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To understand differences in the quality of different conventional japonica rice varieties and variations in metabolites related to rice quality, the quality of three conventional japonica varieties was determined, and the metabolites of the milled rice were investigated using nontargeted metabolomics technology. The results showed that the taste value (TV) of Yangda 4Hao (YD4) was significantly higher than that of Yangda 3Hao (YD3) and Huaidao 5Hao (HD5). The protein content (PC) of HD5 was significantly higher than that of YD3 and YD4. PC was significantly negatively correlated with TV. Ninety-one differential metabolites (59 increased and 32 decreased) were identified between YD3 and HD5. A total of 144 differential metabolites (96 upregulated and 48 downregulated) were identified between YD4 and HD5. A total of 114 differential metabolites (40 increased and 74 decreased) were identified between YD3 and YD4. The metabolites with a high correlation to rice quality were mostly involved in the amino acid metabolism pathway. Amino acid metabolites play an important role in the formation of rice quality. The key metabolites in the synthesis and regulation of metabolic pathways are sucrose, levan, and amylose, which are carbohydrates, and L-glutamine, L-aspartic acid, and L-asparagine, which are amino acid metabolites. It can be seen from this study that the metabolites of sucrose, levan, amylose, L-glutamine, L-aspartic acid, and L-asparagine may be the key metabolites in the quality formation of high-quality rice varieties.

Despite differences in behaviors and living conditions, vertebrate organisms share the great majority of proteins, often with subtle differences in amino acid sequence. Here, we present a simple way to analyze the difference in amino acid occurrence by comparing highly homologous proteins on a sub-proteome level between several vertebrate model organisms. Specifically, we use this method to identify a pattern of amino acid conservation as well as a shift in amino acid occurrence between homeotherms (warm-blooded species) and poikilotherms (cold-blooded species). Importantly, this general analysis and a specific example further establish a correlation, if not likely connection between the thermoadaptation of protein sequences and two of their physical features: a possible change in their protein dynamics and, even more strongly, in their solvation. For poikilotherms, such as frog and fish, the lower body temperature is expected to increase the association of proteins due to a decrease in protein internal dynamics. In order to prevent overly-sticky protein association at low temperatures, the use of amino acids suggests that poikilotherms enhance the solvation of their proteins by favoring polar groups on their protein’s surface. This feature appears to dominate over possible changes in dynamics. The results suggest that a general trend for amino acid choice is part of the mechanism for thermoadaptation of vertebrate organisms at the molecular level.

SIGNIFICANCE: Ergothioneine (ET) is an unusual sulfur-containing amino acid derived from histidine, acquired predominantly from food. Its depletion is associated with deleterious consequences in response to stress stimuli in cell culture models, prompting us to classify it as a vitamin in 2010, which was later supported by in vivo studies. ET is obtained from a variety of foods and is taken up by a selective transporter. ET possesses antioxidant and anti-inflammatory properties that confer cytoprotection. ET crosses the blood–brain barrier and has been reported to have beneficial effects in the brain. In this study, we discuss the cytoprotective and neuroprotective properties of ET, which may be harnessed for combating neurodegeneration and decline during aging. RECENT ADVANCES: The designation of ET as a stress vitamin is gaining momentum, opening a new field of investigation involving small molecules that are essential for optimal physiological functioning and maintenance of health span. CRITICAL ISSUES: Although ET was discovered more than a century ago, its physiological functions are still being elucidated, especially in the brain. As ET is present in most foods, toxicity associated with its deprivation has been difficult to assess. FUTURE DIRECTIONS: Using genetically engineered cells and mice, it may now be possible to elucidate roles of ET. This coupled with advances in genomics and metabolomics may lead to identification of ET function. As ET is a stable antioxidant with anti-inflammatory properties, whose levels decline during aging, supplementing ET in the diet or consuming an ET-rich diet may prove beneficial. Antioxid. Redox Signal. 36, 1306–1317.

Limited data are available on the effects of pregnancy on the maternal metabolome. Therefore, the objective of this study was to use metabolomics analysis to determine pathways impacted by pregnancy followed by targeted confirmatory analysis to provide more powerful conclusions about metabolic alterations during pregnancy. Forty-seven pregnant women, 18–50 years of age were included in this study, with each subject serving as their own control. Plasma samples were collected between 25 and 28 weeks gestation and again ≥3 months postpartum for metabolomics analysis utilizing an HILIC/UHPLC/MS/MS assay with confirmatory targeted specific concentration analysis for 10 of the significantly altered amino acids utilizing an LC/MS assay. Principle component analysis (PCA) on metabolomics data clearly separated pregnant and postpartum groups and identified outliers in a preliminary assessment. Of the 980 metabolites recorded, 706 were determined to be significantly different between pregnancy and postpartum. Pathway analysis revealed three significantly impacted pathways, arginine biosynthesis (p = 2 × 10−5 and FDR = 1 × 10−3), valine, leucine, and isoleucine metabolism (p = 2 × 10−5 and FDR = 2 × 10−3), and xanthine metabolism (p = 4 × 10−5 and FDR = 4 × 10−3). Of these we focused analysis on arginine biosynthesis and branched-chain amino acid (BCAA) metabolism due to their clinical importance and interconnected roles in amino acid metabolism. In the confirmational analysis, 7 of 10 metabolites were confirmed as significant and all 10 confirmed the direction of change of concentrations observed in the metabolomics analysis. The data support an alteration in urea nitrogen disposition and amino acid metabolism during pregnancy. These changes could also impact endogenous nitric oxide production and contribute to diseases of pregnancy. This study provides evidence for changes in both the ammonia-urea nitrogen and the BCAA metabolism taking place during pregnancy.

The recent outbreak of SARS CoV-2 has changed the global scenario of human lives/economy. A significant number of the non-survivors showed cardiac-renal-vasculature dysfunction. A 'cytokine storm' namely, interleukin IL6-IL1 receptors i.e. IL6R-IL1R over-functioning was reported. Here, nigellidine, an indazole-alkaloid and key-component of Nigella Sativa L. (NS) commonly known as black-cumin-seed was analyzed for COVID-19 protein-targeting and IL1R-IL6R inhibition through molecular-docking-study and biochemical-study in experimental-rat to evaluate antioxidative-capacity. The NMR/X-ray-crystallographic/Electron-microscopic structures of COVID-19 Main-protease (6LU7)/Spike-glycoprotein(6vsb)/NSP2(QHD43415_2)/Nucleocapsid(QHD43423), Human IL1R(1itb)-IL6R(1pm9) from PDB were retrieved-analyzed for receptor-ligand interaction. Then those structures were docked with nigellidine using Autodock and Patchdock-server. A brief comparison was made with nigellicine-thymoquinone from N. sativa. Where nigellidine showed highest binding-energy of -6.6 (kcal/mol), ligand-efficiency of -0.3 with COVID19 Nsp2 forming bonds with amino acid CYS240 present in binding-pocket. Nigellidine showed strong interaction with main-protease (BE:-6.38/LE:-0.29). Nigellidine showed affinity to IL1R (-6.23). The NS treated rat showed marked decline in ALP-SGPT-SGOT-malondialdehyde(MDA) than the basal-levels. From the Western-blot and activity-analysis it was observed that Nigellidine (sulfuryl-group-drug) showed no impact on Phenol-catalyzing ASTIV and Steroid-catalyzing estrogen-sulfotransferase expressions and activities in liver-tissue and thus has no influence in sulfation-mediated adverse metabolic-processes. Conclusively, nigellidine has hepato-reno-protective/antioxidant-immunomodulatory/anti-inflammatory activities with inhibit-potentials of COVID-19 proteins. Further validation is necessary.

