Nucleotide Ratio Research Articles

Structure, Evolution, and Mitochondrial Genome Analysis of Mussel Species (Bivalvia, Mytilidae).

Based on the nucleotide sequences of the mitochondrial genome (mitogenome) of specimens taken from two mussel species (Arcuatula senhousia and Mytilus coruscus), an investigation was performed by means of the complex approaches of the genomics, molecular phylogenetics, and evolutionary genetics. The mitogenome structure of studied mussels, like in many other invertebrates, appears to be much more variable than in vertebrates and includes changing gene order, duplications, and deletions, which were most frequent for tRNA genes; the mussel species' mitogenomes also have variable sizes. The results demonstrate some of the very important properties of protein polypeptides, such as hydrophobicity and its determination by the purine and pyrimidine nucleotide ratio. This fact might indirectly indicate the necessity of purifying natural selection for the support of polypeptide functionality. However, in accordance with the widely accepted and logical concept of natural cutoff selection for organisms living in nature, which explains its action against deleterious nucleotide substitutions in the nonsynonymous codons (mutations) and its holding of the active (effective) macromolecules of the polypeptides in a population, we were unable to get unambiguous evidence in favor of this concept in the current paper. Here, the phylogeny and systematics of mussel species from one of the largest taxons of bivalve mollusks are studied, the family known as Mytilidae. The phylogeny for Mytilidae (order Mytilida), which currently has no consensus in terms of systematics, is reconstructed using a data matrix of 26-27 mitogenomes. Initially, a set of 100 sequences from GenBank were downloaded and checked for their gender: whether they were female (F) or male (M) in origin. Our analysis of the new data confirms the known drastic differences between the F/M mitogenome lines in mussels. Phylogenetic reconstructions of the F-lines were performed using the combined set of genetic markers, reconstructing only protein-coding genes (PCGs), only rRNA + tRNA genes, and all genes. Additionally, the analysis includes the usage of nucleotide sequences composed of other data matrices, such as 20-68 mitogenome sequences. The time of divergence from MRCA, estimated via BEAST2, for Mytilidae is close to 293 Mya, suggesting that they originate in the Silurian Period. From all these data, a consensus for the phylogeny of the subfamily of Mytilinae and its systematics is suggested. In particular, the long-debated argument on mussel systematics was resolved as to whether Mytilidae, and the subfamily of Mytilinae, are monophyletic. The topology signal, which was strongly resolved in this paper and in the literature, has refuted the theory regarding the monophyly of Mytilinae.

Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR

Background The COVID-19 pandemic, caused by the SARS-CoV-2, can be effectively managed with diagnostic tools such as RT-qPCR. However, it can produce false-negative results due to viral mutations and RNA secondary structures from the target gene sequence. Methods With High Performance Computing, the complete SARS-CoV-2 genome was obtained from the GenBank/GISAID to generate consensus sequences to design primers/probes for RT-qPCR. ORF8 gene was selected, meanwhile, E and N and RNAse P were according to CDC protocol. Nasopharyngeal swab samples were collected from patients diagnosed with SARS-CoV-2. Total RNA was purified according MagMAX kit, it was used in single, and multiplex RT-qPCR. To avoid templated secondary structures, compensate nucleotide proportions and improve Ct values, a solution composed of tetraethylammonium chloride and dimethyl sulfoxide and other with corresponding to dNTPs proportions in accordance SARS-CoV-2 genome were included. Sensitivity and specificity according to Ct values were determined with the Caret package in R software. Results 126,576 SARS-CoV-2 genomes from January to December 2020 comprised a database. From this, a region near of 5′ ORF8 gene showed three stem-loops was used for primers/FAM-probe. 49 samples were obtained, from them, 22 were positive to gene selected regions. Interestingly, samples from October to November 2020 were positive for all regions, however, in January 2021 different results were observed in ORF8. An improvement in Ct with the adjuvant solutions was determined in all samples with others SARS-CoV-2 primers/probes, for both single and multiplex RT-qPCR. The inclusion of the denaturing solution in single RT-qPCR increased its sensitivity with respect to the commercial method, while in multiplex the opposite was generated. Conclusions Including adjuvant solutions to prevent the formation of RNA secondary structures and the adjustment of the nucleotide ratios of SARS-CoV-2 improved single and multiplex RT-qPCR for viral identification, demonstrating its potential application in health public.

