Synthesis Of Enzyme Protein Research Articles

Effect of Magnetic Field and UV-C Radiation on Postharvest Fruit Properties

This review focuses on the recent information on the effect of different types of magnetic fields (MFs) and ultraviolet radiation (UV-C) on the processes that may finally affect fruit quality and its storage potential. Firstly, the biological effect of MFs on every plant’s growth and development level is described. The magnetic field interacts with a plant’s metabolism and changes the permeability of membranes affecting cells’ homeostasis. It also could affect early seedling development, stimulating enzyme activity and protein synthesis, and later on nutrient and water uptake of adult plants. In some cases, it makes plants more resilient, increasing their tolerance to environmental stresses. Also, MF treatment could lower the disease index of plants, thus improving the internal and external fruit quality indices. The second part of this review focuses on interesting perspectives of using UV-C radiation to reduce postharvest fruit diseases, but also to delay fruit ripening and senescence. The application of UV-C light to combat postharvest infections is associated with two mechanisms of action, such as direct elimination of microorganisms located on the fruit surface and indirect triggering of the plant’s defense reaction. Moreover, the use of hormetic doses of UV-C can additionally increase the nutritional properties of fresh fruit, lead to the accumulation of desired phytochemicals such as polyphenols, for example, to increase anthocyanin or resveratrol content, or elevate antioxidant activity.

Expression and purification of RNA Pseudouridylate synthase putative of malaria parasite (Plasmodium falciparum)

The high morbidity and mortality from malaria infection, especially amongst children under five years of age, necessitates the production of new antimalarial drugs. Plasmodium falciparum RNA pseudouridylate synthase putative (PfRPuSP) is an essential enzyme in protein synthesis, making it a potential drug target. Therefore, this study investigated the cloning, transformation, expression and purification of PfRPuSP. Both (pET28a (+) and pGEX-6P-1) plasmids were utilised for the cloning of PfRPuSP. The heat shock standard method was used for the gene constructs transformation in three strains of E. coli. Terrific and Luria Broth media at 25 °C and 30 °C were utilised to optimise the growth conditions. Nickel-nitrilotriacetate resin and fast protein liquid chromatography were employed for PfRPuSP purification to obtain its pure form. In this study, the best expression of PfRPuSP was achieved using PF3D7_1227900_ pET28a(+) construct in C41(DE3) competent cells overnight at 25 °C in LB medium as depicted by the Western blot results. The PF3D7_1227900_ pGEX-6P-1 construct yielded no desired result after expression and purification. The best medium, buffer, E.coli strain and matrix resin were determined, which can be used as baseline information for further studies and monitoring. These discoveries would be highly valuable in uncovering a way to form and understand the three-dimensional structure of PfRPuSP. This knowledge could lead to the development of medications that can block the enzyme in Plasmodium falciparum, ultimately aiding in eradicating malaria parasites.

2135. Epetraborole in vitro Activity Against Mycobacterium avium complex Recent Clinical Isolates from Japan

Abstract Background Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis. EBO demonstrates potent activity against nontuberculous mycobacteria and is currently under clinical development for treatment of treatment-refractory Mycobacterium avium complex (MAC) lung disease. The objective of this study was to evaluate the in vitro activity of EBO against recent MAC isolates from Japan. Methods Minimal inhibitory concentration (MIC) values for EBO, amikacin (AMK), clarithromycin (CLR), rifabutin (RFB), and ethambutol (EMB) were determined using broth microdilution assays according to Clinical and Laboratory Standards Institute M24-A3 guideline (2018) against 110 MAC clinical isolates collected from Japanese patients in 2020. MAC isolates were of respiratory origin and included 55 M. avium and 55 M. intracellulare isolates. Results EBO (MIC) values ranged from 0.25 - 16 µg/mL and the EBO MIC50 and MIC90 were 2 and 4 µg/mL, respectively (Table 1). The CLR MIC range was 0.125 - > 32 µg/mL and included 4 CLR-resistant isolates (MIC ≥ 32 µg/mL). CLR MIC50 and MIC90 were 1 and 4 µg/mL, respectively. All isolates were susceptible to AMK and had an MIC range of 2 - 32 µg/mL; the MIC50 and MIC90 were 8 and 16 µg/mL, respectively. The EMB MIC range was 2 - > 32 µg/mL; the MIC50 and MIC90 were 4 and 16 µg/mL, and the RFB MIC range was ≤0.03 - 2 µg/mL and MIC50 and MIC90 were 0.06 and 0.25 µg/mL. EBO maintained activity with MIC values of 0.25 - 2 µg/mL against the 4 CLR-resistant isolates. Epetraborole and comparator antimicrobial minimum inhibitory concentration values against 110 recent Mycobacterium avium complex clinical isolates from Japan MIC=minimal inhibitory concentration; MIC50=minimal inhibitory concentration for 50% of isolates tested; MIC90=minimal inhibitory concentration for 90% of isolates tested. Conclusion EBO demonstrated potent in vitro activity against 110 recent MAC isolates collected from Japanese patients. Furthermore, EBO demonstrated in vitro activity against CLR-resistant MAC isolates suggesting that CLR-resistance does not impact EBO activity. These data are similar to results obtained against U.S. isolates and support the continued clinical evaluation of the use of EBO in the treatment of MAC lung disease in Japan. Disclosures Tiffany Keepers White, PhD, AN2 Therapeutics: Employee|AN2 Therapeutics: Stocks/Bonds Satoshi Mitarai, MD, PhD, AN2 Therapeutics, Inc.: Grant/Research Support

Ribosome profiling in the Symbiodiniacean dinoflagellate Fugacium kawagutii shows coordinated protein synthesis of enzymes in different pathways at different times of day.

