Synthetic Activity Research Articles

Dynamics of single-nuclei transcriptomic profiling of adipose tissue from diverse anatomical locations during mouse aging process

Adipose tissue plays critical roles in an individual’s aging process. In this research, we use single-nucleus RNA sequencing to create highly detailed transcriptional maps of subcutaneous adipose tissue and visceral adipose tissue in young and aged mice. We comprehensively identify the various cell types within the white adipose tissue of mice, our study has elucidated seven distinct cell types within this tissue. Further analyses focus on adipocytes, fibro-adipogenic progenitors, and immune cells, revealing age-related declines in the synthetic metabolic activity of adipocytes, diminished immune regulation, and reduced maturation or proliferation of fibroblasts in undifferentiated adipocytes. We confirm the presence of distinct subpopulations of adipocytes, highlighting decreases in adipogenesis subgroups due to aging. Additionally, we uncover a reduction in immune cell subpopulations, driven by age-associated immune system dysregulation. Furthermore, pseudo-time analyses indicate that Adipocyte1 represents the 'nascent' phase of adipocyte development, while Adipocyte2 represents the 'mature' phase. We use cell–cell interaction to explore the age-dependent complexities of the interactions between FAPs and adipocytes, and observed increased expression of the inflammation-related Retn-Tlr4 interaction in older mice, while the anti-inflammatory Angpt1-Tek interaction was only detected in young mice. These transcriptional profiles serve as a valuable resource for understanding the functional genomics underlying metabolic disorders associated with aging in human adipose tissue.

Genetically programmed synthetic cells for thermo-responsive protein synthesis and cargo release

Synthetic cells containing genetic programs and protein expression machinery are increasingly recognized as powerful counterparts to engineered living cells in the context of biotechnology, therapeutics and cellular modelling. So far, genetic regulation of synthetic cell activity has been largely confined to chemical stimuli; to unlock their potential in applied settings, engineering stimuli-responsive synthetic cells under genetic regulation is imperative. Here we report the development of temperature-sensitive synthetic cells that control protein production by exploiting heat-responsive mRNA elements. This is achieved by combining RNA thermometer technology, cell-free protein expression and vesicle-based synthetic cell design to create cell-sized capsules able to initiate synthesis of both soluble proteins and membrane proteins at defined temperatures. We show that the latter allows for temperature-controlled cargo release phenomena with potential implications for biomedicine. Platforms like the one presented here can pave the way for customizable, genetically programmed synthetic cells under thermal control to be used in biotechnology.

Synthesis and Biological Studies of 4‐Methyl-7‐Ethylcoumarin Derivatives Containing Azo Group

خلٌقت مشتقات الجديدة من4-methyl-7-ethylcoumarin التي تحمل مجموعة azo من خلال سلسلة من التفاعلات المتسلسلة واختبار نشاطها البيولوجي. بدءًا من 7-اثيل-4-مثيل كومارين الذي حضٌر من تفاعل ميتا اثيل فينول مع اثيل اسيتو اسيتيت بواسطة تفاعل بكمان بعدها تم نيترة 7-اثيل-4-مثيل كومارين باستخدام حامض النيتريك بوجود H2SO4المركز لإنتاج ايزومر واحد 7-اثيل-4-مثيل -8-نايتروكومارين تحت التبريد في درجة حرار Co (2-5) ، ثم اختزال مجموعة النيترو باستخدام معدن الحديد في وسط حامضي لتكوين امينو كومارين المقابل الذي حوٌل الى أصباغ azo عن طريق تفاعل ملح الديازونيوم الخاص به مع مركبات الفينول المختلفة. شخٌص المركبات المخلقة بواسطة اطياف الماس و FT-IR و 1HNMR و , 13C-NMR والأشعة فوق البنفسجية وتقنية كروموتوكرافيا الطبقة الرقيقة. قيٌمت الفعالية البيولوجية للمركبات المخلقة تجاه نوعين من البكتريا كرام الموجبة والكرام السالبة بتركيز1× M10-3 اظهرت ان المركبات المخلقة الجديدة 4 و 6 تمتلك طيف عريض ضد البكتريا الموجبة والسالبة مقارنة مع الدواء المستخدم كعقار vancomycinبينما كل المركبات اظهرت فعالية وسط تجاه الفطريات مقارنة مع العقار القياسي nystatin . كما اظهرت تلك المركبات المحضرة كمضادات أكسدة قوية جدًا مقارنة بحمض الأسكوربيك كمرجع قياسي .خاصة مركب رقم7 اظهر كفاءة قوية كمضاد للأكسدة لنفس المرجع.

