Ribosome Content Research Articles

Effects of leg immobilization and recovery resistance training on skeletal muscle-molecular markers in previously resistance trained versus untrained adults.

We sought to examine how resistance training (RT) status in young healthy individuals, either well resistance trained (T, n=10) or untrained (UT, n=11), affected molecular markers with leg immobilization followed by recovery RT. All participants underwent two weeks of left leg immobilization via a locking leg brace. Afterwards, all participants underwent eight weeks (3 d/week) of knee extensor focused progressive RT. Vastus lateralis (VL) ultrasound-derived thickness and muscle cross-sectional area were measured at baseline (PRE), immediately after disuse (MID), and after RT (POST) with VL muscle biopsies also being collected at these time points. Both groups presented lower ultrasound derived VL size metrics at MID versus PRE (p<0.001), and values increased in both groups from MID to POST (p<0.05); however, VL size increased from PRE to POST in UT only (p<0.001). Mean and type II myofiber cross-sectional area values were greater at PRE and POST versus MID (p<0.05), with T being greater than UT throughout (P<0.012). In both groups, satellite cell number was not affected by leg immobilization but increased in response to RT (p<0.014), with T being greater than UT throughout (p=0.004). Total RNA (ribosome content) decreased (p=0.010) from PRE to MID, while total RNA and certain endoplasmic reticulum stress proteins increased from MID to POST regardless of training status. Immobilization-induced muscle atrophy and recovery RT hypertrophy outcomes are similar between UT and T participants, and the lack of molecular signature differences between groups supports these findings. However, results are limited to younger adults undergoing non-complicated disuse.

Skeletal muscle ribosome analysis: A comparison of common assay methods and utilization of a novel RiboAb antibody cocktail.

While total RNA concentrations putatively represent ribosome content, there is a need to homologize various quantification approaches. Thus, total RNA concentrations ([RNA]) provided through UV-Vis spectroscopy (UV), fluorometry-only (Fluor), and fluorometry-based microfluidic chip electrophoresis (MFGE) were examined in C2C12 myotubes and mouse skeletal muscle to determine if values aligned with [18S + 28S rRNA] (i.e., criterion ribosome metric). A novel antibody cocktail (termed RiboAb) was also tested and compared to [18S + 28S rRNA] in these models. In myotubes, 24-h IGF-1 treatments increased [18S + 28S rRNA] (~2.0-fold) and [RNA] based on UV (~1.9-fold), Fluor (~2.3 fold), and MFGE (~2.1-fold). In C57BL/6 mice, 10 days of mechanical overload (MOV) elevated plantaris [18S + 28S rRNA] (~1.7-fold) and [RNA] according to UV (~1.5-fold), Fluor (~1.6-fold), and MFGE (~1.8-fold). Myotube and mouse plantaris RiboAb levels were significantly higher with IGF-1 treatments and MOV, respectively, versus controls (1.3-fold and 1.7-fold, respectively), and values correlated with [18S + 28S rRNA] (r = 0.637 and r = 0.853, respectively, p ≤ 0.005). UV, Fluor, and MFGE [RNA] are seemingly valid surrogates of cell/tissue ribosome content, although each method has advantages (e.g., ease of use) and disadvantages (e.g., magnitudes of bias) discussed herein. Finally, the RiboAb cocktail may also represent ribosome content, although this should be further explored in other models.

Genome concentration limits cell growth and modulates proteome composition in Escherichia coli.

Defining the cellular factors that drive growth rate and proteome composition is essential for understanding and manipulating cellular systems. In bacteria, ribosome concentration is known to be a constraining factor of cell growth rate, while gene concentration is usually assumed not to be limiting. Here, using single-molecule tracking, quantitative single-cell microscopy, and modeling, we show that genome dilution in Escherichia coli cells arrested for DNA replication limits total RNA polymerase activity within physiological cell sizes across tested nutrient conditions. This rapid-onset limitation on bulk transcription results in sub-linear scaling of total active ribosomes with cell size and sub-exponential growth. Such downstream effects on bulk translation and cell growth are near-immediately detectable in a nutrient-rich medium, but delayed in nutrient-poor conditions, presumably due to cellular buffering activities. RNA sequencing and tandem-mass-tag mass spectrometry experiments further reveal that genome dilution remodels the relative abundance of mRNAs and proteins with cell size at a global level. Altogether, our findings indicate that chromosome concentration is a limiting factor of transcription and a global modulator of the transcriptome and proteome composition in E. coli. Experiments in Caulobacter crescentus and comparison with eukaryotic cell studies identify broadly conserved DNA concentration-dependent scaling principles of gene expression.

