Normal Protein Synthesis Research Articles

Functional enrichment analysis reveals the involvement of DARS2 in multiple biological pathways and its potential as a therapeutic target in esophageal carcinoma.

The enzyme Aspartyl tRNA synthetase 2 (DARS2) is a crucial enzyme in the mitochondrial tRNA synthesis pathway, playing a critical role in maintaining normal mitochondrial function and protein synthesis. However, the role of DARS2 in ESCA is unclear. Transcriptional data of pan-cancer and ESCA were downloaded from UCSC XENA, TCGA, and GEO databases to analyze the differential expression of DARS2 between tumor samples and normal samples, and its correlation with clinicopathological features of ESCA patients. R was used for GO, KEGG, and GSEA functional enrichment analysis of DARS2 co-expression and to analyze the connection of DARS2 with glycolysis and m6A-related genes. In vitro experiments were performed to assess the effects of interfering with DARS2 expression on ESCA cells. TarBase v.8, mirDIP, miRTarBase, ENCORI, and miRNet databases were used to analyze and construct a ceRNA network containing DARS2. DARS2 was overexpressed in various types of tumors. In vitro experiments confirmed that interfering with DARS2 expression significantly affected the proliferation, migration, apoptosis, cell cycle, and glycolysis of ESCA cells. DARS2 may be involved in multiple biological pathways related to tumor development. Furthermore, correlation and differential analysis revealed that DARS2 may regulate ESCA m6A modification through its interaction with METTL3 and YTHDF1. A ceRNA network containing DARS2, DLEU2/has-miR-30a-5p/DARS2, was successfully predicted and constructed. Our findings reveal the upregulation of DARS2 in ESCA and its association with clinical features, glycolysis pathway, m6A modification, and ceRNA network. These discoveries provide valuable insights into the molecular mechanisms underlying ESCA.

Identification of metallothionein protein in Anodonta woodiana as a biomarker of mercury (Hg) contamination

Abstract. Rahayu SYS, Fadila A, Fahmi MR. 2023. Identification of metallothionein protein in Anodonta woodiana as a biomarker of mercury (Hg) contamination. Nusantara Bioscience 15: 90-94. Heavy metal contamination can affect the survival of aquatic biota and will accumulate in the bodiesorganisms. Moreover, contamination identification at the molecular level can be analyzed using biomarker analysis. Biomarkers are responses measured individually, ranging from enzymes and xenobiotic measurements to organ and overall conditions. Biomarker analysis can be done by checking the metallothionein protein, this expression can be induced by Reactive Oxygen Species (ROS). Metallothionein (MT) has a thiol group with nucleophilic properties. As a result, this group can make Metallothionein able to find metals and free radicals. Therefore, prevention that can be done to reduce contamination at a higher trophic level requires monitoring the molecular level by observing the metallothionein protein. For example, Anodonta woodiana (Rea, 1834) or kijing taiwan induced by HgCl2 aims to characterize their absorption ability in the environment through metallothionein protein. That was conducted by the SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) method. SDS-PAGE electrophoresis results showed that the size of the metallothionein protein was 7 kDa, 15 kDa, and >30 kDa. Size >30 kDa is a group of proteins related to stress control or Heat shock protein (Hsp). The presence of Hsp is due to the body increasing stress protein synthesis and metallothionein to reduce normal protein synthesis. Based on the results obtained, this study revealed that A. woodiana could absorb HgCl2, as evidenced by the metallothionein protein characterization results.

Antibiotic synergist OM19r reverses aminoglycoside resistance in multidrug-resistant Escherichia coli.

