aminoacyl-tRNA Research Articles

Comprehensive Analysis of the Oncogenic Role of Targeting Protein for Xklp2 (TPX2) in Human Malignancies.

Mitosis and spindle assembly require the microtubule-associated protein Xenopus kinesin-like protein 2 (TPX2). Although TPX2 is highly expressed in several malignant tumor forms, little is known about its role in cancer. In this study, we performed the gene set enrichment analysis of TPX2 in 33 types of cancers and an extensive pan-cancer bioinformatic analysis using prognosis, tumor mutational burdens, microsatellite instability, tumor microenvironment, and immune cell infiltration data. According to the differential expression study, TPX2 was found to be overexpressed across all studied cancer types. Based on the survival analysis, increased TPX2 expression was associated with a poor prognosis for most cancers. The TPX2 expression level was confirmed to correlate with the clinical stage, microsatellite instability, and tumor mutational burden across all cancer types. Furthermore, TPX2 expression has been linked to tumor microenvironments and immune cell infiltration, particularly in bladder urothelial carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, stomach adenocarcinoma, and uterine corpus endometrial carcinoma. Finally, the gene set enrichment analysis implicated TPX2 in the regulation of aminoacyl tRNA biosynthesis, which is the most important tumor cell cycle signaling pathway. This comprehensive pan-cancer analysis shows that TPX2 is a prognostic molecular biomarker for most cancers and suggests its potential as an effective therapeutic target for the treatment of these diseases.

Difference in Intestinal Flora and Characteristics of Plasma Metabonomics in Pneumoconiosis Patients.

From the two perspectives of intestinal flora and plasma metabolomics, the mechanism of occurrence and development of pneumoconiosis was explored to provide a new target for the prevention and treatment of pneumoconiosis. In this study, 16S ribosome DNA (16SrDNA) gene sequencing technology was used to analyze the differences in intestinal flora of each research group through operational taxonomic units (OUT) analysis, cluster analysis, principal component analysis (PCA), partial least square discriminant analysis (PLS-DA), Kyoto Encyclopedia of Genes and Genomes (KEGG), and other analytical methods were used to analyze the differences in plasma metabolites between the study groups. Metabonomics analysis showed that the plasma metabolites of pneumoconiosis patients were significantly different from those of normal people. Fold change > 2; vip > 1; p < 0.05 were the screening criteria. In the positive and negative mode, we screened ten types of differential metabolites. These ten metabolites were upregulated to varying degrees in the pneumoconiosis patients. Seven metabolic pathways were obtained by analyzing the metabolic pathways of different metabolites. Among them, the aminoacyl tRNA biosynthesis pathway changed most obviously. The α diversity of two groups of intestinal flora was analyzed using the 16SrDNA technique. The results showed that there was no significant difference in ACE, Chao1, Shannon, or Simpson in the two groups (p > 0.05). Beta diversity analysis showed that there were differences in microbial communities. In pneumoconiosis patients, the abundance of Prevotellaceae increased, and the other nine species decreased. Compared to the control group, the abundance of Prevotellaceae in the intestinal flora of pneumoconiosis increased, and the abundance of the other nine species decreased. Compared to controls, ten substances in the plasma metabolites of pneumoconiosis patients were upregulated. Seven metabolic pathways were obtained by analyzing the metabolic pathways of different metabolites. Among them, the aminoacyl tRNA biosynthesis pathway changed most significantly. This provided a theoretical basis for further study on the pathogenesis, early prevention, and treatment of pneumoconiosis.

Longitudinal Metabolomics and Lipidomics Analyses Reveal Alterations Associated with Envenoming by Bothrops asper and Daboia russelii in an Experimental Murine Model.