Post-exercise hot-water immersion is not effective for ribosome biogenesis in rat skeletal muscle.

Ribosome biogenesis is an important regulator of skeletal muscle hypertrophy induced by repeated bouts of resistance exercise (RE). Hot-water immersion (HWI), a widely used post-exercise recovery strategy, activates the mechanistic target of rapamycin (mTOR) signaling, a key regulator of ribosome biogenesis in skeletal muscle. However, the effect of HWI on skeletal muscle ribosome biogenesis is not well understood. Here, we aimed to investigate the effects of HWI and post-exercise HWI on ribosome biogenesis using a rat RE model. Male Sprague-Dawley rats were randomly assigned to HWI and non-HWI groups. In both groups, the right leg was isometrically exercised using transcutaneous electrical stimulation, while the left leg was used as an internal non-RE control. Following RE, both limbs were immersed in hot water (41.2 ± 0.03°C) for 20 min under isoflurane anesthesia in the HWI group and the gastrocnemius muscles were sampled at 3- and 24-h post-exercise. HWI significantly increased mTOR signaling and c-Myc mRNA expression, whereas post-exercise HWI significantly increased transcription initiation factor-IA mRNA expression. However, neither HWI nor post-exercise HWI enhanced 45S pre-rRNA expression, ribosomal RNA, or ribosomal protein content. In addition, HWI tended to decrease 28S rRNA and 18S rRNA content, widely used markers of ribosome content. These results suggest that HWI as a post-exercise recovery is not effective in activating ribosome biogenesis.NEW & NOTEWORTHY Ribosome biogenesis is crucial in resistance exercise (RE)-induced skeletal muscle hypertrophy. This study examined the effects of hot-water immersion (HWI) on ribosome biogenesis after RE. HWI and post-exercise HWI increased c-Myc and transcription initiation factor-IA mRNA but did not alter ribosomal RNA transcription or ribosomal protein content. HWI tended to decrease 28S and 18S ribosomal RNA. These findings suggest that HWI, as a recovery strategy, does not effectively promote ribosome biogenesis or muscle protein synthesis.

Nitrogen deposition and heathland management cause multi‐element stoichiometric mismatches, reducing insect fitness

Abstract Increased nitrogen and acidifying deposition negatively affect biogeochemical cycles and nutrient stoichiometry, particularly in oligotrophic habitats. In heathlands, management aims to mitigate effects of deposition by topsoil removal (sod‐cutting) or by liming to increase soil pH. However, these management efforts may further exacerbate macro‐ and micronutrient stoichiometric mismatch, with cascading effects on heathland insects. Here, we investigate the physiological mechanisms by which deposition and management affect insects, focusing on nutrient uptake and excretion. We combined a phosphorus (P) addition and liming experiment in previously sod‐cut heathland with laboratory‐rearing experiments using the field cricket (Gryllus campestris L., 1) as a model species. We hypothesized that P addition would benefit cricket performance by reducing food N:P ratio. Conversely, we hypothesized that liming would reduce performance by decreasing food Mn:Mg and/or Fe:Mg ratios. Elemental uptake efficiency, consumption and reproduction, as well as cricket field density, were significantly higher under increased P availability, confirming insect P limitation in the field. Liming decreased food Mn:Mg ratio and reduced reproductive success, eliminating any beneficial effects of P addition. Moreover, cricket stoichiometry better explained growth rates than the amount of food consumed. The best model highlighted cricket N and Mn content, with increasing N content negatively influencing growth, while the opposite was true for Mn. We found no support for liming‐induced Fe limitation. We propose that both P and Mn limit crickets independently. Specifically, P limitation affects growth via reduced ribosomal content, and Mn limitation impacts growth by limiting arginase activity and hence restricting the recycling of amino acids. Our study shows that restoration management can fail to restore habitat quality for insects and may even amplify mismatches in macro and micronutrient stoichiometry. To address this, the addition of not only P but also Mn may be required as follow‐up management, but reducing N‐emission remains imperative as this removes the need for disruptive management interventions. Read the free Plain Language Summary for this article on the Journal blog.

Single-cell heterogeneity in ribosome content and the consequences for the growth laws.