The continued emergence and spread of multidrug-resistant (MDR) bacterial pathogens require a new strategy to improve the efficacy of existing antibiotics. Proline-rich antimicrobial peptides (PrAMPs) could also be used as antibacterial synergists due to their unique mechanism of action. Utilizing a series of experiments on membrane permeability, In vitro protein synthesis, In vitro transcription and mRNA translation, to further elucidate the synergistic mechanism of OM19r combined with gentamicin. A proline-rich antimicrobial peptide OM19r was identified in this study and its efficacy against Escherichia coli B2 (E. coli B2) was evaluated on multiple aspects. OM19r increased antibacterial activity of gentamicin against multidrug-resistance E. coli B2 by 64 folds, when used in combination with aminoglycoside antibiotics. Mechanistically, OM19r induced change of inner membrane permeability and inhibited translational elongation of protein synthesis by entering to E. coli B2 via intimal transporter SbmA. OM19r also facilitated the accumulation of intracellular reactive oxygen species (ROS). In animal models, OM19r significantly improved the efficacy of gentamicin against E. coli B2. Our study reveals that OM19r combined with GEN had a strong synergistic inhibitory effect against multi-drug resistant E. coli B2. OM19r and GEN inhibited translation elongation and initiation, respectively, and ultimately affected the normal protein synthesis of bacteria. These findings provide a potential therapeutic option against multidrug-resistant E. coli.

A novel function for eukaryotic elongation factor 3: Inhibition of stop codon readthrough in yeast

Eukaryotic elongation factor 3 (eEF3) is one of the essential yeast ribosome-associated ATP-binding cassette type F (ABCF) ATPases. Previously, we found that eEF3 stimulates release of mRNA from puromycin-treated polysomes. In this study, we used a cell-free cricket paralysis virus (CrPV) internal ribosome entry site (IRES)-mediated firefly luciferase bicistronic mRNA translation system with yeast S30 extract. When eEF3 was partially removed from the crude extract, the product from the downstream ORF was increased by the readthrough of a UAA stop codon in the upstream ORF. eEF3 enhanced the release of luciferase from the polysome by eukaryotic release factor (eRF)1 and eRF3. These results suggest that eEF3 is a factor that assists eRFs in performing normal protein synthesis termination in yeast.

The response to unfolded proteins in schizophrenia and bipolar disorder

IntroductionSchizophrenia (SCH) and bipolar disorder (BD) are severe mental disordes, which have high incidence (Whiteford et al. Lancet 2013; 381 1575-86) and are the main causes of diasibility in young people (WHO 2022; https://www.who.int/news-room/fact-sheets/detail/mental-disorders).Psycological stress appears in different mental disorders, and this is directly related to oxidative stress (Moller et al. Chem Biol Interact. 1996; 102 17-36)(Pupic-Bakrac et al. 2020; Psychiatr Danub. 32 412-9). Oxidative stress causes reticulum edoplasmic stress (ER stress) and this produces high levels of misfolded proteins. Defective proteins are degraded by the proteasome, but but when the density of misfolded proteins exceeds the capacity of the proteosome, the Unfolded and Misfolded Protein Response (UPR) is triggered through three main pathways: Inositol-requiring enzyme 1α (IRE1α); transcription factor 6 alpha (ATF6α) and protein kinase RNA-Like ER kinase (PERK), trying to recover normal protein synthesis capacity (Bermejo-Millo et al. 2018; Mol Neurobiol. 55 7973-86) (González-Blanco et al. 2022; J Cachexia Sarcopenia Muscle 13 919-31).ObjectivesCharacterizing ER stress and UPR in SCH and BD.MethodsWe studied ER stress and UPR in peripheral blood mononuclear cells (PBMC) from 50 patients with SCH and an equal number of patients with BD compared to their corresponding controls in order to achieve our objectives.Western Blot assay were performed following classical procedure () and the results was normalized to Ponceau as loanding control (Nie et al. 2017; BiochemByophys Resp 12 10-13) (Sander et al. 2019; Anal Biochem 575 44-53). Proteasome activity was assessed using Proteasome Activity Assay Kit (ab107921, Abcam, Cambridge, UK).ResultsER stress was evaluated with BiP/GRP78. Our results showed significantly increased expression in SCH (p<0,01) and BD (p<0,05), being more increased in SCH. Proteasome activity was increased in SCH and BD, being only statistically significant in SQZ (p<0,05). UPR study showed IRE1a cascade significantly activated in SCH (p<0,001) and only slight increased in BD showed without statistical differences. ATF6a pathway is measured by cleavage to active protein (50-kDa). Results showed higher expression in SCH than in BD and controls (p<0,001). In addition, PERK pathway showed higher statistical levels of p-eIF2a/eiF2a ratio in SCH than in BD and control respectively (p<0,05 and p<0,01).ConclusionsOur results showed a greater alteration in SCH than in BD at the level of protein synthesis, which implies a greater toxicity at the cellular level and, therefore, a clear risk for the survival of cells in this pathology.Disclosure of InterestNone Declared

