Protein Production Rate Research Articles

Biosensors Characterisation: Formal methods from the Perspective of Proteome Fractions

Abstract Many studies characterise transcription factors and other regulatory elements to control gene expression in recombinant systems. However, most lack a formal approach to analyse the inherent and context-specific variations of these regulatory components. This study addresses this gap by establishing a formal framework from which convenient methods are inferred to characterise regulatory circuits. We modelled the bacterial cell as a collection of proteome fractions. Deriving the time-dependent proteome fraction, we obtained a general theorem that describes its change as a function of its expression fraction, a specific portion of the total biosynthesis flux of the cell. Formal deduction reveals that when the proteome fraction reaches a maximum, it becomes equivalent to its expression fraction. This equation enables the reliable measurement of the expression fraction through direct protein quantification. In addition, the experimental data demonstrate a linear correlation between protein production rate and specific growth rate over a significant time period. This suggests a constant expression fraction within this window. For an IPTG biosensor, in five cellular contexts, expression fractions determined by the maximum method and the slope method produced strikingly similar dose-response parameters when independently fit to a Hill function. Furthermore, by analysing two more biosensors, for mercury and cumate detection, we demonstrate that the slope method can be applied effectively to various systems. Therefore, the concepts presented here provide convenient methods for obtaining dose-response parameters, clearly defining the time interval of their validity and offering a framework for interpreting typical biosensor outputs in terms of bacterial physiology.

Comparative proteomics uncovers low asparagine content in Plasmodium tRip-KO proteins.

tRNAs are not only essential for decoding the genetic code, but their abundance also has a strong impact on the rate of protein production, folding, and on the stability of the translated messenger RNAs. Plasmodium expresses a unique surface protein called tRip, involved in the import of exogenous tRNAs into the parasite. Comparative proteomic analysis of the blood stage of wild-type and tRip-KO variant of P. berghei parasites revealed that downregulated proteins in the mutant parasite are distinguished by a bias in their asparagine content. Furthermore, the demonstration of the possibility of charging host tRNAs with Plasmodium aminoacyl-tRNA synthetases led us to propose that imported host tRNAs participate in parasite protein synthesis. These results also suggest a novel mechanism of translational control in which import of host tRNAs emerge as regulators of gene expression in the Plasmodium developmental cycle and pathogenesis, by enabling the synthesis of asparagine-rich regulatory proteins that efficiently and selectively control the parasite infectivity.

Genetic and Functional Analyses of Patients with Marked Hypo-High-Density Lipoprotein Cholesterolemia

This study aimed to analyze two cases of marked hypo-high-density lipoprotein (HDL) cholesterolemia to identify mutations in ATP-binding cassette transporter A1 (ABCA1) and elucidate the molecular mechanism by which these novel pathological mutations contribute to hypo-HDL cholesterolemia in Tangier disease. Wild type and mutant expression plasmids containing a FLAG tag inserted at the C-terminus of the human ABCA1 gene were generated and transfected into HEK293T cells. ABCA1 protein expression and cholesterol efflux were evaluated via Western blotting and efflux assay. The difference in the rate of change in protein expression was evaluated when proteolytic and protein-producing systems were inhibited. In case 1, a 20-year-old woman presented with a chief complaint of gait disturbance. Her HDL-C level was only 6.2 mg/dL. Tangier disease was suspected because of muscle weakness, decreased nerve conduction velocity, and splenomegaly. Whole-exome analysis showed compound heterozygosity for a W484* nonsense mutation and S1343I missense mutation, which confirmed Tangier disease. Cholesterol efflux decreased by a mixture of W484* and S1343I mutations. The S1343I mutation decreased the protein production rate but increased the degradation rate, decreasing the protein levels. This patient also had Krabbe disease. The endogenous ABCA1 protein level of macrophage cell decreased by knocking down its internal galactocerebrosidase. Case 2, a 51-year-old woman who underwent tonsillectomy presented with peripheral neuropathy, corneal opacity, and HDL-C of 3.4 mg/dL. Whole-exome analysis revealed compound heterozygosity for R579* and R1572* nonsense mutations, which confirmed Tangier disease. Case 1 is a new ABCA1 mutation with complex pathogenicity, namely, a W484*/S1343I compound heterozygote with marked hypo-HDL cholesterolemia. Analyses of the compound heterozygous mutations indicated that decreases in ABCA1 protein levels and cholesterol efflux activity caused by the novel S1343I mutation combined with loss of W484* protein activity could lead to marked hypo-HDL cholesterolemia. Galactocerebrosidase dysfunction could also be a potential confounding factor for ABCA1 protein function.