Dinoflagellates respond to daily changes in light and dark by changes in cellular metabolism, yet the mechanisms used are still unclear. For example, Fugacium (previously Symbiodinium) kawagutii shows little difference in the transcriptome between day and night suggesting little transcriptional control over gene expression. Here, we have performed ribosome profiling at 2 h intervals over a daily light-dark cycle to assess the degree to which protein synthesis rates might change over the daily cycle. The number of F. kawagutii coding sequences with significant differences in the number of ribosome-protected fragments (RPF) over the 24-h cycle was 2923 using JTK_Cycle and 3655 using ECHO. The majority of the regulated transcripts showed peak translation at the onset of the dark period. The regulated sequences were assigned to different KEGG pathways and transcripts that were translated at roughly the same time were termed concurrently regulated. Both analyses revealed concurrent regulation of many transcripts whose gene products were involved in spliceosome or lysosome biogenesis with peak translation rates around the onset of the dark period, while others, involved in nitrate metabolism and ribosomal proteins, were preferentially translated around the onset of the day phase or the end of the night phase, respectively. In addition, some sequences involved in DNA synthesis were preferentially translated at the end of the day. We conclude that light-dark cycles seem able to synchronize translation of some transcripts encoding proteins involved in a range of different cellular processes, and propose that these changes may help the cells adapt and alter their metabolism as a function of the time of day.

Evaluation of Fermented Soybean Meal to Replace a Portion Fish Meal on Growth Performance, Antioxidant Capacity, Immunity, and mTOR Signaling Pathway of Coho Salmon (Oncorhynchus kisutch).

In this study, we evaluated the effects of fermented soybean meal (FSBM) or/and unfermented SBM replacing a portion of fish meal (FM) on the growth performance, antioxidant capacity, immunity, and mechanistic target of rapamycin (mTOR) signaling pathway of juvenile coho salmon (Oncorhynchus kisutch). Four groups of juvenile coho salmon (initial weight 152.23 ± 3.21 g) in triplicate were fed for 12 weeks on four different iso-nitrogen and iso-lipid experimental diets: G0 diet (28% FM protein, control group), G1 diet (18% FM protein and 10% SBM protein), G2 diet (18% FM protein, 5% SBM protein, and 5% FSBM protein), and G3 diet (18% FM protein and 10% FSBM protein). The main results were compared with the G0 diet; the weight gain rate, specific growth rate, and condition factor of juveniles in G3 were increased significantly (p < 0.05). The content of muscle crude protein, the total protein, glucose, albumin, total cholesterol in serum, and the total antioxidant capacity in the liver of juveniles in G3 was increased significantly (p < 0.05). The activities of pepsin, trypsin, α-amylase, and lipase in the intestine, the superoxide dismutase, catalase, and alkaline phosphatase in the liver of juveniles in G3 were increased significantly (p < 0.05). The expression levels of phosphatidylinositide 3-kinases, serine/threonine kinase, mTOR, and ribosomal protein S6 kinase 1 genes in the liver of juveniles in G3 were upregulated significantly (p < 0.05). The feed coefficient ratio, viscerosomatic index, the contents of muscle moisture, and malondialdehyde in the liver of juveniles in G3 were decreased significantly (p < 0.05). The expression levels of tumor necrosis factor α, interleukin 1β, and interleukin 6 genes in the liver of juveniles in G3 were downregulated significantly (p < 0.05). However, there was no significant effect (p > 0.05) on the survival rate, food intake, and muscle crude lipid and ash of juveniles among the experimental groups. In conclusion, FSBM to replace a portion FM had a positive effect on the growth performance, protein deposition, antioxidant enzyme activity, digestive enzyme activity, protein synthesis, and immune-related genes of juvenile coho salmon.

Morphophysiological and transcriptome analysis reveal that reprogramming of metabolism, phytohormones and root development pathways governs the potassium (K+) deficiency response in two contrasting chickpea cultivars.

