Protein Synthesis Research Articles

The Effects of Multiple Acute Turkesterone Doses on Indirect Measures of Hypertrophy and Metabolic Measures: A Preliminary Investigation

Turkesterone is a naturally occurring plant steroid touted for its medicinal, pharmacological, and biological properties with no reported adverse side effects compared with traditional anabolic androgenic steroids (AAS). However, this ostensible enhancement to increase muscle protein synthesis and facilitate augmented thermogenesis remains undescribed despite uninformed and potentially haphazard consumption. To investigate whether turkesterone enhances insulin-like growth factor-1 (IGF-1) and resting metabolic rate (RMR), eleven apparently healthy males (23.3 ± 2.2) volunteered to participate in the present study with samples collected pre-, 3H post-, and 24H post-ingestion. Subsequent analyses failed to reveal any significant main condition, time, or interaction main effects for serum IGF-1, RMR, lipid, and carbohydrate metabolism (p > 0.05). However, non-significant serum IGF-1 concentrations increased with both turkesterone conditions and remained elevated when compared with placebo. Similarly, RMR remained elevated above baseline across the 3 h assessed. Although these data fail to fully support turkesterone as a potent anabolic supplement, nevertheless, our findings are foundational to persistently tease apart this supplement’s purported ergogenic effects and underscore its favorable hemodynamic and gastrointestinal tolerability profile. Future investigations should, therein, aim to assess turkesterone-mediated IGF-1 increases on long-term whole-muscle growth across several training sessions to further substantiate its efficacy on anabolism.

Activation of platelet-derived growth factor receptors regulate connective tissue growth factor protein levels via the AKT pathway in malignant mesothelioma cells.

The incidence of malignant mesothelioma (MM), a disease linked to refractory asbestos exposure, continues to increase globally, and remains largely resistant to various treatments. Our previous studies have identified a strong correlation between connective tissue growth factor (CTGF) protein expression and MM malignancy, underscoring the importance of understanding CTGF regulation in MM cells. In this study, we demonstrate for the first time that stimulation with platelet-derived growth factor receptor (PDGFR) ligand, PDGF-BB, increases CTGF protein expression levels without affecting CTGF mRNA levels. Inhibition of PDGFR resulted in a reduction of CTGF protein expression, indicating that PDGFR activation is essential in regulating CTGF protein expression in MM cells. PDGF-BB also activated the protein kinase B (AKT) pathway, and inhibition of AKT phosphorylation abolished the PDGFR-induced CTGF protein expression, suggesting that PDGFR acts upstream of CTGF via the AKT pathway. This reinforces the role of CTGF protein as a key regulator of MM malignancy. Additionally, PDGFR activation led to the phosphorylation of mTOR and 4E-BP1, critical regulators of protein synthesis downstream of AKT, suggesting that PDGFR controls CTGF protein expression through the regulation of CTGF mRNA translation.

Protein quality malnutrition

Protein quality refers to the evaluation of a food or a diet based on its amino acid composition, protein digestibility, and protein bioavailability. When these parameters are specified, either through direct measurement or estimation, the amino acids provided by the diet are compared to those required by a healthy individual, and based on this comparison, an adequacy ratio or score is assigned. Two widely used protein quality scoring systems are the protein digestibility-corrected amino acid score (PDCAAS) and the digestible indispensable amino acid score (DIAAS), neither of which account for the dietary source of the protein. In malnourished children, metabolic adaptations reduce the endogenous availability of amino acids and increase the demand for protein synthesis. These increased amino acid requirements are primarily driven by the presence of acute infection and the need for tissue accretion. This review examines two large clinical feeding trials involving moderately malnourished children, where dietary protein quality was carefully measured. The finding s suggest that protein quality scores alone do not reliably predict weight gain or recovery in these children and that consuming milk protein provides distinct advantages over vegetable-based proteins.