Direct single-cell observation of a key Escherichia coli cell-cycle oscillator.

Initiation of DNA replication in Escherichia coli is coupled to cell size via the DnaA protein, whose activity is dependent on its nucleotide-bound state. However, the oscillations in DnaA activity have never been observed at the single-cell level. By measuring the volume-specific production rate of a reporter protein under control of a DnaA-regulated promoter, we could distinguish two distinct cell-cycle oscillators. The first, driven by both DnaA activity and SeqA repression, shows a causal relationship with cell size and divisions, similarly to initiation events. The second one, a reporter of DnaA activity alone, loses the synchrony and causality properties. Our results show that transient inhibition of gene expression by SeqA keeps the oscillation of volume-sensing DnaA activity in phase with the subsequent division event and suggest that DnaA activity peaks do not correspond directly to initiation events.

TIR predictor and optimizer: Web-tools for accurate prediction of translation initiation rate and precision gene design in Saccharomyces cerevisiae.

Translation initiation is the primary determinant of the rate of protein production. The variation in the rate with which this step occurs can cause up to three orders of magnitude differences in cellular protein levels. Several mRNA features, including mRNA stability in proximity to the start codon, coding sequence length, and presence of specific motifs in the mRNA molecule, have been shown to influence the translation initiation rate. These molecular factors acting at different strengths allow precise control of in vivo translation initiation rate and thus the rate of protein synthesis. However, despite the paramount importance of translation initiation rate in protein synthesis, accurate prediction of the absolute values of initiation rate remains a challenge. In fact, as of now, there is no available model for predicting the initiation rate in Saccharomyces cerevisiae. To address this, we train a machine learning model for predicting the in vivo initiation rate in S. cerevisiae transcripts. The model is trained using a diverse set of mRNA transcripts, enabling the comparison of initiation rates across different transcripts. Our model exhibited excellent accuracy in predicting the translation initiation rate and demonstrated its effectiveness with both endogenous and exogenous transcripts. Then, by combining the machine learning model with the Monte-Carlo search algorithm, we have also devised a method to optimize the nucleotide sequence of any gene to achieve a specific target initiation rate. The machine learning model we've developed for predicting translation initiation rates, along with the gene optimization method, are deployed as a web server. Both web servers are accessible for free at the following link: ajeetsharmalab.com/TIRPredictor. Thus, this research advances our fundamental understanding of translation initiation processes, with direct applications in biotechnology.

Preclinical efficacy of carfilzomib in BRAF-mutant colorectal cancer models.