Prediction of side chain conformations of amino acids in proteins (also termed ‘packing’) is an important and challenging part of protein structure prediction with many interesting applications in protein design. A variety of methods for packing have been developed but more accurate ones are still needed. Machine learning (ML) methods have recently become a powerful tool for solving various problems in diverse areas of science, including structural biology. In this work we evaluate the potential of Deep Neural Networks (DNNs) for prediction of amino acid side chain conformations. We formulate the problem as image-to-image transformation and train a U-net style DNN to solve the problem. We show that our method outperforms other physics-based methods by a significant margin: reconstruction RMSDs for most amino acids are about 20% smaller compared to SCWRL4 and Rosetta Packer with RMSDs for bulky hydrophobic amino acids Phe, Tyr and Trp being up to 50% smaller.

Cytochrome P450 monooxygenase (P450 or CYP) plays an important role in the metabolism of insecticides and plant allelochemicals by insects. CYP321B1, a novel Spodoptera litura P450 gene, was identified and characterized. CYP321B1 contains a 1488 bp open reading frame (ORF) that encodes a 495 amino acid protein. In fourth instar larvae, the highest CYP321B1 expression levels were found in the midgut and fat body. In the tannin feeding test, tannin can significantly induce the expression of CYP321B1 in the midgut and fat body of 4th instar larvae. To verify the function of CYP321B1, RNA interference and metabolome analysis were performed. The results showed that silencing CYP321B1 significantly reduced the rate of weight gain under tannin induction. Metabolome analysis showed silencing affected 47 different metabolites, mainly involved in secondary metabolite biosynthesis and amino acid metabolism, including amino acids, lipid fatty acids, organic acids and their derivatives. Henoxyacetic acid and cysteamine are the most highly regulated metabolites, respectively. These findings demonstrate that CYP321B1 plays an important role in tannin detoxification and metabolism. Functional knowledge about metabolite detoxification genes in this major herbivorous insect pest can provide new insights into this biological process and provide new targets for agricultural pest control.

Aminoacyl tRNA synthetases (ARSs) are a group of proteins, acting as transporters to transfer and attach the appropriate amino acids onto their cognate tRNAs for translation. So far, 18 out of 20 cytoplasmic ARSs are reported to be connected to different neuropathy disorders with multi-organ defects that are often accompanied with developmental delays. Thus, it is important to understand functions and impacts of ARSs at the whole organism level. Here, we systematically analyzed the spatiotemporal expression of 14 ars and 2 aimp genes during development in zebrafish that have not be previously reported. Not only in the brain, their dynamic expression patterns in several tissues such as in the muscles, liver and intestine suggest diverse roles in a wide range of development processes in addition to neuronal function, which is consistent with potential involvement in multiple syndrome diseases associated with ARS mutations. In particular, hinted by its robust expression pattern in the brain, we confirmed that aimp1 is required for the formation of cerebrovasculature by a loss-of-function approach. Overall, our systematic profiling data provides a useful basis for studying roles of ARSs during development and understanding their potential functions in the etiology of related diseases.

Uroporphyrinogen III synthetase (UROS) is an important enzyme involved in the structural formation of porphyrin, which is responsible for color intensity. A UROS homolog was isolated and characterized from the pearl mussel, Hyriopsis cumingii (HcUros). The HcUros cDNA was 858 bp in length with an open reading frame that encoded a 285 amino acid protein, which contained a conserved HemD domain. HcUros exhibited widespread tissue distribution, displaying particular enrichment in the mantle and haemolymph of purple mussels, compared to white mussels. In situ hybridization of HcUros in mantle tissue demonstrated that it was specifically expressed in the dorsal epithelial cells of the mantle pallia. Additionally, HcUros expression was upregulated following implantation of the pearl sac to produce pearls. The data suggest that HcUros contributes to shell pigmentation, and nacre formation and coloring in H. cumingii.

Fatty acid desaturase (FADS) generates double bond at a certain position of the corresponding polyunsaturated fatty acids (PUFAs) with high selectivity, the enzyme activity and PUFAs products of which are essential to biological systems and are associated with a variety of physiological diseases. Little is known about the structure of FADSs and their amino acid residues related to catalytic activities. Identifying key residues of Micromonas pusilla delta 6 desaturase (MpFADS6) provides a point of departure for a better understanding of desaturation. In this study, conserved amino acids were anchored through gene consensus analysis, thereby generating corresponding variants by site-directed mutagenesis. To achieve stable and high-efficiency expression of MpFADS6 and its variants in Saccharomyces cerevisiae, the key points of induced expression were optimized. The contribution of conserved residues to the function of enzyme was determined by analyzing enzyme activity of the variants. Molecular modeling indicated that these residues are essential to catalytic activities, or substrate binding. Mutants MpFADS6[Q409R] and MpFADS6[M242P] abolished desaturation, while MpFADS6[F419V] and MpFADS6[A374Q] significantly reduced catalytic activities. Given that certain residues have been identified to have a significant impact on MpFADS6 activities, it is put forward that histidine-conserved region III of FADS6 is related to electronic transfer during desaturation, while histidine-conserved regions I and II are related to desaturation. These findings provide new insights and methods to determine the structure, mechanism and directed transformation of membrane-bound desaturases.

Synonymous codon usage can be influenced by mutations and/or selection, e.g., for speed of protein translation and correct folding. However, this codon bias can also be affected by a general selection at the amino acid level due to differences in the acceptance of the loss and generation of these codons. To assess the importance of this effect, we constructed a mutation–selection model model, in which we generated almost 90,000 stationary nucleotide distributions produced by mutational processes and applied a selection based on differences in physicochemical properties of amino acids. Under these conditions, we calculated the usage of fourfold degenerated (4FD) codons and compared it with the usage characteristic of the pure mutations. We considered both the standard genetic code (SGC) and alternative genetic codes (AGCs). The analyses showed that a majority of AGCs produced a greater 4FD codon bias than the SGC. The mutations producing more thymine or adenine than guanine and cytosine increased the differences in usage. On the other hand, the mutational pressures generating a lot of cytosine or guanine with a low content of adenine and thymine decreased this bias because the nucleotide content of most 4FD codons stayed in the compositional equilibrium with these pressures. The comparison of the theoretical results with those for real protein coding sequences showed that the influence of selection at the amino acid level on the synonymous codon usage cannot be neglected. The analyses indicate that the effect of amino acid selection cannot be disregarded and that it can interfere with other selection factors influencing codon usage, especially in AT-rich genomes, in which AGCs are usually used.