Epidermolytic Hyperkeratosis Type 1 with a New Heterozygous Mutation in KRT1 Gene: A Case Report

Background: Epidermolytic hyperkeratosis (EHK) formerly known as bullous ichthyosiform erythroderma, is a rare autosomal dominant inheritance condition with a prevalence ranging from 1:200,000 to 1:300,000. The underlying etiology of which is a mutation in the genes responsible for keratin proteins synthesis, primarily in KRT1 and KRT10 genes, cataloged under OMIM number 113800. Clinicopathological presentations include blistering in the neonatal period followed by ichthyotic hyperkeratosis in childhood and adolescence, characterized by the hallmark features of generalized dry skin, scaling, hyperkeratosis, and frequently associated with erythroderma. EHK may present with or without palmoplantar keratoderma involvement, depending on the keratin gene variant. Case report: A 24-year-old male of Arab descent was born with diffuse skin hyperkeratotic plates featuring erythematous fissures, covering his entire body, resembling a collodion baby. His birth was full-term via cesarean section without complications, although he later developed developmental dysplasia of the hip. By the time he started primary school, he began experiencing severe thickening of the skin around his joints, reducing flexibility and leading to contractures and significant disfigurement. He is undergoing treatment with acitretin. Genetic testing using Next-Generation Sequencing (NGS) was performed to identify the responsible gene, revealing a novel heterozygous variant in the KRT1 gene (OMIM 139350), while no variant was detected in the KRT10 gene. Conclusion: EHK type 1 manifests with a new heterozygous variant in the KRT1 gene. This rare disorder, associated with variant in genes related to keratin production such as KRT1 and KRT10, results in distinctive skin features present from birth, as well as joint problems and fungal foot infections. Genetic testing has confirmed the presence of a variant in the KRT1 gene.

A rapid, facile, and economical method for the isolation of ribosomes and translational machinery for structural and functional studies.

Ribosomes are essential RNA-protein complexes involved in protein synthesis and quality control. Traditional methods for ribosome isolation are labor intensive, expensive, and require substantial biological material. In contrast, our new method, RAPPL (RNA Affinity Purification using Poly-Lysine), offers a rapid, simple, and cost-effective alternative. This method enriches ribosomes and associated factors from various species and sample types, including cell lysates, whole cells, organs, and whole organisms, and is compatible with traditional isolation techniques. Here, we use RAPPL to facilitate the rapid isolation, functional screening, and structural analysis of ribosomes with associated factors. We demonstrate ribosome-associated resistance mechanisms from patient uropathogeni c Escherichia coli samples and generate a 2.7Å cryoEM structure of ribosomes from Cryptococcus neoformans . By significantly reducing the amount of the starting biological material and the time required for isolation, the RAPPL approach improves the study of ribosomal function, interactions, and antibiotic resistance, providing a versatile platform for academic, clinical, and industrial research on ribosomes. Ribosomes are macromolecular RNA-protein complexes that constitute the central machinery responsible for protein synthesis and quality control in the cell. Ribosomes also serve as a hub for multiple non-ribosomal proteins and RNAs that control protein synthesis. However, the purification of ribosomes and associated factors for functional and structural studies requires a large amount of starting biological material and a tedious workflow. Current methods are challenging as they combine ultracentrifugation, the use of sucrose cushions or gradients, expensive equipment, and multiple hours to days of work. Here, we present a rapid, facile, and cost-effective method to isolate ribosomes from in vivo or in vitro samples for functional and structural studies using single-step enrichment on magnetic beads - RAPPL (RNA Affinity Purification using Poly-Lysine). Using mass spectrometry and western blot analyses, we show that poly-lysine coated beads incubated with E. coli and HEK-293 cell lysates enrich specifically for ribosomes and ribosome-associated factors. We demonstrate the ability of RAPPL to isolate ribosomes and translation-associated factors from limited material quantities, as well as a wide variety of biological samples: cell lysates, cells, organs, and whole organisms. Using RAPPL, we characterized and visualized the different effects of various drugs and translation inhibitors on protein synthesis. Our method is compatible with traditional ribosome isolation. It can be used to purify specific complexes from fractions of sucrose gradients or in tandem affinity purifications for ribosome-associated factors. Ribosomes isolated using RAPPL are functionally active and can be used for rapid screening and in vitro characterization of ribosome antibiotic resistance. Lastly, we demonstrate the structural applications of RAPPL by purifying and solving the 2.7Å cryo-EM structure of ribosomes from the Cryptococcus neoformans , an encapsulated yeast causing cryptococcosis. Ribosomes and translational machinery purified with this method are suitable for subsequent functional or structural analyses and provide a solid foundation for researchers to carry out further applications - academic, clinical, or industrial - on ribosomes.

Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell

Replication, heredity, and evolution are characteristic of Life. We and others have postulated that the reconstruction of a synthetic living system in the laboratory will be contingent on the development of a genetic self-replicator capable of undergoing Darwinian evolution. Although DNA-based life dominates, the in vitro reconstitution of an evolving DNA self-replicator has remained challenging. We hereby emulate in liposome compartments the principles according to which life propagates information and evolves. Using two different experimental configurations supporting intermittent or semi-continuous evolution (i.e., with or without DNA extraction, PCR, and re-encapsulation), we demonstrate sustainable replication of a linear DNA template – encoding the DNA polymerase and terminal protein from the Phi29 bacteriophage – expressed in the ‘protein synthesis using recombinant elements’ (PURE) system. The self-replicator can survive across multiple rounds of replication-coupled transcription-translation reactions in liposomes and, within only ten evolution rounds, accumulates mutations conferring a selection advantage. Combined data from next-generation sequencing with reverse engineering of some of the enriched mutations reveal nontrivial and context-dependent effects of the introduced mutations. The present results are foundational to build up genetic complexity in an evolving synthetic cell, as well as to study evolutionary processes in a minimal cell-free system.

Targeting MuRF1 to Combat Skeletal Muscle Wasting in Cardiac Cachexia: Mechanisms and Therapeutic Prospects.

Cardiac cachexia, the terminal stage of chronic heart failure, is characterized by severe systemic metabolic imbalances and significant weight loss, primarily resulting from skeletal muscle mass depletion. Despite the detrimental consequences, there is no standardized and clinically-approved intervention currently available for cardiac cachexia. In the context of cardiac cachexia, accelerated protein turnover, that is, inhibited protein synthesis and enhanced protein degradation, plays a crucial role in skeletal muscle wasting. This process is primarily mediated by various proteins encoded by atrogenes. Among them, the atrogene Trim63 (tripartite motif family 63) and its encoded protein MuRF1 have been extensively studied. This review article aims to elucidate the pathogenic mechanisms underlying skeletal muscle wasting in cardiac cachexia, describe the biochemical characteristics of MuRF1, and provide an overview of the investigation into MuRF1-targeting inhibitors. The ultimate goal is to offer novel strategies for the clinical treatment for skeletal muscle wasting associated with cardiac cachexia.

Screening of hub genes for sepsis-induced myopathy by weighted gene co-expression network analysis and protein-protein interaction network construction

Sepsis-induced myopathy is one of the serious complications of sepsis, which severely affects the respiratory and peripheral motor systems of patients, reduces their quality of life, and jeopardizes their lives, as evidenced by muscle atrophy, loss of strength, and impaired regeneration after injury. The pathogenesis of sepsis-induced myopathy is complex, mainly including cytokine action, enhances free radical production in muscle, increases muscle protein hydrolysis, and decreases skeletal muscle protein synthesis, etc. The above mechanisms have been demonstrated in existing studies. However, it is still unclear how the overall pattern of gene co-expression affects the pathological process of sepsis-induced myopathy. Therefore, we intend to identify hub genes and signaling pathways. Weighted gene co-expression network analysis was our main approach to study gene expression profiles: skeletal muscle transcriptome in ICU patients with sepsis-induced multi-organ failure (GSE13205). After data pre-processing, about 15,181 genes were used to identify 13 co-expression modules. Then, 16 genes (FEM1B, KLHDC3, GPX3, NIFK, GNL2, EBNA1BP2, PES1, FBP2, PFKP, BYSL, HEATR1, WDR75, TBL3, and WDR43) were selected as the hub genes including 3 up-regulated genes and 13 down-regulated genes. Then, Gene Set Enrichment Analysis was performed to show that the hub genes were closely associated with skeletal muscle dysfunction, necrotic and apoptotic skeletal myoblasts, and apoptosis in sepsis-induced myopathy. Overall, 16 candidate biomarkers were certified as reliable features for more in-depth exploration of sepsis-induced myopathy in basic and clinical studies.

