triglycerides as described above.Cultured bone marrow-derived or thioglycollate-elicited macrophages were kept in serum free media overnight and then incubated with heparin (50 U/ml) for 30 min at 37°C. Lipase activity was measured as described below. For apoE immunoblot analysis, StrataClean beads (Stratagene) were added to the media. The samples were centrifuged, incubated with reducing SDS sample buffer, and heated at 95°C for 3 min. The samples were then resolved on 4–20% Tris-glycine gels.Fasted LRP1+/+ or macLRP1-/- mice were euthanized after 3 weeks on the Western diet or a chow diet. Livers, epididymal fat pads, gastrocnemius and soleus muscles were isolated, and were incubated with heparin (50 U/ml) on ice for 45 min to elute lipoprotein lipase bound to cell surface heparin-sulfate proteoglycans. Lipase activities were determined as previously described .2 Real-Time SYBR Green/ROX PCR Master Mix (SABiosciences). Gene expressions were normalized to beta glucuronidase, (Gusb), hypoxanthine guanine phosphoribosyl transferase (Hprt1) and heat shock protein 90 alpha (Hsp90ab1) housekeeping genes. Data were analyzed based on ΔΔCt fold-change method.Total RNA was extracted from thioglycollate-elicited peritoneal macrophages using Trizol (Invitrogen) reagent as directed by the manufacturer. 1 μg total RNA was used to synthesize cDNA by using the First Strand cDNA Synthesis Kit (SABiosciences). Quantitative real-time PCR was performed on an ABI 7900 HT instrument (Applied Biosystems) by using RTt-test was used to analyze significance between two groups (LRP1+/+ vs macLRP1-/-). In the instance when the influence of two independent variables (Time and genetic variation) on one dependent variable (TG levels) was analyzed, a two way ANOVA analysis was performed using GraphPad PRISM software. In all statistical analysis, p-values less than 0.05 were considered significant. The Figure Legends indicate which test was performed.The two-tailed unpaired Student’s 125I-labeled α2M*. The results reveal a substantially diminished ability of thioglycollate-elicited peritoneal macrophages from the macLRP1-/- mice to internalize this LRP1 ligand, confirming loss of functional activity in macrophages from macLRP1-/- mice  were similar in LRP1+/+ and LRP1-deficient macrophages (Abca2) gene (data not shown).Efflux of cholesterol from macrophages and other cells is mediated by ABCA1 and ABCAG transporters. Prior work in vascular smooth muscle cells revealed that LRP1 modulates levels of ABCA1 by altering the expression of liver X receptors which are key transcriptional regulators of ABCA1 . Thus, wtein Fig –4c and mtein Fig  and 4e irophages . FinallyLipoprotein lipase (LPL) is abundantly expressed in macrophages, and since macrophage-derived LPL plays a significant role in the regulation of serum triglycerides , we measTo determine if genetic deletion of LRP1 in macrophages alters plasma lipoprotein levels, we fed adult LRP1+/+ and macLRP1-/- mice either a chow or Western diet for 6 weeks and then collected the serum after fasting and measured total serum cholesterol and triglyceride levels. We noted a striking and statistically significant increase in plasma cholesterol  and plasTo identify the lipoprotein fraction(s) that are increased in macLRP1-/- mice, plasma samples from fasted mice were subjected to fast protein liquid chromatography (FPLC) gel filtration chromatography. LRP1+/+ or macLRP1-/- mice fed a chow diet displayed no difference in their lipoprotein profile, either measured by cholesterol content or triglyceride content. Mice fed a Western diet displayed significantly elevated VLDL/LDL levels in the macLRP1-/- mice Fig  and 5d.To further characterize the lipoproteins that accumulate in the macLRP1-/- mice, the fraction of lipoproteins with d < 1.019 g/ml was isolated by density gradient centrifugation, and analyzed by SDS-PAGE and immunoblotting. When normalized to protein loading, this analysis revealed no significant changes in the apoE content of the lipoprotein particles Fig  and 6b. Increased accumulation of triglyceride-rich particles in the plasma could result from either increased biosynthesis of VLDL particles from the liver or decreased catabolism. In order to differentiate between the two, we measured the triglyceride biosynthesis rate. This was accomplished by measuring temporal increases in plasma triglycerides under conditions in which triglyceride hydrolysis by lipoprotein lipase is inhibited by injecting the nonionic detergent Triton WR-1339 . The resTo test this possibility, we measured the plasma triglyceride levels following an intragastric bolus of olive oil administration. The results, shown in Lipolysis of VLDL and chylomicron particles at tissue sites is an important component of the processing of these lipoproteins. This process is catalyzed by lipoprotein lipase (LPL), which not only mediates lipolysis of chylomicrons and VLDL particles, but also binds to LRP1 via its carboxyl-terminal domain and facilitates lipoprotein uptake by this receptor –48. Aftein vivo role for LRP1 in mediating cholesterol uptake in macrophages. This was confirmed by demonstrating that macLRP1-/- mice have a significant decrease in macrophage cholesterol content when fed a Western diet. Further, we noted elevated plasma triglyceride and cholesterol levels in macLRP1-/- mice. Together, these data provide compelling in vivo evidence that LRP1 contributes to