Synthetic Modification and Insecticidal Activity of 4-epi-cis-Dihydroagarofuran Derivatives.

To synthesize the fundamental framework of dihydroagarofuran, a novel strategy was devised for constructing the C-ring through a dearomatization reaction using 6-methoxy-1-tetralone as the initial substrate. Subsequently, the dihydroagarofuran skeleton was assembled via two consecutive Michael addition reactions. The conjugated diene and trans-dihydroagarofuran skeleton were modified. The insecticidal activities of 33 compounds against Mythimna separata were evaluated. Compounds 11-5 exhibited an LC50 value of 0.378 mg/mL. The activity exhibited a remarkable 29-fold increase compared to positive control Celangulin V, which was widely recognized as the most renowned natural dihydroagarofuran polyol ester insecticidal active compound. Docking experiments between synthetic compounds and target proteins revealed the shared binding sites with Celangulin V. Structure-activity relationship studies indicated that methyl groups at positions C4 and C10 significantly improved insecticidal activity, while ether groups with linear chains displayed enhanced activity; in particular, the allyl ether group demonstrated optimal efficacy. Furthermore, a three-dimensional quantitative structure-activity relationship model was established to investigate the correlation between the skeletal structure and activity. These research findings provide valuable insights for discovering and developing dihydroagarofuran-like compounds.

Biochemical Characterization of a Marine Pseudoalteromonas citrea-Derived Fatty Acyl-AMP Ligase That Exhibits N-Acyl Amino Acid Synthetic Activity.

Activation of fatty acids as acyl-adenylates by fatty acid-AMP ligase (FAAL) is a well-established process contributing to the formation of various functional natural products. Enzymatic characterization of FAALs is pivotal for unraveling both the catalytic mechanism and its role in specific biosynthetic pathways. In this study, we recombinantly expressed and characterized a novel FAAL derived from marine Pseudoalteromonas citrea (PcFAAL). PcFAAL was a cold-adapted neutral enzyme, demonstrating optimal activity at 30°C and pH 7.5. Notably, its specific activity relied on the presence of Mg2+; however, higher concentrations exceeding 10mM resulted in inhibition of enzyme activity. Various organic solvents, especially water-immiscible organic solvents, demonstrated an activating effect on the activity of PcFAAL on various fatty acids. The specific activity exhibited a remarkable 50-fold increase under 4% (v/v) n-hexane compared to the aqueous system. PcFAAL displayed a broad spectrum of fatty acid substrate selectivity, with the highest specific activity for octanoic acid (C8:0), and the catalytic efficiency (kcat/Km) for octanoic acid was determined to be 1.8nM-1·min-1. Furthermore, the enzyme demonstrated biocatalytic promiscuity in producing a class of N-acyl amino acid natural products, as verified by LC-ESI MS. Results indicated that the PcFAAL exhibits promiscuity towards 10 different kinds of amino acids and further demonstrated their potential value in the biosynthesis of corresponding functional N-acyl amino acids.

Scaffold-Hopping Strategies in Aurone Optimization: A Comprehensive Review of Synthetic Procedures and Biological Activities of Nitrogen and Sulfur Analogues.

Aurones, particular polyphenolic compounds belonging to the class of minor flavonoids and overlooked for a long time, have gained significative attention in medicinal chemistry in recent years. Indeed, considering their unique and outstanding biological properties, they stand out as an intriguing reservoir of new potential lead compounds in the drug discovery context. Nevertheless, several physicochemical, pharmacokinetic, and pharmacodynamic (P3) issues hinder their progression in more advanced phases of the drug discovery pipeline, making lead optimization campaigns necessary. In this context, scaffold hopping has proven to be a valuable approach in the optimization of natural products. This review provides a comprehensive and updated picture of the scaffold-hopping approaches directed at the optimization of natural and synthetic aurones. In the literature analysis, a particular focus is given to nitrogen and sulfur analogues. For each class presented, general synthetic procedures are summarized, highlighting the key advantages and potential issues. Furthermore, the biological activities of the most representative scaffold-hopped compounds are presented, emphasizing the improvements achieved and the potential for further optimization compared to the aurone class.

