Capacity For Protein Synthesis Research Articles

HectD1 controls hematopoietic stem cell regeneration by coordinating ribosome assembly and protein synthesis.

Impaired ribosome function is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. However, how ribosomes are regulated remains poorly understood, as are approaches to restore hematopoietic stem cell (HSC) function loss because of defective ribosome biogenesis. Here we reveal a role of the E3 ubiquitin ligase HectD1 in regulating HSC function via ribosome assembly and protein translation. Hectd1-deficient HSCs exhibit a striking defect in transplantation ability and exvivo maintenance concomitant with reduced protein synthesis and growth rate under stress conditions. Mechanistically, HectD1 ubiquitinates and degrades ZNF622, an assembly factor for the ribosomal 60S subunit. Hectd1 loss leads to accumulation of ZNF622 and the anti-association factor eIF6 on 60S, resulting in 60S/40S joining defects. Importantly, Znf622 depletion in Hectd1-deficient HSCs restored ribosomal subunit joining, protein synthesis, and HSC reconstitution capacity. These findings highlight the importance of ubiquitin-coordinated ribosome assembly in HSC regeneration.

Interactions between Growth of Muscle and Stature: Mechanisms Involved and Their Nutritional Sensitivity to Dietary Protein: The Protein-Stat Revisited.

Childhood growth and its sensitivity to dietary protein is reviewed within a Protein-Stat model of growth regulation. The coordination of growth of muscle and stature is a combination of genetic programming, and of two-way mechanical interactions involving the mechanotransduction of muscle growth through stretching by bone length growth, the core Protein-Stat feature, and the strengthening of bone through muscle contraction via the mechanostat. Thus, growth in bone length is the initiating event and this is always observed. Endocrine and cellular mechanisms of growth in stature are reviewed in terms of the growth hormone-insulin like growth factor-1 (GH-IGF-1) and thyroid axes and the sex hormones, which together mediate endochondral ossification in the growth plate and bone lengthening. Cellular mechanisms of muscle growth during development are then reviewed identifying (a) the difficulties posed by the need to maintain its ultrastructure during myofibre hypertrophy within the extracellular matrix and the concept of muscle as concentric “bags” allowing growth to be conceived as bag enlargement and filling, (b) the cellular and molecular mechanisms involved in the mechanotransduction of satellite and mesenchymal stromal cells, to enable both connective tissue remodelling and provision of new myonuclei to aid myofibre hypertrophy and (c) the implications of myofibre hypertrophy for protein turnover within the myonuclear domain. Experimental data from rodent and avian animal models illustrate likely changes in DNA domain size and protein turnover during developmental and stretch-induced muscle growth and between different muscle fibre types. Growth of muscle in male rats during adulthood suggests that “bag enlargement” is achieved mainly through the action of mesenchymal stromal cells. Current understanding of the nutritional regulation of protein deposition in muscle, deriving from experimental studies in animals and human adults, is reviewed, identifying regulation by amino acids, insulin and myofibre volume changes acting to increase both ribosomal capacity and efficiency of muscle protein synthesis via the mechanistic target of rapamycin complex 1 (mTORC1) and the phenomenon of a “bag-full” inhibitory signal has been identified in human skeletal muscle. The final section deals with the nutritional sensitivity of growth of muscle and stature to dietary protein in children. Growth in length/height as a function of dietary protein intake is described in the context of the breastfed child as the normative growth model, and the “Early Protein Hypothesis” linking high protein intakes in infancy to later adiposity. The extensive paediatric studies on serum IGF-1 and child growth are reviewed but their clinical relevance is of limited value for understanding growth regulation; a role in energy metabolism and homeostasis, acting with insulin to mediate adiposity, is probably more important. Information on the influence of dietary protein on muscle mass per se as opposed to lean body mass is limited but suggests that increased protein intake in children is unable to promote muscle growth in excess of that linked to genotypic growth in length/height. One possible exception is milk protein intake, which cohort and cross-cultural studies suggest can increase height and associated muscle growth, although such effects have yet to be demonstrated by randomised controlled trials.

Control of ribosomal protein synthesis by the Microprocessor complex.