The enzymes involved in H2S homeostasis regulate its production from sulfur-containing amino acids and its oxidation to thiosulfate and sulfate. Two gatekeepers in this homeostatic circuit are cystathionine beta-synthase, which commits homocysteine to cysteine, and sulfide quinone oxidoreductase, which commits H2S to oxidation via a mitochondrial pathway. Inborn errors at either locus affect sulfur metabolism, increasing homocysteine-derived H2S synthesis in the case of CBS deficiency and reducing complex IV activity in the case of SQOR deficiency. In this review, we focus on structural perspectives on the reaction mechanisms and regulation of these two enzymes, which are key to understanding H2S homeostasis in health and its dysregulation and potential targeting in disease.

High protein diet (HPD) is an affordable and positive approach in prevention and treatment of many diseases. It is believed that transcriptional regulation is responsible for adaptation after HPD feeding and Kruppel-like factor 15 (KLF15), a zinc finger transcription factor that has been proved to perform transcriptional regulation over amino acid, lipid and glucose metabolism, is known to be involved at least in part in this HPD response. To gain more insight into molecular mechanisms by which HPD controls expressions of genes involved in amino acid metabolism in the liver, we performed RNA-seq analysis of mice fed HPD for a short period (3 days). Compared to a low protein diet, HPD feeding significantly increased hepatic expressions of enzymes involved in the breakdown of all the 20 amino acids. Moreover, using KLF15 knockout mice and in vivo Ad-luc analytical system, we were able to identify Cth (cystathionine gamma-lyase) as a new target gene of KLF15 transcription as well as Ast (aspartate aminotransferase) as an example of KLF15-independent gene despite its remarkable responsiveness to HPD. These findings provide us with a clue to elucidate the entire transcriptional regulatory mechanisms of amino acid metabolic pathways.

In this study, a phaCR gene encoding PHA synthase was identified in Rhodoligotrophos defluvii which was adjacent to β-ketothiolase encoded by phaAR gene and acetoacetyl-CoA reductase encoded by phaBR gene. Amino acid comparison of PhaCR showed the highest homology of 65.98% with PhaC of R. appendicifer, while its homology with typical class I PHA synthase in Cupriavidus necator was only 42.54%. PHA synthesis genes were then transformed into E. coli harboring phaCABR and phaCRABC which were cultured with 15 g/L glucose respectively, and 20.46 wt% and 16.95 wt% of CDW for poly(3-hydroxybutyrate) (PHB) were accumulated respectively. To further explore the effect of substrate specificity for PHA production, the ptsG gene was then deleted and 15 g/L glucose and 1.5 g/L propionate were co-employed as carbon sources, which enabled the synthesis of poly(3HB-co-3HV) copolymer. As a result, poly(3HB-co-3HV) was accumulated up to 24.74 wt% of CDW, and the highest content of 3-hydroxyvalerate (3HV) was 10.86 mol%. The Td5 was 260 °C, which implied that it possessed good thermal stability, and the Mw of GPC in recombinant strains were between 22 and 26 × 104 g/mol, and the highest PDI was 3.771. The structure of poly (3HB-co-3HV) copolymer was determined through 1H NMR analysis.

Background Pattern matching is a key step in a variety of biological sequence analysis pipelines. The FM-index is a compressed data structure for pattern matching, with search run time that is independent of the length of the database text. Implementation of the FM-index is reasonably complicated, so that increased adoption will be aided by the availability of a fast and flexible FM-index library. Results We present AvxWindowedFMindex (AWFM-index), a lightweight, open-source, thread-parallel FM-index library written in C that is optimized for indexing nucleotide and amino acid sequences. AWFM-index introduces a new approach to storing FM-index data in a strided bit-vector format that enables extremely efficient computation of the FM-index occurrence function via AVX2 bitwise instructions, and combines this with optional on-disk storage of the index’s suffix array and a cache-efficient lookup table for partial k-mer searches. The AWFM-index performs exact match count and locate queries faster than SeqAn3’s FM-index implementation across a range of comparable memory footprints. When optimized for speed, AWFM-index is $$\sim $$ ∼ 2–4x faster than SeqAn3 for nucleotide search, and $$\sim $$ ∼ 2–6x faster for amino acid search; it is also $$\sim $$ ∼ 4x faster with similar memory footprint when storing the suffix array in on-disk SSD storage. Conclusions AWFM-index is easy to incorporate into bioinformatics software, offers run-time performance parameterization, and provides clients with FM-index functionality at both a high-level (count or locate all instances of a query string) and low-level (step-wise control of the FM-index backward-search process). The open-source library is available for download at https://github.com/TravisWheelerLab/AvxWindowFmIndex.

New information is being accumulated for plant-derived oxylipins, such as jasmonic acid (JA) amino acid conjugates. However, these compounds have not being examined for their activity in promoting potato tuber formation. It was found that (−)-JA had the highest activity followed cis-(−)-OPDA, (+)-4, 5-didehydroJA, cis-(+)-OPDA-l-Ile, and (−)-JA-l-Ile, -Leu, -Phe, -Val, although iso-OPDA and 3,7-didehydroJA did not exhibit activity.

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases, generally involving the site-directed mutagenesis of functionally relevant amino acids or the selection of improved inhibitor variants by phage display approaches. Here, we propose a novel approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including arthropod proteases, to appreciate the extent of functional variability among the plant cystatin family. The most, and less, potent of these cystatins were then used as 'donors' of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model 'recipient' inhibitor. In brief, inhibitory activities against Cys proteases strongly differed from one plant cystatin to another, with Ki (papain) values diverging by more than 30-fold and inhibitory rates against arthropod proteases varying by up to 50-fold depending on the enzymes assessed. In line with theoretical assumptions from docking models generated for different Cys protease-cystatin combinations, structural element substitutions had a strong impact on the activity of recipient cystatin SlCYS8, positive or negative depending on the basic inhibitory potency of the donor cystatin. Our data confirm the wide variety of cystatin inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.

Abnormal activation of the mechanistic target of rapamycin (mTOR) signaling is commonly observed in many cancers and attracts extensive attention as an oncology drug discovery target, which is encouraged by the success of rapamycin and its analogs (rapalogs) in treatment of mTORC1-hyperactive cancers in both pre-clinic models and clinical trials. However, rapamycin and existing rapalogs have typically short-lasting partial responses due to drug resistance, thereby triggering our interest to investigate a potential mTORC1 inhibitor that is mechanistically different from rapamycin. Here, we report that hayatine, a derivative from Cissampelos, can serve as a potential mTORC1 inhibitor selected from a natural compound library. The unique properties owned by hayatine such as downregulation of mTORC1 activities, induction of mTORC1's translocation to lysosomes followed by autophagy, and suppression on cancer cell growth, strongly emphasize its role as a potential mTORC1 inhibitor. Mechanistically, we found that hayatine disrupts the interaction between mTORC1 complex and its lysosomal adaptor RagA/C by binding to the hydrophobic loop of RagC, leading to mTORC1 inhibition that holds great promise to overcome rapamycin resistance. Taken together, our data shed light on an innovative strategy using structural interruption-based mTORC1 inhibitors for cancer treatment.