Engineering a computable epiblast for in silico modeling of developmental toxicity

Developmental hazard evaluation is an important part of assessing chemical risks during pregnancy. Toxicological outcomes from prenatal testing in pregnant animals result from complex chemical-biological interactions, and while New Approach Methods (NAMs) based on in vitro bioactivity profiles of human cells offer promising alternatives to animal testing, most of these assays lack cellular positional information, physical constraints, and regional organization of the intact embryo. Here, we engineered a fully computable model of the embryonic disc in the CompuCell3D.org modeling environment to simulate epithelial-mesenchymal transition (EMT) of epiblast cells and self-organization of mesodermal domains (chordamesoderm, paraxial, lateral plate, posterior/extraembryonic). Mesodermal fate is modeled by synthetic activity of the BMP4-NODAL-WNT signaling axis. Cell position in the epiblast determines timing with respect to EMT for 988 computational cells in the computer model. An autonomous homeobox (Hox) clock hidden in the epiblast is driven by WNT-FGF4-CDX signaling. Executing the model renders a quantitative cell-level computation of mesodermal fate and consequences of perturbation based on known biology. For example, synthetic perturbation of the control network rendered altered phenotypes (cybermorphs) mirroring some aspects of experimental mouse embryology, with electronic knockouts, under-activation (hypermorphs) or over-activation (hypermorphs) particularly affecting the size and specification of the posterior mesoderm. This foundational model is trained on embryology but capable of performing a wide variety of toxicological tasks conversing through anatomical simulation to integrate in vitro chemical bioactivity data with known embryology. It is amenable to quantitative simulation for probabilistic prediction of early developmental toxicity.

In planta ANALYSIS OF SALICYLIC ACID MEDIATED MANAGEMENT OF

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a catastrophic bacterial disease of rice, and researchers are facing difficulties in developing the management practices, whereas the continued use of bactericides viz, streptomycin, copper oxychloride leads to the development of resistant Xoo strains. As a consequence, the development of an environmentally safe bactericide for the management of this devastating disease is needed. The present study focused on identification of effective synthetic activators against bacterial leaf blight in rice during their interaction. Survey was conducted to collect different isolates of Xoo and their morphological characters and biochemical properties were determined. All the 10 isolates exhibited circular to flattened slimy yellow colour colonies of the pathogen and showed positive reaction for citrate utilization, lysine utilization and ornithine utilization test. All the ten isolates showed negative reaction for sorbitol utilization. The collected isolates were subjected to PCR for the amplification of 16S-23S intergenic region with the species specific primer and obtained amplicon at ⁓470 bp for the confirmation at molecular level. The virulent isolate was identified by measuring biofilm formation and xanthomonadin pigment which revealed that the isolate ADT 53 strain T3 with the accession number OR121377 recorded maximum OD value of 0.356 for biofilm formation at 570 nm and 0.686 for xanthomonadin pigment at 445 nm. In planta studies revealed that salicylic acid at 100 ppm has the maximum inhibitory effect against bacterial leaf blight disease with the minimal lesion size of 0.7 cm compared to negative control (water). Keywords: Activators, BLB, Biofilm, Characterization, in planta, Xanthomonadin.

Identification and expression of MarCE, a marine carboxylesterase with synthetic ester-degrading activity.

Carboxylic ester hydrolases with the capacity to degrade polyesters are currently highly sought after for their potential use in the biological degradation of PET and other chemically synthesized polymers. Here, we describe MarCE, a carboxylesterase family protein identified via genome mining of a Maribacter sp. isolate from the marine sponge Stelligera stuposa. Based on phylogenetic analysis, MarCE and its closest relatives belong to marine-associated genera from the Cytophaga-Flavobacterium-Bacteroides taxonomic group and appear evolutionarily distinct to any homologous carboxylesterases that have been studied to date in terms of structure or function. Molecular docking revealed putative binding of BHET, a short-chain PET derivative, onto the predicted MarCE three-dimensional structure. The synthetic ester-degrading activity of MarCE was subsequently confirmed by MarCE-mediated hydrolysis of 2 mM BHET substrate, indicated by the release of its breakdown products MHET and TPA, which were measured, respectively, as 1.28 and 0.12 mM following 2-h incubation at 30°C. The findings of this study provide further insight into marine carboxylic ester hydrolases, which have the potential to display unique functional plasticity resulting from their adaptation to complex and fluctuating marine environmentsw.

Substrate-based synthetic strategies and biological activities of 1,3,4-oxadiazole: A review.

