Lower Protein Synthesis Research Articles

Abstract Mo107: Age-Related Impairment of Mitochondrial Protein Turnover Exacerbates Pathogenesis of Heart Failure with Preserved Ejection Fraction in Old Mice

Introduction: Age is recognized as a major risk factor for heart failure with preserved ejection fraction (HFpEF), yet the precise role of aging in HFpEF development remains unknown. Disruption of mitochondrial protein turnover affects the quality and quantity of proteins crucial for energy metabolism. Although mitochondrial dysfunction is a key feature of both aging and HFpEF heart, the influence of age-related disturbances in mitochondrial protein turnover on HFpEF pathogenesis remains unclear. Hypothesis: Age-related impairment of mitochondrial protein turnover intensifies proteostatic perturbations induced by hypertensive and metabolic stress (2-hit stress) to exacerbate pathogenesis of cardiometabolic HFpEF. Methods: Young (3 mo.) and old (19 mo.) C57BL/6J male mice were exposed to high-fat diet and L-NAME to induce HFpEF development. Deuterium oxide (D 2 O) labeling coupled with mass spectrometry was used to investigate bulk and individual mitochondrial protein synthesis. The changes in individual mitochondrial protein abundance and protein degradation pathways were also studied. Results: Old mice presented exacerbated HFpEF characteristics compared to young mice, with higher mortality and worsened cardiac and extra-cardiac phenotypes. Regardless of age, mice subjected to 2-hit stress had 24% higher bulk mitochondrial protein synthesis compared to controls. Old control and HFpEF mice showed 17% lower mitochondrial protein synthesis compared to younger counterparts, potentially hindering the replacement of dysfunctional mitochondrial proteins. Aging reduced the synthesis rates of 30 out of 115 proteins synthesized more rapidly with HFpEF, including those involved in mitochondrial metabolism. Despite slower synthesis of numerous mitochondrial proteins with age, the overall abundance of these proteins remained unchanged or elevated compared to young counterparts, indicating impaired protein degradation. We found that aging and 2-hit stress differentially inhibited degradation pathways, including autophagy and the ubiquitin-proteasome system, further impairing protein turnover. Conclusion: Aging impairs mitochondrial protein turnover in cardiometabolic HFpEF and contributes to poorer disease outcomes.

BMAL1 and CLOCK proteins exhibit differential association with mitochondrial dynamics, protein synthesis pathways and muscle strength in human muscle.

Murine models lacking CLOCK/BMAL1 proteins in skeletal muscle (SkM) present muscle deterioration and mitochondria abnormalities. It is unclear whether humans with lower levels of these proteins in the SkM have similar alterations. Here we evaluated the association between BMAL1 and CLOCK protein mass with mitochondrial dynamics parameters and molecular and functional SkM quality markers in males. SkM biopsies were taken from the vastus lateralis of 16 male (non-athletes, non-obese and non-diabetic) subjects (8-9 a.m.). The morphology of mitochondria and their interaction with the sarcoplasmic reticulum (mitochondria-SR) were determined using transmission electron microscopy images. Additionally, protein abundance of the OXPHOS complex, mitochondria fusion/fission regulators, mitophagy and signalling proteins related to muscle protein synthesis were measured. To evaluate the quality of SkM, the cross-sectional area and maximal SkM strength were also measured. The results showed that BMAL1 protein mass was positively associated with mitochondria-SR distance, mitochondria size, mitochondria cristae density and mTOR protein mass. On the other hand, CLOCK protein mass was negatively associated with mitochondria-SR interaction, but positively associated with mitochondria complex III, OPA1 and DRP1 protein mass. Furthermore, CLOCK protein mass was positively associated with the protein synthesis signalling pathway (total mTOR, AKT and P70S6K protein mass) and SkM strength. These findings suggest that the BMAL1 and CLOCK proteins play different roles in regulating mitochondrial dynamics and SkM function in males, and that modulation of these proteins could be a potential therapeutic target for treating muscle diseases. KEY POINTS: In murine models, reductions in BMAL1 and CLOCK proteins lead to changes in mitochondria biology and a decline in muscle function. However, this association has not been explored in humans. We found that in human skeletal muscle, a decrease in BMAL1 protein mass is linked to smaller intermyofibrillar mitochondria, lower mitochondria cristae density, higher interaction between mitochondria and sarcoplasmic reticulum, and reduced mTOR protein mass. Additionally, we found that a decrease in CLOCK protein mass is associated with a higher interaction between mitochondria and sarcoplasmic reticulum, lower protein mass of OPA1 and DRP1, which regulates mitochondria fusion and fission, lower protein synthesis signalling pathway (mTOR, AKT and P70S6K protein mass), and decreased skeletal muscle strength. According to our findings in humans, which are supported by previous studies in animals, the mitochondrial dynamics and skeletal muscle function could be regulated differently by BMAL1 and CLOCK proteins. As a result, targeting the modulation of these proteins could be a potential therapeutic approach for treating muscle diseases and metabolic disorders related to muscle.