The stringent response is critical for the survival of Mycobacterium tuberculosis (Mtb) under nutrient starvation. The mechanism is mediated by a GTP pyrophosphokinase known as Rel, containing N-terminal synthetase and hydrolase domains and C-terminal regulatory domains, which include the TGS domain (ThrRS, GTPase, and SpoT proteins) that has been proposed to activate the synthetase domain via interaction with deacylated tRNA. Here, we present the NMR solution structure of the Mtb Rel TGS domain (MtRel TGS), consisting of five antiparallel β-strands and one helix-loop-helix motif. The interaction of MtRel TGS with deacylated tRNA is shown, indicating the critical amino acids of MtRel TGS in tRNA binding, and presenting the first structural evidence of MtRel TGS binding to deacylated tRNA in solution in the absence of the translational machinery. Ministry of Education (MOE) We would like to thank the Singapore Ministry of Education Academic Research Fund Tier 1 (Rg137/15)for the funding to GG.

Yarrowia lipolytica is used as a model in this study to screen the potential candidates for inflating the innate lipid content of the cell. This study focuses on reducing the lipid degradation that occurs by the β-oxidation process and discursively increasing the innate lipid content. Acyl-CoA oxidase-1, the primary and initial enzyme involved in the lipid degradation pathway, was selected as a target and blocked using various lipid analogous compounds. The blocking study was carried out using molecular docking and dynamic studies using computation tools. The largest active site pocket located around the Phe-394 amino acid of the target protein is taken as a site for docking. The molecular docking was performed for the selected compounds (citric acid, Finsolv, lactic acid, oxalic acid, Tween-80 and Triton X-100) and the docking results were compared with the outcome of the standard molecule (octadecatrienoic acid). Citric acid, Finsolv, Tween-80 and Triton X-100 were found to be the potential candidates for blocking the target molecule in the static condition using docking studies, revealing a minimum binding energy requirement than the standard molecule. They were further taken for a dynamics study using GROMACS software. The RMSD, RMSF, number of hydrogen bond interactions and radius of gyration of the complex molecules were studied in a dynamic approach for 100 ns. Citric acid has been found to be the potential hit compound to block acyl-CoA oxidase-1 enzyme with its maximum hydrogen interaction and minimum fluctuations. It also revealed out the minimum total energy requirement for the complex formation.

Background Amyloids are insoluble fibrillar aggregates that are highly associated with complex human diseases, such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes. Recently, many studies reported that some specific regions of amino acid sequences may be responsible for the amyloidosis of proteins. It has become very important for elucidating the mechanism of amyloids that identifying the amyloidogenic regions. Accordingly, several computational methods have been put forward to discover amyloidogenic regions. The majority of these methods predicted amyloidogenic regions based on the physicochemical properties of amino acids. In fact, position, order, and correlation of amino acids may also influence the amyloidosis of proteins, which should be also considered in detecting amyloidogenic regions. Results To address this problem, we proposed a novel machine-learning approach for predicting amyloidogenic regions, called ReRF-Pred. Firstly, the pseudo amino acid composition (PseAAC) was exploited to characterize physicochemical properties and correlation of amino acids. Secondly, tripeptides composition (TPC) was employed to represent the order and position of amino acids. To improve the distinguishability of TPC, all possible tripeptides were analyzed by the binomial distribution method, and only those which have significantly different distribution between positive and negative samples remained. Finally, all samples were characterized by PseAAC and TPC of their amino acid sequence, and a random forest-based amyloidogenic regions predictor was trained on these samples. It was proved by validation experiments that the feature set consisted of PseAAC and TPC is the most distinguishable one for detecting amyloidosis. Meanwhile, random forest is superior to other concerned classifiers on almost all metrics. To validate the effectiveness of our model, ReRF-Pred is compared with a series of gold-standard methods on two datasets: Pep-251 and Reg33. The results suggested our method has the best overall performance and makes significant improvements in discovering amyloidogenic regions. Conclusions The advantages of our method are mainly attributed to that PseAAC and TPC can describe the differences between amyloids and other proteins successfully. The ReRF-Pred server can be accessed at http://106.12.83.135:8080/ReRF-Pred/.

A novel esterase (est3S) gene, 1026 bp in size, was cloned from a metagenomic library made of uncultured microorganisms from the contents of cow rumen. The esterolytic enzyme (Est3S) is composed of 342 amino acids and shows the highest identity with EstGK1 (71.7%) and EstZ3 (63.78%) esterases from the uncultured bacterium. The Est3S did not cluster in any up-to-date classes (I to XVIII) of esterase and lipase. Est3S protein molecular weight was determined to be 38 kDa by gel electrophoresis and showed optimum activity at pH 7.0 and 40 °C and is partially resistant to organic solvents. Est3S activity was enhanced by K+, Na+, Mg2+, and Ca2+ and its highest activity was observed toward the short-chain p-nitrophenyl esters. Additionally, Est3S can degrade chlorpyrifos (CP) and methyl parathion (70% to 80%) in an hour. A mutated Est3S (Ser132-Ala132) did not show any activity toward CP and ester substrates. Notably, the GHS132QG motif is superimposed with the homolog esterase and cutinase-like esterase. Therefore, Ser132 is the critical amino acid like other esterases. The Est3S is relatively stable with ester compounds, and the methyl parathion complex was confirmed by molecular dynamics simulation. Novelty statement A novel esterase gene (est3S) expressing esters and organophosphorus insecticide degradation traits was isolated from the uncultured bacterium in the contents of cow rumen. The Est3S protein did not cluster in any up-to-date classes (I to XVIII) of esterase/lipase proteins. Est3S was stable with the ligands up to 100 ns during the molecular dynamic simulations.

Background: Proteins are biomolecules that consist of sequences of amino acids (primary structure) which can further interact and cause the backbone to fold into more complex arrangements (secondary and tertiary structures). Any chemical alterations of the molecules after the translation of the messenger RNA code into a protein primary sequence are known as posttranslational modifications (PTMs). PTMs may affect the protein’s functionality; thus it is necessary to identify them. PTMs of particular interest to the pharmaceutical industry include deamidation, oxidation, deglycosylation and isomerization, which may occur due to environmental stressors. However, they have proved challenging to identify quickly. Electronic and vibrational spectroscopies have proved valuable tools for studying higher-order structure and stability of proteins. Materials & Methods: In this work, circular dichroism (CD), infrared absorbance (IR) and Raman spectroscopies were applied to characterize antibody (mAb NIP 228) PTMs as a result of different stressors. Mass spectrometry was used to confirm the identity of modifications including the targeted ones. Room temperature CD showed that the secondary structure was the same after all treatments, and temperature-controlled CD showed how protein stability was affected by modifications. Both Raman and IR analysis detected small differences between the reference and deglycosylated proteins, and clearly indicated the presence of other PTMs. Conclusion: This work required some novel computational approaches to pre-process Raman and IR spectra and a review of the band assignments for proteins existing in the literature.