The five-membered 1,3,4-oxadiazole heterocyclic ring has received considerable attention because of its unique bio-isosteric properties and an unusually wide spectrum of biological activities. After a century since 1,3,4-oxadiazole was discovered, its uncommon potential attracted medicinal chemist's attention, leading to the discovery of a few presently accessible drugs containing 1,3,4-oxadiazole units, and a large number of patents have been granted on research related to 1,3,4-oxadiazole. It is worth noting that interest in 1,3,4-oxadiazoles' biological applications has doubled in the last few years. Herein, this review presents a comprehensive overview of the recent achievements in the synthesis of 1,3,4-oxadiazole-based compounds and highlights the major advances in their biological applications in the last 10 years, as well as brief remarks on prospects for further development. We hope that researchers across the scientific streams will benefit from the presented review articles for designing their work related to 1,3,4-oxadiazoles.

Obtaining a biodegradable biocatalyst – study on lipase immobilization on spent coffee grounds as potential carriers

Coffee waste can be reused as matrices for enzyme immobilization, as it contains various organic compounds able to adsorb catalytic proteins. In this study, spent coffee grounds were used as a support in the immobilization process in their native form and after being pretreated with hexane, ethanol, and sulphuric acid solution, respectively. Microbial lipases from Aspergillus oryzae, Thermomyces lanuginosus, and Rhizomucor miehei were adsorbed on the carrier, and as a reference, the abovementioned lipases were also immobilized on a synthetic matrix - Lewatit VP OC 1600. The research investigated the impact of the purification step on the immobilization process and the full characteristics of the obtained biocatalysts. The hydrolytic and synthetic activities of the immobilized enzyme preparations were tested, as well as substrate specificity, recovery, and temperature and pH activity profiles. SEM and FTIR analyses were also performed. The results showed that the chemical composition of coffee waste influenced the activity of the obtained biocatalysts, and the lack of hemicellulose caused a reduction in lipolytic activity. The study reveals that spent coffee grounds can be a potential support for enzyme immobilization, and lipases adsorbed on them have improved properties such as hydrolytic or synthetic activity, and stability in pH.

Synthesis of Fluorinated Nucleosides/Nucleotides and Their Antiviral Properties.

The FDA has approved several drugs based on the fluorinated nucleoside pharmacophore, and numerous drugs are currently in clinical trials. Fluorine-containing nucleos(t)ides offer significant antiviral and anticancer activity. The insertion of a fluorine atom, either in the base or sugar of nucleos(t)ides, alters its electronic and steric parameters and transforms the lipophilicity, pharmacodynamic, and pharmacokinetic properties of these moieties. The fluorine atom restricts the oxidative metabolism of drugs and provides enzymatic metabolic stability towards the glycosidic bond of the nucleos(t)ide. The incorporation of fluorine also demonstrates additional hydrogen bonding interactions in receptors with enhanced biological profiles. The present article discusses the synthetic methodology and antiviral activities of FDA-approved drugs and ongoing fluoro-containing nucleos(t)ide drug candidates in clinical trials.

Identification and structure-based engineering of a dipeptidase CpPepD from Clostridium perfringens for the synthesis of l-carnosine

l-Carnosine (l-Car), an endogenous dipeptide presents in muscle and brain tissues of various vertebrates, has a wide range of application values. The enzymatic preparation of l-Car is a promising synthetic method because it avoids the protection and deprotection steps. In the present study, a dipeptidase gene (CpPepD) from Clostridium perfringens with high l-Car synthetic activity was cloned and characterized. In an effort to improve the performance of this enzyme, we carried out site saturation mutagenesis using CpPepD as the template. By the o-phthalaldehyde (OPA)-derived high throughput screening method, mutant A171S was obtained with 2.2-fold enhanced synthetic activity. The enzymatic properties of CpPepD and mutant A171S were investigated. Under the optimized conditions, 63.94 mM (14.46 g L−1) or 67.02 mM (15.16 g L−1) l-Car was produced at the substrate concentrations of 6 M β-Ala and 0.2 M l-His using wild-type or mutant A171S enzyme, respectively. Although the mutation enhanced the enzyme activity, the reaction equilibrium was barely affected.