A comparative study of tissue protein synthesis rates in an Antarctic, Harpagifer antarcticus and a temperate, Lipophrys pholis teleost

The affect of temperature on tissue protein synthesis rates has been reported in temperate and tropical, but not Antarctic fishes. Previous studies have generally demonstrated low growth rates in Antarctic fish species in comparison to temperate relatives and elevated levels of protein turnover. This study investigates how low temperatures effect tissue protein synthesis and hence tissue growth in a polar fish species. Groups of Antarctic, Harpagifer antarcticus and temperate, Lipophrys pholis, were acclimated to a range of overlapping water temperatures and protein synthesis was measure in white muscle (WM), liver and gastrointestinal tract (GIT). WM protein synthesis rates increased linearly with temperature in both species (H. antarcticus 0.16–0.23%.d−1, L. pholis, 0.31–0.76%.d−1), while liver (H. antarcticus 0.24–0.27%.d−1, L. pholis, 0.44–1.03%.d−1) and GIT were unaffected by temperature in H. antarcticus but increased non-linearly in L.pholis (H. antarcticus 0.22–0.26%.d−1, L. pholis, 0.40–0.86%.d−1). RNA to protein ratios were unaffected by temperature in H. antarcticus but increased weakly, in L.pholis WM and liver. In L.pholis, RNA translational efficiency increased significantly with temperature in all tissues, but only in liver in H. antarcticus. At the overlapping temperature of 3 °C, protein synthesis (WM 26%, Liver, 39%, GIT, 35%) and RNA translational efficiency (WM 273%, Liver, 271%, GIT, 300%) were significantly lower in H. antarcticus than L.pholis, while RNA to protein ratios were significantly higher (WM 270%, Liver 170%, GIT 186%). Tissue specific effects of temperature are detectable in both species. This study provides the first evidence, that tissue protein synthesis rates are constrained in Antarctic fishes.

Lower basal and postprandial muscle protein synthesis after 2 weeks single-leg immobilization in older men: No protective effect of anti-inflammatory medication.

Muscle inactivity may reduce basal and postprandial muscle protein synthesis (MPS) rates in humans. Anti-inflammatory treatment alleviates the MPS impairments in younger individuals. The present study explored the influence of nonsteroidal anti-inflammatory drugs (NSAIDs) upon MPS during a period of inactivity in older humans. Eighteen men (age 60-80 years) were allocated to ibuprofen (1200 mg/day, Ibu) or control (Plc) groups. One lower limb was cast immobilized for 2 weeks. Postabsorptive and postprandial MPS was measured before and after the immobilization by L-[ring-13 C6 ]-phenylalanine infusion. The protein expression of select anabolic signaling molecules was investigated by western blot. Basal (0.038 ± 0.002%/h and 0.039 ± 0.005%/h, Plc and Ibu, respectively) and postprandial (0.064 ± 0.004%/h and 0.067 ± 0.010%/h, Plc and Ibu, respectively) MPS rate were higher pre-immobilization compared to basal (0.019 ± 0.005%/h and 0.020 ± 0.010%/h, Plc and Ibu, respectively) and postprandial (0.033 ± 0.005%/h and 0.037 ± 0.006%/h, Plc and Ibu, respectively) MPS rate post-immobilization (p < 0.001). NSAID treatment did not affect the suppression of MPS (p > 0.05). The anabolic signaling were in general reduced after immobilization (p < 0.05). These changes were unaffected by NSAID treatment (p > 0.05). Basal and postprandial MPS dropped markedly after 2 weeks of lower limb immobilization. NSAID treatment neither influenced the reduction in MPS nor the anabolic signaling after immobilization in healthy older individuals.

Post-prandial muscle protein synthesis rates following the ingestion of pea-derived protein do not differ from ingesting an equivalent amount of milk-derived protein in healthy, young males.