Serine/threonine-protein kinase B-raf (BRAF) mutations are found in 8-15% of colorectal cancer patients and identify a subset of tumors with poor outcome in the metastatic setting. We have previously reported that BRAF-mutant human cells display a high rate of protein production, causing proteotoxic stress, and are selectively sensitive to the proteasome inhibitors bortezomib and carfilzomib. In this work, we tested whether carfilzomib could restrain the growth of BRAF-mutant colorectal tumors not only by targeting cancer cells directly, but also by promoting an immune-mediated antitumor response. In human and mouse colorectal cancer cells, carfilzomib triggered robust endoplasmic reticulum stress and autophagy, followed by the emission of immunogenic-damage-associated molecules. Intravenous administration of carfilzomib delayed the growth of BRAF-mutant murine tumors and mobilized the danger-signal proteins calreticulin and high mobility group box 1 (HMGB1). Analyses of drug-treated samples revealed increased intratumor recruitment of activated cytotoxic T cells and natural killers, concomitant with the downregulation of forkhead box protein P3 (Foxp3)+ T-cell surface glycoprotein CD4 (CD4)+ T cells, indicating that carfilzomib promotes reshaping of the immune microenvironment of BRAF-mutant murine colorectal tumors. These results will inform the design of clinical trials in BRAF-mutant colorectal cancer patients.

Proteome efficiency of metabolic pathways in Escherichia coli increases along the nutrient flow.

Understanding the allocation of the cellular proteome to different cellular processes is central to unraveling the organizing principles of bacterial physiology. Proteome allocation to protein translation itself is maximally efficient, i.e., it represents the minimal allocation of dry mass able to sustain the observed protein production rate. In contrast, recent studies on bacteria have demonstrated that the concentrations of many proteins exceed the minimal level required to support the observed growth rate, indicating some heterogeneity across pathways in their proteome efficiency. Here, we systematically analyze the proteome efficiency of metabolic pathways, which together account for more than half of the Escherichia coli proteome during exponential growth. Comparing the predicted minimal and the observed proteome allocation to different metabolic pathways across growth conditions, we find that the protein abundance in the most costly biosynthesis pathways-those for amino acid biosynthesis and cofactor biosynthesis-is regulated for near-optimal efficiency. Overall, proteome efficiency increases along the carbon flow through the metabolic network; proteins involved in pathways of nutrient uptake and central metabolism tend to be highly over-abundant, while proteins involved in anabolic pathways and in protein translation are much closer to the expected minimal abundance across conditions. Our work thus provides a bird's-eye view of metabolic pathway efficiency, demonstrating systematic deviations from optimal cellular efficiency at the network level. IMPORTANCE Protein translation is the most expensive cellular process in fast-growing bacteria, and efficient proteome usage should thus be under strong natural selection. However, recent studies show that a considerable part of the proteome is unneeded for instantaneous cell growth in Escherichia coli. We still lack a systematic understanding of how this excess proteome is distributed across different pathways as a function of the growth conditions. We estimated the minimal required proteome across growth conditions in E. coli and compared the predictions with experimental data. We found that the proteome allocated to the most expensive internal pathways, including translation and the synthesis of amino acids and cofactors, is near the minimally required levels. In contrast, transporters and central carbon metabolism show much higher proteome levels than the predicted minimal abundance. Our analyses show that the proteome fraction unneeded for instantaneous cell growth decreases along the nutrient flow in E. coli.

Antibiofilm activity of bromelain from pineapple against Staphylococcus aureus

Bromelain is a set of proteolytic enzymes usually obtained from pineapple (Ananas comosus). Although bromelain has distinguished therapeutic properties, little is known about its proteolytic potential against opportunistic pathogens related to wound healing complications, such as Staphylococcus aureus. This study aimed to investigate the antibiofilm and antibacterial activity of bromelain in 43 clinical strains of S. aureus isolated from chronic wounds and blood cultures. Bromelain’s activity against S. aureus biofilm in vitro was assessed by analyzing biofilm formation in cultures grown in the presence of 1% bromelain and biofilm destruction after the addition of 1% bromelain to mature biofilms. Proteinase K and sodium metaperiodate were also added to mature biofilms in parallel to compare their activity with that of bromelain and, together with exopolysaccharide and protein production rate assays, to determine the chemical composition of the biofilm extracellular matrix of selected strains of S. aureus. Bromelain was also evaluated for its DNase activity and impact on cellular hydrophobicity and auto-aggregation. Mueller-Hinton agar dilution was used to determine bromelain minimal inhibitory concentration (MIC). Biofilm assays showed that 1% bromelain significantly inhibits S. aureus biofilm formation (p = 0.0157) by up to 4-fold and destroys its mature biofilms (p < 0.0001) by up to 6.4-fold, both compared to the control grown without bromelain. Biofilms of methicillin-resistant S. aureus strains isolated from chronic wounds were the most affected by bromelain treatment. No antibacterial activity was detected with bromelain MIC assays and the proteolytic activity of bromelain was identified as the main antibiofilm mechanism of the enzyme, though its DNase activity may also contribute. The epithelial therapeutic properties of bromelain combined with its antibiofilm activity against S. aureus make it a promising alternative to compose the therapeutic arsenal for the control of S. aureus biofilms in the context of wound care.