Longitudinal metabolomics and lipidomics analyses were carried out on the blood plasma of mice injected intramuscularly with venoms of the viperid species Bothrops asper or Daboia russelii. Blood samples were collected 1, 3, 6, and 24 h after venom injection, and a control group of non-envenomed mice was included. Significant perturbations in metabolomics and lipidomics were observed at 1, 3, and 6 h, while values returned close to those of control mice by 24 h, hence reflecting a transient pattern of metabolic disturbance. Both venoms induced significant changes in amino acids, as well as in several purines and pyrimidines, and in some metabolites of the tricarboxylic acid cycle. KEGG analysis of metabolic pathways that showed those with the greatest change included aminoacyl tRNA synthesis and amino acid biosynthesis and metabolism pathways. With regard to lipid metabolism, there was an increase in triglycerides and some acyl carnitines and a concomitant drop in the levels of some phospholipids. In addition, envenomed mice had higher levels of cortisol, heme, and some oxidative stress markers. The overall pattern of metabolic changes in envenomed mice bears similarities with the patterns described in several traumatic injuries, thus underscoring a metabolic response/adaptation to the injurious action of the venoms.

Validation of metabolomic and lipidomic analyses of human tears using ultra-high-performance liquid chromatography tandem mass spectrometry

To facilitate application in ophthalmological and systemic diseases, there is a need to standardize preanalytical and analytical steps for metabo-lipidomics in human tears. We assessed different methods for each step of the workflow, from sampling to omics profiles acquisition, to provide the largest metabo-lipidomic coverage with the most robust analytical criteria in human tears. We compared reproducibility according to different extraction methods, two sampling techniques, three volumes (2 μL, 5 μL, 10 μL) and eye laterality using ultra-high-performance liquid chromatography coupled with tandem high-resolution mass spectrometry for metabolomic and lipidomic application. The effect of age on the tear metabo-lipidome was also investigated in healthy subjects. The extraction method using methanol/water provided the best results for Schirmer strip metabolomics, while Folch extraction was superior for lipidomics, whatever the sampling method used. When comparing both sampling methods, microcapillary glass tube was superior to Schirmer strip for metabolomics but comparable for lipidomics. The 5 μL volume provided a satisfying metabo-lipidomic coverage. There was no significant difference in tear metabo-lipidome between both eyes in healthy subjects. While most metabolites and lipids where not influenced by age, the phenylalanine-tyrosine-tryptophan pathway, aminoacyl t-RNA biosynthesis, and alanine-aspartate-glutamate metabolism were the 3 principal pathways associated with the 15 most variable metabolites according to age. The current findings will contribute to improve metabo-lipidomic workflow in human tears for the identification of new biomarkers. Preanalytical and analytical standardization is mandatory in order to perform better between-study comparisons and increase the chances of transferring laboratory findings into clinical practice.

A multidisciplinary approach to the diagnosis of antisynthetase syndrome.

Antisynthetase syndrome is a subtype of idiopathic inflammatory myopathy, strongly associated with the presence of interstitial lung disease. Diagnosis is made by identifying myositis-specific antibodies directed against aminoacyl tRNA synthetase, and relevant clinical and radiologic features. Given the multisystem nature of the disease, diagnosis requires the careful synthesis of subtle clinical and radiological features with the interpretation of specialized autoimmune serological testing. This is provided in a multidisciplinary environment with input from rheumatologists, respiratory physicians, and radiologists. Differentiation from other idiopathic interstitial lung diseases is key; treatment and prognosis differ between patients with antisynthetase syndrome and idiopathic interstitial lung disease. In this review article, we look at the role of the multidisciplinary team and its individual members in the initial diagnosis of the antisynthetase syndrome, including the role of physicians, radiologists, and the wider team.

House feeding system improves the estrus rate in yaks (Bos grunniens) by increasing specific fecal microbiota and myo-inositol content in serum.