Plant-derived proteins have received considerable attention as an alternative to animal-derived proteins. However, plant-derived proteins are considered to have less anabolic properties when compared with animal-derived proteins. The lower muscle protein synthesis rates following ingestion of plant- compared with animal-derived protein have been attributed to the lower essential amino acid content of plant-derived proteins and/or their specific amino acid deficiencies. This study aimed to compare post-prandial muscle protein synthesis rates following the ingestion of 30g pea-derived protein with 30g milk-derived protein in healthy, young males. In a randomized, double-blind, parallel-group design, 24 young males (24 ± 3 y) received a primed continuous L-[ring-13C6]-phenylalanine infusion after which they ingested 30g pea (PEA) or 30g milk-derived protein (MILK). Blood and muscle biopsies were collected frequently for 5h to assess post-prandial plasma amino acid profiles and subsequent post-prandial muscle protein synthesis rates. MILK increased plasma essential amino acid concentrations more than PEA over the 5h post-prandial period (incremental area under curve 151 ± 31 vs 102 ± 15mmol∙300min∙L-1, respectively; P < 0.001). Ingestion of both MILK and PEA showed a robust muscle protein synthetic response with no significant differences between treatments (0.053 ± 0.013 and 0.053 ± 0.017%∙h-1, respectively; P = 0.96). Post-prandial muscle protein synthesis rates following the ingestion of 30g pea-derived protein do not differ from the response following ingestion of an equivalent amount of milk-derived protein. International Clinical Trials Registry Platform (NTR6548; 27-06-2017).

One Week of Single-Leg Immobilization Lowers Muscle Connective Protein Synthesis Rates in Healthy, Young Adults.

Short periods of limb immobilization lower myofibrillar protein synthesis rates. Within skeletal muscle, the extracellular matrix of connective proteins is recognized as an important factor determining the capacity to transmit contractile force. Little is known regarding the impact of immobilization and subsequent recovery on muscle connective protein synthesis rates. This study examined the impact of 1 wk of leg immobilization and 2 wk of subsequent ambulant recovery on daily muscle connective protein synthesis rates. Thirty healthy, young (24 ± 5 yr) men were subjected to 7 d of one-legged knee immobilization followed by 14 d of ambulant recovery. Deuterium oxide ingestion was applied over the entire period, and muscle biopsy samples were collected before immobilization, after immobilization, and after recovery to measure muscle connective protein synthesis rates and mRNA expression of key extracellular matrix proteins (collagen I, collagen III), glycoproteins (fibronectin, tenascin-C), and proteoglycans (fibromodulin, and decorin). A two-way repeated-measures (time-leg) ANOVA was used to compare changes in muscle connective protein synthesis rates during immobilization and recovery. During immobilization, muscle connective protein synthesis rates were lower in the immobilized (1.07 ± 0.30%·d -1 ) compared with the nonimmobilized (1.48 ± 0.44%·d -1 ; P < 0.01) leg. When compared with the immobilization period, connective protein synthesis rates in the immobilized leg increased during subsequent recovery (1.48 ± 0.64%·d -1 ; P < 0.01). After recovery, skeletal muscle collagen I, collagen III, fibronectin, fibromodulin, and decorin mRNA expression increased when compared with the postimmobilization time point (all P < 0.001). One week of leg immobilization lowers muscle connective protein synthesis rates. Muscle connective protein synthesis rates increase during subsequent ambulant recovery, which is accompanied by increased mRNA expression of key extracellular matrix proteins.

L-Isoleucine reverses hyperammonemia-induced myotube mitochondrial dysfunction and post-mitotic senescence

Perturbations in the metabolism of ammonia, a cytotoxic endogenous metabolite, occur in a number of chronic diseases, with consequent hyperammonemia. Increased skeletal muscle ammonia uptake causes metabolic, molecular, and phenotype alterations including cataplerosis of (loss of tricarboxylic acid cycle (TCA) cycle intermediate) α-ketoglutarate (αKG), mitochondrial oxidative dysfunction, and senescence-associated molecular phenotype (SAMP). L-Isoleucine (Ile) is an essential, branched-chain amino acid (BCAA) that simultaneously provides acetyl-CoA as an oxidative substrate and succinyl-CoA for anaplerosis (providing TCA cycle intermediates). Our multiomics analyses in myotubes and skeletal muscle from hyperammonemic mice and human patients with cirrhosis showed perturbations in BCAA transporters and catabolism. We, therefore, determined if Ile reverses hyperammonemia-induced impaired mitochondrial oxidative function and SAMP. Studies were performed in differentiated murine C2C12 myotubes that were early passage, late passage (senescent), or those depleted of LAT1/SLC7A5 and human induced pluripotent stem cell-derived myotubes (hiPSCM). Ile reverses hyperammonemia-induced reduction in the maximum respiratory capacity, complex I, II, and III functions in early passage murine myotubes and hiPSCM. Consistently, low ATP content and impaired global protein synthesis (high energy requiring cellular process) during hyperammonemia are reversed by Ile in murine myotubes and hiPSCM. Lower abundance of critical regulators of protein synthesis in mTORC1 signaling, and increased phosphorylation of eukaryotic initiation factor 2α are also reversed by Ile. Genetic depletion studies showed that Ile responses are independent of the amino acid transporter LAT1/SLC7A5. Our studies show that Ile reverses the hyperammonemia-induced impaired mitochondrial oxidative function, cataplerosis, and SAMP in a LAT1/SLC7A5 transporter-independent manner.