Brassinolide promotes interaction between chloroplasts and mitochondria during the optimization of photosynthesis by the mitochondrial electron transport chain in mesophyll cell protoplasts of Arabidopsis thaliana.

The current experimental data unveils the role of brassinolide (BL), a phytohormone of class brassinosteroids (BRs), in augmenting the cross-talk between the mitochondrial electron transport chain (mETC) and chloroplasts to strengthen the efficiency of the Calvin-Benson cycle (CBC) for higher assimilation of carbon dioxide in the mesophyll cell protoplasts (MCP) of Arabidopsis thaliana. The outcome of total respiration (TR) and photosynthetic carbon assimilation (PCA) was monitored as O2 uptake under dark and NaHCO3-dependent O2 evolution under light, respectively, after pre-incubation of MCP at a broad spectrum of BL concentration from 0.05 pM to 5 pM at 25 °C and optimum light intensity of 1000 μmol m-2 s-1. The addition of optimal concentration (0.5 pM) of BL to MCP stimulated the (i) TR, (ii) PCA, and (iii) para-benzoquinone-dependent O2 evolution (PSII activity). Further, in response to BL, the enzyme activity or transcript levels of redox-regulated CBC enzymes and glucose-6-phosphate raised considerably. Also, the addition of BL to MCP remarkably accelerated the capacity of the cytochrome oxidase (COX) and alternative oxidase (AOX) pathways concurrently with an increase in total cellular pyruvate and reactive oxygen species (ROS) levels. Besides, malate valve components (Malate, Chl-MDH, M-MDH) increased in response to BL. At the same time, the cellular redox ratios of pyridine nucleotides (NADPH and NADH) were kept low in the presence of BL. However, BL could not keep up the CBC activity of photosynthesis along with its associated light-activated enzymes/transcripts when mETC through COX or AOX pathway is restricted by antimycin A (AA) or salicylhydroxamic acid (SHAM), respectively. In contrast, adding BL to MCP under restricted mETC showed aggravation in total cellular ROS, pyruvate, malate, and redox ratio of pyridine nucleotides with a concomitant increase in transcripts associated with malate valve and antioxidant systems. These results suggest that BL enhances the PCA by coordinating in cross-talk of chloroplasts and mitochondria to regulate the cellular redox ratio or ROS through the involvement of COX and AOX pathways along with the malate valve and antioxidant systems.

Regulation of Adenine Nucleotide Metabolism by Adenylate Kinase Isozymes: Physiological Roles and Diseases.

Adenylate kinase (AK) regulates adenine nucleotide metabolism and catalyzes the ATP + AMP ⇌ 2ADP reaction in a wide range of organisms and bacteria. AKs regulate adenine nucleotide ratios in different intracellular compartments and maintain the homeostasis of the intracellular nucleotide metabolism necessary for growth, differentiation, and motility. To date, nine isozymes have been identified and their functions have been analyzed. Moreover, the dynamics of the intracellular energy metabolism, diseases caused by AK mutations, the relationship with carcinogenesis, and circadian rhythms have recently been reported. This article summarizes the current knowledge regarding the physiological roles of AK isozymes in different diseases. In particular, this review focused on the symptoms caused by mutated AK isozymes in humans and phenotypic changes arising from altered gene expression in animal models. The future analysis of intracellular, extracellular, and intercellular energy metabolism with a focus on AK will aid in a wide range of new therapeutic approaches for various diseases, including cancer, lifestyle-related diseases, and aging.

Brome mosaic virus detected in Kansas wheat co-infected with other common wheat viruses.