Ribosome biogenesis in eukaryotes requires the coordinated production and assembly of 80 ribosomal proteins and four ribosomal RNAs (rRNAs), and its rate must be synchronized with cellular growth. Here, we showed that the Microprocessor complex, which mediates the first step of microRNA processing, potentiated the transcription of ribosomal protein genes by eliminating DNA/RNA hybrids known as R-loops. Nutrient deprivation triggered the nuclear export of Drosha, a key component of the Microprocessor complex, and its subsequent degradation by the E3 ubiquitin ligase Nedd4, thereby reducing ribosomal protein production and protein synthesis. In mouse erythroid progenitors, conditional deletion of Drosha led to the reduced production of ribosomal proteins, translational inhibition of the mRNA encoding the erythroid transcription factor Gata1, and impaired erythropoiesis. This phenotype mirrored the clinical presentation of human "ribosomopathies." Thus, the Microprocessor complex plays a pivotal role in synchronizing protein synthesis capacity with cellular growth rate and is a potential drug target for anemias caused by ribosomal insufficiency.

Reduced rDNA transcription diminishes skeletal muscle ribosomal capacity and protein synthesis in cancer cachexia.

Muscle wasting in cancer is associated with deficits in protein synthesis, yet, the mechanisms underlying this anabolic impairment remain poorly understood. The capacity for protein synthesis is mainly determined by the abundance of muscle ribosomes, which is in turn regulated by transcription of the ribosomal (r)RNA genes (rDNA). In this study, we investigated whether muscle loss in a preclinical model of ovarian cancer is associated with a reduction in ribosomal capacity and was a consequence of impaired rDNA transcription. Tumor bearing resulted in a significant loss in gastrocnemius muscle weight and protein synthesis capacity, and was consistent with a significant reduction in rDNA transcription and ribosomal capacity. Despite the induction of the ribophagy receptor NUFIP1 mRNA and the loss of NUFIP1 protein, in vitro studies revealed that while inhibition of autophagy rescued NUFIP1, it did not prevent the loss of rRNA. Electrophoretic analysis of rRNA fragmentation from both in vivo and in vitro models showed no evidence of endonucleolytic cleavage, suggesting that rRNA degradation may not play a major role in modulating muscle ribosome abundance. Our results indicate that in this model of ovarian cancer-induced cachexia, the ability of skeletal muscle to synthesize protein is compromised by a reduction in rDNA transcription and consequently a lower ribosomal capacity. Thus, impaired ribosomal production appears to play a key role in the anabolic deficits associated with muscle wasting in cancer cachexia.

Fibroblast involvement in cardiac remodeling and repair under ischemic conditions.

Cardiac fibroblasts play a main role in the physiological turnover of the extracellular matrix, as well as its pathological remodeling. A study was performed on a batch of 23 cases who died of various cardiac complications secondary to scarring myocardial infarctions. The aim of the study was to assess the fibroblast involvement in cardiac repair under ischemic conditions after myocardial infarction. Tissue myocardial samples from the left ventricle were taken from these cases for microscopy examination, in order to investigate the type and degree of fibrosis as well as the presence of cardiac interstitial fibroblasts. Multiple series of histological sections were also performed and examined, along with immunohistochemical analysis. The fibroblasts were diffusely distributed in the interstitium among the residual cardiomyocytes, showing variable expression of vimentin and smooth muscle actin. During cardiac remodeling, there was a successive interstitial deposition, first of reticulin fibers and then of collagen fibers, leading to interstitial fibrosis and myocardial replacement. There was a correlation between vimentin and smooth muscle actin expression and collagen deposition. Fibrosis with cardiac remodeling is based on maintaining proliferation capacity of the fibroblast and its capacity of protein synthesis in the extracellular matrix. Under hypoxic ischemic conditions, followed by myocardial infarction, the fibroblast switches phenotype and transdifferentiate into myofibroblast, contributing to the healing by secreting extracellular matrix proteins and collagen deposition, with subsequent cardiac remodeling and regulation of the micro-environment metabolism.

Metabolomic profiling reveals the effects of early-life lactoferrin intervention on protein synthesis, energy production and antioxidative capacity in the liver of suckling piglets.