Lower muscle protein synthesis in humans with obesity concurrent with lower expression of muscle IGF1 splice variants.

This study tested the hypothesis that expression of insulin-like growth factor 1 (IGF-1) protein and mRNA splice variants is lower in skeletal muscle of humans with obesity who have a lower mixed-muscle protein fractional synthesis rate (MMP-FSR) when compared with individuals without obesity. The study included nine participants with obesity (OB, mean [SD], BMI = 35 [3] kg/m2 , MMP-FSR = 0.06%/h [0.02%/h]) and nine participants without obesity (W-OB, BMI = 24 [3] kg/m2 , MMP-FSR = 0.08%/h [0.02%/h]; for both BMI and MMP-FSR p < 0.05). MMP-FSR and mitochondrial protein FSR were measured following an overnight fast. Along with lower MMP-FSR, OB participants displayed lower mitochondrial protein FSR (p = 0.03) compared with W-OB participants. Expression of IGF-1 (p = 0.04) and IGF-1 receptor (p < 0.01) proteins was lower in muscle of OB participants. In addition, OB participantshad lower (p < 0.05) mRNA expression of IGF1 variants Eb and Ec. This study demonstrates that lower protein synthesis in muscle of humans with obesity occurs concurrently with lower expression of muscle IGF-1 and IGF-1 receptor proteins, as well as lower mRNA expression of the IGF1 splice variants. These findings indicate that lower protein synthesis observed in muscle of humans with obesity may result from diminished muscle IGF1 gene expression.

Females display relatively preserved muscle quality compared with males during the onset and early stages of C26-induced cancer cachexia.

Cancer cachexia is clinically defined by involuntary weight loss >5% in <6 mo, primarily affecting skeletal muscle. Here, we aimed to identify sex differences in the onset of colorectal cancer cachexia with specific consideration to skeletal muscle contractile and metabolic functions. Eight-weeks old BALB/c mice (69 males, 59 females) received subcutaneous C26 allografts or PBS vehicle. Tumors were developed for 10-, 15-, 20-, or 25 days. Muscles and organs were collected, in vivo muscle contractility, protein synthesis rate, mitochondrial function, and protein turnover markers were assessed. One-way ANOVA within sex and trend analysis between sexes were performed, P < 0.05. Gastrocnemius and tibialis anterior (TA) muscles became atrophic in male mice at 25 days, whereas female mice exhibited no significant differences in muscle weights at endpoints despite presenting hallmarks of cancer cachexia (fat loss, hepatosplenomegaly). We observed lowered muscle contractility and protein synthesis concomitantly to muscle mass decay in males, with higher proteolytic markers in muscles of both sexes. mRNA of Opa1 was lower in TA, whereas Bnip3 was higher in gastrocnemius after 25 days in male mice, with no significant effect in female mice. Our data suggest relative protections to skeletal muscle in females compared with males despite other canonical signs of cancer cachexia and increased protein degradation markers; suggesting we should place onus upon nonmuscle tissues during early stages of cancer cachexia in females. We noted potential protective mechanisms relating to skeletal muscle contractile and mitochondrial functions. Our findings underline possible heterogeneity in onset of cancer cachexia between biological sexes, suggesting the need for sex-specific approaches to treat cancer cachexia.NEW & NOTEWORTHY Our study demonstrates biological-sex differences in phenotypic characteristics of cancer cachexia between male and female mice, whereby females display many common characteristics of cachexia (gonadal fat loss and hepatosplenomegaly), protein synthesis markers alterations, and common catabolic markers in skeletal muscle despite relatively preserved muscle mass in early-stage cachexia compared with males. Mechanisms of cancer cachexia appear to differ between sexes. Data suggest need to place onus of early cancer cachexia detection and treatment on nonmuscle tissues in females.