Wheat breeders are developing new virus-resistant varieties; however, it is assumed that only a few viruses or well-known viruses are present in the field. New sequencing technology is allowing for better determination of natural field virus populations. For three years, 2019-2021, Kansas wheat field surveys were conducted to determine the constituents of natural field virus populations using nanopore sequencing. During analysis, brome mosaic virus (BMV) was identified for the first time in Kansas but was in association with other wheat viruses. Brome mosaic virus was identified from 29 out of 47 different Kansas counties sampled and 44% of the total samples. BMV was found co-infected with wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) in 27.8% of the samples, with WSMV only (13.9%) and co-infected with WSMV + TriMV + High Plains wheat mosaic emaravirus (HPWMoV) (13.9%). RNA genomes of Kansas BMV isolates had 99.4 to 100% nucleotide and amino acid sequence identity, respectively, to each other. RNA2a possessed relatively high divergence (π = 0.01) compared to RNA1a and RNA3a (π = 0.004). Coding regions of all BMV RNAs were considered negative for purifying selection pressure as nonsynonymous and synonymous nucleotide ratio was less than one (dNs/dS >1). The identification of BMV in Kansas virus populations adds another layer of complexity to plant breeding. This work provides information to improve tools to aid in monitoring, detecting, and determining the variation within BMV.

The Mitogenome Structure of Righteye Flounders (Pleuronectidae): Molecular Phylogeny and Systematics of the Family in East Asia

This paper reports the first complete sequence of the mitochondrial genome (mitogenome) of the yellow-striped flounder Pseudopleuronectes herzensteini (Pleuronectoidei: Pleuronectidae). Mitogenome evolution, and molecular phylogenetic reconstruction based on four to six techniques, including coalescent analysis, were performed for flatfish. The genome size of the specimen sampled was 16,845 bp, including 13 protein-coding genes, 22 tRNA genes, 12S, and 16S rRNA genes, and the control region, CR. The composition and arrangement of the genes are similar to those in other teleost fish, including the second mitogenome reported in this paper. The frequency of A, C, G, and T nucleotides in the P. herzensteini mitogenome is 27%, 29.2%, 17.6%, and 26.2%, respectively. The ratio of complementary nucleotides in the mitogenome of this and other species of the family was A+T:G+C (53.2: 46.8%) and do not deviate significantly from the expected equilibrium proportion. The submission to the global database (GenBank) of two new mitogenomes along with 106 analyzed GenBank sequences will contribute to phylogenetic studies of flounders at the family and suborder levels. Based on 26 and 108 nucleotide sequences of protein-coding genes (PCGs), we investigated the molecular phylogeny of flounders and performed analysis for two sets of sequences, including those of members of the family Pleuronectidae and the suborder Pleuronectoidei and estimated their importance in establishing the taxonomy at these two levels. Data obtained by up to six techniques of multigene phylogenetic reconstructions support monophyly within the family Pleuronectidae with high statistical confidence; however, conclusions regarding the phylogenetics at the suborder level require further investigation. Our results also revealed paraphyletic and weakly supported branches that are especially numerous at the suborder level; thus, there is a clear need for taxonomic revisions at the suborder, and possibly family levels. Genetic distance analysis reveals the suitability for DNA barcoding of species specimens at single genes as well as at whole mitogenome data.

GetSequenceInfo: a suite of tools allowing to get genome sequence information from public repositories

BackgroundBiological sequences are increasing rapidly and exponentially worldwide. Nucleotide sequence databases play an important role in providing meaningful genomic information on a variety of biological organisms.ResultsThe getSequenceInfo software tool allows to access sequence information from various public repositories (GenBank, RefSeq, and the European Nucleotide Archive), and is compatible with different operating systems (Linux, MacOS, and Microsoft Windows) in a programmatic way (command line) or as a graphical user interface. getSequenceInfo or gSeqI v1.0 should help users to get some information on queried sequences that could be useful for specific studies (e.g. the country of origin/isolation or the release date of queried sequences). Queries can be made to retrieve sequence data based on a given kingdom and species, or from a given date. This program allows the separation between chromosomes and plasmids (or other genetic elements/components) by arranging each component in a given folder. Some basic statistics are also performed by the program (such as the calculation of GC content for queried assemblies). An empirically designed nucleotide ratio is calculated using nucleotide information in order to tentatively provide a “NucleScore” for studied genome assemblies. Besides the main gSeqI tool, other additional tools have been developed to perform various tasks related to sequence analysis.ConclusionThe aim of this study is to democratize the use of public repositories in programmatic ways, and to facilitate sequence data analysis in a pedagogical perspective. Output results are available in FASTA, FASTQ, Excel/TSV or HTML formats. The program is freely available at: https://github.com/karubiotools/getSequenceInfo. getSequenceInfo and supplementary tools are partly available through the recently released Galaxy KaruBioNet platform (http://calamar.univ-ag.fr/c3i/galaxy_karubionet.html).