MaxCal can infer models from coupled stochastic trajectories of gene expression and cell division

Gene expression is inherently noisy due to small numbers of proteins and nucleic acids inside a cell. Likewise, cell division is stochastic, particularly when tracking at the level of a single cell. The two can be coupled when gene expression affects the rate of cell division. Single-cell time-lapse experiments can measure both fluctuations by simultaneously recording protein levels inside a cell and its stochastic division. These information-rich noisy trajectory data sets can be harnessed to learn about the underlying molecular and cellular details that are often not known a priori. A critical question is: How can we infer a model given data where fluctuations at two levels—gene expression and cell division—are intricately convoluted? We show the principle of maximum caliber (MaxCal)—integrated within a Bayesian framework—can be used to infer several cellular and molecular details (division rates, protein production, and degradation rates) from these coupled stochastic trajectories (CSTs). We demonstrate this proof of concept using synthetic data generated from a known model. An additional challenge in data analysis is that trajectories are often not in protein numbers, but in noisy fluorescence that depends on protein number in a probabilistic manner. We again show that MaxCal can infer important molecular and cellular rates even when data are in fluorescence, another example of CST with three confounding factors—gene expression noise, cell division noise, and fluorescence distortion—all coupled. Our approach will provide guidance to build models in synthetic biology experiments as well as general biological systems where examples of CSTs are abundant.

The endoplasmic reticulum acetyl-CoA transporter AT-1 plays a role in the protective unfolded protein response in the exocrine pancreas

Pancreatic acinar cells are responsible for the synthesis and secretion of digestive enzymes. In order to sustain high rates of secretory protein production, acinar cells rely on adaptive mechanisms to mitigate endoplasmic reticulum (ER) stress and maintain secretory homeostasis. The adaptive unfolded protein response (UPR), mediated by the IRE1-XBP1s axis, is essential for acinar function and protects against cellular injury. Recent work has demonstrated that the ER acetyl-CoA transporter, AT-1, is necessary for nascent secretory protein acetylation and export out of the ER and is regulated by IRE1-XBP1s. Furthermore, AT-1 is rapidly downregulated during experimental pancreatitis. To investigate the role of AT-1 in exocrine pancreas function, we generated inducible, acinar-specific AT-1 knockout mice (Ela-Cre AT-1-/-). Loss of AT-1 produced a chronic pancreatitis phenotype characterized by inflammation, immune cell infiltration, tissue fibrosis, and aberrant intracellular activation of the proteolytic digestive enzyme trypsin. Despite the histological and biochemical dysfunction observed, Ela-Cre AT-1-/- mice grow normally, have typical lifespans, and do not lose pancreatic mass. Interestingly, XBP1s expression is significantly upregulated in Ela-Cre AT-1-/- and may confer a protective effect with loss of AT-1. Our data suggests a role for AT-1 in the IRE1-XBP1s axis and highlights the previously undescribed role of protein acetylation in exocrine pancreas physiology. Given the numerous potential downstream effects of dysregulated acetyl-CoA transport, this model will advance our understanding of pancreatic function and disease. NIH This is the full abstract presented at the American Physiology Summit 2023 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.