Potassium (K+) is an essential macronutrient for plant growth and development. K+ deficiency hampers important plant processes, such as enzyme activation, protein synthesis, photosynthesis and stomata movement. Molecular mechanism of K+ deficiency tolerance has been partly understood in model plants Arabidopsis, but its knowledge in legume crop chickpea is missing. Here, morphophysiological analysis revealed that among five high yielding desi chickpea cultivars, PUSA362 shows stunted plant growth, reduced primary root growth and low K+ content under K+ deficiency. In contrast, PUSA372 had negligible effect on these parameters suggesting that PUSA362 is K+ deficiency sensitive and PUSA372 is a K+ deficiency tolerant chickpea cultivar. RNA-seq based transcriptome analysis under K+ deficiency revealed a total of 820 differential expressed genes (DEG's) in PUSA362 and 682 DEGs in PUSA372. These DEGs belongs to different functional categories, such as plant metabolism, signal transduction components, transcription factors, ion/nutrient transporters, phytohormone biosynthesis and signalling, and root growth and development. RNA-seq expression of randomly selected 16 DEGs was validated by RT-qPCR. Out of 16 genes, 13 showed expression pattern similar to RNA-seq expression, that verified the RNA-seq expression data. Total 258 and 159 genes were exclusively up-regulated, and 386 and 347 genes were down-regulated, respectively in PUSA362 and PUSA372. 14 DEGs showed contrasting expression pattern as they were up-regulated in PUSA362 and down-regulated in PUSA372. These include somatic embryogenesis receptor-like kinase 1, thaumatin-like protein, ferric reduction oxidase 2 and transcription factor bHLH93. Nine genes which were down-regulated in PUSA362 found to be up-regulated in PUSA372, including glutathione S-transferase like, putative calmodulin-like 19, high affinity nitrate transporter 2.4 and ERF17-like protein. Some important carbohydrate metabolism related genes, like fructose-1,6-bisphosphatase and sucrose synthase, and root growth related Expansin gene were exclusively down-regulated, while an ethylene biosynthesis gene 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO1) was up-regulated in PUSA362. Interplay of these and several other genes related to hormones (auxin, cytokinin, GA etc.), signal transduction components (like CBLs and CIPKs), ion transporters and transcription factors might underlie the contrasting response of two chickpea cultivars to K+ deficiency. In future, some of these key genes will be utilized in genetic engineering and breeding programs for developing chickpea cultivars with improved K+ use efficiency (KUE) and K+ deficiency tolerance traits.

1716. In Vitro Drug-Drug Interaction Evaluation of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor

Abstract Background Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. EBO is being developed for the treatment of Mycobacterium avium complex lung disease patients and will be used in combination with other drugs. Therefore, EBO and its major circulating metabolite M3 were evaluated in a comprehensive drug-drug interactions (DDI) risk assessment. Methods Stability of EBO was evaluated in human liver microsomes, hepatocytes and recombinant CYP enzymes. The inhibitory potential of EBO (0.03-100 μM) and M3 (1-1000 μM) on cytochrome P450 (CYP) activities was assessed using human hepatic microsomes. The CYP induction potential of EBO (25-200 μM), M3 was evaluated and compared to prototypical inducers in human hepatocytes (three donors) and mRNA. Fold increase in mRNA expression was utilized to investigate the CYP induction potential of EBO (0.3-100 µM) and M3 (1-250 µM). Stably transfected cell lines that expressed individual transporters were used to determine whether EBO or M3 were substrates or inhibitors for these proteins. Results In vitro studies with microsomes, hepatocytes and recombinant cytochrome P450 (CYP) enzymes indicated that EBO was a poor substrate for major CYP enzymes; drug interactions with epetraborole as victim are considered unlikely. Neither EBO nor its major metabolite M3 was a potent reversible or time-dependent inhibitor of major CYP enzymes. Half maximal inhibitory concentration (IC50) values for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 were &amp;gt;100 µM. EBO was not an inducer of CYP1A2 mRNA in human hepatocytes from three donors, while it was a weak inducer of CYP2B6 and CYP3A4. No induction of mRNA was observed in human hepatocytes at concentrations relevant to planned clinical doses. EBO is unlikely to be a substrate for P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, MATE1 and MATE2K. EBO was an in vitro substrate for OCT2, a transporter involved in active renal secretion. At clinically relevant concentrations, neither EBO nor M3 inhibited major human efflux or uptake transporters. Conclusion At clinically relevant concentrations of EBO and its major circulating metabolite M3, there is a low risk of victim or perpetrator DDI. Disclosures Afshin Shafiee, PhD, AN2 Therapeutics: Stocks/Bonds Bradley K. Wong, PhD, AN2 Therapeutics: Advisor/Consultant Sanjay Chanda, PhD, AN2 Therapeutics: Stocks/Bonds.

1698. Pharmacokinetics/pharmacodynamics of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, and High Intracellular Penetration in the Intracellular Hollow Fiber System Model of Mycobacterium avium Complex Lung Disease