This study aimed to determine the effects of an early-life lactoferrin (LF) intervention on liver metabolism in suckling piglets. Sixty newborn piglets with an average initial body weight (BW) of 1.51 ± 0.05 kg were assigned to a control (CON) group and an LF group. At age 1 to 7 days, the piglets in the LF group were orally administered LF solution (0.5 g per kg BW daily), whereas the piglets in the CON group were orally administered the same dose of physiological saline. Plasma, jejunum and liver samples were collected on days 8 and 21. The LF piglets showed a decreased plasma urea nitrogen level on day 8 and an increased plasma albumin level on day 21. Pathway analysis of the metabolomic profiles showed that the LF treatment affected amino acid metabolism in the liver. In addition, the LF treatment upregulated the gene expression levels of proteolytic enzymes and amino acid transporters (APA, APN, EAAC1, Pept1, CAT1, B0AT1 and ASCT2) in the jejunum, and it enhanced the phosphorylation levels of mTOR and p70S6K in the liver. The LF treatment also upregulated the expression of a β-oxidation-related gene (CPT1) and affected the tricarboxylic acid cycle in the liver on day 21. Furthermore, the LF piglets showed a decreased level of malondialdehyde and increased levels of GSH, GSH-Px and GCLC in the liver mitochondria. Overall, the early-life LF intervention affected the protein synthesis, energy production and antioxidative capacity in the liver of the neonatal piglets.

Rates of gene conversions between Escherichia coli ribosomal operons.

Due to their universal presence and high sequence conservation, ribosomal RNA (rRNA) sequences are used widely in phylogenetics for inferring evolutionary relationships between microbes and in metagenomics for analyzing the composition of microbial communities. Most microbial genomes encode multiple copies of rRNA genes to supply cells with sufficient capacity for protein synthesis. These copies typically undergo concerted evolution that keeps their sequences identical, or nearly so, due to gene conversion, a type of intragenomic recombination that changes one copy of a homologous sequence to exactly match another. Widely varying rates of rRNA gene conversion have previously been estimated by comparative genomics methods and using genetic reporter assays. To more directly measure rates of rRNA intragenomic recombination, we sequenced the seven Escherichia coli rRNA operons in 15 lineages that were evolved for ∼13,750 generations with frequent single-cell bottlenecks that reduce the effects of selection. We identified 38 gene conversion events and estimated an overall rate of intragenomic recombination within the 16S and 23S genes between rRNA copies of 3.6 × 10−4 per genome per generation or 8.6 × 10−6 per rRNA operon per homologous donor operon per generation. This rate varied only slightly from random expectations at different sites within the rRNA genes and between rRNA operons located at different positions in the genome. Our accurate estimate of the rate of rRNA gene conversions fills a gap in our quantitative understanding of how ribosomal sequences and other multicopy elements diversify and homogenize during microbial genome evolution.

Cytosolic and Membrane-Associated Ribosomes’ mRNA Profiles are Differentially Regulated by Learning and Sleep in Hippocampal Neurons

The hippocampus plays an essential role in consolidating transient experiences into long-lasting memories. Memory consolidation can be facilitated by post-learning sleep, although the underlying cellular mechanisms are largely unknown. We took an unbiased approach to this question using a mouse model of hippocampally-mediated, sleep-dependent memory consolidation (contextual fear memory; CFM), which is known to be disrupted by post-learning sleep loss. Translating ribosome affinity purification (TRAP) was used to quantify ribosome-associated RNAs in different subcellular compartments (cytosol and membrane) and in different hippocampal cell populations (either whole hippocampus, Camk2a+ excitatory neurons, or highly active neurons expressing phosphorylated ribosomal subunit S6 [pS6+]). Using RNA-seq, we examined how these transcript profiles change as a function of sleep vs. sleep deprivation (SD), and as a function of prior learning (contextual fear conditioning; CFC). To our surprise, we found that while many mRNAs on cytosolic ribosomes were altered by sleep loss, almost none were altered by learning. Of the few changes in cytosolic ribosomal transcript abundance following CFC, almost all were occluded by subsequent SD. This effect was particularly pronounced in pS6+ neurons with the highest level of neuronal activity following CFC, suggesting SD-induced disruption of post-learning transcript changes in putative “engram” neurons. In striking contrast, far fewer transcripts on membrane-bound (MB) ribosomes were altered by SD, and many more mRNAs (and lncRNAs) were altered MB ribosomes as a function of prior learning. For hippocampal neurons, cellular pathways most significantly affected by CFC were involved in structural remodeling. Comparisons of post-CFC MB transcript profiles between freely-sleeping and SD mice implicated changes in cellular metabolism in Camk2a+ neurons, and increased protein synthesis capacity in pS6+ neurons, as biological processes disrupted by post-learning sleep loss. Together these findings suggest that disruptive effects of sleep loss on hippocampal memory consolidation are both cell type- and subcellular compartment-specific.