Physiology of trans-translation deficiency in Bacillus subtilis - a comparative proteomics study.

trans-Translation is the most effective ribosome rescue system known in bacteria. While it is essential in some bacteria, Bacillus subtilis possesses two additional alternative ribosome rescue mechanisms that require the proteins BrfA or RqcH. To investigate the physiology of trans-translation deficiency in the model organism B.subtilis, we compared the proteomes of B. subtilis168 and a ΔssrA mutant in the mid-log phase using gel-free label-free quantitative proteomics. In chemically defined medium, the growth rate of the ssrA deletion mutant was 20% lower than that of B.subtilis 168. An 35 S-methionine incorporation assay demonstrated that protein synthesis rates were also lower in the ΔssrA strain. Alternative rescue factors were not detected. Among the 34 proteins overrepresented in the mutant strain were eight chemotaxis proteins. Indeed, both on LB agar and minimal medium the ΔssrA strain showed an altered motility and chemotaxis phenotype. Despite the lower growth rate, in the mutant proteome ribosomal proteins were more abundant while proteins related to amino acid biosynthesis were less abundant than in the parental strain. This overrepresentation of ribosomal proteins coupled with a lower protein synthesis rate and down-regulation of precursor supply reflects the slow ribosome recycling in the trans-translation-deficient mutant.

The cost of competency?

Curtailed protein translation ensures stemness and multipotency in embryonic and adult tissue-specific stem cells. In this issue of Cell, a study led by Zhao and colleagues uncovered increased susceptibility of hematopoietic stem cells (HSC) to iron-dependent programmed necrotic cell death (ferroptosis) as a consequence of low protein synthesis.

Adaptive exhaustion during prolonged intermittent hypoxia causes dysregulated skeletal muscle protein homeostasis.

Nocturnal hypoxaemia, which is common in chronic obstructive pulmonary disease (COPD) patients, is associated with skeletal muscle loss or sarcopenia, which contributes to adverse clinical outcomes. In COPD, we have defined this as prolonged intermittent hypoxia (PIH) because the duration of hypoxia in skeletal muscle occurs through the duration of sleep followed by normoxia during the day, in contrast to recurrent brief hypoxic episodes during obstructive sleep apnoea (OSA). Adaptive cellular responses to PIH are not known. Responses to PIH induced by three cycles of 8h hypoxia followed by 16h normoxia were compared to those during chronic hypoxia (CH) or normoxia for 72h in murine C2C12 and human inducible pluripotent stem cell-derived differentiated myotubes. RNA sequencing followed by downstream analyses were complemented by experimental validation of responses that included both unique and shared perturbations in ribosomal and mitochondrial function during PIH and CH. A sarcopenic phenotype characterized by decreased myotube diameter and protein synthesis, and increased phosphorylation of eIF2α (Ser51) by eIF2α kinase, and of GCN-2 (general controlled non-derepressed-2), occurred during both PIH and CH. Mitochondrial oxidative dysfunction, disrupted supercomplex assembly, lower activity of Complexes I, III, IV and V, and reduced intermediary metabolite concentrations occurred during PIH and CH. Decreased mitochondrial fission occurred during CH. Physiological relevance was established in skeletal muscle of mice with COPD that had increased phosphorylation of eIF2α, lower protein synthesis and mitochondrial oxidative dysfunction. Molecular and metabolic responses with PIH suggest an adaptive exhaustion with failure to restore homeostasis during normoxia. KEY POINTS: Sarcopenia or skeletal muscle loss is one of the most frequent complications that contributes to mortality and morbidity in patients with chronic obstructive pulmonary disease (COPD). Unlike chronic hypoxia, prolonged intermittent hypoxia is a frequent, underappreciated and clinically relevant model of hypoxia in patients with COPD. We developed a novel, in vitro myotube model of prolonged intermittent hypoxia with molecular and metabolic perturbations, mitochondrial oxidative dysfunction, and consequent sarcopenic phenotype. In vivo studies in skeletal muscle from a mouse model of COPD shared responses with our myotube model, establishing the pathophysiological relevance of our studies. These data lay the foundation for translational studies in human COPD to target prolonged, nocturnal hypoxaemia to prevent sarcopenia in these patients.

Muscle characteristics comparison and targeted metabolome analysis reveal differences in carcass traits and meat quality of three pig breeds.