Inhibition of ATR Reverses a Mitochondrial Respiratory Insufficiency.

Diseases that affect the mitochondrial electron transport chain (ETC) often manifest as threshold effect disorders, meaning patients only become symptomatic once a certain level of ETC dysfunction is reached. Cells can invoke mechanisms to circumvent reaching their critical ETC threshold, but it is an ongoing challenge to identify such processes. In the nematode Caenorhabditis elegans, severe reduction of mitochondrial ETC activity shortens life, but mild reduction actually extends it, providing an opportunity to identify threshold circumvention mechanisms. Here, we show that removal of ATL-1, but not ATM-1, worm orthologs of ATR and ATM, respectively, key nuclear DNA damage checkpoint proteins in human cells, unexpectedly lessens the severity of ETC dysfunction. Multiple genetic and biochemical tests show no evidence for increased mutation or DNA breakage in animals exposed to ETC disruption. Reduced ETC function instead alters nucleotide ratios within both the ribo- and deoxyribo-nucleotide pools, and causes stalling of RNA polymerase, which is also known to activate ATR. Unexpectedly, atl-1 mutants confronted with mitochondrial ETC disruption maintain normal levels of oxygen consumption, and have an increased abundance of translating ribosomes. This suggests checkpoint signaling by ATL-1 normally dampens cytoplasmic translation. Taken together, our data suggest a model whereby ETC insufficiency in C. elegans results in nucleotide imbalances leading to the stalling of RNA polymerase, activation of ATL-1, dampening of global translation, and magnification of ETC dysfunction. The loss of ATL-1 effectively reverses the severity of ETC disruption so that animals become phenotypically closer to wild type.

Genomic signatures of protease and reverse transcriptase genes from HIV-1 subtype C isolated from first-line ART patients in India.

Genomic signatures of the protease and reverse transcriptase gene of HIV-1 from HIV infected North Indian patients who were under ART from 1 to ≤ 7 years were analyzed. The DNA from plasma samples of 9 patients and RNA from 57 patients were isolated and subjected to amplification for the protease and reverse transcriptase gene of HIV-1 subtype C. Then sequencing was carried out following the WHO dried blood spot protocol. The drug resistance mutation patterns were analyzed using the HIV Drug Resistance Database, Stanford University, USA. Lamivudine-associated drug-resistance mutations such as M184V/M184I, nevirapine-associated drug resistance mutations Y181C and H221Y, and efavirenz-associated drug resistance mutations M230I were observed in reverse transcriptase gene of archived DNA of two HIV-1 infected patients. No mutation was observed in the remaining 7 patients. Various computational tools and websites like viral epidemiological signature pattern analysis (VESPA), hyper mutation, SNAP version 2.1.1, and entropy were utilized for the analysis of the signature pattern of amino acids, hyper mutation, selection pressure, and Shannon entropy in the protease and reverse transcriptase gene sequences of the 9 archived DNA, 56 protease gene and 51 reverse transcriptase gene from the HIV-1 DNA amplified sequences of RNA. The HIV-1 Subtype-C (Gene bank accession number: AB023804) and first isolate HXB2 (Gene bank accession number: K03455.1) was taken as reference sequence. The signature amino acid sequences were identified in the protease and reverse transcriptase gene, no hyper mutation, highest entropy was marked in the amino acid positions and synonymous to non-synonymous nucleotide ratio was calculated in the protease and reverse transcriptase gene of 9 archived DNA sequences, 56 protease and 51 reverse transcriptase gene sequences of HIV-1 Subtype C isolates.