Paenilamicins bind to a unique site on the ribosome to inhibit protein synthesis.

Paenilamicins bind to a unique site on the ribosome to inhibit protein synthesis.

AMBRA1 controls the translation of immune-specific genes in T lymphocytes

T cell receptor (TCR) engagement causes a global cellular response that entrains signaling pathways, cell cycle regulation, and cell death. The molecular regulation of mRNA translation in these processes is poorly understood. Using a whole-genome CRISPR screen for regulators of CD95 (FAS/APO-1)-mediated T cell death, we identified AMBRA1, a protein previously studied for its roles in autophagy, E3 ubiquitin ligase activity, and cyclin regulation. T cells lacking AMBRA1 resisted FAS-mediated cell death by down-regulating FAS expression at the translational level. We show that AMBRA1 is a vital regulator of ribosome protein biosynthesis and ribosome loading on select mRNAs, whereby it plays a key role in balancing TCR signaling with cell cycle regulation pathways. We also found that AMBRA1 itself is translationally controlled by TCR stimulation via the CD28-PI3K-mTORC1-EIF4F pathway. Together, these findings shed light on the molecular control of translation after T cell activation and implicate AMBRA1 as a translational regulator governing TCR signaling, cell cycle progression, and T cell death.

N6-Methyladenosine RNA Modification Regulates the Differential Muscle Development in Large White and Ningxiang Pigs.

N6-methyladenosine (m6A) is the most common modification in eukaryotic RNAs. Growing research indicates that m6A methylation is crucial for a multitude of biological processes. However, research on the m6A modifications in the regulation of porcine muscle growth is lacking. In this study, we identified differentially expressed genes in the neonatal period of muscle development between Large White (LW) and NingXiang (NX) pigs and further reported m6A methylation patterns via MeRIP-seq. We found that m6A modification regulates muscle cell development, myofibrils, cell cycle, and phosphatase regulator activity during the neonatal phase of muscle development. Interestingly, differentially expressed genes in LW and NX pigs were mainly enriched in pathways involved in protein synthesis. Furthermore, we performed a conjoint analysis of MeRIP-seq and RNA-seq data and identified 27 differentially expressed and m6A-modified genes. Notably, a typical muscle-specific envelope transmembrane protein, WFS1, was differentially regulated by m6A modifications in LW and NX pigs. We further revealed that the m6A modification accelerated the degradation of WFS1 in a YTHDF2-dependent manner. Noteworthy, we identified a single nucleotide polymorphism (C21551T) within the last exon of WFS1 that resulted in variable m6A methylation, contributing to the differing WFS1 expression levels observed in LW and NX pigs. Our study conducted a comprehensive analysis of the m6A modification on NX and LW pigs during the neonatal period of muscle development, and elucidated the mechanism by which m6A regulates the differential expression of WFS1 in the two breeds.

Development and applications of enzymatic peptide and protein ligation.