This study investigated the molecular basis for differences in meat yield and quality between Duroc, Taoyuan black (TB), and Xiangcun black (XB) pigs. The results show that TB pigs have lower carcass weight, lean percentage, pH decline, and glycolytic potential but have higher fat percentage, water- holding capacity, intramuscular fat content, antioxidant capacity, and percentage of slow-twitch fibers than the Duroc pigs. Moreover, muscles of TB pigs have lower protein synthesis and lipolysis gene expression than the muscles of Duroc pigs. Targeted metabolome analysis indicates that 24 metabolites significantly differ among these three pig breeds. Correlation analysis suggests that L-malic acid and β-alanine contents in muscle are closely related to meat quality. These findings suggest that the excellent meat quality of TB pigs is closely related to muscle metabolism and fiber characteristics, while lower protein synthesis and lipolysis may contribute to less meat yield.

Escherichia coli protein synthesis is limited by mRNA availability rather than ribosomal capacity during phosphate starvation.

Protein synthesis is the most energetically costly process in the cell. Consequently, it is a tightly regulated process, and regulation of the resources allocated to the protein synthesis machinery is at the heart of bacterial growth optimization theory. However, the molecular mechanisms that result in dynamic downregulation of protein synthesis in response to nutrient starvation are not well described. Here, we first quantify the Escherichia coli response to phosphate starvation at the level of accumulation rates for protein, RNA and DNA. Escherichia coli maintains a low level of protein synthesis for hours after the removal of phosphate while the RNA contents decrease, primarily as a consequence of ribosomal RNA degradation combined with a reduced RNA synthesis rate. To understand the molecular basis for the low protein synthesis rate of phosphate-starved cells, template mRNA for translation was overproduced in the form of a highly induced long-lived mRNA. Remarkably, starved cells increased the rate of protein synthesis and reduced the rate of ribosomal RNA degradation upon mRNA induction. These observations suggest that protein synthesis in phosphate-starved cells is primarily limited by the availability of template, and does not operate at the maximum capacity of the ribosomes. We suggest that mRNA limitation is an adaptive response to phosphate starvation that prevents the deleterious consequences of overcommitting resources to protein synthesis. Moreover, our results support the model that degradation of ribosomal RNA occurs as a consequence of the availability of idle ribosomal subunits.

Review on microbially synthesized nanoparticles: a potent biocontrol agent against Aedes species of mosquito

Aedes aegypti is a principal vector of dengue, chikungunya and Zika illnesses worldwide. Because effective vaccinations or medications for the prevention and/or treatment for these illnesses are not available, vector management has been embraced as the primary method of reducing their transmission. The strategy that is often used for controlling Aedes populations is chemical insecticides; however, despite their efficiency, extensive usage of these pesticides has resulted in high operating costs, the establishment of resistant populations and undesirable non-target impacts. Therefore, the use of nanoparticles derived from multiple synthesis processes as new insecticides has recently attracted the interest of researchers. This study provides an overview of the current understanding concerning the method of action of nanoparticles against mosquitoes. Metal nanoparticles strongly influence antioxidant and detoxifying enzymes in insects, causing oxidative stress and cell death. These nanoparticles similarly inhibited acetylcholinesterase activity. Metal-based nanoparticles can potentially bind to S and P units in proteins and nucleic acids respectively resulting in decreased membrane permeability, organelle and enzyme denaturation which leads to cell death. Furthermore, they upregulate and downregulate critical insect genes, lower protein synthesis and gonadotrophin release, resulting in developmental and reproductive failure. This review critically examines insect nanotoxicology research trends and highlights the ecotoxicological effects of practical usage of nanoparticles as insecticides.

Preterm birth alters the feeding-induced activation of Akt signaling in the muscle of neonatal piglets.

Postnatal lean mass accretion is commonly reduced in preterm infants. This study investigated mechanisms involved in the blunted feeding-induced activation of Akt in the skeletal muscle of preterm pigs that contributes to lower protein synthesis rates. On day 3 following cesarean section, preterm and term piglets were fasted or fed an enteral meal. Activation of Akt signaling pathways in skeletal muscle was determined. Akt1 and Akt2, but not Akt3, phosphorylation were lower in the skeletal muscle of preterm than in term pigs (P < 0.05). Activation of Akt-positive regulators, PDK1 and mTORC2, but not FAK, were lower in preterm than in term (P < 0.05). The formation of Akt complexes with GAPDH and Hsp90 and the abundance of Ubl4A were lower in preterm than in term (P < 0.05). The abundance of Akt inhibitors, PHLPP and SHIP2, but not PTEN and IP6K1, were higher in preterm than in term pigs (P < 0.05). PP2A activation was inhibited by feeding in term but not in preterm pigs (P < 0.05). Our results suggest that preterm birth impairs regulatory components involved in Akt activation, thereby limiting the anabolic response to feeding. This anabolic resistance likely contributes to the reduced lean accretion following preterm birth. The Akt-mTORC1 pathway plays an important role in the regulation of skeletal muscle protein synthesis in neonates. This is the first evidence to demonstrate that, following preterm birth, the postprandial activation of positive regulators of Akt in the skeletal muscle is reduced, whereas the activation of negative regulators of Akt is enhanced. This anabolic resistance of Akt signaling in response to feeding likely contributes to the reduced accretion of lean mass in premature infants. These results may provide potential novel molecular targets for intervention to enhance lean growth in preterm neonates.