A Novel Method for Long-Term Analysis of Lactic Acid and Ammonium Production in Non-growing Lactococcus lactis Reveals Pre-culture and Strain Dependence.

In various (industrial) conditions, cells are in a non-growing but metabolically active state in which de novo protein synthesis capacity is limited. The production of a metabolite by such non-growing cells is dependent on the cellular condition and enzyme activities, such as the amount, stability, and degradation of the enzyme(s). For industrial fermentations in which the metabolites of interest are mainly formed after cells enter the stationary phase, the investigation of prolonged metabolite production is of great importance. However, current batch model systems do not allow prolonged measurements due to metabolite accumulation driving product-inhibition. Here we developed a protocol that allows high-throughput metabolic measurements to be followed in real-time over extended periods (weeks). As a validation model, sugar utilization and arginine consumption by a low density of translationally blocked Lactococcus lactis was designed in a defined medium. In this system L. lactis MG1363 was compared with its derivative HB60, a strain described to achieve higher metabolic yield through a shift toward heterofermentative metabolism. The results showed that in a non-growing state HB60 is able to utilize more arginine than MG1363, and for both strains the decay of the measured activities were dependent on pre-culture conditions. During the first 5 days of monitoring a ∼25-fold decrease in acidification rate was found for strain HB60 as compared to a ∼20-fold decrease for strain MG1363. Such measurements are relevant for the understanding of microbial metabolism and for optimizing applications in which cells are frequently exposed to long-term suboptimal conditions, such as microbial cell factories, fermentation ripening, and storage survival.

Breath Biopsy Assessment of Liver Disease Using an Exogenous Volatile Organic Compound-Toward Improved Detection of Liver Impairment.

INTRODUCTION:Liver cirrhosis and its complication — hepatocellular carcinoma (HCC) — have been associated with increased exhaled limonene. It is currently unclear whether this increase is more strongly associated with the presence of HCC or with the severity of liver dysfunction.METHODS:We compared the exhaled breath of 40 controls, 32 cirrhotic patients, and 12 cirrhotic patients with HCC using the Breath Biopsy platform. Breath samples were analyzed by thermal desorption–gas chromatography–mass spectrometry. Limonene levels were compared between the groups and correlated to bilirubin, albumin, prothrombin time international normalized ratio, and alanine aminotransferase.RESULTS:Breath limonene concentration was significantly elevated in subjects with cirrhosis-induced HCC (M: 82.1 ng/L, interquartile range [IQR]: 16.33–199.32 ng/L) and cirrhosis (M: 32.6 ng/L, IQR: 6.55–123.07 ng/L) compared with controls (M: 6.2 ng/L, IQR: 2.62–9.57 ng/L) (P value = 0.0005 and 0.0001, respectively) with no significant difference between 2 diseased groups (P value = 0.37). Levels of exhaled limonene correlated with serum bilirubin (R2 = 0.25, P value = 0.0016, r = 0.51), albumin (R2 = 0.58, P value = 5.3e-8, r = −0.76), and international normalized ratio (R2 = 0.29, P value = 0.0003, r = 0.51), but not with alanine aminotransferase (R2 = 0.01, P value = 0.36, r = 0.19).DISCUSSION:Exhaled limonene levels are primarily affected by the presence of cirrhosis through reduced liver functional capacity, as indicated by limonene correlation with blood metrics of impaired hepatic clearance and protein synthesis capacity, without further alterations observed in subjects with HCC. This suggests that exhaled limonene is a potential non-invasive marker of liver metabolic capacity (see Visual abstract, Supplementary Digital Content 1, http://links.lww.com/CTG/A388).

CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody in Escherichia coli.

Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to increase the production of a single‐domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways and found that the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the production of two versions of an expression‐optimized sdAb. Proteomics analysis of the sdAb‐producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome‐associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake‐flask fermentation where the product yield was improved 2.6‐fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best‐performing pyrG growth decoupling strain.

A year in the salt marsh: Seasonal changes in gill protein expression in the temperate intertidal mussel Geukensia demissa

Organisms living in temperate and polar regions experience extensive seasonal changes in the physical and biotic environment, including temperature, insolation, and food availability, among other factors. Sessile intertidal organisms respond to such seasonal fluctuations largely through physiological and biochemical means, because their behavioral responses are severely limited. In this study, we used a proteomic approach to examine changes in seasonal protein expression of gill from the intertidal mussel Geukensia demissa, a keystone species of the western Atlantic salt marsh, over the course of one year. Gill tissue of mussels collected in summer had the greatest number of proteins significantly increased in abundance (37 of 592 spots detected on two-dimensional polyacrylamide gels), although autumn mussels revealed a comparable proportion of up-regulated proteins (31 spots). In contrast, the number of proteins changing in abundance in winter and spring mussels were substantially smaller (15 and 9, respectively). Identification of these proteins revealed both expected and unanticipated changes to the proteome. Maintenance of gill cilia dominates in the summer when filter-feeding is most active, as evidenced by cytoskeletal proteins such as tektin-4 and tubulin isoforms; a signal of protection from heat stress is also present in summer (e.g., heat shock cognate 70). In autumn oxidative stress protection (peroxiredoxin-5 and manganese-containing superoxide dismutase) and aerobic ATP synthetic capacity (ATP synthase subunits a and delta) appear to increase. In winter a signal of cold-induced oxidative stress is apparent (Mn-SOD and NADP-dependent isocitrate dehydrogenase), perhaps in association with heavy metal toxicity and exposure to pathogens. Gill tissue from spring shows relatively little environmental acclimatization, other than a possible increase in protein synthesis capacity.

Pretreatment with Retro-2 protects cells from death caused by ricin toxin by retaining the capacity of protein synthesis.

The current study explores the detoxification effect of Retro-2 on ricin toxin (RT) cytotoxicity, as well as the mechanisms underlying such effects, to provide a basis for follow-up clinical applications of Retro-2. The mouse-derived mononuclear/macrophage cell line, RAW264.7, was used to evaluate the detoxification effect of Retro-2 on RT by detecting cell viability, capacity for protein synthesis and the expression of cytokines, as well as endoplasmic reticulum stress (ERS)-related mRNA. The results indicated that many cells died when challenged with concentrations of RT ≥50ng/mL. The protein synthesis capacity of cells decreased when challenged with 200ng/mL RT for 2hours. Furthermore, the synthesis and release of many cytokines decreased, while the expression of cytokines or ERS-related mRNA increased when challenged with 200ng/mL of RT for 12 or more hours. However, cell viability, capacity for protein synthesis and release levels of many cytokines were higher, while the expression levels of cytokine, or ERS-related mRNA, were lower in cells pretreated with 20μm Retro-2 and challenged with RT, compared with those that had not been pretreated with Retro-2. In conclusion, Retro-2 retained the capacity for protein synthesis inhibited by RT, alleviated ERS induced by RT and increased the viability of cells challenged with RT. Retro-2 shows the potential for clinical applications.

Zones of biosynthesis.

Morral, Stanisavljevic et al. show that colorectal cancer (CRC) cell hierarchy is based on the capacity to perform biosynthesis. Undifferentiated CRC cells that have high capacity for protein synthesis were found immediately adjacent to the stroma, and this capacity was lost upon differentiation.