Synthetic β-hairpin antimicrobial peptides (AMPs) offer a useful source for the development of novel antimicrobial agents. β-hairpin peptides generally consist of two side strands bridged by a reverse turn. In literature, most studies focused on the modifications of the side strands to manipulate the stability and activity of β-hairpin peptides, and much less is known about the impact of the turn region. By designing a series of de novo β-hairpin peptides with identical side strands but varied turns, we demonstrated that mutations of only 2 to 4 amino acids at the turn region could impart a wide range of antimicrobial profiles among synthetic β-hairpin AMPs. BTT2–4 and BTT6 displayed selective potency against Gram-negative bacteria, with minimum inhibitory concentrations (MICs) of 4–8 µM. In contrast, BTT1 exhibited broad-spectrum activity, with MICs of 4–8 µM against both Gram-positive and Gram-negative strains. Additionally, BTT1 was potent against methicillin-resistant Staphylococcus aureus (MRSA) and colistin-resistant Enterobacterales. The antimicrobial potency of BTT1 persisted after 14 days of serial passage. Mechanistic studies revealed that interactions between lipopolysaccharide (LPS) and the peptides were critical to their membranolytic activity against the bacterial inner membrane. Aside from folding stability, we observed that a degree of conformational flexibility was required for disruptive membrane interactions. Statement of significance By examining the significance of the turn region of β-hairpin peptides, we present valuable knowledge to the design toolkit of novel antimicrobial peptides as alternative therapeutics to overcome antibiotic resistance. Our de novo designed synthetic peptides displayed selective activity against Gram-negative bacteria and potent activity against clinically relevant antibiotic-resistant strains (e.g. colistin-resistant Enterobacterales and methicillin-resistant Staphylococcus aureus). The bactericidal activity of our peptides was shown to be robust in the presence of proteolytic trypsin and saline, conditions that could suppress peptide activity. Our peptides were also determined to be non-cytotoxic against a human cell line.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality, nevertheless, this set still exhibits limitations. The incorporation of noncanonical amino acids into proteins can enlarge its functional scope. Although proofreading will counteract the charging of tRNAs with other amino acids than the canonical ones, the translation machinery may still accept noncanonical amino acids as surrogates and incorporate them at the canonically prescribed locations within the protein sequence. Here, we use a cell-free expression system to demonstrate the full replacement of l-lysine by l-hydroxylysine at all lysine sites of recombinantly produced GFP. In vivo, as a main component of collagen, post-translational l-hydroxylysine generation enables the formation of cross-links. Our work represents a first step towards in vitro production of (modified) collagens, more generally of proteins that can easily be crosslinked.

Arsenic is toxic for plants. Our previous results showed that the application of proline enhanced the sensitivity of tobacco BY-2 cells to arsenate. In order to clarify the enhancement mechanism, we investigated the effects of other amino acids on the arsenate-stressed BY-2 cells. Glutamate at up to 10 m m did not affect the cell growth in the absence or presence of arsenate. Arginine at up to 10 m m did not affect the growth in the absence of arsenate but arginine at 10 m m enhanced the inhibition of the cell growth by arsenate. Alanine at up to 10 m m did not affect the cell growth under nonstressed condition but alanine at 10 m m significantly improved the cell growth under arsenate stress. These results suggest that alanine mitigates arsenate stress in BY-2 cells and that arginine like proline enhances the sensitivity of BY-2 cells to arsenate.

A anemia falciforme é uma patologia caracterizada pela alteração do aminoácido ácido glutâmico pela valina derivada de uma substituição da timina pela adenina no sexto códon no DNA do cromossomo 11. Essa substituição ocorre na cadeia beta da molécula de hemoglobina, dando origem a uma molécula anômala nomeada de hemoglobina S (HbS). A HbS formada na ausência de oxigenação apresenta caráter insolúvel e, mediante a isso, ocorre a formação de fibras polimerizadas, resultando na produção das hemácias em foice. Este estudo objetiva a análise e descrição dos aspectos bioquímicos e hematológicos da anemia falciforme. Para tal, foi realizada uma revisão bibliográfica com busca por artigos científicos, livros e meio eletrônico, publicados entre o período de 2010 a 2020.Resultante desse processo, identificou-se a correspondência bioquímica e hematológicarelacionadas ao fluxo sanguíneo lentificado, oclusão vascular e o surgimento de lesões orgânicas que causam episódios inflamatórios, caracterizando as principais ocorrências da patologia.

Motivation The objective is to diagnose dynamics perturbations caused by amino-acid mutations as prerequisite to assess protein functional health or drug failure, simply using network models of protein X-ray structures. Results We find that the differences in the allocation of the atomic interactions of each amino acid to 1D, 2D, 3D, 4D structural levels between variants structurally robust, recover experimental dynamic perturbations. The allocation measure validated on two B-pentamers variants of AB5 toxins having 17 mutations, also distinguishes dynamic perturbations of pathogenic and non-pathogenic Transthyretin single-mutants. Finally, the main proteases of the coronaviruses SARS-CoV and SARS-CoV-2 exhibit changes in the allocation measure, raising the possibility of drug failure despite the main proteases structural similarity. Availability and implementation The Python code used for the production of the results is available at github.com/lorpac/protein_partitioning_atomic_contacts. The authors will run the analysis on any PDB structures of protein variants upon request. Supplementary information Supplementary data are available at Bioinformatics online.

Dopamine is a neurotransmitter hormone for pleasure and reward. It is synthesized only in the brain cells of human and animals and responsible for the regulation of behavior, mood, memory, cognitive, flexible movement, the body weight, and other important functions. The Dopamine Receptor D3 (DRD3) is the most important receptor for dopamine. In this investigation, expression of DRD3 gene was studied in rabbits fed on supplemented diet of dry and fresh faba bean (Vicia faba L. Sakha 3). DRD3 gene (≈ 1200 bp) in control and treated rabbits were PCR amplified, sequenced and aligned with reference gene (Acc. No XM_017346708.1). High genetic similarity values were detected among all sequences. DRD3 gene sequences of control, fresh and dry faba bean fed rabbits were deposited in the GenBank with accession numbers MZ714134, MZ714135 and MZ714136 respectively. Direct estimation of blood phenylalanine (Phe) amino acid indicated that feeding rabbits on dry faba bean reflected the highest level of Phe in the rabbit’s blood. Quantitative RT-qPCR analysis showed that DRD3 gene was over expressed after feeding rabbits on dry faba bean form compared with feeding on green form and control. Thus, diet rich with phenylalanine like Sakha3 (dry and fresh forms) enhance gene expression of DRD3 gene. However, diet doesn't affect the DRD3 gene sequence and structure. In a conclusion, our findings indicated a direct effect of faba bean supplemented diet on increasing DRD3 expression levels which improve the life quality for human.

Metalloproteases represent a class of extracellular proteases found in Vibrio anguillarum that can generate toxic and pathogenic effects in turbot (Scophthalmus maximus). The toxicological effect partly results from oxidative damage due to the production of excessive reactive oxygen species (ROS). Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) are major antioxidant enzymes induced by various oxidative stresses and can scavenge peroxides generated in cells. To evaluate the effects of metalloprotease-induced ROS on the antioxidation defense mechanism of S. maximus head kidney cells, the cDNA of CAT gene (designated as SmCAT) was cloned and characterized. SmCAT comprises a 1 584-bp coding sequence that encodes a protein containing 527 amino acids with a poly(A) tail. Bioinformatics analysis revealed an active site signature sequence, a heme-ligand signature sequence, and three catalytic amino acid residues. The deduced SmCAT amino acid sequence shares a sequence similarity of 66.1%–92.4% with those of other species. Phylogenetic analysis revealed that SmCAT is classified with CAT of other fishes. Quantitative real-time PCR analysis showed that SmCAT was extensively expressed in all tested tissues, especially in blood. The expression of SmCAT, SmMnSOD, and SmGPx were inhibited significantly in head kidney cells treated with metalloprotease from 12 to 24 h. In 6 to 24 h metalloprotease-treated groups compared to that of the untreated group, it was found that the production of ROS was markedly increased, and the mitochondrial membrane potential was decreased considerably. Hoechst 33342 staining revealed the presence of apoptotic bodies when the cells were incubated with 8.0 or 40.0 μg/mL metalloprotease for 12 and 24 h. Hence, the toxic effects of metalloprotease are associated with the down-regulation of antioxidant enzyme expression and increased ROS levels, which trigger the activation of apoptosis in the head kidney cells of turbot. Our findings provide a better understanding on the mechanism of metalloprotease-induced apoptosis in fish.