Chemical synthesis of complex peptides and proteins continues to play increasingly important roles in industry and academia, where strategies for covalent ligation of two or more peptide fragments to produce longer peptides and proteins in convergent manners have become critical. In recent decades, efficient and site-selective ligation strategies mediated by exploiting the biocatalytic capacity of nature's diverse toolkit (i.e., enzymes) have been widely recognized as a powerful extension of existing chemical strategies. In this review, we present a chronological overview of the development of proteases, transpeptidases, transglutaminases, and ubiquitin ligases. We survey the different properties between the ligation reactions of various enzymes, including the selectivity and efficiency of the reaction, the ligation "scar" left in the product, the type of amide bond formed (natural or isopeptide), the synthetic availability of the reactants, and whether the enzymes are orthogonal to another. This review also describes how the inherent specificity of these enzymes can be exploited for peptide and protein ligation.

Zinc deficiency in ruminants and its management: A brief review

Minerals are organic substances found in nature and living things. In ruminants, minerals are one of the crucial components of the body. One of the mineral elements that is very important for the body is Zinc (Zn). The prominent role of Zn in ruminants is the synthesis and degradation of carbohydrates, fats, proteins, and nucleic acids. Ruminants can experience Zn deficiency by showing clinical symptoms such as dermatitis, anorexia and parakeratosis, stunted growth, and nail damage. In contrast, ruminants can also experience Zn poisoning with various clinical symptoms such as general weakness, diarrhea, hemolytic anemia, and decreased body weight. Current Zn deficiency can be overcome by adding commercial supplement products containing Zn. This paper aims to add brief insights into the benefits and treatment of Zn deficiency in ruminants.

Reprogramming the genetic code with flexizymes.

In the canonical genetic code, the 61 sense codons are assigned to the 20 proteinogenic amino acids. Advancements in genetic code manipulation techniques have enabled the ribosomal incorporation of nonproteinogenic amino acids (npAAs). The critical molecule for translating messenger RNA (mRNA) into peptide sequences is aminoacyl-transfer RNA (tRNA), which recognizes the mRNA codon through its anticodon. Because aminoacyl-tRNA synthetases (ARSs) are highly specific for their respective amino acid-tRNA pairs, it is not feasible to use natural ARSs to prepare npAA-tRNAs. However, flexizymes are adaptable aminoacylation ribozymes that can be used to prepare diverse aminoacyl-tRNAs at will using amino acids activated with suitable leaving groups. Regarding recognition elements, flexizymes require only an aromatic ring in either the leaving group or side chain of the activated amino acid, and the conserved 3'-end CCA of the tRNA. Therefore, flexizymes allow virtually any amino acid to be charged onto any tRNA. The flexizyme system can handle not only L-α-amino acids with side chain modifications but also various backbone-modified npAAs. This Review describes the development of flexizyme variants and discusses their structure and mechanism and their applications in genetic code reprogramming for the synthesis of unique peptides and proteins.

Integrative Human Genetic and Cellular Analysis of the Pathophysiological Roles of AnxA2 in Alzheimer's Disease.

Alzheimer's disease (AD) is an intractable and progressive neurodegenerative disease. Amyloid beta (Aβ) aggregation is the hallmark of AD. Aβ induces neurotoxicity through a variety of mechanisms, including interacting with membrane receptors to alter downstream signaling, damaging cellular or organelle membranes, interfering with protein degradation and synthesis, and inducing an excessive immune-inflammatory response, all of which lead to neuronal death and other pathological changes associated with AD. In this study, we extracted gene expression profiles from the GSE5281 and GSE97760 microarray datasets in the GEO (Gene Expression Omnibus) database, as well as from the Human Gene Database. We identified differentially expressed genes in the brain tissues of AD patients and healthy persons. Through GO, KEGG, and ROC analyses, annexin A2 (AnxA2) was identified as a putative target gene. Notably, accumulating evidence suggests that intracellular AnxA2 is a key regulator in various biological processes, including endocytosis, transmembrane transport, neuroinflammation, and apoptosis. Thus, we conducted a series of cell biology experiments to explore the biological function of AnxA2 in AD. The results indicate that AnxA2 gene knockdown primarily affects oxidative phosphorylation, cell cycle, AD, protein processing in the endoplasmic reticulum, SNARE interactions in vesicular transport, and autophagy. In SH-SY5Y cells secreting Aβ42, AnxA2 gene knockdown exacerbated Aβ42-induced cytotoxicity, including cell death, intracellular ROS levels, and neuronal senescence, altered cell cycle, and reduced ATP levels, suggesting its critical role in mitochondrial function maintenance. AnxA2 gene knockdown also exacerbated the inhibitory effect of Aβ42 on cell migration. AnxA2 overexpression reduced the inflammatory response induced by Aβ42, while its absence increased pro-inflammatory and decreased anti-inflammatory responses. Furthermore, AnxA2 gene knockdown facilitated apoptosis and decreased autophagy. These results indicated potential pathophysiological roles of AnxA2 in AD.