Grazing (G) yaks (Bos grunniens) are generally of low fertility, which severely limits the income of local pastoralists. However, we recently found that yaks had a 52% higher estrus rate in house feeding (HF) than in G. Gas chromatography-mass spectrometry (GC-MS) and 16S rRNA gene sequencing were used to analyze serum metabolites and fecal microbiota of 20 rutting yaks in the G and HF systems, respectively, to explain this phenomenon. The results showed that 73 total metabolites differed significantly (p < 0.05 and VIP > 1) between the G and HF systems. In the HF system, 53 were upregulated and 20 were downregulated compared with the G system. Organic oxygen compounds, organic acids and their derivatives, and lipids and lipid-like molecules were the most common differential metabolites. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapper revealed that 25 metabolic signaling pathways differed significantly between the two systems. The top three enriched pathways included central carbon metabolism in cancer, aminoacyl–tRNA biosynthesis, and ABC transporters. The 16S rRNA gene sequencing data showed no significant differences in Chao 1 index between the two systems. According to principal component analysis (PCA), the HF and G systems were distinctly and separately clustered in terms of fecal microbiota distribution. The G system showed significantly higher abundances of Firmicutes. The HF system showed significantly higher abundances of Alistipes, Treponema, and Rikenellaceae_ RC9_ gut_ group. Pearson's correlation analysis and core network analysis revealed that Rikenellaceae_RC9_ gut_ group, Alistipes, and Treponema were positively correlated with myo-inositol and formed the core bacteria. In summary, the HF system promoted the estrus rate and changed the composition of yak fecal microbiota and serum metabolites. Increased estrus rate might be obtained due to enhanced myo-inositol content in yak serum via the HF system. Correlation analysis suggested that myo-inositol content might also be partly increased via yak-specific fecal microbiota, contributing to the estrus rate. These findings could lead to a novel therapeutic strategy for G yaks due to their low estrus rate.

Aminoacyl transfer ribonucleic acid synthetase complex-interacting multifunctional protein 1 induces microglial activation and M1 polarization via the mitogen-activated protein kinase/nuclear factor-kappa B signaling pathway.

Activation of microglia, which is the primary immune cell of the central nervous system, plays an important role in neuroinflammation associated with several neuronal diseases. Aminoacyl tRNA synthetase (ARS) complex-interacting multifunctional protein 1 (AIMP1), a structural component of the multienzyme ARS complex, is secreted to trigger a pro-inflammatory function and has been associated with several inflammatory diseases. However, the effect of AIMP1 on microglial activation remains unknown. AIMP1 elevated the expression levels of activation-related cell surface markers and pro-inflammatory cytokines in primary and BV-2 microglial cells. In addition to the AIMP1-mediated increase in the expression levels of M1 markers [interleukin (IL)-6, tumor necrosis factor-α, and IL-1β], the expression levels of CD68, an M1 cell surface molecule, were also increased in AIMP-1-treated microglial cells, while those of CD206, an M2 cell surface molecule, were not, indicating that AIMP1 triggers the polarization of microglial cells into the M1 state but not the M2 state. AIMP1 treatment induced the phosphorylation of mitogen-activated protein kinases (MAPKs), while MAPK inhibitors suppressed the AIMP1-induced microglial cell activation. AIMP1 also induced the phosphorylation of the nuclear factor-kappa B (NF-κB) components and nuclear translocation of the NF-κB p65 subunit in microglial cells. Furthermore, c-Jun N-terminal kinase (JNK) and p38 inhibitors markedly suppressed the AIMP1-induced phosphorylation of NF-κB components as well as the nuclear translocation of NF-κB p65 subunit, suggesting the involvement of JNK and p38 as upstream regulators of NF-κB in AIMP1-induced microglial cell activation. The NF-κB inhibitor suppressed the AIMP1-induced M1 polarization of the microglial cells. Taken together, AIMP1 effectively induces M1 microglial activation via the JNK and p38/NF-κB-dependent pathways. These results suggest that AIMP1 released under stress conditions may be a pathological factor that induces neuroinflammation.

Phenotype microarray analysis reveals the biotransformation of Fusarium oxysporum f.sp. lycopersici influenced by Bacillus subtilis PBE-8 metabolites.

Herein, Bacillus subtilis PBE-8's biocontrol efficacy was evaluated through physiological and metabolic approaches against Fusarium oxysporum f.sp. lycopersici (FOL). The study elaborates on PBE-8's cell-free filtrate (CFF) antifungal activity through mycelial growth inhibition, metabolite profiling, and substrates utilization patterns. Additionally, under different CFF concentrations, reduction in spore count (94%-55%), biomass (50%), and cytoplasmic bulbous protrusions in mycelia were also observed. Furthermore, the effect of bacterial CFF on FOL metabolism was confirmed through GC-MS. CFF suppresses the concentration of aliphatic amino acids like L-valine, L-leucine, L-Isoleucine, glycine, and fatty acids such as linoleic acid and α- linolenic acid during the co-culturing conditions, which are essential for pathogenicity and resistance against host's systemic acquired resistance. The phenotype microarray assay revealed that CFF-treated FOL shows phenotype loss in 507 (56.58%) out of 896 substrates. Among 507, twenty-seven substrates showed significant phenotype loss, among which four substrates such as L-glutamic acid, L-glutamine, ammonia, and L-arginine are common in different crucial metabolic pathways of FOL, like alanine, aspartate, and glutamate metabolism, arginine and proline, carbon metabolism, arginine biosynthesis, nitrogen metabolism, amino-acyl tRNA synthesis, and biosynthesis of amino acids. The results suggest that PBE-8 CFF has certain antifungal metabolites that hinder the fungal metabolic pathways.