Abstract Background Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. Dose-effect studies in the intracellular hollow fiber system model of Mycobacterium avium complex (MAC) lung disease, or HFS-MAC, have demonstrated that EBO is highly bactericidal. In this study we used dose fractionation in the HFS-MAC system to find the best pharmacokinetic/ pharmacodynamic (PK/PD) index that was associated with EBO microbial kill and acquired microbial resistance (AMR). Methods A dose-fractionation design was used to dose 30 HFS-MAC units. ED20, ED50 and ED80 doses were administered as one of 4 dose schedules of twice daily, once daily, twice weekly, or once weekly at a half-life of 10.4h. Total MAC burden and EBO-resistant MAC burden were determined every 7 days by culturing HFS-MAC contents on Middlebrook 7H10 agar. For drug concentrations, the central compartment of each HFS-MAC unit was sampled throughout the 28 days, and after the final dose, intracellular (IC) PK in the MAC-infected monocytes were established. EBO PK/PD index parameters including 0-168h area under the concentrations-time curve (AUC0-168), peak concentration, or % of time concentration persists above the intracellular MIC (%TMIC), which was 0.5 mg/L, were modeled versus total MAC burden using the inhibitory sigmoid maximal effect (Emax) model. The PK/PD index was chosen using Akaike Information Criteria (AIC). For AMR, EBO PK/PD index exposure value versus EBO-resistant MAC burden were modeled using a quadratic function, and the best model was chosen using AIC. Results The measured EBO concentrations demonstrated PKs shown in Figure 1. The median EBO IC versus extracellular (IC/EC) elimination rate constants were 0.018h-1 versus 0.061h-1 (p&amp;lt; 0.0001), which means slower IC EBO elimination, leading to IC/EC to AUC ratio of 11. Based on AIC scores in Table 1, the PK/PD driver for microbial kill was unequivocally AUC on each sampling day. For AMR the PK/PD driver was also AUC. Table 1.Corrected Akaike Information Criteria (AIC) Scores for PK/PD Indicesa-The intracellular EBO MIC was 0.5 mg/LFigure 1.Epetraborole concentrations achieved in the HFS-MAC during the 168-hour dosing interval. Epetraborole concentrations were measured for each of the four dosing schedules, and are shown together with compartmental pharmacokinetics modeling during the 168-hour dosing interval. A. Twice a day dosing. B. Once a day dosing. C. Once every 3.5 days. D. Once every 7 days. Conclusion The PK/PD index best associated with EBO microbial kill and AMR was AUC. Disclosures Shruti Athale, PhD, Praedicare Inc.: Stocks/Bonds Moti Chapagain, MD, PhD, Praedicare Inc.: Stocks/Bonds Jotam Pasipanodya, MD, PhD, Praedicare Inc.: Employee MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Tawanda Gumbo, MD, Hydronium Biopharma: Ownership Interest|Hydronium Biopharma: Stocks/Bonds|Praedicare Africa Pvt Ltd: Board Member|Praedicare Africa Pvt Ltd: Ownership Interest|Praedicare Africa Pvt Ltd: Stocks/Bonds|Praedicare Inc.: Patents|Praedicare Inc.: Ownership Interest|Praedicare Inc.: Stocks/Bonds.

1704. Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, Demonstrates Potent Efficacy and Improves Efficacy of Standard of Care Regimen Against Mycobacterium avium complex in a Chronic Mouse Lung Infection Model

Abstract Background Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. These studies evaluated oral doses (PO) of EBO against 5 M. avium complex (MAC) strains in a chronic mouse infection model either as monotherapy or in combination with standard of care [SOC; clarithromycin (CLR), rifabutin (RFB), ethambutol (EMB)]. Methods A pilot chronic efficacy study against M. avium 2285R evaluated EBO at 1, 10, 30, 100, 300 and 500 mg/kg PO once daily (QD) compared to 250 mg/kg CLR PO QD. C57BL/6 mice were infected with a pulmonary aerosol of 1x1011 CFU. Treatment was administered for 56 days starting on day 28 post-infection. The bacterial burden (CFU) in lungs was evaluated on days 1, 28 and 84 post-infection by plating serial dilutions of homogenates on Middlebrook 7H11 charcoal agar plates. An additional 4 strains of MAC were evaluated with EBO doses of 100, 200, 300 or 400 mg/kg QD compared with the SOC therapy for MAC (CLR 250 mg/kg, RFB 100 mg/kg, EMB 100 mg/kg) QD and SOC plus EBO 200mg/kg QD. Oral exposures of EBO were determined in a group of uninfected mice (Table 1).Table 1:C57BL/6 Murine Pharmacokinetic Parameters Results In a study with M. avium 2285R, a biofilm-forming strain, EBO at all doses tested was significantly better than CLR dosed at 250 mg/kg (Figure 1), and no CFU were detected on agar plates containing EBO (16 mg/L). In subsequent studies, SOC was compared to EBO in 4 additional MAC strains (Figure 2). Efficacy of EBO monotherapy was better than SOC against M. avium ATCC 700898, while it was as good as SOC with M. intracellulare 1956, M. intracellulareDNA00055, and M. intracellulare DNA00111 with CFU reductions ranging from 2 - 4.8 log10 compared to day 28 controls. In all four strains tested, 200 mg/kg EBO, which approximates the human oral equivalent dose of 500 mg, combined with SOC increased bacterial killing from 1.4 - 3.0 log10 CFU compared to SOC alone resulting in total lung CFU reductions of 4.6 - 5.6 log10. Figure 1Figure 2 Conclusion In this chronic mouse lung infection model, no EBO resistance development was detected with M. avium 2285R at day 84. EBO demonstrated potent in vivo efficacy against 5 MAC strains and significantly improved efficacy when combined with SOC, supporting further clinical development for EBO. Disclosures Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn Shoen, PhD, AN2 Therapeutics: Grant/Research Support Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds.