Single-cell RNA sequencing reveals the suppressive effect of PPP1R15A inhibitor Sephin1 in antitumor immunity

Protein phosphatase 1 regulatory subunit 15A (PPP1R15A) is an important factor in the integrated stress response (ISR) in mammals and may play a crucial role in tumorigenesis. In our studies, we found an inhibitor of PPP1R15A, Sephin1, plays a protumorigenic role in mouse tumor models. By analyzing the single-cell transcriptome data of the mouse tumor models, we found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in antitumor immune cell types and lower expression levels of cytotoxicity-related genes. In addition, Tcell receptor (TCR) repertoire analysis demonstrated that the clonal expansion of tumor-specific Tcells was inhibited by Sephin1. A special TCR+ macrophage subtype in tumor was identified to be significantly depleted upon Sephin1 treatment, implying its key antitumor role. These results suggest that PPP1R15A has the potential to be an effective target for tumor therapy.

A possible connection between reactive oxygen species and the unfolded protein response in lens development: From insight to foresight.

The lens is a relatively special and simple organ. It has become an ideal model to study the common developmental characteristics among different organic systems. Lens development is a complex process influenced by numerous factors, including signals from the intracellular and extracellular environment. Reactive oxygen species (ROS) are a group of highly reactive and oxygen-containing molecules that can cause endoplasmic reticulum stress in lens cells. As an adaptive response to ER stress, lens cells initiate the unfolded protein response (UPR) to maintain normal protein synthesis by selectively increasing/decreasing protein synthesis and increasing the degradation of misfolded proteins. Generally, the UPR signaling pathways have been well characterized in the context of many pathological conditions. However, recent studies have also confirmed that all three UPR signaling pathways participate in a variety of developmental processes, including those of the lens. In this review, we first briefly summarize the three stages of lens development and present the basic profiles of ROS and the UPR. We then discuss the interconnections between lens development and these two mechanisms. Additionally, the potential adoption of human pluripotent stem-cell-based lentoids in lens development research is proposed to provide a novel perspective on future developmental studies.

Plasma bupivacaine levels (total and free/unbound) during epidural infusion in liver resection patients: a prospective, observational study

IntroductionLiver resection patients may be at an increased risk of local anesthetic (LA) toxicity because the liver is essential for metabolizing LA and producing proteins (mainly α1-acid glycoprotein (AAG)) that...

Gene expression study alerted to possible impairment in Daphnia magna individuals as a consequence of exposure to sublethal concentrations of prochloraz

In the present study, Daphnia magna individuals were exposed for 21 days to 87, 130, 170, 230 and 380 μg/L of prochloraz. The expression of genes related to lipid metabolism (fabd), oxidative stress (cat and gst), heat shock proteins synthesis (hsp70 and hsp90), haemoglobin synthesis (hgb1 and hgb2), metallothioneins synthesis (mt-a, mt-b and mt-c), and vitellogenin synthesis (vgt1 y vgt2) were analyzed. Results showed that some gene expression in D. magna was altered as a consequence of the individual exposure to the fungicide. The genes fabd, vtg1 and vtg2, cat and gst resulted unaltered by the exposure of the daphnids to different fungicide concentrations. However, daphnid exposure to 380 μg/L of prochloraz resulted in an overexpression (p < 0.05) of hsp70 gene which indicated an alteration of the normal protein synthesis and its integrity maintenance. On the other hand, mt-b gene resulted significantly underexpressed (p < 0.05) in daphnids exposed to the lowest fungicide concentrations (87, 130 and 170 μg/L, respectively). In addition, hgb1 and hgb2 genes which are related with the haemoglobin synthesis weresignificantly overexpressed (p < 0.05). Results showed that hgb1 gene was overexpressed almost 100 times more in daphnids exposed for 21 days to 170, 230 and 380 μg/L than control values. However, many authors advocate for an association of these gene expression changes with the presence of contaminants in the medium, in fact they could be used as a good indicator of early contamination at low concentrations of toxicants.

