In-Silico Analysis &In-Vivo Results Concur on Glutathione Depletion in Glyphosate Resistant GMO Soy, Advancing a Systems Biology Framework for Safety Assessment of GMOs

This study advances previous efforts towards development of computational systems biology, in silico, methods for biosafety assessment of genetically modified organisms (GMOs). C1 metabolism is a critical molecular system in plants, fungi, and bacteria. In our previous research, critical molecular systems of C1 metabolism were identified and modeled using CytoSolve?, a platform for in silico analysis. In addition, multiple exogenous molecular systems affecting C1 metabolism such as oxidative stress, shikimic acid metabolism, glutathione biosynthesis, etc. were identified. Subsequent research expanded the C1 metabolism computational models to integrate oxidative stress, suggesting glutathione (GSH) depletion. Recent integration of data from the EPSPS genetic modification of Soy, also known as Roundup Ready Soy (RRS), with C1 metabolism predicts similar GSH depletion and HCHO accumulation in RRS. The research herein incorporates molecular systems of glutathione biosynthesis and glyphosate catabolism to expand the extant in silico models of C1 metabolism. The in silico results predict that Organic Soy will have a nearly 250% greater ratio of GSH and GSSG, a measure of glutathione levels, than in RRS that are glyphosate-treated glyphosate-resistant Soy versus the Organic Soy. These predictions also concur with in vivo greenhouse results. This concurrence suggests that these in silico models of C1 metabolism may provide a viable and validated platform for biosafety assessment of GMOs, and aid in selecting rational criteria for informing in vitro and in vivo efforts to more accurately decide in the problem formulation phase whose parameters need to be assessed so that conclusion on “substantial equivalence” or material difference of a GMO and its non-GMO counterpart can be drawn on a well-grounded basis.

Immobilization of β-glucuronidase:biocatalysis of glycyrrhizin to 18β-glycyrrhetinic acid and in-silico lead finding

The non-covalent immobilization ofβ-glucuronidase enzyme obtained from Rhizopus oryzae was carried out by entrapment in natural fiber(papaya and coconut).The bioconversion capability of immobilized enzyme was analyzed based on conversion of glycyrrhizin to 18β-glycyrrhetinic acid under different conditions.The hydrolytic activity of theβ-glucuronidase enzyme was highly depended on the microbial source and matrix,in which enzyme was immobilized.R.oryzaeβ-glucuronidase immobilized in papaya fibers produced the highest GA content(13.170μg/mL)at 10 h of reaction.However R.oryzaeβ-glucuronidase immobilized in coconut fibers produced the highest GA content(21.425μg/mL)at 15 h of reaction.Online Molinspiration software was used to predict drug like molecular properties of the 18β-glycyrrhetinic acid,and software suggested that the compounds had potential of becoming the orally active molecules.Therefore,in silico studies were conducted on proposed 18β-glycyrrhetinic acid to select the best possible drug candidates based on drug properties and bioactivity score of the compounds.

Characterization of SARS-COV-2 main protease inhibitory peptides from Ulva prolifera proteins

The main protease(M^(pro))is essential for the replication of SARS-COV-2 and therefore represents a promising anti-viral target.In this study,we screened M^(pro)inhibitory peptides from Ulva prolifera protein on in-silico proteolysis.Cytotoxicity analysis using the online toxic prediction tool ToxinPred revealed that all the peptides were non-cytotoxic.The hexapeptide(SSGFID)exhibited high M^(pro)inhibitory activity in molecular docking and its IC_(50)value was 139.40±0.82μmol/L in vitro according to fluorescence resonance energy transfer assay(FRET).Quantitative real-time(qRT-)PCR results show that SSGFID could stimulate the expression of mitosis-related factors,including nuclear factor-κB,cyclin D1,and cyclin-dependent kinase 4,to promote the proliferation of mice splenocytes.Stability study revealed that SSGFID showed resistance against pepsin and trypsin but lost D(Asp)after pretreatment at121℃ for 15 min.Besides,SSGFID was mainly transported through the Caco-2 cell monolayer by the peptide transporter PepT1 and passive-mediated transport during the transport study.Unfortunately,the peptide was also degraded by Caco-2 intracellular enzymes,and the transfer rate of intact peptide was4.2%.Furthermore,Lineweaver–Burk plots demonstrated that SSGFID possessed a mixed inhibitory characteristic with M^(pro).Our study indicated the potential of Ulva prolifera as antiviral and immuneenhancing functional food ingredients and nutraceuticals.