Population Genetic Analysis of Euaresta bullans (Wiedemann, 1830) (Diptera: Tephritidae) on Xanthium spinosum L.

This study was based on Euaresta bullans (Wiedemann, 1830) (Diptera: Tephritidae) specimens collected from seven different provinces of Turkey (Amasya, Çorum, Kahramanmaraş, Kayseri, Samsun, Sinop and Yozgat). Euaresta bullans is monophage on Xanthium spinosum L. and were swept from over the host, which is a highly invasive plant in agricultural areas. Xanthium spinosum originated from South America and has spread throughout the world as well as in Turkey. In this study, we investigated population structure of E. bullans based on mitochondrial cytochrome oxidase I (COI) gene data. We evaluated 105 adult specimens, 79 successful samples from sequence products were analysed, and 17 haplotypes were identified. Calculated nucleotide ratios of E. bullans populations (C: 16.7%; T: 38.93%; A: 29.05% and G: 15.8%), revealed that the base compounds were directed towards Adenine and Thymine (67, 98%). As a result of the study, the genetic distance between populations was found to be 0.09% - 0.046%. This showed that genetic variation among individuals of the E. bullans specimens is very low. According to the neutrality test (Tajima's D and Fu's Fs), result was p<0.05, it was determined that the populations showed a strong spread. Although E. bullans specimens have spread in different habitat characteristics and different geographic regions, they show a similar rate between populations. In particular, the high dependence of the host and the presence of the host in different habitats provide wide tolerance for this species in terms of environmental differences. Finally, it was found that gene flow continued between E. bullans populations and was not affected by any geographic and ecological barrier. Euaresta bullans have high populations on the host plant and results of this study support that it can be an effective biological control agent on X. spinosum in different regions.

The Protein‐Templated Synthesis of Enzyme‐Generated Aptamers

AbstractInspired by the chemical synthesis of molecularly imprinted polymers, we demonstrated for the first time, the protein‐target mediated synthesis of enzyme‐generated aptamers (EGAs). We prepared pre‐polymerisation mixtures containing different ratios of nucleotides, an initiator sequence and protein template and incubated each mixture with terminal deoxynucleotidyl transferase (TdT). Upon purification and rebinding of the EGAs against the target, we observed an enhancement in binding of templated‐EGAs towards the target compared to a non‐templated control. These results demonstrate the presence of two primary mechanisms for the formation of EGAs, namely, the binding of random sequences to the target as observed in systematic evolution of ligands by exponential enrichment (SELEX) and the dynamic competition between TdT enzyme and the target protein for binding of EGAs during synthesis. The latter mechanism serves to increase the stringency of EGA‐based screening and represents a new way to develop aptamers that relies on rational design.

The Protein-Templated Synthesis of Enzyme-Generated Aptamers.

Inspired by the chemical synthesis of molecularly imprinted polymers, we demonstrated for the first time, the protein‐target mediated synthesis of enzyme‐generated aptamers (EGAs). We prepared pre‐polymerisation mixtures containing different ratios of nucleotides, an initiator sequence and protein template and incubated each mixture with terminal deoxynucleotidyl transferase (TdT). Upon purification and rebinding of the EGAs against the target, we observed an enhancement in binding of templated‐EGAs towards the target compared to a non‐templated control. These results demonstrate the presence of two primary mechanisms for the formation of EGAs, namely, the binding of random sequences to the target as observed in systematic evolution of ligands by exponential enrichment (SELEX) and the dynamic competition between TdT enzyme and the target protein for binding of EGAs during synthesis. The latter mechanism serves to increase the stringency of EGA‐based screening and represents a new way to develop aptamers that relies on rational design.

Saliva Metabolomics in Dry Mouth Patients with Head and Neck Cancer or Sjögren's Syndrome.