MECHANISMS OF THE KEEPING AND CHANGE OF FORMS AND SIZES OF THE CELL NUCLEI (REVIEW)

The size and shape of the cell nucleus are the frequently used parameters in the studies of Russian and foreign-states authors, not only as necessary for calculating the nuclear-cytoplasmic ratio of a cell in ontogenesis, differentiation, and pathological processes, but also having values as such. However, in discussions, two extreme points of view are expressed on the value of information about the shape and, especially, about the size of the nucleus. According to the first point of view, the morphometry of the size and shape of the cell nucleus without measuring the cytoplasm with the subsequent calculation of the nuclear-cytoplasmic ratio does not make any sense, and the data obtained do not carry significant information. Proponents of the second point of view consider the cell nucleus as a labile and significant indicator of the morphological and functional state of the cell, the size and shape of which change during normal aging, pathological conditions, proliferation, gene expression, and protein synthesis. In this regard, a meta-analysis of modern scientific literature devoted to the study of the mechanisms of maintaining and changing the size and shape of the cell nucleus was carried out. The data obtained were subjected to an analytical study in order to formulate and explain the structures, factors and mechanisms of maintenance, changes in the size, shape of the cell nucleus. Based on the analysis of data from Russian and foreign-states sources, it can be confidently stated that the amount of DNA in the nucleus is not the only factor that determines its size and shape, but also the structure and modification of chromatin can affect nuclear morphology. It can be considered proven that the leading structures of the cell that determine the size and shape of the cell nucleus are the cytoskeleton, the complex of nuclear pores, the nuclear lamina, the endoplasmic reticulum, and the factors are nuclear-cytoplasmic exchange and osmolarity. Further study of the structures and factors affecting the size and shape of the nucleus, establishing the relationship between its morphology and processes occurring at the tissue and cellular levels, promises to provide new approaches to the diagnosis, prevention and treatment of a number of diseases.

PSVII-7 Prematurity alters the regulation of Akt signaling in skeletal muscle of piglets

Abstract Objectives: Postnatal growth faltering is common after preterm birth. Recently we showed that premature birth in piglets impairs normal postnatal weight gain and skeletal muscle protein synthesis compared to piglets born at term. This response is associated with a reduction in the feeding-induced activation of Akt and subsequent downstream signaling, despite no change in insulin receptor activation. The aim of this study was to identify key regulators of Akt responsible for the blunted anabolic response in preterm muscle. Methods: Piglets delivered by cesarean section 11 d (preterm/PT) or 2 d (term/T) before term birth were fed by total parenteral nutrition. On day 3, after 4 h fasting, piglets were fasted one additional h or fed orally a sow milk replacer (per kg body weight: 31.5 kcal, 1.3 g carbohydrate, 2.7 g amino acids BW, 1.6 g lipid). Positive and negative regulators of Akt activity in longissimus dorsi muscle were determined by Western blot. Results: Phosphorylation of Akt1 and Akt2, but not Akt3, was lower in PT than in T pigs (P &amp;lt; 0.05). Phosphorylation of Akt activators, PDK1 and mTORC2, and the abundance of Ubl4A, a positive regulator of Akt, were lower in PT than in T (P &amp;lt; 0.05). Abundance of Akt inhibitors, PHLPP and SHIP2, but not PTEN, was higher in PT than in T (P &amp;lt; 0.05). Activation of the Akt phosphatase, PP2A, was unaffected by feeding in PT but inhibited by feeding in T pigs (P &amp;lt; 0.05). Conclusions: These results show that the feeding-induced activation of positive regulators of Akt is reduced by preterm birth, whereas the activation of negative regulators is enhanced. Our findings suggest that premature birth impairs the activation of Akt that is essential for channeling dietary nutrients for anabolism and likely contributes to the postnatal growth faltering of prematurity. Research Support: NIH and USDA.