Across species and environments, the ribosome content of cell populations correlates with population growth rate. The robustness and universality of this correlation have led to its classification as a "growth law." This law has fueled theories about how evolution selects for microbial organisms that maximize their growth rate based on nutrient availability, and it has informed models about how individual cells regulate their growth rates and ribosomal content. However, due to methodological limitations, this growth law has rarely been studied at the level of individual cells. While populations of fast-growing cells tend to have more ribosomes than populations of slow-growing cells, it is unclear whether individual cells tightly regulate their ribosome content to match their environment. Here, we employ recent groundbreaking single-cell RNA sequencing techniques to study this growth law at the single-cell level in two different microbes, S. cerevisiae (a single-celled yeast and eukaryote) and B. subtilis (a bacterium and prokaryote). In both species, we observe significant variation in the ribosomal content of single cells that is not predictive of growth rate. Fast-growing populations include cells exhibiting transcriptional signatures of slow growth and stress, as do cells with the highest ribosome content we survey. Broadening our focus to non-ribosomal transcripts reveals subpopulations of cells in unique transcriptional states suggestive that they have evolved to do things other than maximize their rate of growth. Overall, these results indicate that single-cell ribosome levels are not finely tuned to match population growth rates or nutrient availability and cannot be predicted by a Gaussian process model that assumes measurements are sampled from a normal distribution centered on the population average. This work encourages the expansion of growth law and other models that predict how growth rates are regulated or how they evolve to consider single-cell heterogeneity. To this end, we provide extensive data and analysis of ribosomal and transcriptomic variation across thousands of single cells from multiple conditions, replicates, and species.

Ageing-associated long non-coding RNA extends lifespan and reduces translation in non-dividing cells

Genomes produce widespread long non-coding RNAs (lncRNAs) of largely unknown functions. We characterize aal1 (ageing-associated lncRNA), which is induced in quiescent fission yeast cells. Deletion of aal1 shortens the chronological lifespan of non-dividing cells, while ectopic overexpression prolongs their lifespan, indicating that aal1 acts in trans. Overexpression of aal1 represses ribosomal-protein gene expression and inhibits cell growth, and aal1 genetically interacts with coding genes functioning in protein translation. The aal1 lncRNA localizes to the cytoplasm and associates with ribosomes. Notably, aal1 overexpression decreases the cellular ribosome content and inhibits protein translation. The aal1 lncRNA binds to the rpl1901 mRNA, encoding a ribosomal protein. The rpl1901 levels are reduced ~2-fold by aal1, which is sufficient to extend lifespan. Remarkably, the expression of the aal1 lncRNA in Drosophila boosts fly lifespan. We propose that aal1 reduces the ribosome content by decreasing Rpl1901 levels, thus attenuating the translational capacity and promoting longevity. Although aal1 is not conserved, its effect in flies suggests that animals feature related mechanisms that modulate ageing, based on the conserved translational machinery.

DRMY1 promotes robust morphogenesis in Arabidopsis by sustaining the translation of cytokinin-signaling inhibitor proteins

Robustness is the invariant development of phenotype despite environmental changes and genetic perturbations. In the Arabidopsis flower bud, four sepals robustly initiate and grow to a constant size to enclose and protect the inner floral organs. We previously characterized the mutant development-related myb-like 1 (drmy1), where 3-5 sepals initiate variably and grow to different sizes, compromising their protective function. The molecular mechanism underlying this loss of robustness was unclear. Here, we show that drmy1 has reduced TARGET OF RAPAMYCIN (TOR) activity, ribosomal content, and translation. Translation reduction decreases the protein level of ARABIDOPSIS RESPONSE REGULATOR7 (ARR7) and ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), two cytokinin-signaling inhibitors that are normally rapidly produced before sepal initiation. The resultant upregulation of cytokinin signaling disrupts robust auxin patterning and sepal initiation. Our work shows that the homeostasis of translation, a ubiquitous cellular process, is crucial for the robust spatiotemporal patterning of organogenesis.

Grain albumin content is affected by 1BL/1RS translocation and alters the processing quality of wheat (Triticum aestivum L.).