Relation of Tea Ingestion to Salivary Redox and Flow Rate in Healthy Subjects

The biochemistry of human saliva can be altered by food intake. The benefits of tea drinking were extensively studied but the influence of tea ingestion on human saliva has not been revealed. The work aimed to investigate the immediate and delayed effect of vine tea, oolong tea and black tea intake on certain salivary biochemistry and flow rate. The saliva samples of healthy subjects were collected before, after and 30 min after tea ingestion. The chemical compositions and antioxidant capacity of tea samples were analyzed to correlate with salivary parameters. Principal component analysis indicated that the effects of vine tea consumption were dominated by increasing salivary flow rate (SFR), production rate of total protein (TPC), thiol (SH), malondialdehyde, catalase activity and antioxidant capacity (FRAP) in saliva. The antioxidant profile of studied tea samples (FRAP, polyphenols, flavonoids) was positively correlated with salivary SFR, TPC, SH and FRAP but negatively correlated with salivary uric acid concentration in saliva.

Modeling mRNA Translation With Ribosome Abortions.

We derive a deterministic mathematical model for the flow of ribosomes along a mRNA called the ribosome flow model with extended objects and abortions (RFMEOA). This model incorporates important cellular features such as every ribosome covers several codons and they may detach from various regions along the track due to more realistic biological situations including phenomena of ribosome-ribosome collisions. We prove that the ribosome density profile along the mRNA in the RFMEOA and in particular, the protein production rate converge to a unique steady-state. Simulations of the RFMEOA demonstrate a surprising result that an increase in the initiation rate may sometimes lead to a decrease in the production rate. We believe that this model could be helpful to provide insight into the effects of premature termination on the protein expression and be useful for understanding and re-engineering the translation process.

On the gain of entrainment in the n-dimensional ribosome flow model.

The ribosome flow model (RFM) is a phenomenological model for the flow of particles along a one-dimensional chain of n sites. It has been extensively used to study ribosome flow along the mRNA molecule during translation. When the transition rates along the chain are time-varying and jointly T-periodic the RFM entrains, i.e. every trajectory of the RFM converges to a unique T-periodic solution that depends on the transition rates, but not on the initial condition. In general, entrainment to periodic excitations like the 24 h solar day or the 50 Hz frequency of the electric grid is important in numerous natural and artificial systems. An interesting question, called the gain of entrainment (GOE) in the RFM, is whether proper coordination of the periodic translation rates along the mRNA can lead to a larger average protein production rate. Analysing the GOE in the RFM is non-trivial and partial results exist only for the RFM with dimensions n = 1, 2. We use a new approach to derive several results on the GOE in the general n-dimensional RFM. Perhaps surprisingly, we rigorously characterize several cases where there is no GOE, so to maximize the average production rate in these cases, the best choice is to use constant transition rates all along the chain.

Differential production rates of cytosolic and transmembrane GFP reporters in C. elegans L3 larval uterine cells.

Transgene driven protein expression is an important tool for investigating developmental mechanisms in C. elegans . Here, we have assessed protein production rates and levels in L3 larval uterine cells (UCs). Using ubiquitous promoter driven cytosolic and transmembrane tethered GFP, fluorescence recovery after photobleaching, and quantitative fluorescence analysis, we reveal that cytosolic GFP is produced at an ~two-fold higher rate than transmembrane tethered GFP and accumulates at ~five-fold higher levels in UCs. We also provide evidence that cytosolic GFP in the anchor cell, a specialized UC that mediates uterine-vulval connection, is more rapidly degraded through an autophagy-independent mechanism.

Translation in the cell under fierce competition for shared resources: a mathematical model.