LC and LC–MS/MS studies for the identification and characterization of degradation products of acebutolol

The aim of the present investigation was to demonstrate an approach involving use of liquid chromatography(LC) and liquid chromatography-mass spectrometry(LC–MS) to separate, identify and characterize very small quantities of degradation products(DPs) of acebutolol without their isolation from the reaction mixtures. The drug was subjected to oxidative, hydrolytic, thermal and photolytic stress conditions as per International Conference on Harmonization(ICH) guideline Q1 A(R2). Among all the stress conditions the drug was found to be labile in hydrolytic(acidic & basic) and photolytic stress conditions, while it was stable in water-induced hydrolysis, oxidative and thermal stress conditions. A total of four degradation products were formed. A C18 column was employed for the separation of all the DPs on a gradient mode by using high-performance liquid chromatography(HPLC). All the DPs were characterized with the help of their fragmentation pattern and the masses obtained upon LC–MS/MS and MSnanalysis. All the hitherto unknown degradation products were identified as 1-(2-(2-hydroxy-3-(isopropylamino)propoxy)-5-(amino)phenyl)ethanone(DP-I), N-(4-(2-hydroxy-3-(isopropylamino)propoxy)-3-acetylphenyl)acrylamide(DP-II), 1-(2-(2-hydroxy-3-(isopropylamino)propoxy)-5-(hydroxymethylamino)phenyl)ethanone(DP-III) and 1-(6-(2-hydroxy-3-(isopropylamino)propoxy)-2,3-dihydro-2-propylbenzo[d]oxazol-5-yl)ethanone(DP-IV). Finally the in-silico carcinogenicity and hepatotoxicity predictions of the drug and all the DPs were performed by using toxicity prediction softwares viz., TOPKAT, LAZAR and Discovery Studio ADMET. The results of in-silico toxicity studies revealed that acebutolol(0.967) and DP-I(0.986) were found to be carcinogenic, while acebutolol(0.490) and DP-IV(0.437) were found to be hepatotoxic.

Reversed-phase fused-core HPLC modeling of peptides

Different fused-core stationary phase chemistries（C18,Amide,Phenyl-hexyl and Peptide ES-C18） were used for the analysis of 21 structurally representative model peptides.In addition,the effects of the mobile phase composition（ACN or MeOH as organic modifier;formic acid or acetic acid,as acidifying component） on the column selectivity,peak shape and overall chromatographic performance were evaluated.The RP-amide column,combined with a formic acid-acetonitrile based gradient system,performed as best.A peptide reversed-phase retention model is proposed,consisting of 5 variables：log SumAA,log Sv,clog P,log nHDon and log nHAcc.Quantitative structure-retention relationship（QSRR） models were constructed for 16 different chromatographic systems.The accuracy of this peptide retention model was demonstrated by the comparison between predicted and experimentally obtained retention times,explaining on average 86% of the variability.Moreover,using an external set of 5 validation peptides,the predictive power of the model was also demonstrated.This peptide retention model includes the novel in-silico calculated amino acid descriptor,AA,which was calculated from log P,3D-MoRSE,RDF and WHIM descriptors.