Hematopoietic Stem Cells Depend upon Aggrephagy to Maintain Protein Homeostasis and Self-Renewal Activity

Hematopoietic stem cells (HSCs) regenerate blood and immune cells throughout life. To preserve their health and longevity, HSCs are particularly dependent on maintaining protein homeostasis (proteostasis). HSCs maintain proteostasis partly by sustaining low protein synthesis rates that limit the biogenesis of misfolded proteins. Nevertheless, HSCs ultimately accumulate misfolded proteins that must be eliminated to preserve stem cell fitness. However, how HSCs purge misfolded proteins to maintain proteostasis is mostly unknown. We found that in contrast to most cell types that utilize the proteasome to degrade misfolded proteins, HSCs depend on aggrephagy, a selective form of autophagy, to maintain proteostasis in vivo. Young adult HSCs exhibit unusually high autophagic activity, and genetically disabling autophagy results in significant accumulation of protein aggregates and impaired HSC function. Furthermore, we determined that mouse and human HSCs preferentially express Bag3, a critical stress response gene that promotes aggrephagy and transport of misfolded proteins to aggresomes. Aggresomes are cytosolic inclusion bodies containing protein aggregates that typically form under stress conditions to help maintain proteostasis. Surprisingly, we found that the vast majority of HSCs contain aggresomes, even under steady state conditions in vivo. Conditional deletion of Bag3 causes HSCs to accumulate protein aggregates and impairs their self-renewal activity in vivo. Finally, we determined that old adult HSCs exhibit diminished autophagic flux and become increasingly reliant on the proteasome to degrade misfolded proteins. Overall, these studies demonstrate that protein degradation pathways are uniquely configured in HSCs to preserve proteostasis and fitness, and that disruptions in proteostasis network activity contribute to cellular and molecular changes in HSCs during aging.

Protein Homeostasis Is Dynamically Regulated throughout Hematopoietic Stem Cell Ontogeny

Hematopoietic stem cells (HSCs) are required for establishing hematopoiesis during development and for maintaining lifelong regeneration of blood cells. As demands for blood cell production change throughout ontogeny, there is a commensurate change in HSC biology. Fetal, neonatal, and adult HSCs exhibit differences in gene expression, developmental potential, and self-renewal activity. Thus, fetal, neonatal, and adult HSCs are, at least functionally, distinct populations. We recently found that fetal HSCs exhibit substantially higher rates of protein synthesis as compared to adult HSCs, which depend on unusually low protein synthesis rates to limit the biogenesis of misfolded and unfolded proteins to preserve protein homeostasis (proteostasis) and fitness. This raised questions of whether fetal HSCs experience significant proteotoxic stress in vivo and how proteostasis is regulated throughout HSC development. Remarkably, we found that fetal HSCs restrict accumulation of unfolded proteins to levels similar to those observed in adult HSCs, despite having nearly 7-fold higher protein synthesis rates. Surprisingly, however, we determined that unfolded protein abundance spikes dramatically within HSCs at birth. This transient burst of proteotoxic stress is followed by rapid restoration of proteostasis in neonates, as unfolded protein abundance declines precipitously, ultimately reaching unusually low levels by P14 before moderately rising again in adulthood. In contrast to the dynamic oscillation of unfolded protein abundance, protein synthesis rates progressively decline throughout HSC ontogeny. Fetal HSCs synthesize significantly more protein per hour than HSCs from newborn and postnatal mice, which in turn have nearly 3-fold and 2-fold higher protein synthesis rates than adult HSCs, respectively. Strikingly, however, postnatal declines in HSC protein synthesis occur gradually and non-uniformly. While fetal and adult HSCs have narrow distributions of protein synthesis rates, neonatal HSCs exhibit significant heterogeneity, suggesting an uncoordinated transition to achieving low protein synthesis rates and proteostasis in adulthood. Taken together, these findings indicate that HSC ontogeny is associated with rapid, dynamic, and robust changes in proteostasis activity, and that fetal, neonatal, and adult HSCs utilize distinct mechanisms to regulate proteostasis in vivo. The regulation of proteostasis is thus a key distinguishing feature of HSC ontogeny that could provide a framework to uncover how pathogenic changes disrupt hematopoietic development and function in an age-specific manner. Furthermore, harnessing developmental proteostasis mechanisms could open new avenues to restore HSC fitness in response to various stressors and blood disorders that emerge throughout life.