During translation, mRNAs 'compete' for shared resources. Under stress conditions, during viral infection and also in high-throughput heterologous gene expression, these resources may become scarce, e.g. the pool of free ribosomes is starved, and then the competition may have a dramatic effect on the global dynamics of translation in the cell. We model this scenario using a network that includes m ribosome flow models (RFMs) interconnected via a pool of free ribosomes. Each RFM models ribosome flow along an mRNA molecule, and the pool models the shared resource. We assume that the number of mRNAs is large, so many ribosomes are attached to the mRNAs, and the pool is starved. Our analysis shows that adding an mRNA has an intricate effect on the total protein production. The new mRNA produces new proteins, but the other mRNAs produce less proteins, as the pool that feeds these mRNAs now has a smaller abundance of ribosomes. As the number of mRNAs increases, the marginal utility of adding another mRNA diminishes, and the total protein production rate saturates to a limiting value. We demonstrate our approach using an example of insulin protein production in a cell-free system.

Targeting Transcriptional and Translational Hindrances in a Modular T7RNAP Expression System in Engineered Pseudomonas putida.

The T7 RNA polymerase is considered one of the most popular tools for heterologous gene expression in the gold standard biotechnological host Escherichia coli. However, the exploitation of this tool in other prospective hosts, such as the biotechnologically relevant bacterium Pseudomonas putida, is still very scarce. The majority of the existing T7-based systems in P.putida show low expression strengths and possess only weak controllability. A fundamental understanding of these systems is necessary in order to design robust and predictable biotechnological processes. To fill this gap, we established and characterized a modular T7 RNA polymerase-based system for heterologous protein production in P.putida, using the enhanced Green Fluorescent Protein (eGFP) as an easy-to-quantify reporter protein. We have effectively targeted the limitations associated with the initial genetic setup of the system, such as slow growth and low protein production rates. By replacing the T7 phage-inherent TΦ terminator downstream of the heterologous gene with the synthetic tZ terminator, growth and protein production rates improved drastically, and the T7 RNA polymerase system reached a productivity level comparable to that of an intrinsic RNA polymerase-based system. Furthermore, we were able to show that the system was saturated with T7 RNA polymerase by applying a T7 RNA polymerase ribosome binding site library to tune heterologous protein production. This saturation indicates an essential role for the ribosome binding sites of the T7 RNA polymerase since, in an oversaturated system, cellular resources are lost to the synthesis of unnecessary T7 RNA polymerase. Eventually, we combined the experimental data into a model that can predict the eGFP production rate with respect to the relative strength of the ribosome binding sites upstream of the T7 gene.

Cytidine-containing tails robustly enhance and prolong protein production of synthetic mRNA in cell and in vivo

Synthetic mRNAs are rising rapidly as alternative therapeutic agents for delivery of proteins. However, the practical use of synthetic mRNAs has been restricted by their low cellular stability as well as poor protein production efficiency. The key roles of poly(A) tail on mRNA biology inspire us to explore the optimization of tail sequence to overcome the aforementioned limitations. Here, the systematic substitution of non-A nucleotides in the tails revealed that cytidine-containing tails can substantially enhance the protein production rate and duration of synthetic mRNAs both invitro and invivo. Such C-containing tails shield synthetic mRNAs from deadenylase CCR4-NOT transcription complex, as the catalytic CNOT proteins, especially CNOT6L and CNOT7, have lower efficiency in trimming of cytidine. Consistently, these enhancement effects of C-containing tails were observed on all synthetic mRNAs tested and were independent of transfection reagents and cell types. As the C-containing tails can be used along with other mRNA enhancement technologies to synergically boost protein production, we believe that these tails can be broadly used on synthetic mRNAs to directly promote their clinical applications.

Effects of DNA template preparation on variability in cell-free protein production.