Little information is available regarding the region of Cry toxins involved in binding to their major receptors, the ATP-binding cassette (ABC) transporters. We analyzed which Cry1Aa amino acid residues contribute to binding to Bombyx mori ABC transporter C2 (BmABCC2). Several two oxidized double-cysteine substitution mutant toxins were made. In these, two amino acids at distant positions on toxin loop α8 and loop 2 or loop 2 and loop 3 were substituted with cysteine residues and crosslinked. These mutants exhibited a marked reduction in binding affinity to BmABCC2, suggesting that the binding site comprises complex cavities formed by loops α8, 2, and 3. Loop swapping between Cry1Aa and other BmABCC2-incompatible toxins indicated that loop 2 acts as a binding affinity-generating part of Cry1Aa toxin. Using single amino acid substitution mutants, the results of surface plasmon resonance (SPR) analysis and response assays with BmABCC2-expressing Sf9 cells indicated that Y366, R367, R368, and L447 in the Cry1Aa root and base region of loops 2 and 3 play important roles in binding. Furthermore, SPR analyses of these mutants suggested that a two-state binding model fits best the data obtained. Moreover, complex cavities and the above-mentioned amino acid residues contribute to the generation of multiple binding points and high-affinity binding. Finally, we found that the binding site of B. mori cadherin-like protein consists of complex cavities comprising loops 1, 2, and 3, partially overlapping that of BmABCC2, suggesting that the loop region of Cry1Aa toxin acts as a promiscuous binding site.

Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC50 values of 12.9 and 8.6 ng/mL, with a linear range of 6.9–24.0 and 4.7–16.0 ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC50 of 3.5 ng/mL for LAS and 4.1 ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2–92.7%(CV

Numerous deaths worldwide have been caused by the coronavirus pandemic and are currently progressing with successive mutations and a lack of appropriate and definitive treatment. One of the drug targets to control the replication of the virus and treat this disease is to block the ion channel of the virus. This will lead to its death by disturbing the internal balance of the virus. Natural compounds such as alkaloids are usually known as effective compounds due to their medicinal characteristics and easy access to their sources. To this end, more than 3,200 natural alkaloid structures interacted with pentameric ion channels. Alkaloid compounds established significant and stable interactions with the channel. More clearly and in more detail, six alkaloid compounds with the best pharmacokinetics and binding affinity of less than-10.52 kcal/mol were selected as hit and suitable compounds for virus control. The compound of psammaplysin U (NA-1) with a binding affinity of-13.52 kcal/mol and binding free energy of-82.21 kcal/mol established hydrogen interactions with the amino acid of Val 25 in the B chain of the ion channel, which placed the compound at the top of the selected compounds. The molecular dynamics simulation of the ligand-protein complex in the 100 ps trajectory showed that the principal interactions were hydrogen and halogen bonding with the amino acids of Val 25 and Thr 30 in the B chain and A chain, respectively, which could be a suitable inhibitor to combat the COVID-19. © 2021 by the authors.

Spike (S) protein has been recognized as a promising molecular target for diagnostic, vaccines and antiviral drugs development for COVID-19. In this study, we analyzed the most predominant mutations in the S protein of Brazilian isolates and predicted the effect of these amino acid alterations to protein conformation. A total of 25,924 sequences were obtained from GISAID for five regions of Brazilian territory (Midwest, North, Northeast, South, and Southeast), according to exclusion criteria. Most of the SARS-CoV-2 isolates belongs to the G clade and showed a large occurrence of D614G, N501Y and L18F substitutions. Prediction effects of these amino acid substitutions on the structure dynamics of the spike protein indicated a positive ΔΔG values and negative ΔΔSVib in most cases which is associated to structural stabilization and flexibility reduction of the S protein. Mutations E484K, N501Y and K417N belong to several SARS-CoV-2 variants of concern such as Alpha, Beta, Gamma and Delta, and showed high incidence among Brazilian isolates. These mutations have been described to increase RBD affinity to ACE-2 host and abolishment of RBD affinity to potent neutralizing ant-RBD. The increase in rates of infection and reinfection requires continuous genomic surveillance studies in order to characterize emerging mutations and monitor vaccine efficacy, and thus consideration structural data and dynamics in the observed phenotypes.Communicated by Ramaswamy H. Sarma.

Three-dimensional structures of SARS-CoV-2 and other coronaviral proteins archived in the Protein Data Bank were used to analyze viral proteome evolution during the first six months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48,000 viral proteome sequences showed how each one of the 29 viral study proteins have undergone amino acid changes. Structural models computed for every unique sequence variant revealed that most substitutions map to protein surfaces and boundary layers with a minority affecting hydrophobic cores. Conservative changes were observed more frequently in cores versus boundary layers/surfaces. Active sites and protein-protein interfaces showed modest numbers of substitutions. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for six drug discovery targets and four structural proteins comprising the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and functional interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

Enzymatic amino acid assays are important in physiological research and clinical diagnostics because abnormal amino acid concentrations in biofluids are associated with various diseases. L-histidine decarboxylase from Photobacterium phosphoreum (PpHDC) is a pyridoxal 5′-phosphate-dependent enzyme and a candidate for use in an L-histidine quantitation assay. Previous cysteine substitution experiments demonstrated that the PpHDC C57S mutant displayed improved long-term storage stability and thermostability when compared with those of the wild-type enzyme. In this study, combinational mutation experiments of single cysteine substitution mutants of PpHDC were performed, revealing that the PpHDC C57S/C101V/C282V mutant possessed the highest thermostability. The stabilizing mechanism of these mutations was elucidated by solving the structures of PpHDC C57S and C57S/C101V/C282V mutants by X-ray crystallography. In the crystal structures, two symmetry-related PpHDC molecules form a domain-swapped homodimer. The side chain of S57 is solvent exposed in the structure, indicating that the C57S mutation eliminates chemical oxidation or disulfide bond formation with a free thiol group, thereby providing greater stability. Residues 101 and 282 form hydrophobic interactions with neighboring hydrophobic residues. Mutations C101V and C282V enhanced thermostability of PpHDC by filling a cavity present in the hydrophobic core (C101V) and increasing hydrophobic interactions.

The Stability and electronic properties of bio-hybrid molecules are investigated in the framework of the first-principles density functional theory. The site-specific interactions between (ZnO)12 n...