Synthetic BZLF1-targeted transcriptional activator for efficient lytic induction therapy against EBV-associated epithelial cancers

The unique virus-cell interaction in Epstein-Barr virus (EBV)-associated malignancies implies targeting the viral latent-lytic switch is a promising therapeutic strategy. However, the lack of specific and efficient therapeutic agents to induce lytic cycle in these cancers is a major challenge facing clinical implementation. We develop a synthetic transcriptional activator that specifically activates endogenous BZLF1 and efficiently induces lytic reactivation in EBV-positive cancer cells. A lipid nanoparticle encapsulating nucleoside-modified mRNA which encodes a BZLF1-specific transcriptional activator (mTZ3-LNP) is synthesized for EBV-targeted therapy. Compared with conventional chemical inducers, mTZ3-LNP more efficiently activates EBV lytic gene expression in EBV-associated epithelial cancers. Here we show the potency and safety of treatment with mTZ3-LNP to suppress tumor growth in EBV-positive cancer models. The combination of mTZ3-LNP and ganciclovir yields highly selective cytotoxic effects of mRNA-based lytic induction therapy against EBV-positive tumor cells, indicating the potential of mRNA nanomedicine in the treatment of EBV-associated epithelial cancers.

Overexpression of Slc22a18 facilitates fat accumulation in mice

We previously reported that solute carrier family 22 member 18 (Slc22a18) regulates lipid accumulation in 3T3-L1 adipocytes. Here, we provide additional evidence derived from experiments with adenoviral vector expression and genetic manipulation of mice. In primary cultured rat hepatocytes, adenoviral overexpression of mouse Slc22a18 increased triglyceride accumulation and triglyceride synthetic activity, which was decreased in an adenoviral knockdown experiment. Adenoviral overexpression of mouse Slc22a18 in vivo caused massive fatty liver in mice, even under normal dietary conditions. Conversely, adenoviral knockdown of mouse Slc22a18 reduced hepatic lipid accumulation induced by a high-glucose and high-sucrose diet. We created Slc22a18 knockout mice, which grew normally and showed no obvious spontaneous phenotypes. However, compared with control littermates, the knockout mice exhibited decreased hepatic triglyceride content under refeeding conditions, significantly reduced epididymal fat mass, and tended to have lower liver weight in conjunction with leptin deficiency. Finally, we created transgenic mice overexpressing rat Slc22a18 in an adipose-specific manner, which had increased body weight and epididymal fat mass primarily because of increased adipocyte cell volume. In these transgenic mice, a positive correlation was observed between adiposity and the expression levels of the rat Slc22a18 transgene. Taken together, these results indicate that Slc22a18 has positive effects on lipid accumulation in vivo.

Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination

DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, DSB repair pathway choice occurs at the level of DNA end resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate end resection and repair by homologous recombination (HR). However, inability of CDK phospho-mimetic mutants to bypass this cell cycle regulation, suggests that additional cell cycle regulators may be important. Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that end resection and HR require activation by DDK. Mechanistically, DDK phosphorylates at least two resection nucleases in budding yeast: the Mre11 activator Sae2, which promotes resection initiation, as well as the Dna2 nuclease, which promotes resection elongation. Notably, synthetic activation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair pathway selection.

Chlorogenic Acid Derivatives: Structural Modifications, Drug Design, and Biological Activities: A Review.

Phenolic acids have recently gained considerable attention because of their numerous practical, biological, and pharmacological benefits. Various polyphenolic compounds are widely distributed in plant sources. Flavonoids and phenolic acids are the two main polyphenolic compounds that many plants contain abundant polyphenols. Chlorogenic acid, one of the most abundant phenolic acids, has various biological activities, but it is chemically unstable and degrades into other compounds or different enzymatic processes. In this review, we have studied many publications about CA and its derivatives. CA derivatives were classified into three categories in terms of structure and determined each part's effects on the body. The biological evaluations, structure-activity relationship, and mechanism of action of CA derivatives were investigated. The search databases for this review were ScienceDirect, Scopus, Pub- Med and google scholar. Many studies have reported that CA derivatives have demonstrated several biological effects, including anti-oxidant, anti-inflammatory, anti-microbes, anti-mutation, anti-carcinogenic, anti-viral, anti-hypercholesterolemia, anti-hypertensive, anti-bacterial, and hypoglycemic actions. The synthesis of new stable CA derivatives can enhance its metabolic stability and biological activity. The present study represented different synthetic methods and biological activities of CA derivatives. These compounds showed high antioxidant activity across a wide range of biological effects. Our goal was to help other researchers design and develop stable analogs of CA for the improvement of its metabolic stability and the promotion of its biological activity.