Structural basis for shape-selective recognition and aminoacylation of a D-armless human mitochondrial tRNA

Human mitochondrial gene expression relies on the specific recognition and aminoacylation of mitochondrial tRNAs (mtRNAs) by nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs). Despite their essential role in cellular energy homeostasis, strong mutation pressure and genetic drift have led to an unparalleled sequence erosion of animal mtRNAs. The structural and functional consequences of this erosion are not understood. Here, we present cryo-EM structures of the human mitochondrial seryl-tRNA synthetase (mSerRS) in complex with mtRNASer(GCU). These structures reveal a unique mechanism of substrate recognition and aminoacylation. The mtRNASer(GCU) is highly degenerated, having lost the entire D-arm, tertiary core, and stable L-shaped fold that define canonical tRNAs. Instead, mtRNASer(GCU) evolved unique structural innovations, including a radically altered T-arm topology that serves as critical identity determinant in an unusual shape-selective readout mechanism by mSerRS. Our results provide a molecular framework to understand the principles of mito-nuclear co-evolution and specialized mechanisms of tRNA recognition in mammalian mitochondrial gene expression.

Systems biology approach delineates critical pathways associated with disease progression in rheumatoid arthritis

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease leading to inflammation, cartilage cell death, synoviocyte proliferation, and increased and impaired differentiation of osteoclasts and osteoblasts leading to joint erosions and deformities. Transcriptomics, proteomics, and metabolomics datasets were analyzed to identify the critical pathways that drive the RA pathophysiology. Single nucleotide polymorphisms (SNPs) associated with RA were analyzed for the functional implications, clinical outcomes, and blood parameters later validated by literature. SNPs associated with RA were grouped into pathways that drive the immune response and cytokine production. Further gene set enrichment analysis (GSEA) was performed on gene expression omnibus (GEO) data sets of peripheral blood mononuclear cells (PBMCs), synovial macrophages, and synovial biopsies from RA patients showed enrichment of Th1, Th2, Th17 differentiation, viral and bacterial infections, metabolic signalling and immunological pathways with potential implications for RA. The proteomics data analysis presented pathways with genes involved in immunological signaling and metabolic pathways, including vitamin B12 and folate metabolism. Metabolomics datasets analysis showed significant pathways like amino-acyl tRNA biosynthesis, metabolism of amino acids (arginine, alanine aspartate, glutamate, glutamine, phenylalanine, and tryptophan), and nucleotide metabolism. Furthermore, our commonality analysis of multi-omics datasets identified common pathways with potential implications for joint remodeling in RA. Disease-modifying anti-rheumatic drugs (DMARDs) and biologics treatments were found to modulate many of the pathways that were deregulated in RA. Overall, our analysis identified molecular signatures associated with the observed symptoms, joint erosions, potential biomarkers, and therapeutic targets in RA. Communicated by Ramaswamy H. Sarma

The first apicoplast tRNA thiouridylase plays a vital role in the growth of Toxoplasma gondii

Toxoplasmosis caused by the protozoan Toxoplasma gondii is one of the most common parasitic diseases in humans and almost all warm-blooded animals. Lys, Glu, and Gln-specific tRNAs contain a super-modified 2-thiourea (s2U) derivatives at the position 34, which is essential for all living organisms by maintaining the structural stability and aminoacylation of tRNA, and the precision and efficiency of codon recognition during protein translation. However, the enzyme(s) involved in this modification in T. gondii remains elusive. In this report, three putative tRNA-specific 2-thiolation enzymes were identified, of which two were involved in the s2U34 modification of tRNALys, tRNAGlu, and tRNAGln. One was named TgMnmA, an apicoplast-located tRNA-specific 2-thiolation enzyme in T. gondii. Knockout of TgMnmA showed that this enzyme is important for the lytic cycle of tachyzoites. Loss of TgMnmA also led to abnormities in apicoplast biogenesis and severely disturbed apicoplast genomic transcription. Notably, mice survived from the infection with 10 TgMnmA-KO RH tachyzoites. These findings provide new insights into s2U34 tRNA modification in Apicomplexa, and suggest TgMnmA, the first apicoplast tRNA thiouridylase identified in all apicomplexans, as a potential drug target.