We have been developing bacterial expression systems for human mucin-type O-glycosylation on therapeutic proteins, which is initiated by the addition of α-linked GalNAc to serine or threonine residues by enzymes in the GT-27 family of glycosyltransferases. Substrate preference across different isoforms of this enzyme is influenced by isoform-specific amino acid sequences at the site of glycosylation, which we have exploited to engineer production of Core 1 glycan structures in bacteria on human therapeutic proteins. Using RP-HPLC with a novel phenyl bonded phase to resolve intact protein glycoforms, the effect of sequon mutation on O-glycosylation initiation was examined through in vitro modification of the naturally O-glycosylated human interferon α-2b, and a sequon engineered human growth hormone. As part of the development of our glycan engineering in the bacterial expression system we are surveying various orthologues of critical enzymes to ensure complete glycosylation. Here we present an in vitro enzyme kinetic profile of three related GT-27 orthologues on natural and engineered sequons in recombinant human interferon α2b and human growth hormone where we show a significant change in kinetic properties with the amino acid changes. It was found that optimizing the protein substrate amino acid sequence using Isoform Specific O-Glycosylation Prediction (ISOGlyP, http://isoglyp.utep.edu/index.php) resulted in a measurable increase in kcat/KM, thus improving glycosylation efficiency. We showed that the Drosophila orthologue showed superior activity with our human growth hormone designed sequons compared with the human enzyme.

The endophytic fungus Phomopsis liquidambaris is characterized as a plant growth-promoting agent under salt stress, but its mechanism is unknown. Herein, 1-aminocyclopropane-1-carboxylate deaminase (ACCD) from the strain was confirmed that it had the ability of utilizing 1-aminocyclopropane-1-carboxylate as the sole nitrogen source. The full-length ACCD gene was 1152 bp, which encodes a mature protein of 384 amino acids with a molecular mass of 41.53 kDa. The ACCD activity was 3.9-fold in 3 mmol L-1 ACC by qRT-PCR under salt stress comparing with no salt tress. Ethylene production was increased to 34.55-70.60% and reduced the growth of rice by 23-69.73% under salt stress. Inoculation of P. liquidambaris increased root-shoot length, fresh and dry weight, and overall growth of stressed rice seedlings. ACC accumulation, ACC synthase and ACC oxidase activities increased in salt-treated rice seedlings, while they were significantly reduced when P. liquidambaris was inoculated into rice by qRT-PCR. It therefore can be concluded that P. liquidambaris can be used as a plant growth promoting fungus against salt stress and other biotic or abiotic stresses.

Small heat shock proteins (sHSPs) function as ATP-independent chaperones that preserve cellular proteostasis under stressful conditions. In this study, Cshsp19.0, which encodes a new small heat shock protein, was isolated and characterized from Chilo suppressalis (Walker) to better understand the contribution of sHSPs to insect development and stress tolerance. The full-length Cshsp19.0 cDNA was 697 bp and encoded a 19.0 kDa protein with an isoelectric point of 5.95. Phylogenetic analysis and amino acid alignments indicated that Cshsp19.0 is a member of the sHSP family. Cshsp19.0 was expressed at maximal levels in foreguts and showed the least amount of expression in fat bodies. Expression analysis in different developmental stages of C. suppressalis revealed that Cshsp19.0 was most highly expressed in 1st instar larvae. Furthermore, Cshsp19.0 was upregulated when insects were exposed to heat and cold stress for a 2-h period. There were significant differences in the male and female pupae in response to humidity; Cshsp19.0 expression increased in male pupae as RH increased, whereas the inverse pattern was observed in female pupae. Larvae exhibited a lower rate of survival when Cshsp19.0 was silenced by a nanomaterial-promoted RNAi method. The results confirm that Cshsp19.0 functions to increase environmental stress tolerance and regulates physiological activities in C. suppressalis.

Background N-acetyltransferase 13 (NAT13) is a probable catalytic component of the ARD1A-NARG1 complex possessing alpha (N-terminal) acetyltransferase activity. Results In this study, a full-length complementary DNA (cDNA) encoding Schistosoma japonicum NAT13 (SjNAT13) was isolated from schistosome cDNAs. The 621 bp open reading frame of SjNAT13 encodes a polypeptide of 206 amino acids. Real-time PCR analysis revealed SjNAT13 expression in all tested developmental stages. Transcript levels were highest in cercariae and 21-day-old worms, and higher in male adult worms than female adult worms. The rSjNAT13 protein induced high levels of anti-rSjNAT13 IgG antibodies. In two independent immunoprotection trials, rSjNAT13 induced 24.23% and 24.47% reductions in the numbers of eggs in liver. RNA interference (RNAi) results showed that small interfering RNA (siRNA) Sj-514 significantly reduced SjNAT13 transcript levels in worms and decreased egg production in vitro. Conclusions Thus, rSjNAT13 might play an important role in the development and reproduction of schistosomes.

The effects of trehalose (Tre), a non-reducing disaccharide, on metabolic changes, antioxidant status, and salt tolerance in Dunaliella bardawil cells were investigated. Algal suspensions containing 1, 2, and 3 M NaCl were treated with 5 mM Tre. While the content of pigments, reducing sugars, proteins, glycerol, and ascorbate pool accumulated with increasing salinity, the content of non-reducing sugars, starch, amino acids, proline, hydrogen peroxide, and lipid peroxidation level decreased significantly. Tre-treated cells showed a decrease in pigments content, reducing sugars, starch, proteins, amino acids, proline, glycerol, and the activity of non-specific peroxidase and polyphenol oxidase, but an increase in non-reducing sugars, oxidized ascorbate, and ascorbate peroxidase activity occurred unchanged in the ascorbate pool. However, the density and fresh weight of the cells remained statistically unchanged in all Tre-treated and untreated cultures. These results suggest that D. bardawil cells potentially tolerate different salt levels by accumulating metabolites, whereas Tre treatment changes carbon partitioning and significantly reduces beneficial metabolites without altering salt tolerance. Therefore, the regulation of carbon partitioning rather than the amount of assimilated carbon may play an important role in inducing salinity tolerance of D. bardawil. However, Tre is not able to enhance the salt tolerance of halotolerants and is even economically damaging due to the reduction of unique metabolites such as glycerol and β-carotene.

G-protein signaling and ubiquitin-dependent degradation are both involved in grain development in rice, but how these pathways are coordinated in regulating this process is unknown. Here, we show that Chang Li Geng 1 (CLG1), which encodes an E3 ligase, regulates grain size by targeting the Gγ protein GS3, a negative regulator of grain length, for degradation. Overexpression of CLG1 led to increased grain length, while overexpression of mutated CLG1 with changes in three conserved amino acids decreased grain length. We found that CLG1 physically interacts with and ubiquitinats GS3which is subsequently degraded through the endosome degradation pathway, leading to increased grain size. Furthermore, we identified a critical SNP in the exon 3 of CLG1 that is significantly associated with grain size variation in a core collection of cultivated rice. This SNP results in an amino acid substitution from Arg to Ser at position 163 of CLG1 that enhances the E3 ligase activity of CLG1 and thus increases rice grain size. Both the expression level of CLG1 and the SNP CLG1163S may be useful variations for manipulating grain size in rice.