A rare case of bilateral keratoconus and granular corneal dystrophy managed with contact lens

Keratoconus (KC) is a bilateral, non-inflammatory, progressive thinning disorder of the cornea that results in high irregular astigmatism, distortion, and poor vision. Granular corneal dystrophy (GCD) is an autosomal dominant stromal dystrophy with onset in childhood that involves the entire cornea. KC associated with GCD is a rare combination that could occur due to abnormal synthetic activity of keratocytes and degeneration of defective basal epithelial cells. This case report shows the management of KC associated with GCD – an uncommon condition.

ГЕПАТОПРОТЕКТОРНАЯ АКТИВНОСТЬ ПРОИЗВОДНОГО 2-АМИНОЭТАНСУЛЬФОНОВОЙ КИСЛОТЫ ПРИ ЛЕКАРСТВЕННОМ ПОРАЖЕНИИ ПЕЧЕНИ В ЭКСПЕРИМЕНТЕ

We studied the hepatoprotective activity of 2-aminoethanesulfonic acid derivative (laboratory code LBK-527) in antibiotic-associated liver injury. The studies were performed on 18 laboratory rats (Wistar line), divided into four groups: 1st - intact animals, 2nd - control group with tetracycline drug-induced liver injury (TDLI), 2nd group - experimental with DILI and injected LBK- 527 (28 mg/kg), group 3 – experimental with DILI, injected N-adenosyl-S-methionine for 14 days. DILI was induced by intragastric administration of tetracycline at a dose of 500 mg/kg for 5 days. Animals were removed from the experiment on 7 and 14 days and studied activity of aspartic (AST) and alanine (ALT) aminotransferases, total protein (TP), total bilirubin (TB), alkaline phosphatase (ALP), and creatinine (C). The liver was taken from all animals, weighed, and the macro- and microstructure was assessed, stained with hemotoxylin and eosin according to the standard method using a Nikon Eclipes Ni light microscope (Japan). Antibiotic-associated liver injury occurs of the cholestatic type and is characterized by an increase in ALP, TB and the ALT/ALP ratio by two times, decomposition of the hepatic beams, moderate necrosis, fatty degeneration of hepatocytes by 7 days. The administration of LBK-527 and N-adenosyl-S-methionine reduced the toxic effect of tetracycline on the liver, which was manifested in a decrease in the activity of cytolytic icholestatic syndromes, preservation of the beam structure of the liver, degeneration and congestion in the hepatic veins. By day 14, in the control group of animals there was a decrease in the activity of intracellular enzymatic systems of hepatocytes, but complete restoration of the liver structure did not occur in comparison with the intact group. Compound LBK-527 by day 14 The area of the cytoplasm, nucleus and nuclear-cytoplasmic ratio approached the values of the intact group. The appearance of multinucleated hepatocytes indicated the activation of the synthetic activity of the liver and the launch of regeneration mechanisms. Thus, we can conclude that the pharmaceutical composition under study has a hepatoprotective effect.

Methionyl-tRNA synthetase synthetic and proofreading activities are determinants of antibiotic persistence.

Bacterial antibiotic persistence is a phenomenon where bacteria are exposed to an antibiotic and the majority of the population dies while a small subset enters a low metabolic, persistent, state and are able to survive. Once the antibiotic is removed the persistent population can resuscitate and continue growing. Several different molecular mechanisms and pathways have been implicated in this phenomenon. A common mechanism that may underly bacterial antibiotic persistence is perturbations in protein synthesis. To investigate this mechanism, we characterized four distinct metG mutants for their ability to increase antibiotic persistence. Two metG mutants encode changes near the catalytic site of MetRS and the other two mutants changes near the anticodon binding domain. Mutations in metG are of particular interest because MetRS is responsible for aminoacylation both initiator tRNAMet and elongator tRNAMet indicating that these mutants could impact translation initiation and/or translation elongation. We observed that all the metG mutants increased the level of antibiotic persistence as did reduced transcription levels of wild type metG. Although, the MetRS variants did not have an impact on MetRS activity itself, they did reduce translation rates. It was also observed that the MetRS variants affected the proofreading mechanism for homocysteine and that these mutants' growth is hypersensitive to homocysteine. Taken together with previous findings, our data indicate that both reductions in cellular Met-tRNAMet synthetic capacity and reduced proofreading of homocysteine by MetRS variants are positive determinants for bacterial antibiotic persistence.