The etiology of dry mouth conditions is multi-faceted. Patients radiated after head and neck cancer (HNC) and those with primary Sjögren’s syndrome (pSS) share many of the same symptoms despite different causes. With the aim of better understanding the pathophysiology and biochemical processes behind dry mouth with different etiologies, we investigated the metabolic profile of 10 HNC patients, 9 pSS patients and 10 healthy controls using high-performance liquid chromatography-high resolution mass spectrometry (HPLC-MS) metabolomics. Principal component analysis (PCA) revealed different metabolic profiles when comparing all subjects included in the study. Both patient groups showed higher ratios of several pyrimidine nucleotides and nucleosides when compared to controls. This finding may indicate that purinergic signaling plays a role in dry mouth conditions. Moreover, significantly increased levels of DL-3-aminoisobutyric acid were found in HNC patients when compared to controls, and a similar tendency was observed in the pSS patients. Furthermore, a dysregulation in amino acid metabolism was observed in both patient groups. In conclusion, metabolomics analysis showed separate metabolic profiles for HNC and pSS patients as compared to controls that could be useful in diagnostics and for elucidating the different pathophysiologies. The demonstrated dysregulation of pyrimidine nucleotides and levels of metabolites derived from amino acids in the patient groups should be studied further.

Contributors to Cancer Susceptibility, Development and Treatment: Cyclic Nucleotides, Steroids and Autophagy Modulators

Cell autophagy and apoptosis processes are of interest in drug development and contribute to the chemotherapy outcomes of patients receiving cancer treatment. The functional roles of cyclic nucleotides in cells include maintenance of metabolic homeostasis. cGMP and steroid compounds participate in apoptotic and autophagic events, and modulate the function of multi-drug resistance proteins. Endogenous steroid and cyclic nucleotide ratios change with ageing and this may initiate detrimental changes in cell function. This study uses a computational chemistry approach to investigate molecular similarity within chemotherapeutic and steroid compound structures. Modulators of autophagy/apoptosis and endogenous steroid structures all demonstrate molecular similarity to the structure of cGMP. Relative molecular similarity within these structures facilitates additive and synergistic treatment effects. Endogenous steroids are natural modulators of autophagy and apoptosis; concentration changes consequently have the potential to impact cancer risks.

Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides.

Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.

Nucleotide degradation, biogenic amine level and microbial contamination as quality indicators of cold-stored rainbow trout (Oncorhynchus mykiss) gravad

The extent to which the transformation of nucleotides, biogenic amines, and microbiological changes affect the quality and shelf life of vacuum packaged low processed rainbow trout (Oncorhynchus mykiss) gravad during storage at 7 ± 1 °C for 42 days was investigated. Although total viable counts increased slowly up to 6 log CFU g−1 at the end of storage, coliform bacteria disappeared. The histamine concentration and the biogenic amine index increased up to 45.2 ± 1.62 mg kg−1 and 100 mg kg−1 respectively. The highest concentration of inosine monophosphate was achieved in freshly prepared gravad, whereas the hypoxanthine level increased with storage time up to 28 days. Among nucleotide ratios, the G value is more suitable for the determination of gravad quality than K, Ki and H values. Once the gravad obtained the limit of acceptability by the panelists (35 days) the G value rose to 470%.

Genetic data of the ITS1-5.8S rDNA sequences of small liver flukes (Opisthorchiidae) from cats in Northern Vietnam

Domestic cats and humans are favorite final hosts for some hepatic trematodes of Opisthorchiidae. The present study examined the sequence variation in the nuclear ribosomal DNA (rDNA) region, including the internal transcribed spacer (ITS1) and the 5.8S gene, of two human opisthorchiid species Clonorchis sinensis (Cobbold, 1875) Looss, 1907 and Opisthorchis viverrini (Poirier, 1886) Stiles &amp; Hassal, 1896, in cats sampled in Northern Vietnam. The length of 30 partial ITS1‒5.8S rDNA sequences of C. sinensis from different hosts and localities varied from 761 to 766 bp. The results showed completely identity for all investigated 5.8S rDNA sequences of C. sinensis. In contrast, the variations were revealed in the complete ITS1 sequences of C. sinensis. Three polymorphic sites, indel and new sites with intragenomic polymorphism were detected. The length of the complete ITS1 rDNA region ranged from 656 to 661 bp. There are no substitutions downstream of the 5 bp insertion, and the different ratio of nucleotide in polymorphic sites with presence/absence of insertion leads to the existence of 6 genotypes in the investigated samples. The major genotype was obtained in 70% of sequences, other genotypes were presented by 1, 2 or 3 sequences. Nucleotide diversity in whole sample of C. sinensis was 0.00122 ± 0.00073. Partial sequences of ITS1‒5.8S rDNA of O. viverrini were 745 bp in length, and no difference was observed. Phylogenetic analyses by neighbor joining phylogram inferred from ITS1‒5.8S rDNA of 3 sequences of O. viverrini and 30 sequences of C. sinensis were formed two separately monophyletic clades.