Background The tick Dermacentor silvarum Olenev (Acari: Ixodidae) is a vital vector tick species mainly distributed in the north of China and overwinters in the unfed adult stage. The knowledge of the mechanism that underlies its molecular adaptation against cold is limited. In the present study, genes of hsp70 and hsp90 cDNA, named Dshsp70 and Dshsp90, and tubulin were cloned and characterized from D. silvarum, and their functions in cold stress were further evaluated. Methods The genome of the heat shock proteins and tubulin of D. silvarum were sequenced and analyzed using bioinformatics methods. Each group of 20 ticks were injected in triplicate with Dshsp90-, Dshsp70-, and tubulin-derived dsRNA, whereas the control group was injected with GFP dsRNA. Then, the total RNA was extracted and cDNA was synthesized and subjected to RT-qPCR. After the confirmation of knockdown, the ticks were incubated for 24 h and were exposed to − 20 °C lethal temperature (LT50), and then the mortality was calculated. Results Results indicated that Dshsp70 and Dshsp90 contained an open reading frame of 345 and 2190 nucleotides that encoded 114 and 729 amino acid residues, respectively. The transcript Dshsp70 showed 90% similarity with that identified from Dermacentor variabilis, whereas Dshsp90 showed 85% similarity with that identified from Ixodes scapularis. Multiple sequence alignment indicates that the deduced amino acid sequences of D. silvarum Hsp90, Hsp70, and tubulin show very high sequence identity to their corresponding sequences in other species. Hsp90 and Hsp70 display highly conserved and signature amino acid sequences with well-conserved MEEVD motif at the C-terminal in Hsp90 and a variable C-terminal region with a V/IEEVD-motif in Hsp70 that bind to numerous co-chaperones. RNA interference revealed that the mortality of D. silvarum was significantly increased after injection of dsRNA of Dshsp70 (P = 0.0298) and tubulin (P = 0.0448), whereas no significant increases were observed after the interference of Dshsp90 (P = 0.0709). Conclusions The above results suggested that Dshsp70 and tubulin play an essential role in the low-temperature adaptation of ticks. The results of this study can contribute to the understanding of the survival and acclimatization of overwintering ticks. Graphical abstract

Pyrrolysyl-tRNA synthetase (PylRS) is an enzyme of some methanogenic Archaea for the natural incorporation of pyrrolysine into proteins. The discovery of PylRS as a natural tool for genetic code expansion paved the way for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins, with versatile side chains useful in biotechnology. Almost 20 years after the discovery, we describe the journey which led to three distinct classes of PylRSs with unique ncAA recognitions.

Ribosomal protein S3 (rpS3), a member of 40S small ribosomal subunit, is a multifunctional protein with various extra-ribosomal functions including DNA repair endonuclease activity and is secreted from cancer cells. Therefore, antibodies with high specificity against rpS3 protein could be useful cancer biomarkers. In this study, polyclonal antibody (pAb) and monoclonal antibodies (mAbs) were raised against rpS3 protein and epitope mapping was performed for each antibody; the amino acid residues of rpS3 were scanned from amino acid 185 to 243 through peptide scanning to reveal the epitopes of each mAb. Results showed that pAb R2 has an epitope from amino acid 203 to 230, mAb M7 has an epitope from amino acid 213 to 221, and mAb M8 has an epitope from amino acid 197 to 219. Taken together, novel mAbs and pAb against rpS3 were raised and mapped against rpS3 with different specific epitopes.

Trypanosomiases and leishmaniases are neglected tropical diseases that have been spreading to previously non-affected areas in recent years. Identification of new chemotherapeutics is needed as there are no vaccines and the currently available treatment options are highly toxic and often ineffective. The causative agents for these diseases are the protozoan parasites of the Trypanosomatidae family, and they alternate between invertebrate and vertebrate hosts during their life cycles. Hence, these parasites must be able to adapt to different environments and compete with their hosts for several essential compounds, such as amino acids, vitamins, ions, carbohydrates, and lipids. Among these nutrients, lipids and fatty acids (FAs) are essential for parasite survival. Trypanosomatids require massive amounts of FAs, and they can either synthesize FAs de novo or scavenge them from the host. Moreover, FAs are the major energy source during specific life cycle stages of T. brucei, T. cruzi, and Leishmania. Therefore, considering the distinctive features of FAs metabolism in trypanosomatids, these pathways could be exploited for the development of novel antiparasitic drugs. In this review, we highlight specific aspects of lipid and FA metabolism in the protozoan parasites T. brucei, T. cruzi, and Leishmania spp., as well as the pathways that have been explored for the development of new chemotherapies.

Angiopoietin-like protein 8 (Angptl8) is a new constituent of angiopoietin-related protein family. In the present study, Landrace × Yorkshire pigs, C57BL/6 J mice, and Wistar rat were used as experimental animals. Using normal PCR, T-A cloning, RACE, and other experimental techniques, full length of porcine Angptl8 gene was cloned. Through real-time PCR, Angptl8 expression was detected in assorted tissues (kidney, heart, spleen, liver, lung, abdominal fat, and back longest muscle) of different animals. The present result showed that the full-length of porcine Angptl8 was 875 bp. Its ORF region contained 597 bp, from 21 bp to 618 bp, it had high conservation. The protein of Angptl8 had 198 amino acids. Its relative molecular mass was 21,988.3 Da. Its theoretical isoelectric point was 6.35. There was a highly hydrophobic region near the 10th amino acid site of the Angptl8 gene, while 177th and 187th were highly hydrophilic. Angptl8 protein may be not a transmembrane protein. The first to the 19th amino acids on the N-terminal were signal peptides, and the first 18th–19th amino acid sites may be the signal peptide shear site. In addition, Angptl8 in healthy pigs was mainly expressed in the liver. Its expression in heart and adipose tissue was minimum. Angptl8 of both mouse and rat was enriched in the fat and liver; mouse and rat minimum Angptl8 expression was in muscle and spleen, respectively. The present results suggest that there may exist species difference in tissue-specific expression of Angptl8.

Graphic abstract Papain-like protease (nsp-3; non-structural protein) of novel corona virus is an ideal target for developing drugs as it plays multiple important functions for viral growth and replication. For instance, role of nsp-3 has been recognized in cleavage of viral polyprotein; furthermore, in infected host it weakens the immune system via downregulating the production of type I interferon. This downregulation is promoted by removal of ubiquitin-like interferon-stimulated gene 15 protein (ISG15) from interferon-responsive factor 3 (IRF3) protein. Among known inhibitors of SARS-CoV-PLpro GRL0617 is by far the most effective inhibitor. As PLpro of SARS-CoV2 is having more than 80% similarity with SARS-CoV-PLpro, GRL0617 is reported to be effective even against SARS-CoV2. Owing to this similarity, certain key amino acids remain the same/conserved in both proteins. Among conserved amino acids Tyr268 for SARS-CoV2 and Tyr269 for SARS-CoV produce important hydrophobic interactions with aromatic rings of GRL0617. Here, in this study antibacterial compounds were collected from ZINC database, and they were filtered to select compounds that are having similar structural features as GRL0617. This filtered library of compound was then docked with SARS-CoV and CoV2-PLpro. Five hits were noted that were able to interact with Tyr268 (SARS-CoV2) and Tyr269 (SARS-CoV). Further, best hit 2-(2-((benzofuran-2-carboxamido)methyl)-5-methoxy-1H-indol-1-yl)acetic acid (ZINC44459905) was studied using molecular dynamic simulation where stability of protein–ligand complex as well as stability of produced interactions was noted.