CCRG-01. KAT5 ACTIVITY REGULATES G0-LIKE STATES IN HUMAN GLIOMAS

Abstract Glioblastoma, the most aggressive type of primary brain cancer, benefits little from standard of care therapy. Over the past few decades not much advancement has been made in improving the recurrence-free survival after treatment, with a median survival period of 15 month from diagnostic. G0-like states are thought to act a reservoir for tumor recurrence after treatment in glioblastoma. Targeting G0 states in glioblastoma poses an attractive therapeutic approach, however, little progress has been made in the field, likely due to poorly understood mechanisms that regulate G0 ingress and egress. To find regulators of G0 states, we performed a genome-wide CRISPR-Cas9 screen of patient-derived glioblastoma stem cells. We identified KAT5, a histone acetyltransferase coding gene which is the catalytic subunit of the histone acetyltransferase complex NuA4, as an important candidate for regulating G0 ingress and egress. We found that in primary gliomas, KAT5-low cells display G0-like properties, while overall KAT5 activity increases from low to high grade tumors. To study G0 states in glioblastoma, we have engineered an inducible KAT5 system that functions as a fully tunable model system, where we can control expression of KAT5 and, therefore, effectively control glioma cell entry and exit from G0-like states. We provide evidence that G0-like states are characterized by hypoacetylated histones and low protein synthesis rates, which remarkably induces shifts in cell state reminiscent of dedifferentiation and acquisition of stem-like behavior. Our work demonstrates that regulation of G0 like states may be coupled to the generation of tumor heterogeneity and, more importantly, suggests a potential strategy whereby inhibiting KAT5 activity could effectively "down grade" GBM tumors by lengthening residence time in G0-like states, significantly increasing survival times. For these studies we will present a variety of techniques, including single cell RNA-seq to characterize cellular subpopulations in tumors and GBM stem-like cell cultures.

Anabolic Characterization Of Immortalized Murine L6, And Canine Myok9 Skeletal Myotubes

Healthy skeletal muscle is an important site of metabolic regulation. Metabolic dysregulation of skeletal muscle contributes to the development of obesity, the metabolic syndrome, and muscle degeneration. Interest in developing an in vitro canine model has been increasing as canine skeletal muscle is more pathologically similar to human skeletal muscle than rodent. In vitro models are useful to control variables, reduce clinical costs, and assess molecular characteristics of tissue. Therefore, characterization of Myok9 myotube anabolic function and comparison to an established cell line is necessary to understand metabolic function of this novel cell line for its use in the study of skeletal muscle dysfunction and disease. PURPOSE: To describe anabolic signaling and determine fractional synthesis rates of novel Myok9 myotubes when compared to established murine L6 myotubes. METHODS: L6 and Myok9 myoblasts were grown in vitro in standard culture medium until 70% confluent. Differentiation was induced by switching to a low serum medium. Media containing 4% deuterium oxide (stable isotope label) supplemented with the appropriate pharmacological treatment (DMSO, rapamycin, and torin1) was added to myotubes during the final 24-hours of the 7-day differentiation period. Cells and media samples were harvested for fractional synthesis rates, and protein content of key anabolic markers using Western blotting techniques. RESULTS: Myofibrillar protein synthesis rates (MPS) were similar between species, but slightly lower in canine versus rat cells. Both cell lines exhibited significantly lower MPS in the myotubes treated with torin1. The phosphorylated to total protein ratios of 4EBP1 were significantly lower in both L6 and Myok9 myotubes treated with torin1. The phosphorylated to total protein ratios of p70S6K were significantly lower in L6 and Myok9 myotubes treated with rapamycin or torin1. CONCLUSION: Similar anabolic responses to pharmacologic inhibition occurred in both L6 and Myok9 myotubes. Interestingly, MPS rates in Myok9 myotubes were slightly lower compared to L6 myotubes. These results suggest Myok9 cell line could be used in the development of pharmacologic treatments of skeletal muscle dysfunction or disease while remaining cognizant of lower overall protein synthesis rates.