Combined Yeast Cultivation and Pectin Hydrolysis as an Effective Method of Producing Prebiotic Animal Feed from Sugar Beet Pulp

An effective and ecological method for liberation of pectin-derived oligosaccharides (POS) from sugar beet pulp (SBP) was developed using enzymatic and microorganism-mediated biomass conversion. The POS may be applied in the production of prebiotic feed additives. Various yeast strains were screened for their capacity for protein synthesis and monosaccharide assimilation. Combined yeast cultivation and pectin hydrolysis were found to be an effective method of producing prebiotics. Separate enzymatic hydrolysis and fermentation of SBP resulted in the release of 3.6 g of POS per 100 g d.w., whereas the yield of POS acquired after the combined process was 17.9% higher, giving 4.2 g of POS per 100 g d.w. Introducing the yeast into the process improved hydrolysis performance due to lower enzyme inhibition by mono- and disaccharides. The prebiotic effect of the POS was assessed by in vitro fermentation using individual cultures of gastrointestinal bacteria. The POS in the SBP hydrolysate effectively promoted the growth of lactobacilli and bifidobacteria. A large increase in adherence to Caco-2 cells in the presence of POS was noted for beneficial Lactobacillus brevis strains, whereas pathogenic bacteria and yeast (C. albicans, C. lusitanie, C. pelliculosa), responsible for infections in breeding animals, showed much weaker adhesion.

Biological interaction levels of zinc oxide nanoparticles; lettuce seeds as case study

BackgroundSeed germination is a critical stage in plant life, and recent practices use nanomaterials for the improvement of plant seed germination indices. This study was conducted to assess the effect of laboratory prepared zinc oxide nanoparticles on the physiological and biochemical changes of lettuce seeds. MethodsLettuce seeds were soaked in a suspension of moderately polydisperse zinc oxide nanoparticles at two different concentrations (25 ppm or 50 ppm) and shaken for 3 h at 25 °C. Seeds treatment was followed subsequently by two to three days drying at ambient conditions. Treated seeds were stored for 3–4 weeks, at ambient conditions and then tested for germination in petri dishes. Germination was observed on daily basis and seedling length was measured. After imbibition and before the start of the visible germination, seeds were examined for topography and surface analysis using the scanning electron microscope and zinc uptake was measured by using the atomic absorption spectrometry and the energy dispersive X-ray. The pattern of mobilization of biomolecules was analyzed to detect any differences among different seed groups. ResultsThere was no loss of viability for the nanoparticles treated seeds. Indeed their germination was enhanced and their biomass increased. The activated performance of the nanoparticles imbibed seeds has been found to be correlated with an increased level of Zn inside lettuce seeds. The recorded measurements show a significant enhancement of seedling length. Interaction of zinc oxide nanoparticles with lettuce seeds mediates a variation in the biochemical processes. Changes detected in treated seeds were as following: reduced levels of the total carbohydrates (including simple saccharides and polysaccharides), higher capacity of protein synthesis, an elevated level of starch as well as an increased activity of antioxidant enzymes. Discussion and conclusionLettuce seeds primed with ZnO nanoparticles were found not only to maintain seed viability but even to exhibit a detectable level of germination enhancement compared to the control seeds. Overall, the promoted response of lettuce seeds during early stages of seed growth is encouraging for the application of ZnO NPs for seed priming for better germination indices.

Macromolecular crowding links ribosomal protein gene dosage to growth rate in Vibrio cholerae

BackgroundIn fast-growing bacteria, the genomic location of ribosomal protein (RP) genes is biased towards the replication origin (oriC). This trait allows optimizing their expression during exponential phase since oriC neighboring regions are in higher dose due to multifork replication. Relocation of s10-spc-α locus (S10), which codes for most of the RP, to ectopic genomic positions shows that its relative distance to the oriC correlates to a reduction on its dosage, its expression, and bacterial growth rate. However, a mechanism linking S10 dosage to cell physiology has still not been determined.ResultsWe hypothesized that S10 dosage perturbations impact protein synthesis capacity. Strikingly, we observed that in Vibrio cholerae, protein production capacity was independent of S10 position. Deep sequencing revealed that S10 relocation altered chromosomal replication dynamics and genome-wide transcription. Such changes increased as a function of oriC-S10 distance. Since RP constitutes a large proportion of cell mass, lower S10 dosage could lead to changes in macromolecular crowding, impacting cell physiology. Accordingly, cytoplasm fluidity was higher in mutants where S10 is most distant from oriC. In hyperosmotic conditions, when crowding differences are minimized, the growth rate and replication dynamics were highly alleviated in these strains.ConclusionsThe genomic location of RP genes ensures its optimal dosage. However, besides of its essential function in translation, their genomic position sustains an optimal macromolecular crowding essential for maximizing growth. Hence, this could be another mechanism coordinating DNA replication to bacterial growth.