Small molecule compounds targeting DNA binding domain of STAT3 for inhibition of tumor growth and metastasis

Background Signal transducer and activator of transcription 3 （STAT3） is constitutively activated in malignant tumors and has important roles in multiple aspects of cancer aggressiveness. Thus targeting STAT3 promi- ses to be an attractive strategy for treatment of advanced metastatic tumors. Although many STAT3 inhibitors targe- ting the SH2 domain have been reported, few have moved into clinical trials. Targeting the DNA-binding domain （DBD） of STAT3, however, has been avoided due to its ＇ undruggable＇ nature and potentially limited selectivity. Aim This study aims at developing effective and specific inhibitors targeting DNA binding domain of STAT3. Methods： This study reported an improved in-silico approach targeting the DBD of STAT3 that resulted in a small- molecule STAT3 inhibitor （inS3-54） and describe an extensive structure and activity-guided hit optimization and mechanistic characterization effort, which led to identification of an improved lead compound （inS3-54A18） with increased specificity and pharmacological properties. Results： InS3-54A18 not only binds directly to the DBD and inhibits the DNA-binding activity of STAT3 both in vitro and in situ but also effectively inhibits the constitutive and interleukin-6-stimulated expression of STAT3 downstream target genes. InS3-54A18 is completely soluble in an oral formulation and effectively inhibits lung xenograft tumor growth and metastasis with little adverse effect on animals. Conclusion： InS3-54A18 may serve as a potential candidate for further development as anticancer therapeutics tar-geting the DBD of human STAT3 and DBD of transcription factors may not be ＇ undruggable＇ as previously thought.

A stability-indicating LC–MS/MS method for zidovudine: Identification,characterization and toxicity prediction of two major acid degradation products

Zidvovudine(AZT) is a nucleoside analogue reverse transcriptase inhibitor(NRTI), a class of anti-retroviral drug. A stability-indicating assay method for AZT was developed in line with ICH guideline. Successful separation of AZT and its degradation products was achieved by gradient elution mode on reverse phase C_(18) column using 10 mM ammonium acetate: acetonitrile as the mobile phase at 0.8 mL/min flow rate, 25 μL injection volume, 30 °C column temperature and 285 nm detection wavelength. Two major acid degradation products were identified and characterized by liquid chromatography–electrospray ionization mass spectrometry(LC–ESI/MS/MS) and accurate mass measurements. The probable mechanisms for the formation of degradation products were identified based on a comparison of the fragmentation pattern of the [M + H]^+ions of AZT and its degradation products. One of the degradation products, DP-1, was isolated by semi-preparative high performance liquid chromatography(HPLC) using Waters XBridge Prep C_(18)(250 mm×10 mm, 5 μm).Degradation products showed higher toxicity compared to the drug in some models assessed by TOPKAT software. The method validation was performed with respect to robustness, specificity, linearity, precision and accuracy as per ICH guideline Q2(R1).

Novel Point Mutations in 3'-Untranslated Region of GATA4 Gene Are Associated with Sporadic Non-syndromic Atrial and Ventricular Septal Defects

Objective:Transcription factor GATA4 has significant roles in embryonic heart development.Mutations of GATA4 appear to be responsible for a wide variety of congenital heart defects(CHD).Despite the high prevalence of GATA4 mutations in CHD phenotypes,extensive studies have not been performed.The 3'-untranslated region(3'-UTR)of tho GATA4 gene comprises regulatory motifs and microRNA binding sites that are critical for the appropriate gene expression,nuclear transportation,and regulation of translation,and stability of mRNA.This study aimed to evaluate the association between mutations in the 3'-UTR of the GATA4 gene and CHD risk among Iranian patients.

Computational Analysis of a Series of Chlorinated Chalcone Derivatives

A systematic conceptual density functional theory (DFT) analysis was performed on a series of chlorinated chalcones to study the effect of electron distribution on antimicrobial activity. In our previous work, a series of 16 chlorinated chalcones were synthesized to determine the antimicrobial effects of varying the location of the halogen substituent on each aromatic ring of the chalcone. Herein is reported a DFT investigation of those 16 chalcones and a comparison of quantum chemical properties to their antimicrobial activity. DFT global chemical reactivity descriptors (chemical hardness/softness, chemical potential/electronegativity, and electrophilicity) and local reactivity descriptors (Fukui functions and dual descriptor) were calculated for all compounds using Spartan’18 software. All calculations were carried out at the B3LYP/6-31G* level of theory. Reactivity analysis of the Fukui dual descriptor calculations reveals sites of nucleophilic and electrophilic attack. These in-silico results provide a foundation for further synthetic optimization of the chalcone skeleton to serve as novel antimicrobial agents.