Effects of salinomycin and ethanamizuril on the three microbial communities in vivo and in vitro.

The fate of a drug is not only the process of drug metabolism in vivo and in vitro but also the homeostasis of drug-exposed microbial communities may be disturbed. Anticoccidial drugs are widely used to combat the detrimental effects of protozoan parasites in the poultry industry. Salinomycin and ethanamizuril belong to two different classes of anticoccidial drugs. The effect of salinomycin and ethanamizuril on the microbiota of cecal content, manure compost, and soil remains unknown. Our results showed that although both salinomycin and ethanamizuril treatments suppressed some opportunistic pathogens, they failed to repair the great changes in chicken cecal microbial compositions caused by coccidia infection. Subsequently, the metabolite5 profiling of cecal content by LC-MS/MS analyses confirmed the great impact of coccidia infection on chicken cecum and showed that histidine metabolism may be the main action pathway of salinomycin, and aminoacyl tRNA biosynthesis may be the major regulatory mechanism of ethanamizuril. The microbial community of manure compost showed a mild response to ethanamizuril treatment, but ethanamizuril in soil could promote Actinobacteria reproduction, which may inhibit other taxonomic bacteria. When the soil and manure were exposed to salinomycin, the Proteobacteria abundance of microbial communities showed a significant increase, which suggested that salinomycin may improve the ability of the microbiota to utilize carbon sources. This hypothesis was confirmed by a BIOLOG ECO microplate analysis. In the animal model of coccidia infection, the treatment of salinomycin and ethanamizuril may reconstruct a new equilibrium of the intestinal microbiota. In an in vitro environment, the effect of ethanamizuril on composting and soil microbiota seems to be slight. However, salinomycin has a great impact on the microbial communities of manure composting and soil. In particular, the promoting effect of salinomycin on Proteobacteria phylum should be further concerned. In general, salinomycin and ethanamizuril have diverse effects on various microbial communities.

From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris

BackgroundExpansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-l-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale.ResultsCo-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L−1 and 15 mg L−1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry.ConclusionsPichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads.

Time-resolved transcriptome analysis of Scenedesmus obliquus HTB1 under 10% CO2 condition.

Certain microalgal species can grow under high CO2 concentrations providing potential for mitigating CO2 pollution in flue gas produced by power plants. Microalga Scenedesmus obliquus strain HTB1 was isolated from the Chesapeake Bay and grow rapidly in a high level of CO2 . However, little is known about the molecular responses of HTB1 to high CO2 levels. Here, we investigated how HTB1 responds to 10% CO2 using the time-resolved transcriptome analysis. Gene expression profiles indicated that HTB1 responds quickly (in 2 h) and becomes adaptive within 12 h when exposed to 10% CO2 . Interestingly, certain genes of light-harvesting, chlorophyll synthesis and carbon fixation (i.e. rbcS) were up-regulated at 10% CO2 , and these functional responses are consistent with the increased photosynthesis efficiency and algal biomass under 10% CO2 . Nitrate assimilation was strongly enhanced, with amino acid biosynthesis and aminoacyl tRNA biosynthesis genes being markedly up-regulated, indicating that HTB1 actively takes up nitrogen and accelerates protein synthesis at 10% CO2 . Carbon metabolism including fatty acid biosynthesis and TCA cycle was enhanced at 10% CO2 , supporting the earlier observation of increased lipid content of Scenedesmus sp. under high CO2 levels. Interestingly, key genes like RuBisCO (rbcL) and carbonic anhydrase in carboxysomes did not respond actively to 10% CO2 , implying that exposure to 10% CO2 has little impact on the carbon concentrating mechanisms and CO2 fixation of the Calvin cycle. It appears that HTB1 can grow rapidly at 10% CO2 without significant metabolic changes in carbon fixation and ATP synthesis.