Grain albumin is highly nutritious and closely related to the processing quality of wheat. However, few studies have explored the grain albumin content (GAC) in wheat. This study aims to uncover quantitative trait loci (QTLs) linked to wheat GAC by analyzing a doubled haploid (DH) population derived from common wheat cultivars ShanNong23 and ZhouMai17. We detected six QTLs controlling GAC on chromosomes 1B, 5A, and 6D, with individual QTL explaining 5.78% to 22.29% of the GAC variation. The effect of QGac.cau-1B.1 on GAC is attributed to the presence of the 1BL/1RS translocation, indicating that the 1BL/1RS translocation increase of GAC compared with the non-1BL/1RS translocation lines. The higher GAC observed in 1BL/1RS lines could be primarily attributed to the increased accumulation of omega-secalin, omega-gliadin, low molecular weight glutenin subunit and ribosomal protein content. Additionally, we also found that the SDS-sedimentation value of whole wheat flour was decreased by adding albumin solution. These results advance our understanding of the genetic basis of GAC and offer novel perspectives for enhancing wheat quality through genetic enhancements.

Relative rDNA copy number is not associated with resistance training-induced skeletal muscle hypertrophy and does not affect myotube anabolism in vitro.

Ribosomal DNA (rDNA) copies exist across multiple chromosomes and inter-individual variation in copy number is speculated to influence the hypertrophic response to resistance training. Thus, we examined if rDNA copy number was associated with resistance training-induced skeletal muscle hypertrophy. Participants (n=53 males, 21±1 years old; n=29 females, 21±2 years old) performed 10-12 weeks of full-body resistance training. Hypertrophy outcomes were determined, as was relative rDNA copy number from pre-intervention vastus lateralis (VL) biopsies. Pre- and post-intervention VL biopsy total RNA was assayed in all participants, and mRNA/rRNA markers of ribosome content and biogenesis were also assayed in the 29 females prior to training, 24 hours following training bout 1, and in the basal state after 10 weeks of training. Across all participants, no significant associations were evident between relative rDNA copy number and training-induced changes in whole body lean mass (r = -0.034, p=0.764), vastus lateralis thickness (r = 0.093, p=0.408), mean myofiber cross-sectional area (r = -0.128, p=0.259), or changes in muscle RNA concentrations (r = 0.026, p=0.818), and these trends were similar when examining each gender. However, all Pol-I regulon mRNAs as well as 45S pre-rRNA, 28S rRNA and 18S rRNA increased 24 hours following the first training bout in females. Follow-up studies using LHCN-M2 myotubes demonstrated a reduction in relative rDNA copy number induced by bisphenol A (BPA) did not significantly affect insulin-like-growth factor-induced myotube hypertrophy. These findings suggest relative rDNA copy number is not associated with myofiber hypertrophy.

The hallmarks of a tradeoff in transcriptomes that balances stress and growth functions.

Fast growth phenotypes are achieved through optimal transcriptomic allocation, in which cells must balance tradeoffs in resource allocation between diverse functions. One such balance between stress readiness and unbridled growth in E. coli has been termed the fear versus greed (f/g) tradeoff. Two specific RNA polymerase (RNAP) mutations observed in adaptation to fast growth have been previously shown to affect the f/g tradeoff, suggesting that genetic adaptations may be primed to control f/g resource allocation. Here, we conduct a greatly expanded study of the genetic control of the f/g tradeoff across diverse conditions. We introduced 12 RNA polymerase (RNAP) mutations commonly acquired during adaptive laboratory evolution (ALE) and obtained expression profiles of each. We found that these single RNAP mutation strains resulted in large shifts in the f/g tradeoff primarily in the RpoS regulon and ribosomal genes, likely through modifying RNAP-DNA interactions. Two of these mutations additionally caused condition-specific transcriptional adaptations. While this tradeoff was previously characterized by the RpoS regulon and ribosomal expression, we find that the GAD regulon plays an important role in stress readiness and ppGpp in translation activity, expanding the scope of the tradeoff. A phylogenetic analysis found the greed-related genes of the tradeoff present in numerous bacterial species. The results suggest that the f/g tradeoff represents a general principle of transcriptome allocation in bacteria where small genetic changes can result in large phenotypic adaptations to growth conditions.IMPORTANCETo increase growth, E. coli must raise ribosomal content at the expense of non-growth functions. Previous studies have linked RNAP mutations to this transcriptional shift and increased growth but were focused on only two mutations found in the protein's central region. RNAP mutations, however, commonly occur over a large structural range. To explore RNAP mutations' impact, we have introduced 12 RNAP mutations found in laboratory evolution experiments and obtained expression profiles of each. The mutations nearly universally increased growth rates by adjusting said tradeoff away from non-growth functions. In addition to this shift, a few caused condition-specific adaptations. We explored the prevalence of this tradeoff across phylogeny and found it to be a widespread and conserved trend among bacteria.