Molecular docking investigation of the amantadine binding to the enzymes upregulated or downregulated in Parkinson's disease.

Parkinson’s disease (PD) is a progressive neurodegenerative disease. Levodopa in combination with amantadine has a demonstrated efficacy in motility impairment. An extensive investigation of some enzymes described to be upregulated or downregulated in PD was made – adenylate kinase (AK), adenine phosphoribosyltransferase (APRT), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), nucleoside-diphosphate kinase 3 (NDK3), purine nucleoside phosphorylase 1 (PNP1), and ecto-5’-nucleotidase (NT5E). Also, creatine kinase (CK) was included in the study because it is one of the main enzymes involved in the regulation of the nucleotide ratio in energy metabolism. To date, there is no proven link between amantadine treatment of PD and these enzymes. Because there are many AKs isoforms modified in PD, the AK was the first investigated. The molecular docking experiments allow the analysis of the selective binding of amantadine – unionized (with –NH2 group) and ionized form (with –NH3+ group) – to the AKs’ isoforms implicated in PD. Using available X-ray 3D structures of human AKs in closed-conformation, it was demonstrated that there are notable differences between the interactions of the two forms of amantadine for the zebrafish AK1 (5XZ2), human AK2 (2C9Y), human AK5 (2BWJ), and AK from B.stearothermophilus. The cytosolic human AK1 and human AK2 mostly interact with ionized amantadine by AMP binding residues. The human AK5 interaction with ionized amantadine does not involve the residues from the catalytic site. Among other enzymes tested in the present study, APRT revealed the best results in respect of binding amantadine ionized form. The results offer a new perspective for further investigation of the connections between amantadine treatment of PD and some enzymes involved in purine metabolism.

Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells.

Metabolons, multiprotein complexes consisting of sequential enzymes of a metabolic pathway, are proposed to be biosynthetic "hotspots" within the cell. However, experimental demonstration of their presence and functions has remained challenging. We used metabolomics and in situ three-dimensional submicrometer chemical imaging of single cells by gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) to directly visualize de novo purine biosynthesis by a multienzyme complex, the purinosome. We found that purinosomes comprise nine enzymes that act synergistically, channeling the pathway intermediates to synthesize purine nucleotides, increasing the pathway flux, and influencing the adenosine monophosphate/guanosine monophosphate ratio. Our work also highlights the application of high-resolution GCIB-SIMS for multiplexed biomolecular analysis at the level of single cells.

Signs of a metabolon in action

Biosynthetic Enzymes Eukaryotic cells have a heterogeneous cytoplasm, with compartments large and small, membrane bound or not. Enzymes that catalyze the de novo synthesis of purine nucleotides, which are needed in rapidly dividing cells, are known to assemble into loosely associated, multienzyme structures called purinosomes, but the extent to which these structures are metabolically active has been less certain. Pareek et al. performed metabolomics to trace how purines are synthesized within purinosomes and used sophisticated mass spectrometry imaging to directly observe hotspots of metabolic activity within frozen HeLa cells (see the Perspective by Alexandrov). They found evidence for metabolic channeling between enzymes, which limits equilibration of intermediates formed in purinosomes with the bulk cellular metabolite pool. This process occurs specifically within purinosomes associated with mitochondria, because the input metabolites, glycine, aspartate, and formate, come from mitochondrial metabolism. Such channeling may help cells control the ratio and abundance of purine nucleotides. Science , this issue p. [283][1]; see also p. [241][2] [1]: /lookup/doi/10.1126/science.aaz6465 [2]: /lookup/doi/10.1126/science.abb3094