Molecular Docking Studies of Alpinia galanga Phytoconstituents for Psychostimulant Activity

Dopamine is a neurotransmitter responsible for sending signals from the central nervous system. It allows human beings to stay attentive and focused. Caffeine, the most widely consumed psychoactive substance in the world, is known to improve alertness by enhancing dopamine signaling in the brain. EnXtra®, an Alpinia galanga extract has been clinically proven to promote alertness however the mechanism for such action required elucidation. The current study was designed to explore the mechanism for the neurocognitive enhancing property of EnXtra®by the in-silico interaction of its potential compounds with various targets involved in such process namely Dopamine and Acetylcholinesterase (AchE). As evident by the outcomes of the study, active compounds of EnXtra®can block the dopamine reuptake thereby increasing the dopamine levels which further can enhance the visuospatial performance and mental clarity, leading to improved mental alertness. At the same time, its strong effect on Acetylcholinesterase receptors is indicative of its nootropic potential.

Comprehensive relative quantitative metabolomics analysis of lycopodium alkaloids in different tissues of Huperzia serrata

Qian ceng Ta,the whole plant of Huperzia serrata,is an important landscape and medicinal herbs and contains abundant bioactive lycopodium alkaloids.Although the structures of more than 100 lycopodium alkaloids in Huperzia serrata have been isolated and identified,the content and distribution of these alkaloids in different tissues are still unclear.In current study,an ultra-performance liquid chromatography-mass spectrometry based comprehensive metabolomics strategy was developed,including the extraction,separation,identification,and statistical analysis.The results showed that different types lycopodium alkaloids could be separated at different time-windows,which was helpful for further metabolite identification.Peak4388 and peak3954 were metabolite biomarkers for the different tissues according to the principle component analysis and partial least squares-discriminant analysis model.A computational tool based in-house database was also built up and used for putative identification.Of the 2354 true peaks after four-step filtration,118 peaks were putatively identified as lycopodium alkaloids by using in-house database,and four of which was identified by authentic standards.Alternatively,another computational software was used to predict the fragmentation pattern,to dereplicate the structure of identified peaks,and identified the peak3585 to N-methylhuperzine A.The integration of both computational tools could be used for more metabolites identification.

外源性化合物心脏毒性体外研究新进展

外源性化合物导致的心脏毒性问题一直是毒理学研究中的一个非常重要的关注点。心脏毒性体外研究可以降低研究成本,缩短实验周期,在心脏毒性研究中起重要作用。传统的心脏毒性体外研究方法常存在通量低、操作复杂、预测准确性不好等缺点。近年来,随着科学技术的不断发展,新的心脏毒性研究技术和方法不断出现,包括实时x CELLigence细胞分析技术、微电极阵列技术、扫描离子电导显微镜技术、新型线粒体膜电位检测技术、in-silico模型及新型心肌损伤生物标志物等。本文主要就以上心脏毒性研究的新技术和方法进行了综述。

Integrated “Generate, Make, and Test” for Formulated Products using Knowledge Graphs

In the multi-billion dollar formulated product industry, state of the art continues to rely heavily on experts during the "generate, make and test" steps of formulation design. We propose automation aids to each step with a knowledge graph of relevant information as the central artifact. The generate step usually focuses on coming up with new recipes for intended formulation. We propose to aid the experts who generally carry out this step manually by providing a recommendation system and a templating system on top of the knowledge graph. Using the former, the expert can create a recipe from scratch using historical formulations and related data. With the latter, the expert starts with a recipe template created by our system and substitutes the requisite constituents to form a recipe. In the current state of practice, the three steps mentioned above operate in a fragmented manner wherein observations from one step do not aid other steps in a streamlined manner. Instead of manually operated labs for the make and test steps, we assume automated or robotic labs and in-silico testing, respectively. Using two formulations, namely face cream and an exterior coating, we show how the knowledge graph may help integrate and streamline the communication between the generate, the make, and the test steps. Our initial exploration shows considerable promise.