Transcriptome sequencing reveals improved ammonia nitrogen tolerance in Zebra II strain of the Manila clam Ruditapes philippinarum

In this research, we identified genes associated with ammonia nitrogen (TAN) stress response and resistance in juveniles of the Zebra II strain and a wild population of the Manila clam Ruditapes philippinarum. Both groups were subjected to a 96 h acute toxicity test using TAN concentrations of 17.617 ± 0.634 and 16.670 ± 0.7 mg/l, respectively. We then collected samples, conducted transcriptome sequencing and screened the sequences for differentially expressed genes (DEGs) related to TAN stress response. We identified 2908 and 2861 DEGs in the Zebra II and wild clam groups, respectively, and the two groups had 626 DEGs in common. The verified DEGs had less of a detoxification effect in the wild population than that in the Zebra II group. Gene Ontology database analysis showed that Zebra II juveniles were mainly enriched in protein phosphorylation, purine nucleoside binding, and kinase activity, whereas the wild population juveniles were primarily enriched in oxidases activity, organic acid metabolic processes, and extracellular regions. Kyoto Encyclopedia of Genes and Genomes pathway analysis mainly highlighted aminoacyl tRNA biosynthesis in Zebra II juveniles and sphingolipid metabolism, FOXO signaling, biosynthesis of aminoacyl tRNA, and other pathways in the wild population. These results show that the toxic effect of TAN on the Manila clam is related to a variety of pathways, which are mainly related to immune response, inflammatory response, metabolic pathways, and nerve conduction. This study provides basic data and theoretical reference for revealing the molecular regulation mechanism of the improved TAN tolerance of Zebra II strain as compared with the wild population of Ruditapes philippinarum.

Crystal structures of glutamyl-tRNA synthetase from Elizabethkingia anopheles and E. meningosepticum.

Elizabethkingia bacteria are globally emerging pathogens that cause opportunistic and nosocomial infections, with up to 40% mortality among the immunocompromised. Elizabethkingia species are in the pipeline of organisms for high-throughput structural analysis at the Seattle Structural Genomics Center for Infectious Disease (SSGCID). These efforts include the structure-function analysis of potential therapeutic targets. Glutamyl-tRNA synthetase (GluRS) is essential for tRNA aminoacylation and is under investigation as a bacterial drug target. The SSGCID produced, crystallized and determined high-resolution structures of GluRS from E. meningosepticum (EmGluRS) and E.anopheles (EaGluRS). EmGluRS was co-crystallized with glutamate, while EaGluRS is an apo structure. EmGluRS shares ∼97% sequence identity with EaGluRS but less than 39% sequence identity with any other structure in the Protein Data Bank. EmGluRS and EaGluRS have the prototypical bacterial GluRS topology. EmGluRS and EaGluRS have similar binding sites and tertiary structures to other bacterial GluRSs that are promising drug targets. These structural similarities can be exploited for drug discovery.

Free Energy Landscape of the Adenylation Reaction of the Aminoacylation Process at the Active Site of Aspartyl tRNA Synthetase.

The process of protein biosynthesis is initiated by the aminoacylation process where a transfer ribonucleic acid (tRNA) is charged by the attachment of its cognate amino acid at the active site of the corresponding aminoacyl tRNA synthetase enzyme. The first step of the aminoacylation process, known as the adenylation reaction, involves activation of the cognate amino acid where it reacts with a molecule of adenosine triphosphate (ATP) at the active site of the enzyme to form the aminoacyl adenylate and inorganic pyrophosphate. In the current work, we have investigated the adenylation reaction between aspartic acid and ATP at the active site of the fully solvated aspartyl tRNA synthetase (AspRS) from Escherichia coli in aqueous medium at room temperature through hybrid quantum mechanical/molecular mechanical (QM/MM) simulations combined with enhanced sampling methods of well-tempered and well-sliced metadynamics. The objective of the present work is to study the associated free energy landscape and reaction barrier and also to explore the effects of active site mutation on the free energy surface of the reaction. The current calculations include finite temperature effects on free energy profiles. In particular, apart from contributions of interaction energies, the current calculations also include contributions of conformational, vibrational, and translational entropy of active site residues, substrates, and also the rest of the solvated protein and surrounding water into the free energy calculations. The present QM/MM metadynamics simulations predict a free energy barrier of 23.35 and 23.5 kcal/mol for two different metadynamics methods used to perform the reaction at the active site of the wild type enzyme. The free energy barrier increases to 30.6 kcal/mol when Arg217, which is an important conserved residue of the wild type enzyme at its active site, is mutated by alanine. These free energy results including the effect of mutation compare reasonably well with those of kinetic experiments that are available in the literature. The current work also provides molecular details of structural changes of the reactants and surroundings as the system dynamically evolves along the reaction pathway from reactant to the product state through QM/MM metadynamics simulations.