DNA templates for protein production remain an unexplored source of variability in the performance of cell-free expression (CFE) systems. To characterize this variability, we investigated the effects of two common DNA extraction methodologies, a postprocessing step and manual versus automated preparation on protein production using CFE. We assess the concentration of the DNA template, the quality of the DNA template in terms of physical damage and the quality of the DNA solution in terms of purity resulting from eight DNA preparation workflows. We measure the variance in protein titer and rate of protein production in CFE reactions associated with the biological replicate of the DNA template, the technical replicate DNA solution prepared with the same workflow and the measurement replicate of nominally identical CFE reactions. We offer practical guidance for preparing and characterizing DNA templates to achieve acceptable variability in CFE performance.

Dynamics and growth rate implications of ribosomes and mRNAs interaction in E. coli

Understanding how cells grow and adapt under various nutrient conditions is pivotal in the study of biological stoichiometry. Recent studies provide empirical evidence that cells use multiple strategies to maintain an optimal protein production rate under different nutrient conditions. Mathematical models can provide a solid theoretical foundation that can explain experimental observations and generate testable hypotheses to further our understanding of the growth process. In this study, we generalize a modeling framework that centers on the translation process and study its asymptotic behaviors to validate algebraic manipulations involving the steady states. Using experimental results on the growth of E. coli under C-, N-, and P-limited environments, we simulate the expected quantitative measurements to show the feasibility of using the model to explain empirical evidence. Our results support the findings that cells employ multiple strategies to maintain a similar protein production rate across different nutrient limitations. Moreover, we find that the previous study underestimates the significance of certain biological rates, such as the binding rate of ribosomes to mRNA and the transition rate between different ribosomal stages. Furthermore, our simulation shows that the strategies used by cells under C- and P-limitations result in a faster overall growth dynamics than under N-limitation. In conclusion, the general modeling framework provides a valuable platform to study cell growth under different nutrient supply conditions, which also allows straightforward extensions to the coupling of transcription, translation, and energetics to deepen our understanding of the growth process.

The Role of Comorbidities on Aging-Induced Changes in Muscle Health and Amino Acid Kinetics

ObjectivesAging and comorbidities are known to influence amino acid metabolism, contributing to reduced muscle and cognitive dysfunction in older adults. Whether these aging-induced manifestations remain after correction for the presence of comorbidities is unclear. MethodsOlder adults were selected from the MEDIT database. The subjects were divided into two age groups: 264 young old (age 50–70 y: n = 149 with at least 1 comorbidity based on Charlson comorbidity index (CCI > 0) and n = 115 with CCI = 0), and 196 middle old (age 70–85 y: n = 119 with CCI > 0 and n = 77 with CCI = 0). Body composition was assessed by DXA, respiratory and skeletal muscle strength by mouth pressures and dynamometry, and cognition and mood by assessments and questionnaires. Postabsorptive kinetics of multiple amino acids were measured by pulse stable isotope administration and subsequent blood sampling. Plasma amino acid concentrations and enrichments were analyzed by LC-MS/MS to measure whole-body protein production (WBP) and conversion rates. Statistics are done with ANCOVA with sex and BMI and age group as independents. Results expressed as means [95% CI]. ResultsThe middle old had lower lean mass on whole body level and in the extremities (p < 0.0001), and lower respiratory (p = 0.05) and handgrip (p = 0.005) strength compared to the young old, independent of muscle health lowering effects of CCI. Although no differences were observed in cognition and mood between the groups, an age x CCI interaction was found for depression, anxiety (p = 0.03), and lean mass extremity (p = 0.04). The middle old had lower values for WBP of tau-methylhistidine (p = 0.01), hydroxyproline (P = 0.008), and taurine (p = 0.004), and plasma glutamine concentration (P = 0.016). In addition, the middle old showed higher glutamine to glutamate conversion (p = 0.02). Age x CCI interaction was observed for plasma concentration of tyrosine (p = 0.04). No differences were observed in plasma concentrations and WBP of phenylalanine and the branched-chain amino acids (leucine, valine, and isoleucine) between the groups. ConclusionsMuscle loss and weakness and specific disturbances in amino acid kinetics due to aging exist even after correction for the presence of comorbidities. Funding SourcesNone