Chemotherapeutic drugs negatively affect myoblast proliferation and myotube anabolism by reducing rDNA transcription

Cancer cachexia negatively affects quality of life and contributes to increased morbidity and mortality in a wide variety of cancer types. Emerging evidence suggests that, in addition to tumor derived factors, chemotherapeutic drugs may also play a role in muscle wasting. The goal of this study was to determine the cellular mechanisms underpinning skeletal muscle depletion by chemotherapy drugs. Treatment of myoblasts or myotubes with Paclitaxel (PTX) or Doxorubicin (DXR) reduced cell viability in a dose dependent manner with myoblasts displaying more susceptibility than myotubes regardless of the drug. At non‐lethal doses, PTX or DXR induced significant reductions in myoblast proliferation (47%, p<0.01 and 43%, p<0.05 respectively). In myoblasts, PTX and DXR caused a reduction in rDNA transcription of 24% (p<0.0001) and 19% (p<0.0005) relative to control and a concomitant reduction in rRNA of 16% (p<0.0001) and 18% (p<0.0001), respectively. In myotubes, PTX or DXR reduced rDNA transcription by 21% (p<0.0001) and 30% (p<0.0001) compared to control, and consequently rRNA was 20% (p<0.01) and 17% (p<0.05) lower after the respective drug treatments. The reduction in rRNA reduced translational capacity in myotubes as evidenced by lower global protein synthesis of 52% (p<0.001) and 47% (p<0.001) following exposure to PTX or DXR. These results indicate that chemotherapeutic agents may affect muscle mass by impairing the ability of myogenic cells to proliferate, and by reducing the capacity for protein synthesis in myofibers via a reduction in rDNA transcription.Support or Funding InformationSupported by NIH grant AR073385

Monosomes actively translate synaptic mRNAs in neuronal processes.

To accommodate their complex morphology, neurons localize messenger RNAs (mRNAs) and ribosomes near synapses to produce proteins locally. However, a relative paucity of polysomes (considered the active sites of translation) detected in electron micrographs of neuronal processes has suggested a limited capacity for local protein synthesis. In this study, we used polysome profiling together with ribosome footprinting of microdissected rodent synaptic regions to reveal a surprisingly high number of dendritic and/or axonal transcripts preferentially associated with monosomes (single ribosomes). Furthermore, the neuronal monosomes were in the process of active protein synthesis. Most mRNAs showed a similar translational status in the cell bodies and neurites, but some transcripts exhibited differential ribosome occupancy in the compartments. Monosome-preferring transcripts often encoded high-abundance synaptic proteins. Thus, monosome translation contributes to the local neuronal proteome.

Optimal background colour for rearing Asian seabass (Lates calcarifer)

Background colour influences the growth performance and feed utilization of various fish species. In the current study, the effects of different aquarium backgrounds (transparent, white, black, red, green and blue) on growth performance, feed utilization, digestive enzyme activities, flesh quality, carcass composition and haematological parameters were investigated in Asian seabass (Lates calcarifer). The fish (16.17 ± 0.02 g initial body weight) were distributed into eighteen glass aquaria (6 treatments × 3 replications) with the various background colours and reared for 10 weeks. At the end of the experiment, positive growth performance and feed utilization were observed in fish reared against transparent, white, black or green backgrounds, while negative effects were noted for fish reared on the remaining treatments (p < .05). Modulation of the main digestive enzymes was observed across the six treatments, indicating different strategies for utilizing nutrients by the fish. The activity ratio of trypsin to chymotrypsin was highest in the fish reared against a black background, followed by a white background. Flesh quality, in terms of protein synthesis capacity and protein turnover rate, indicated a significant improvement in the fish reared against white, black and green backgrounds, while the contents of myosin and actin were similar across the six colour treatments. Carcass composition and haematological parameters showed no negative effects in either of the preferred treatments. These findings indicate that the most suitable background colours for rearing Asian seabass are black, followed by white and green, while transparent, red or blue backgrounds are unsuitable.