Epigenetic Alterations in Cryopreserved Human Spermatozoa: Suspected Potential Functional Defects.

Background: Gene set enrichment analysis (GSEA) was conducted on raw data, and alternative splicing (AS) events were found after mRNA sequencing of human spermatozoa. In this study, we aimed to compare unknown micro-epigenetics alternations in fresh and cryopreserved spermatozoa to evaluate the effectivity of cryopreservation protocols. Methods: Spermatozoa were divided into three groups: fresh spermatozoa (group 1), cryoprotectant-free vitrified spermatozoa (group 2), and conventionally frozen spermatozoa (group 3). Nine RNA samples (three replicates in each group) were detected and were used for library preparation with an Illumina compatible kit and sequencing by the Illumina platform. Results: Three Gene Ontology (GO) terms were found to be enriched in vitrified spermatozoa compared with fresh spermatozoa: mitochondrial tRNA aminoacylation, ATP-dependent microtubule motor activity, and male meiotic nuclear division. In alternative splicing analysis, a number of unknown AS events were found, including functional gene exon skipping (SE), alternative 5′ splice sites (A5SS), alternative 3′ splice sites (A3SS), mutually exclusive exon (MXE), and retained intron (RI). Conclusions: Cryopreservation of spermatozoa from some patients can agitate epigenetic instability, including increased alternative splicing events and changes in crucial mitochondrial functional activities. For fertilization of oocytes, for such patients, it is recommended to use fresh spermatozoa whenever possible; cryopreservation of sperm is recommended to be used only in uncontested situations.

Biochemistry of Aminoacyl tRNA Synthetase and tRNAs and Their Engineering for Cell-Free and Synthetic Cell Applications.

Cell-free biology is increasingly utilized for engineering biological systems, incorporating novel functionality, and circumventing many of the complications associated with cells. The central dogma describes the information flow in biology consisting of transcription and translation steps to decode genetic information. Aminoacyl tRNA synthetases (AARSs) and tRNAs are key components involved in translation and thus protein synthesis. This review provides information on AARSs and tRNA biochemistry, their role in the translation process, summarizes progress in cell-free engineering of tRNAs and AARSs, and discusses prospects and challenges lying ahead in cell-free engineering.

Transcriptional and Translational Dynamics of Zika and Dengue Virus Infection.

Zika virus (ZIKV) and dengue virus (DENV) are members of the Flaviviridae family of RNA viruses and cause severe disease in humans. ZIKV and DENV share over 90% of their genome sequences, however, the clinical features of Zika and dengue infections are very different reflecting tropism and cellular effects. Here, we used simultaneous RNA sequencing and ribosome footprinting to define the transcriptional and translational dynamics of ZIKV and DENV infection in human neuronal progenitor cells (hNPCs). The gene expression data showed induction of aminoacyl tRNA synthetases (ARS) and the translation activating PIM1 kinase, indicating an increase in RNA translation capacity. The data also reveal activation of different cell stress responses, with ZIKV triggering a BACH1/2 redox program, and DENV activating the ATF/CHOP endoplasmic reticulum (ER) stress program. The RNA translation data highlight activation of polyamine metabolism through changes in key enzymes and their regulators. This pathway is needed for eIF5A hypusination and has been implicated in viral translation and replication. Concerning the viral RNA genomes, ribosome occupancy readily identified highly translated open reading frames and a novel upstream ORF (uORF) in the DENV genome. Together, our data highlight both the cellular stress response and the activation of RNA translation and polyamine metabolism during DENV and ZIKV infection.