Skeletal muscle preservation in arctic ground squirrels during hibernation season

Reduced skeletal loading leads to muscle atrophy in humans and most mammals. By contrast, hibernating mammals demonstrate limited loss of skeletal muscle mass and strength by the end of winter after being physically inactive for several months. The present study objective was to establish potential underlying processes for the lack of muscle loss during the hibernation season of arctic ground squirrels (AGS). Our hypothesis was that early in the the hibernation season, protective mechanisms of AGS are not yet mobilized and muscles show signs of atrophy, while compensatory processes for preventing muscle loss appear at a later time point. Quadriceps muscles of juvenile male AGS were collected shortly before hibernation, and on 2, 6, 10-12 and 16-22 weeks during hibernation. Pre-hibernating animals were used as controls. Fiber cross-sectional area (CSA) and fiber type composition were determined with immunohistochemistry. Amount of total RNA, ribosomal RNA content and expression levels of genes involved in 2 pathways of protein degradation, ubiquitination and autophagy, were analyzed by real-time PCR, to evaluate molecular mechanisms of muscle maintenance. One-Way ANOVA was used to statistically analyze the group differences. We found that CSA was not different between the groups (P &gt; 0.05). CSA frequency distribution curves did not reveal any shift to a smaller or larger size range. No difference was detected in myofiber composition between the hibernation groups compared to the control. Total RNA and ribosomal RNA content were not significantly different between the groups during hibernation. Muscle atrophy marker FBXO32 (Atrogin-1, MAFBX) and autophagy related genes MAP1LC3A and BECN1 did not show different level of expression between the groups. Only another muscle atrophy marker TRIM63 (MURF-1, Iris Ring Finger Protein) was significantly over-expressed at the time point of 2 weeks of hibernation. These results indicate that throughout the hibernation season, AGS preserve muscle fiber CSA showing limited signs of muscle atrophy only during the first weeks of hibernation, and yet-to-be determined processes exist to suppress protein degradation in AGS muscles during hibernation. The work was supported by Center of Biomedical Research Excellence under grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glucose ingestion before and after resistance training sessions does not augment ribosome biogenesis in healthy moderately trained young adults

PurposeResistance training-induced skeletal muscle hypertrophy seems to depend on ribosome biogenesis and content. High glucose treatment may augment ribosome biogenesis through potentiating resistance training-induced adaptations. This was investigated with total RNA and ribosomal RNA abundances as main outcomes, with relevant transcriptional/translational regulators (c-Myc/UBF/rpS6) as a secondary outcome.MethodsSixteen healthy, moderately trained individuals [male/female, n = 9/7; age, 24.1 (3.3)] participated in a within-participant crossover trial with unilateral resistance training (leg press and knee extension, 3 sets of 10 repetitions maximum) and pre- and post-exercise ingestion of either glucose (3 × 30 g, 90 g total) or placebo supplements (Stevia rebaudiana, 3 × 0.3 g, 0.9 g total), together with protein (2 × 25 g, 50 g total), on alternating days for 12 days. Six morning resistance exercise sessions were conducted per condition, and the sessions were performed in an otherwise fasted state. Micro-biopsies were sampled from m. vastus lateralis before and after the intervention.ResultsGlucose ingestion did not have beneficial effects on resistance training-induced increases of ribosomal content (mean difference 7.6% [− 7.2, 24.9], p = 0.34; ribosomal RNA, 47S/18S/28S/5.8S/5S, range 7.6–37.9%, p = 0.40–0.98) or levels of relevant transcriptional or translational regulators (c-MYK/UBF/rpS6, p = 0.094–0.292). Of note, both baseline and trained state data of total RNA showed a linear relationship with UBF; a ∼14% increase in total RNA corresponded to 1 SD unit increase in UBF (p = 0.003).ConclusionGlucose ingestion before and after resistance training sessions did not augment ribosomal RNA accumulation during twelve days of heavy-load resistance training in moderately trained young adults.