1713. In Vitro Activities of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, Against Mycobacterium avium Complex Isolates

Abstract Background Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. We evaluated the effects of select culture conditions on MIC determinations of EBO against isolates of M. avium complex (MAC), as well as EBO MIC90 results with Middlebrook 7H9 broth compared to those with cation-adjusted Mueller Hinton Broth (CAMHB) for 51 MAC isolates. Methods Six strains of MAC were used to test the in vitro activity of EBO in different conditions in a broth microdilution (BMD) assay. Activity was compared in Middlebrook 7H9 and CAMHB with 5% OADC from different manufacturers. The effects of glycerol, cations, oxyrase, varying pH levels, and increasing inoculum sizes were tested. Finally, EBO in vitro activity was tested for 51 MAC isolates in a BMD assay in both Middlebrook 7H9 and CAMHB with 5% OADC. Results In general, manipulation of select culture conditions caused very little variation in EBO MIC values for the 6 MAC strains except for increasing the inoculum from ∼105 to 107 CFU/mL, which caused an approximately 64x increase in the MIC. Since 1 MAC isolate out of 6 was affected by the addition of casitone, we tested 51 MAC isolates in both the minimal media Middlebrook 7H9 and the complex media CAMHB. EBO had a narrow MIC range in both broths, 0.25-8 mg/L for all isolates. The EBO modal MIC, MIC50 and MIC90 for the entire MAC panel of 51 isolates was 2 mg/L, 2 mg/L, and 8 mg/L for CAMHB and 1 mg/L, 1 mg/L, and 4 mg/L for Middlebrook 7H9, respectively (Table 1). Three clarithromycin-resistant isolates had EBO MIC values of 0.5 mg/L, 1 mg/L, and 2 mg/L suggesting that clarithromycin resistance does not affect EBO in vitro activity. In addition, amikacin resistance as determined using the Clinical Laboratory Standards Institute (CLSI) IV amikacin breakpoint (MIC ≥ 64 mg/L) had no noticeable effect on EBO MIC values. Table 1EBO In Vitro Activity Against 51 Isolates of Mycobacterium avium complex Conclusion The MIC distribution for the 51 MAC isolates tested was similar in both media types, indicating that CAMHB can be used to test EBO MAC susceptibilities per CLSI guidelines. Clarithromycin- and amikacin-resistant isolates demonstrated no cross-resistance with EBO. Disclosures Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn M. Shoen, PhD, AN2 Therapeutics: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support.

1697. Dose-response Studies of the Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, Epetraborole, in the Intracellular Hollow Fiber System Model of Mycobacterium avium complex Lung Disease

Abstract Background Based on meta-analyses the current standard-of-care regimen (SOC) for Mycobacterium avium complex (MAC) lung disease achieves a sustained sputum conversion rate of ∼54% at best. Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. To identify EBO exposure-effect parameters we used the intracellular hollow fiber system model of intracellular pulmonary MAC (HFS-MAC). Methods EBO was administered once daily at 8 different doses to HFS-MAC replicates for 28 days to achieve a half-life (t½) of 10.4h and the 0-24h area under the concentration-time curves (AUC0-24) that cover the observed AUC values in humans. The SOC combination of clarithromycin (CLR), ethambutol (EMB), and rifabutin (RFB) at human intrapulmonary pharmacokinetic (PK) concentrations was used. The central compartment of each HFS-MAC unit was sampled throughout the 28 days to assess PK parameters as was the peripheral compartment for total bacterial burden and the EBO-resistant bacterial burden. EBO AUC versus total MAC burden was modeled using the inhibitory sigmoid maximal effect (Emax) model. EBO AUC versus EBO-resistant MAC burden was modeled using a quadratic function. Results Measured EBO concentrations demonstrated a t1/2 of 10h. For SOC, the AUC for CLR, EMB, and RFB was 60 mg*h/L (t1/2=6h), 39 mg*h/L (t1/2=8h), and 1.5 mg*h/L (t1/2=45h), respectively, similar to human lung concentrations. Changes in MAC burden over 28 days (Fig. 1) show that highest EBO exposures matched SOC until day 14. The exposure versus effect on each sampling day is shown in Fig. 2. The EBO AUC mediating 50% of Emax (EC50) was an AUC of 22 mg*h/L (95% confidence interval [CI]: 16-70), and the EC80 was an AUC of 47.5 mg*h/L (CI: 34.6-151.2). The relationship between AUC and EBO-resistant subpopulation (Fig. 3) shows that an AUC0-24 of 47.5 mg*h/L, the same as the EC80 for microbial kill, was associated with resistance suppression, as was the addition of SOC to EBO. Figure 1.Changes in bacterial burden over the 28-day dosing period. Bacterial burden on serial samples from all HFS-MAC units and replicates over 28days are shown. The highest epetraborole exposures demonstrated the same effectiveness as the three drug SOC until day 14, after which it failed as resistance emerged. On the other hand adding epetraborole at an AUC of 43.6 mg*h/L to the SOC led to much faster kill (i.e, higher kill slopes) than SOC or the epetraborole monotherapy at the same AUC. Figure 2.Epetraborole exposure versus effect on bacterial burden. Shown are inhibitory sigmoid Emax curves for epetraborole AUC versus log10 CFU/mL on each sampling day, as well as various extents of microbial kill such as stasis (holding burden similar to day 0), 1 log CFU/mL kill and 2 log CFU/mL kill. Beyond day 3, epetraborole killed &amp;gt; 1log CFU/mL, which means it was rapidly bactericidal. Figure 3.Epetraborole exposure versus epetraborole-resistant subpopulation. Drug-resistant burden is shown as log10 CFU/mL. The addition of SOC to EBO totally suppressed emergence of any resistance on all days. Conclusion EBO monotherapy with an AUC0-24 &amp;gt; 16.9 mg*h/L killed &amp;gt; 1.0 log10 CFU/mL of MAC compared to day 0. At the EC80, EBO killed at least 2.0 log10 CFU/mL, thus was highly bactericidal. EBO plus SOC demonstrated resistance suppression. Disclosures Moti Chapagain, MD, PhD, Praedicare Inc.: Stocks/Bonds Shruti Athale, PhD, Praedicare Inc.: Stocks/Bonds Jotam Pasipanodya, MD, PhD, Praedicare Inc.: Employee MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Tawanda Gumbo, MD, Hydronium Biopharma: Ownership Interest|Hydronium Biopharma: Stocks/Bonds|Praedicare Africa Pvt Ltd: Board Member|Praedicare Africa Pvt Ltd: Ownership Interest|Praedicare Africa Pvt Ltd: Stocks/Bonds|Praedicare Inc.: Patents|Praedicare Inc.: Ownership Interest|Praedicare Inc.: Stocks/Bonds.