TRNA and tsRNA: From Heterogeneity to Multifaceted Regulators.

As the most ancient RNA, transfer RNAs (tRNAs) play a more complex role than their constitutive function as amino acid transporters in the protein synthesis process. The transcription and maturation of tRNA in cells are subject to stringent regulation, resulting in the formation of tissue- and cell-specific tRNA pools with variations in tRNA overall abundance, composition, modification, and charging levels. The heterogeneity of tRNA pools contributes to facilitating the formation of histocyte-specific protein expression patterns and is involved in diverse biological processes. Moreover, tRNAs can be recognized by various RNase under physiological and pathological conditions to generate tRNA-derived small RNAs (tsRNAs) and serve as small regulatory RNAs in various biological processes. Here, we summarize these recent insights into the heterogeneity of tRNA and highlight the advances in the regulation of tRNA function and tsRNA biogenesis by tRNA modifications. We synthesize diverse mechanisms of tRNA and tsRNA in embryonic development, cell fate determination, and epigenetic inheritance regulation. We also discuss the potential clinical applications based on the new knowledge of tRNA and tsRNA as diagnostic and prognostic biomarkers and new therapeutic strategies for multiple diseases.

Multi-omics reveals the molecular mechanisms of rapid growth in distant hybrid fish

Distant hybridization usually results in heterosis. In previous studies, we obtained an improved fish (WR-II, 2n = 100) with rapid growth through distant hybridization. To investigate the molecular mechanisms underlying the rapid growth of WR-II, the intestinal structure, microbiome and metabolome of the gut and the transcriptome of muscle were compared between WR-II and its parents (WCC and RCC, 2n = 100). The results revealed that WR-II had more intestinal folds, and the height, width and goblet cell density of the intestinal folds were greater than those of its parents. The absolute abundances of the probiotics Ralstonia and Salinivibrio were significantly greater in WR-II than in the parents (p < 0.05), and the probiotic Cupriavidus was found in only WR-II. A total of 481 and 718 differentially abundant metabolites (DMs) were identified in WR-II vs. WCC and WR-II vs. RCC, respectively. These DMs were enriched mainly in pathways related to metabolism, protein synthesis and development, such as fumaric acid, sphingosine, betaine and 5′-adenylic acid (AMP). A total of 5680 and 11,795 differentially expressed genes (DEGs) were identified in WR-II vs. WCC and WR-II vs. RCC, respectively. These DEGs were enriched mainly in pathways related to protein synthesis and substance metabolism. Growth-related genes such as irs1, irs2, foxo1, igf1r, and akt were upregulated in WR-II. Multi-omics analysis revealed that Salinivibrio can promote the level of AMP, which then regulates the expression of irs1 and foxo1, thus promoting the growth of WR-II. This study revealed the mechanism of rapid growth of WR-II from the probiotics, metabolites and genes, which provides evidence for the molecular mechanism of heterosis.