A connection between the ribosome and two S. pombe tRNA modification mutants subject to rapid tRNA decay.

tRNA modifications are crucial in all organisms to ensure tRNA folding and stability, and accurate translation. In both the yeast Saccharomyces cerevisiae and the evolutionarily distant yeast Schizosaccharomyces pombe, mutants lacking certain tRNA body modifications (outside the anticodon loop) are temperature sensitive due to rapid tRNA decay (RTD) of a subset of hypomodified tRNAs. Here we show that for each of two S. pombe mutants subject to RTD, mutations in ribosomal protein genes suppress the temperature sensitivity without altering tRNA levels. Prior work showed that S. pombe trm8Δ mutants, lacking 7-methylguanosine, were temperature sensitive due to RTD, and that one class of suppressors had mutations in the general amino acid control (GAAC) pathway, which was activated concomitant with RTD, resulting in further tRNA loss. We now find that another class of S. pombe trm8Δ suppressors have mutations in rpl genes, encoding 60S subunit proteins, and that suppression occurs with minimal restoration of tRNA levels and reduced GAAC activation. Furthermore, trm8Δ suppression extends to other mutations in the large or small ribosomal subunit. We also find that S. pombe tan1Δ mutants, lacking 4-acetylcytidine, are temperature sensitive due to RTD, that one class of suppressors have rpl mutations, associated with minimal restoration of tRNA levels, and that suppression extends to other rpl and rps mutations. However, although S. pombe tan1Δ temperature sensitivity is associated with some GAAC activation, suppression by an rpl mutation only modestly inhibits GAAC activation. We propose a model in which ribosomal protein mutations result in reduced ribosome concentrations, leading to both reduced ribosome collisions and a reduced requirement for tRNA, with these effects having different relative importance in trm8Δ and tan1Δ mutants. This model is consistent with our results in S. cerevisiae trm8Δ trm4Δ mutants, known to undergo RTD, fueling speculation that this model applies across eukaryotes.

Ultrastructural and morphological studies on variables affecting Escherichia coli with selected commercial antibiotics

BackgroundMany studies reported the effects of antibiotic exposure on E. coli bacterial growth and cell modification. However, scarce descriptive information on ultrastructural effects upon exposure of commercial antibiotics. MethodsThis study described the morphological and ultrastructural alterations caused by selected antibiotics (amoxicillin-clavulanate, ceftriaxone, polymyxin B, colistin, gentamicin, and amikacin) that targeted cell wall, plasma membrane, and cytoplasmic density, and also proteins synthesis. We determined extracellular morphological changes of exposure through scanning electron microscopy (FESEM) and intracellular activities through transmission electron microscopy (TEM) investigation. ResultsFESEM and TEM micrograph of E. coli exposed with selected antibiotics shows ultrastructural changes in beta-lactam class (amoxicillin-clavulanate, ceftriaxone) elongated the cells as the cell wall was altered as it inhibits bacterial cell wall synthesis, polymyxin class (polymyxin B, colistin) had plasmid and curli-fimbriae as it breaking down the plasma/cytoplasmic membrane, and aminoglycoside class (gentamicin, and amikacin) reduced ribosome concentration as it inhibits bacterial protein synthesis by binding to 30 s ribosomes. ConclusionMorphological and ultrastructural alterations of E. coli’s mechanism of actions were translated and depicted. This study could be reference for characterization studies for morphological and ultrastructural of E. coli upon exposure to antimicrobial agents.

Low baseline ribosome-related gene expression and resistance training-induced declines in ribosome-related gene expression are associated with skeletal muscle hypertrophy in young men and women.

Ribosomes are essential cellular machinery for protein synthesis. It is hypothesised that ribosome content supports muscle growth and that individuals with more ribosomes have greater increases in muscle size following resistance training (RT). Aerobic conditioning (AC) also elicits distinct physiological adaptations; however, no measures of ribosome content following AC have been conducted. We used ribosome-related gene expression as a proxy measure for ribosome content and hypothesised that AC and RT would increase ribosome-related gene expression. Fourteen young men and women performed 6 weeks of single-legged AC followed by 10 weeks of double-legged RT. Muscle biopsies were taken following AC and following RT in the aerobically conditioned (AC+RT) and unconditioned (RT) legs. No differences in regulatory genes (Ubf, Cyclin D1, Tif-1aandPolr-1b) involved in ribosomal biogenesis or ribosomal RNA (45S, 5.8S, 18Sand28S rRNAs) expression were observed following AC and RT, except for c-Myc (RT > AC+RT) and 5S rRNA (RT < AC+RT at pre-RT) with 18Sexternal transcribed spacer and 5.8S internal transcribed spacerexpression decreasing from pre-RT to post-RT in the RT leg only. When divided for change in leg-lean soft tissue mass (ΔLLSTM) following RT, legs with the greatest ΔLLSTM had lower expression in 11/13 measured ribosome-related genes before RT and decreased expression in 9/13 genes following RT. These results indicate that AC and RT did not increase ribosome-related gene expression. Contrary to previous research, the greatest increase in muscle mass was associated with lower changes in ribosome-related gene expression over the course of the 10-week training programme. This may point to the importance of translational efficiency rather than translational capacity (i.e.ribosome content) in mediating long-term exercise-induced adaptations in skeletal muscle.