1712. In Vitro Activity of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, in Drug Combinations Against Nontuberculous Mycobacteria Including Resistance Frequency and MIC Characterization of Mycobacterium avium ATCC 700898 Epetraborole-resistant Mutants

Abstract Background Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria (NTM). The standard of care therapy for Mycobacterium avium complex (MAC) lung disease consists of a combination of a macrolide, ethambutol (EMB) and a rifamycin. A total of 5 strains of MAC, M. abscessus ATCC 19977 and M. peregrinum ATCC 700686 were tested in checkerboard assays to evaluate the effects of combining EBO with other agents. The spontaneous resistance frequency (RF) was determined for EBO singly and in combination against M. avium ATCC 700898. Methods The effects of combining EBO with clarithromycin (CLR), rifabutin (RBT), EMB, amikacin (AMK) or bedaquiline (BDQ) were evaluated in 7 strains of NTM. Synergy, additive effects, indifference or antagonism was characterized in the checkerboard assay using EUCAST criteria. The RF of M. avium ATCC 700898 at 2x, 4x and 8x the MIC (8 mg/L) of EBO was determined, as was the RF of EBO combined with CLR, RBT, AMK or EMB. MICs of selected EBO mutants were determined against AMK, BDQ, CLR, RBT, EMB, and clofazimine (CFZ) and the mutants were further characterized. Results The RF of EBO ranged from 1.58x10-7 to 8.48x10-9 when selected on 2 - 8x agar MIC. The MIC for EBO increased 32- &amp;gt; 256-fold for the resistant mutants; however, the MICs for the other drugs tested against EBO-resistant strains did not change significantly. The activity of EBO was not antagonized, and was mainly indifferent to the addition of CLR, RBT, AMK, or BDQ for all the NTM strains tested. Synergy with EMB was observed with 2 strains and additivity with 2 additional strains of MAC. The addition of EMB (24 mg/L), CLR (32 mg/L), RFB (2 mg/L) or AMK (128 mg/L) to agar plates containing 2x MIC of EBO lowered the RF to at least &amp;lt; 2.13x10-10 (Table 1). Table 1.In Vitro Resistance Frequency for the Components of SOC with and without EBO in Mycobacterium avium ATCC 700898 Conclusion In the checkerboard studies, no evidence of antagonism was observed with any strain or EBO combination; interactions were largely indifferent. EBO combined with EMB in MAC resulted in synergy or additive effects in the checkerboard assay. The addition of EMB, CLR, RFB or AMK to EBO led to a &amp;gt; 700-fold reduction in RF. Activity of other drugs was not impacted by EBO resistance suggesting that cross-resistance did not occur. Disclosures Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn M. Shoen, PhD, AN2 Therapeutics: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support.

Proteomic Analysis of Alfalfa (Medicago sativa L.) Roots in Response to Rhizobium Nodulation and Salt Stress.

(1) Background: Alfalfa is an important legume forage throughout the world. Although alfalfa is considered moderately tolerant to salinity, its production and nitrogen-fixing activity are greatly limited by salt stress. (2) Methods: We examined the physiological changes and proteomic profiles of alfalfa with active nodules (NA) and without nodules (NN) under NaCl treatment. (3) Results: Our data suggested that NA roots showed upregulation of the pathways of abiotic and biotic stress responses (e.g., heat shock proteins and pathogenesis-related proteins), antioxidant enzyme synthesis, protein synthesis and degradation, cell wall degradation and modification, acid phosphatases, and porin transport when compared with NN plants under salt stress conditions. NA roots also upregulated the processes or proteins of lipid metabolism, heat shock proteins, protein degradation and folding, and cell cytoskeleton, downregulated the DNA and protein synthesis process, and vacuolar H+-ATPase proteins under salt stress. Besides, NA roots displayed a net H+ influx and low level of K+ efflux under salt stress, which may enhance the salt tolerance of NA plants. (4) Conclusions: The rhizobium symbiosis conferred the host plant salt tolerance by regulating a series of physiological processes to enhance stress response, improve antioxidant ability and energy use efficiency, and maintain ion homeostasis.