Sheep models of F508del and G542Xcystic fibrosismutations show cellular responses to human therapeutics.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The F508del and G542X are the most common mutations found in US patients, accounting for 86.4% and 4.6% of all mutations, respectively. The F508del causes deletion of the phenylalanine residue at position 508 and is associated with impaired CFTR protein folding. The G542X is a nonsense mutation that introduces a stop codon into the mRNA, thus preventing normal CFTR protein synthesis. Here, we describe the generation of CFTRF508del / F508del and CFTRG542X / G542X lambs using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). First, we introduced either F508del or G542X mutations into sheep fetal fibroblasts that were subsequently used as nuclear donors for SCNT. The newborn CF lambs develop pathology similar to CFTR −/− sheep and CF patients. Moreover, tracheal epithelial cells from the CFTRF508del / F508del lambs responded to a human CFTR (hCFTR) potentiator and correctors, and those from CFTRG542X / G542X lambs showed modest restoration of CFTR function following inhibition of nonsense‐mediated decay (NMD) and aminoglycoside antibiotic treatments. Thus, the phenotype and electrophysiology of these novel models represent an important advance for testing new CF therapeutics and gene therapy to improve the health of patients with this life‐limiting disorder.

Review: Modification of Nanocellulose as Conjugate of Infection-Causing Antibacterial Hydrogel

Infection is the process of entering and reproducing microorganisms such as bacteria, viruses, fungi, and parasites that cause tissue injury. Some of the common types of bacteria that play a role in wound infection are Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Staphylococcus epidermis. The antibacterial able to inhibit bacterial growth by inhibiting cell wall biosynthesis, increasing the permeability of the bacterial cytoplasmic membrane, and interfering with the normal bacterial protein synthesis. The aim of this review article is to conduct a study of nanocellulose as an antibacterial hydrogel conjugate. The method used is to summarize information from various recent journals related to nanocellulose, nanocellulose modification, nanocellulose-based hydrogels, and their application as antibacterial. Some journals from primary sources such as the PMC system (PubMed Central), National Library of Medicine (NIH), Science Direct, Elsevier, Nature, ACS Chemical Society, and several other sources. Nanocellulose consists of β-1, 4-glucose, and there are three hydroxyls active at the C2, C3, and C6 positions of the pyranose attachment. Nanocellulose can respond by the reaction of oxidation, esterification, or etherification, by adding a new functional group. Nanocellulose can become nanocellulose nanocrystals (CNC), cellulose nanofibers (NFC), and nanocellulose bacteria (BNC). Nanocellulose formulated in the form of hydrogels and combined with antibiotics will increase the effectiveness in reducing the risk of infection that is resistant to antibiotics.

Evaluation of the optimum dietary essential amino acid pattern for adult pacu (Piaractus mesopotamicus)

The optimum levels of essential amino acids (EAAs) are required to be maintained in fish feed for obtaining normal body protein synthesis and muscle growth. This study aimed to determine the optimum dietary EAA pattern for adult pacu (Piaractus mesopotamicus) by the amino acid (AA) deletion method. A total of 165 adult fish of 1109.83 ± 14.26 g (initial body weight ± standard deviation) were equally distributed among 11 treatments in a recirculation aquaculture system. Each of the 11 treatments consisted of three randomly arranged replicates (tanks) and 5 fish were stocked per tank. During the 60-day experimental period, fish were fed with 11 diets including a control diet (CD) and 10 EAA limiting diets (LDs) two times daily until apparent satiation. The body nitrogen (N) retention and weight gain in fish fed with EAA LDs was lower (P < 0.05) than the CD. The feed efficiency in fish fed with CD was similar (P > 0.05) to most EAA LDs, except arginine and histidine LDs. The specific growth rate in fish fed with tryptophan, arginine and histidine LDs was different (P < 0.05) as compared to the CD. Based on the present N retention data obtained through the AA deletion method, the optimum pattern of dietary EAAs estimated in relation to lysine requirement (100%) was as follows: arginine 72.16%; phenylalanine 40.83%; histidine 30.15%; isoleucine 53.01%; leucine 81.71%; methionine 24.97%; threonine 58.20%; tryptophan 9.13% and valine 54.69%.