Microorganisms in subarctic soils are depleted of ribosomes under short-, medium-, and long-term warming.

Physiological responses of soil microorganisms to global warming are important for soil ecosystem function and the terrestrial carbon cycle. Here, we investigate the effects of weeks, years, and decades of soil warming across seasons and time on the microbial protein biosynthesis machineries (i.e. ribosomes), the most abundant cellular macromolecular complexes, using RNA:DNA and RNA:MBC (microbial biomass carbon) ratios as proxies for cellular ribosome contents. We compared warmed soils and non-warmed controls of 15 replicated subarctic grassland and forest soil temperature gradients subject to natural geothermal warming. RNA:DNA ratios tended to be lower in the warmed soils during summer and autumn, independent of warming duration (6weeks, 8-14years, and > 50years), warming intensity (+3°C, +6°C, and +9°C), and ecosystem type. With increasing temperatures, RNA:MBC ratios were also decreasing. Additionally, seasonal RNA:DNA ratios of the consecutively sampled forest showed the same temperature-driven pattern. This suggests that subarctic soil microorganisms are depleted of ribosomes under warm conditions and the lack of consistent relationships with other physicochemical parameters besides temperature further suggests temperature as key driver. Furthermore, in incubation experiments, we measured significantly higher CO2 emission rates per unit of RNA from short- and long-term warmed soils compared to non-warmed controls. In conclusion, ribosome reduction may represent a widespread microbial physiological response to warming that offers a selective advantage at higher temperatures, as energy and matter can be reallocated from ribosome synthesis to other processes including substrate uptake and turnover. This way, ribosome reduction could have a substantial effect on soil carbon dynamics.

Bone Marrow Microenvironment and Hematopoietic Cell Types Are Differentially Affected By Transcriptomic Quality Control Filtering Metrics in Single Cell Data

A crucial step prior to analysis of single cell transcriptomic data includes filtering out low-quality (e.g., dying) cells and artifacts (e.g., empty droplets). Choosing appropriate quality control parameters leads to correct cell assignment, so that the data for downstream analysis is high-quality and is representative of all processed cell types. These metrics include high and low cut-offs for mitochondrial content, total number of genes, number of different genes, and ribosomal content per cell. They have been found to vary based on cell and tissue type, as well biological variables such as age and disease, and technical variables such as sequencing technology. The aim of this study was to investigate the variation of these quality control metrics in a data set that included stem, progenitor, and differentiated cell types of the hematopoietic, mesenchymal, and endothelial lineages. Furthermore, we set out to determine whether applying standard cut-offs may differentially affect different cell types. We calculated individual filtering parameters for each cell type using the R package ddqc (Subramanian et al. Genome Biol. 2022). Thus, any biological variability based on mitochondrial, ribosomal, and gene counts was accounted for. Notably, distribution of measurements for each of the above quality control metrics varied by cluster, and certain cell-type means were adjacent to commonly used cut-off values. For instance, we observed higher content of mitochondrial genes in osteoblasts, sinusoidal endothelial cells, a subset of adipocyte-primed mesenchymal cells (MSPC), as well as hematopoietic progenitor cells, pre-B, and B cells. On the other hand, subsets of erythroblasts, neutrophils, as well as arteriolar endothelial cells had the lowest mitochondrial content. In addition, lower total gene counts and lower gene complexity were found in adipocyte-primed MSPCs, neutrophils, and megakaryocyte-erythrocyte progenitors, while higher counts were found in granulocyte-monocyte progenitor cells, megakaryocyte/erythroid-primed multipotent progenitor cells and subsets of erythroblasts. Applying universal cutoff values to this data set would greatly reduce, and some cases, eliminate cell types that have high mitochondrial content or lower gene counts and complexity, while leaving those cell types on the opposite spectrum unaffected. This would also skew proportions of cells in downstream analyses, thus affecting biological interpretation of the results. Overall, our results demonstrate biological variability of the quality control metric distributions in a single cell transcriptomic data set that includes diverse bone marrow niche and hematopoietic cell types. This reveals novel biological information regarding distinct cell types as well as cautions that applying standard filtering paraments on such a data set would eliminate some cell types and bias downstream analysis for others. Optimization of filtering criteria will be important when broadening single cell analysis to multiplexed approaches incorporating transcriptome, genome, epigenome, and proteome-level data.