349 Dietary Zinc and Ractopamine Elicit Changes in Gene Expression of Zinc Transporters in the Muscle of Finishing Beef Steers

Abstract Twenty-four Angus cross steers (467 ± 13 kg) were used to assess the effects of supplemental Zn and ractopamine (RAC; Actogain, Zoetis) on muscle gene expression. Steers were housed in pens (6 steers/pen) with GrowSafe bunks and randomly assigned to treatments (6 steers/treatment) which included 0 (CON), 60 (LO), 120 (MED), or 180 (HI) mg supplemental Zn (Availa-Zn, Zinpro)/kg dry matter. Dietary Zn treatments were initiated on d 0 and RAC supplementation (300 mg/steer/d) began on d 53. Blood and muscle (longissimus thoracis) samples were collected from all steers on d -4, 48, and 67. Plasma Zn concentrations were determined via inductively coupled plasma-mass spectrometry and muscle gene expression was determined via the Fluidigm Biomark HD system. Data were analyzed using ProcMixed of SAS (fixed effect = treatment; experimental unit = steer); treatments were compared using orthogonal linear and quadratic contrast statements. The LO treatment was removed from gene expression analyses due to poor reads. There was a tendency for a quadratic effect on d 48 and 67 plasma Zn (P = 0.10) where plasma was greater in Zn-supplemented animals than CON. Minimal effects of Zn supplementation were observed on muscle gene expression prior to the start of RAC. However, 14 d after the start of RAC, the expression of several genes involved in Zn storage and transport (MT1A, SLC39A7, SLC39A8, SLC39A9, SLC39A10, SLC39A13) linearly decreased with increasing Zn supplementation (P ≤ 0.08). These effects were mainly driven by an increase in gene expression for CON steers, suggesting RAC influences intracellular Zn trafficking or demand. Several of these transporters are located on organelles responsible for Zn-dependent processes like protein synthesis and metalation of enzymes. Therefore, increasing Zn supplementation prior to RAC feeding may support beta-agonist induced muscle growth in beef steers.

Aminoacyl-tRNA Synthetases, Indispensable Players in Lung Tumorigenesis.

Being an essential enzyme in protein synthesis, the aminoacyl-tRNA synthetases (aaRSs) have a conserved function throughout evolution. However, research has uncovered altered expressions as well as interactions of aaRSs, in league with aaRS-interacting multi-functional proteins (AIMPs), forming a multi-tRNA synthetase complex (MSC) and divulging into their roles outside the range of protein synthesis. In this review, we have directed our focus into the rudimentary structure of this compact association and also how these aaRSs and AIMPs are involved in the maintenance and progression of lung cancer, the principal cause of most cancer-related deaths. There is substantial validation that suggests the crucial role of these prime housekeeping proteins in lung cancer regulation. Here, we have addressed the biological role that the three AIMPs and the aaRSs play in tumorigenesis, along with an outline of the different molecular mechanisms involved in the same. In conclusion, we have introduced the potentiality of these components as possible therapeutics for the evolution of new-age treatments of lung tumorigenesis.

Improving growth performance of rice cultivars IR64 and Mentik Wangi in drought stress with applying nano Zn

Increasing temperatures due to climate change caused irregular rainfall in some regions. Changes in rainfall initiate drought to increase every year and negatively impact vital human activities, including agricultural sectors. Drought stress is becoming increasingly important in agriculture because it affects changes in physiological function and leads to a severe decline in crop production. Nano Zn is one of the solutions for drought stress which has a role in enzyme activation, protein synthesis, and carbohydrate metabolism. This study aims to determine the effect of applying nano Zn on rice growth performance in drought stress conditions. The method used was a non-factorial Completely Randomized Design with analysis of variance followed by DMRT at 5% level. The levels of Zn concentration were 0; 0.5; 1; 1.5; 2 ppm Zn. Two varieties were used, ‘IR 64’ as a hybrid cultivar and ‘Mentik Wangi’ as a local cultivar in Indonesia. The variables observed were plant height (cm), number of tillers, number of leaves, biomass weight (g), and root dry weight (g). The result showed that applying nano Zn can increase rice growth due to drought stress on the various variables. The higher the nano Zn dosage, the higher plant height, number of tillers, number of leaves, biomass, and root dry weight by 3-6 %, 2-33 %, 6-23 %, 8-28 %, and 3-34 % respectively compared without Zn. These results can be considered a solution for environmental conditions affected by drought stress by paying attention to Zn concentration level.

Mini-TrpRS is essential for IFNγ-induced monocyte-derived giant cell formation

Truncated tryptophanyl-tRNA synthetase (mini-TrpRS), like any other aminoacyl-tRNA synthetases, canonically functions as a protein synthesis enzyme. Here we provide evidence for an additional signaling role of mini-TrpRS in the formation of monocyte-derived multinuclear giant cells (MGCs). Interferon-gamma (IFNγ) readily induced monocyte aggregation leading to MGC formation with paralleled marked upregulation of mini-TrpRS. Small interfering (si)RNA, targeting mini-TrpRS in the presence of IFNγ prevented monocyte aggregation. Moreover, blockade of mini-TrpRS, either by siRNA, or the cognate amino acid and decoy substrate D-Tryptophan to prevent mini-TrpRS signaling, resulted in a marked reduction in expression of the purinergic receptor P2X 7 (P2RX7) in monocytes activated by IFNγ. Our findings identify mini-TrpRS as a critical signaling molecule in a mechanism by which IFNγ initiates monocyte-derived giant cell formation.