Coordinate Regulation of Ribosome and tRNA Biogenesis Controls Hypoxic Injury and Translation

The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to efficiently produce proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment, as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans. In a screen for mutations protecting C.elegans from hypoxic stress, we isolated multiple genes impacting protein synthesis: a ribosomal RNA helicase gene, tRNA biosynthesis genes, and a gene controlling amino acid availability. To define better the mechanisms by which these genes impact protein synthesis, we performed a second screen for suppressors of the conditional developmental arrest phenotype of the RNA helicase mutant and identified genes involved in ribosome biogenesis. Surprisingly, these suppressor mutations restored normal hypoxic sensitivity and protein synthesis to the tRNA biogenesis mutants, but not to the mutant reducing amino acid uptake. Proteomic analysis demonstrated that reduced tRNA biosynthetic activity produces a selective homeostatic reduction in ribosomal subunits, thereby offering a mechanism for the suppression results. Our study uncovers an unrecognized higher-order-translation regulatory mechanism in a metazoan whereby ribosome biogenesis genes communicate with genes controlling tRNA abundance matching the global rate of protein synthesis with available resources.

Overexpression of budding yeast protein phosphatase Ppz1 impairs translation

The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.

Integrated Proteomics and Metabolomics Analysis Provides Insights into Ganoderic Acid Biosynthesis in Response to Methyl Jasmonate in Ganoderma Lucidum.

Ganoderma lucidum is widely recognized as a medicinal basidiomycete. It was previously reported that the plant hormone methyl jasmonate (MeJA) could induce the biosynthesis of ganoderic acids (GAs), which are the main active ingredients of G. lucidum. However, the regulatory mechanism is still unclear. In this study, integrated proteomics and metabolomics were employed on G. lucidum to globally identify differences in proteins and metabolites under MeJA treatment for 15 min (M15) and 24 h (M24). Our study successfully identified 209 differential abundance proteins (DAPs) in M15 and 202 DAPs in M24. We also identified 154 metabolites by GC–MS and 70 metabolites by LC–MS in M24 that are involved in several metabolic pathways. With an in-depth analysis, we found some DAPs and metabolites that are involved in the oxidoreduction process, secondary metabolism, energy metabolism, transcriptional and translational regulation, and protein synthesis. In particular, our results reveal that MeJA treatment leads to metabolic rearrangement that inhibited the normal glucose metabolism, energy supply, and protein synthesis of cells but promoted secondary metabolites, including GAs. In conclusion, our proteomics and metabolomics data further confirm the promoting effect of MeJA on the biosynthesis of GAs in G. lucidum and will provide a valuable resource for further investigation of the molecular mechanisms of MeJA signal response and GA biosynthesis in G. lucidum and other related species.

Identifying and Pharmacologically Correcting the Molecular Pathophysiology of Contractures in Neonatal Brachial Plexus Injury