Key Anabolic Markers in Human M. Soleus after 21-Day Head-Down Tilt Bedrest

Prolonged bed rest can have a significant negative effect on skeletal muscle, leading to muscle wasting and reduced strength. This process can occur in as little as 10 days in healthy individuals, with the loss of muscle mass and strength being particularly pronounced during the first week of immobilization. Head-down tilt bed rest (HDT) is a method used to simulate the physiological changes that occur in weightlessness during spaceflight. This technique involves lying in bed with the head tilted downward. This paper is dedicated to the analysis of key anabolic markers of the soleus muscle during 21 days of HDT BR. The HDT BR experiment was conducted at the Institute of Biomedical Problems, Russian Academy of Sciences. Six healthy male volunteers, aged 25–35 years, were subjected to 21 days of strict bed rest with a tilt angle of –6°. A needle biopsy of the m.soleus was performed using the Bergström method before the start of HDT BR and on day 21 of HDT BR. The biopsy material was immediately frozen in liquid nitrogen for further Western blot and PCR analysis. Examination of mTORC1 substrates showed a significant decrease in p70 and 4EBP1 phosphorylation after HDT BR. We also observed a significant decrease in the phosphorylation of another ribosomal kinase, p90RSK, a significant increase in eEF2 phosphorylation and an increase in eEF2k mRNA expression. In addition, the phosphorylation of AMPK and its substrate ACC decreased after HDT BR. The data obtained in this work support the hypothesis that a decrease in protein synthesis, together with an increase in proteolysis, contributes to the development of human m. soleus atrophy after 21 days of HDT BR.

Versatile Dual Reporter to Identify Ribosome Pausing Motifs Alleviated by Translation Elongation Factor P.

Protein synthesis is influenced by the chemical and structural properties of the amino acids incorporated into the polypeptide chain. Motifs containing consecutive prolines can slow the translation speed and cause ribosome stalling. Translation elongation factor P (EF-P) facilitates peptide bond formation in these motifs, thereby alleviating stalled ribosomes and restoring the regular translational speed. Ribosome pausing at various polyproline motifs has been intensively studied using a range of sophisticated techniques, including ribosome profiling, proteomics, and in vivo screening, with reporters incorporated into the chromosome. However, the full spectrum of motifs that cause translational pausing in Escherichia coli has not yet been identified. Here, we describe a plasmid-based dual reporter for rapid assessment of pausing motifs. This reporter contains two coupled genes encoding mScarlet-I and chloramphenicol acetyltransferase to screen motif libraries based on both bacterial fluorescence and survival. In combination with a diprolyl motif library, we used this reporter to reveal motifs of different pausing strengths in an E. coli strain lacking efp. Subsequently, we used the reporter for a high-throughput screen of four motif libraries, with and without prolines at different positions, sorted by fluorescence-associated cell sorting (FACS) and identify new motifs that influence the translational efficiency of the fluorophore. Our study provides an in vivo platform for rapid screening of amino acid motifs that affect translational efficiencies.

A Novel Peptide from VP1 of EV-D68 Exhibits Broad-Spectrum Antiviral Activity Against Human Enteroviruses

Enteroviruses have been a historical concern since the identification of polioviruses in humans. Wild polioviruses have almost been eliminated, while multiple species of non-polio enteroviruses and their variants co-circulate annually. To date, at least 116 types have been found in humans and are grouped into the species Enterovirus A–D and Rhinovirus A–C. However, there are few available antiviral drugs, especially with a universal pharmaceutical effect. Here, we demonstrate that peptide P25 from EV-D68 has broad antiviral activity against Ev A–D enteroviruses in vitro. P25, derived from the HI loop and β-I sheet of VP1, operates through a conserved hydrophilic motif -R---K-K--K- and the hydrophobic F near the N-terminus. It could prevent viral infection of EV-A71 by competing for the heparan sulfate (HS) receptor, and binding and stabilizing virions by suppressing the release of the viral genome. P25 also inhibited the generation of infectious viral particles by reducing viral protein synthesis. The molecular docking revealed that P25 might bind to the pocket opening area, a potential target for broad-spectrum antivirals. Our findings implicate the multiple antiviral effects of peptide P25, including blocking viral binding to the HS receptor, impeding viral genome release, and reducing progeny particles, which could be a novel universal anti-enterovirus drug candidate.