Effects of corticosteroids and platelet-rich plasma on synoviocytes in IL-1ß-induced inflammatory condition

ABSTRACT Aims Corticosteroid injections are used to treat shoulder pain. Platelet-rich plasma (PRP) is known to have anti-inflammatory and anabolic effects, as well as cytoprotective effects against corticosteroids. Thus, this study was to investigate the effects of co-treatment of corticosteroid and PRP on anti-inflammatory and matrix homeostasis of synoviocytes in IL-1ß-induced inflammatory conditions. Materials and Methods Synoviocytes were cultured with 1 ng/mL IL-1β, 1 μM dexamethasone, and 10% (vol/vol) Platelet-poor plasma (PPP), PRP200, PRP1000, and PRP4000 X 103/μL. Gene expressions of pro-inflammatory and anti-inflammatory cytokines, degradative enzymes, and their inhibitors were evaluated and protein synthesis of degradative enzymes and their inhibitors were also examined. Results Corticosteroid modulated anti-inflammatory and pro-inflammatory cytokines, and subsequent PRP treatment did not interfere with the effect of a corticosteroid and modulated the gene expressions of cytokines such as TNF-α and IL-4, which were not regulated by the corticosteroid alone. Gene expressions and protein expressions of degradative enzymes and their inhibitors were suppressed by corticosteroid. Additional PRPs did not alter the gene expression and protein regulated by the corticosteroid and inhibited the gene expression of ADAMTS-5 and protein synthesis of MMP-9 and ADAMTS-5, which were not modulated by the corticosteroid alone. Conclusion Corticosteroid regulated the inflammation and synovial homeostasis. When PRP and the corticosteroid were used together, it exhibited synergistic effects on synoviocytes by regulating the parts that were not controlled by corticosteroid alone while not interfering with the effects of the corticosteroid in an inflammatory condition.

Rice albino 1, encoding a glycyl-tRNA synthetase, is involved in chloroplast development and establishment of the plastidic ribosome system in rice

The chloroplast is an important organelle that performs photosynthesis as well as biosynthesis and storage of many metabolites. Aminoacyl-tRNA synthetases (aaRSs) are key enzymes in protein synthesis. However, the relationship between chloroplast development and aaRSs still remains unclear. In this study, we isolated a rice albino 1 (ra1) mutant through methane sulfonate (EMS) mutagenesis of rice japonica cultivar Ningjing 4 (Oryza sativa L.), which displayed albinic leaves in seedling stage due to abnormal chloroplast development. Compared with wild type (WT), ra1 showed significantly decreased levels of chlorophylls (Chl) and carotenoids (Car) in 2-week-old seedlings, which also showed obvious plastidic structural defects including abnormal thylakoid membrane structures and more osmiophilic particles. These defects caused albino phenotypes in seedlings. Map-based cloning revealed that RA1 gene encodes a glycyl-tRNA synthetase (GlyRS), which was confirmed by genetic complementation and knockout by Crispr/Cas9 technology. Sequence analysis showed that a single base mutation (T to A) occurred in the sixth exon of RA1 and resulted in a change from Isoleucine (Ile) to Lysine (Lys). Real-time PCR analyses showed that RA1 expression levels were constitutive in most tissues, but most abundant in the leaves and stems. By transient expression in Nicotiana benthamiana, we found that RA1 protein was localized in the chloroplast. Expression levels of chlorophyll biosynthesis and plastid development related genes were disordered in the ra1 mutant. RNA analysis revealed biogenesis of chloroplast rRNAs was abnormal in ra1. Meanwhile, western blotting showed that synthesis of proteins associated with plastid development was significantly repressed. These results suggest that RA1 is involved in early chloroplast development and establishment of the plastidic ribosome system in rice.

A pharmacologic increase in activity of plasma transaminase derived from small intestine in animals receiving an acyl CoA: Diacylglycerol transferase (DGAT) 1 inhibitor

Acyl CoA: diacylglycerol acyltransferase (DGAT) 1 is an enzyme that catalyzes the re-synthesis of triglycerides (TG) from free fatty acids and diacylglycerol. JTT-553 is a DGAT1 inhibitor and exhibits its pharmacological action (inhibition of re-synthesis of TG) in the enterocytes of the small intestine leading to suppression of a postprandial elevation of plasma lipids. After repeated oral dosing JTT-553 in rats and monkeys, plasma transaminase levels were increased but there were neither changes in other hepatic function parameters nor histopathological findings suggestive of hepatotoxicity. Based on the results of exploratory studies for investigation of the mechanism of the increase in transaminase levels, plasma transaminase levels were increased after dosing JTT-553 only when animals were fed after dosing and a main factor in the diet contributing to the increase in plasma transaminase levels was lipids. After dosing JTT-553, transaminase levels were increased in the small intestine but not in the liver, indicating that the origin of transaminase increased in the plasma was not the liver but the small intestine where JTT-553 exhibits its pharmacological action. The increase in small intestinal transaminase levels was due to increased enzyme protein synthesis and was suppressed by inhibiting fatty acid-transport to the enterocytes. In conclusion, the JTT-553-related increase in plasma transaminase levels is considered not to be due to release of the enzymes from injured cells into the circulation but to be phenomena resulting from enhancement of enzyme protein synthesis in the small intestine due to the pharmacological action of JTT-553 in this organ.