Purpose: Neonatal brachial plexus injury (NBPI) causes disabling contractures that cannot be fully prevented or corrected, largely because their pathophysiology is incompletely understood. Research in a mouse model has shown that contractures result at least in part from impaired postnatal muscle growth, but the mechanism of this impaired muscle growth is unknown. The current work uses a mouse model to experimentally assess and correct muscle protein imbalance (synthesis versus degradation) as a mechanism of impaired muscle growth and contractures following NBPI.&#x0D; Methods: Unilateral global (C5-T1) NBPIs were surgically created in 5-day-old mice, which permanently denervates the forelimb and reliably causes shoulder and elbow contractures 4 weeks post-NBPI. Protein synthesis was measured in denervated and contralateral control elbow flexor muscles within 4 weeks post-NBPI by incorporation of puromycin, a nucleoside analog, and by expression of major structural and contractile proteins by RNA-sequencing and Western blot. Protein degradation was similarly measured by K48-linkage specific polyubiquitin, an indicator of protein degradation by the proteasome pathway, and by expression of protein degradation markers by RNA-sequencing. Subsequently, to test the ability of proteasome inhibition to prevent contractures, bortezomib, a 20S proteasome inhibitor, was co-administrated with [Gly14]-Humanin G ([Gly14]-HN, to limit bortezomib toxicity) systemically for 4 weeks post-NBPI. Saline and [Gly14]-HN alone were administered separately as controls. Shoulder and elbow contractures were measured 4 weeks post-NBPI, and denervated and control elbow flexor muscles were assayed for 20S proteasome activity, volume and cross-sectional area (CSA) by microCT, and sarcomere length by DIC microscopy.&#x0D; Results: NBPI did not reduce muscle protein synthesis, measured either by puromycin incorporation or by RNA and protein levels of all major structural and contractile proteins. However, NBPI increased protein degradation, as indicated by a doubling of K48-linkage specific polyubiquitin and increased expression of Trim63/MurF1, a driver of proteasome-mediated protein degradation. Bortezomib + [Gly14]-HN effectively prevented contractures following NBPI compared to saline and [Gly14]-HN alone (p&lt;0.001). Bortezomib blunted the denervation-induced increase in proteasome activity (p&lt;0.001), and rescued muscle growth in volume (p&lt;0.0001), CSA (p&lt;0.001), and sarcomere length (p=0.03).&#x0D; Conclusion: Contractures following NBPI are associated with increased muscle protein degradation counteracting normal protein synthesis. Inhibition of the proteasome pathway of protein degradation improves growth of denervated muscle and prevents contractures following NBPI.&#x0D; Significance: This study identifies a pathophysiologic mechanism of impaired muscle growth and contracture formation following neonatal brachial plexus injury, and demonstrates the first ever successful pharmacologic strategy to prevent these contractures by targeting a causative molecular mechanism.

Whole-blood transcriptomic responses to lumacaftor/ivacaftor therapy in cystic fibrosis

BackgroundCystic fibrosis (CF) remains without a definitive cure. Novel therapeutics targeting the causative defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are in clinical use. Lumacaftor/ivacaftor is a CFTR modulator approved for patients homozygous for the CFTR variant p.Phe508del, but there are wide variations in treatment responses preventing prediction of patient responses. We aimed to determine changes in gene expression related to treatment initiation and response. MethodsWhole-blood transcriptomics was performed using RNA-Seq in 20 patients with CF pre- and 6 months post-lumacaftor/ivacaftor (drug) initiation and 20 non-CF healthy controls. Correlation of gene expression with clinical variables was performed by stratification via clinical responses. ResultsWe identified 491 genes that were differentially expressed in CF patients (pre-drug) compared with non-CF controls and 36 genes when comparing pre-drug to post-drug profiles. Both pre- and post-drug CF profiles were associated with marked overexpression of inflammation-related genes and apoptosis genes, and significant under-expression of T cell and NK cell-related genes compared to non-CF. CF patients post-drug demonstrated normalized protein synthesis expression, and decreased expression of cell-death genes compared to pre-drug profiles, irrespective of clinical response. However, CF clinical responders demonstrated changes in eIF2 signaling, oxidative phosphorylation, IL-17 signaling, and mitochondrial function compared to non-responders. Top overexpressed genes (MMP9 and SOCS3) that decreased post-drug were validated by qRT-PCR. Functional assays demonstrated that CF monocytes normalized calcium (increases MMP9 expression) concentrations post-drug. ConclusionsTranscriptomics revealed differentially regulated pathways in CF patients at baseline compared to non-CF, and in clinical responders to lumacaftor/ivacaftor.

A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System.

We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription-translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.