Resistance training diminishes mitochondrial adaptations to subsequent endurance training in healthy untrained men.

We investigated the effects of performing a period of resistance training (RT) on the performance and molecular adaptations to a subsequent period of endurance training (ET). Twenty-five young adults were divided into an RT+ET group (n=13), which underwent 7weeks of RT followed by 7weeks of ET, and an ET-only group (n=12), which performed 7weeks of ET. Body composition, endurance performance and muscle biopsies were collected before RT (T1, baseline for RT+ET), before ET (T2, after RT for RT+ET and baseline for ET) and after ET (T3). Immunohistochemistry was performed to determine fibre cross-sectional area (fCSA), myonuclear content, myonuclear domain size, satellite cell number and mitochondrial content. Western blots were used to quantify markers of mitochondrial remodelling. Citrate synthase activity and markers of ribosome content were also investigated. RT improved body composition and strength, increased vastus lateralis thickness, mixed and type II fCSA, myonuclear number, markers of ribosome content, and satellite cell content (P<0.050). In response to ET, both groups similarly decreased body fat percentage (P<0.0001) and improved endurance performance (e.g. , and speed at which the onset of blood lactate accumulation occurred, P<0.0001). Levels of mitochondrial complexes I-IV in the ET-only group increased 32-66%, while those in the RT+ET group increased 1-11% (time, P<0.050). Additionally, mixed fibre relative mitochondrial content increased 15% in the ET-only group but decreased 13% in the RT+ET group (interaction, P=0.043). In conclusion, RT performed prior to ET had no additional benefits to ET adaptations. Moreover, prior RT seemed to impair mitochondrial adaptations to ET. KEY POINTS: Resistance training is largely underappreciated as a method to improve endurance performance, despite reports showing it may improve mitochondrial function. Although several concurrent training studies are available, in this study we investigated the effects of performing a period of resistance training on the performance and molecular adaptations to subsequent endurance training. Prior resistance training did not improve endurance performance and impaired most mitochondrial adaptations to subsequent endurance training, but this effect may have been a result of detraining from resistance training.

In situ cell division and mortality rates of SAR11, SAR86, Bacteroidetes, and Aurantivirga during phytoplankton blooms reveal differences in population controls.

Net growth of microbial populations, that is, changes in abundances over time, can be studied using 16S rRNA fluorescence in situ hybridization (FISH). However, this approach does not differentiate between mortality and cell division rates. We used FISH-based image cytometry in combination with dilution culture experiments to study net growth, cell division, and mortality rates of four bacterial taxa over two distinct phytoplankton blooms: the oligotrophs SAR11 and SAR86, and the copiotrophic phylum Bacteroidetes, and its genus Aurantivirga. Cell volumes, ribosome content, and frequency of dividing cells (FDC) co-varied over time. Among the three, FDC was the most suitable predictor to calculate cell division rates for the selected taxa. The FDC-derived cell division rates for SAR86 of up to 0.8/day and Aurantivirga of up to 1.9/day differed, as expected for oligotrophs and copiotrophs. Surprisingly, SAR11 also reached high cell division rates of up to 1.9/day, even before the onset of phytoplankton blooms. For all four taxonomic groups, the abundance-derived net growth (-0.6 to 0.5/day) was about an order of magnitude lower than the cell division rates. Consequently, mortality rates were comparably high to cell division rates, indicating that about 90% of bacterial production is recycled without apparent time lag within 1 day. Our study shows that determining taxon-specific cell division rates complements omics-based tools and provides unprecedented clues on individual bacterial growth strategies including bottom-up and top-down controls. IMPORTANCE The growth of a microbial population is often calculated from their numerical abundance over time. However, this does not take cell division and mortality rates into account, which are important for deriving ecological processes like bottom-up and top-down control. In this study, we determined growth by numerical abundance and calibrated microscopy-based methods to determine the frequency of dividing cells and subsequently calculate taxon-specific cell division rates in situ. The cell division and mortality rates of two oligotrophic (SAR11 and SAR86) and two copiotrophic (Bacteroidetes and Aurantivirga) taxa during two spring phytoplankton blooms showed a tight coupling for all four taxa throughout the blooms without any temporal offset. Unexpectedly, SAR11 showed high cell division rates days before the bloom while cell abundances remained constant, which is indicative of strong top-down control. Microscopy remains the method of choice to understand ecological processes like top-down and bottom-up control on a cellular level.