cholesterol accumulation in macrophages. Interestingly, CD36 and SR-A, two receptors implicated in macrophage cholesterol uptake, also show elevated cholesterol and triglyceride levels when Cd36-/- and Msr1-/- mice on an apoE null background are fed a Western diet [The results obtained in the current investigation document an important ern diet . Thus, iboth the LDL receptor and LRP1 are deleted in the liver. Thus genetic deletion of either hepatic LRP1 alone [The role of LRP1 in chylomicron remnant catabolism is well established , althougP1 alone  or the LP1 alone  does notP1 alone . FurtherP1 alone , and stuP1 alone .Msr1) and CD36 (Cd36) have been implicated in this process [In contrast to the hepatic uptake of lipoproteins, the mechanisms by which macrophages take up lipoprotein particles are not as well understood. Macrophage scavenger receptors have been identified that participate in the uptake and internalization of modified forms of LDL –56, and  process ,8. Despi process ,58, abun process  revealin process , and hav process .in vivo replacement of resident macrophages, such as Kupffer cells, upon bone marrow transplantation is a relatively slow process taking several months [The findings of elevated triglyceride-rich lipoprotein levels in the plasma of macLRP1-/- mice fed a Western diet are somewhat suprising since our results differ from ealier work on macLRP1-/- mice. For example, Hu et al.  generatel months .The mechanism by which macrophage LRP1 influences the levels of plasma triglyceride-rich lipoproteins is not clear at this time, but several possibilities exist. First, macrophages may play a direct role in the clearance of remnant lipoproteins. In this regard, it is interesting to highlight the studies of Hussain et al. ,65 who dDespite the increased cholesterol content of LRP1-expressing macrophages, studies have revealed that macrophage LRP1 protects the vasculature from the development of atherosclerosis –28 and fin vivo role for macrophages and macrophage LRP1 in modulating cholesterol metabolism. The data further reveal that LRP1 is one of the major receptors that contribute to lipoprotein uptake in macrophages leading to foam cell formation. It is well known that the development of atherosclerosis is a complex process involving multiple genes, and defining the potential of LRP1 to regulate these processes may assist in identifying molecules which could have significant therapeutic potential.In summary, the current investigation has confirmed an important


MTOR are clustered in various regulatory domains in ccRCC. We focused our attention on a prominent cluster of hyperactivating mutations in the FAT (FRAP–ATM–TTRAP) domain of mTOR in ccRCC that led to an increase in both mTORC1 and mTORC2 activities and led to an increased proliferation of cells [RHEB (Ras homolog enriched in brain) in ccRCC patients that exclusively increased mTORC1 activity. Interestingly, mutations in the FAT domain of MTOR and in RHEB remained sensitive to rapamycin, though several of these mutations demonstrated residual mTOR kinase activity after treatment with rapamycin at clinically relevant doses. Overall, our data suggests that point mutations in the mTOR pathway may lead to downstream mTOR hyperactivation through multiple different mechanisms to confer a proliferative advantage to a tumor cell.Renal cell carcinoma (RCC) has multiple subtypes and may occur in hereditary and sporadic forms. Sporadic renal cell carcinomas are most commonly clear cell cancers (80%). Metastatic disease is found at presentation in almost 30% of patients with renal cell carcinoma and treatment of RCC metastases is greatly different from the treatment regimens of the primary tumor. Currently, several FDA approved therapies exist for metastatic clear cell RCC (ccRCC) which includes two rapamycin analogs-everolimus and temsirolimus. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase and catalytic subunit of two biochemically distinct complexes called mTORC1 and mTORC2. Recently published TCGA data report aberrations in the PI3K/AKT/mTOR pathway in up to 28% of RCC cases [of cells . Severalin vitro as they have a distinct advantage in that they inhibit 4E-BP1 phosphorylation at rapamycin resistant sites and also block AKT phosphorylation at Ser473 [Given the central role of mTORC1 as a downstream target of PI3K activity, there exists a clear rationale for targeting mTORC1 in cancer and using rapalogs clinically. Unfortunately, the effectiveness of rapamycin as a single agent therapy is fraught with several limitations. mTORC1 promotes IRS-1 degradation  implyingt Ser473 . Signifiin vitro data suggests that these hyperactivating mutations confer relative resistance to rapalog therapy and these findings may have dosing implications for patients with ccRCC. These findings may be highly relevant from a clinical point of view, as MTOR mutations could serve as biomarker predicting tumor responses to mTOR allosteric inhibitors and explain acquired resistance to this class of drugs in humans.While most of mTOR targeted therapies including everolimus and temsirolimus target mTORC1, mTORC2 is emerging as a pivotal player in many cancers. Defining mTORC2's role in the cellular milieu has been more challenging compared to mTORC1 because of its insensitivity to acute rapamycin treatment. As mTORC2 is a key regulator of cell proliferation and metabolic reprogramming of tumor cells , there i