Reproducibility and Reliability Assays of the Gene

Background Reliability and reproducibility are key metrics for gene expression assays. This report assesses the utility of the correlation coefficient in the analysis of reproducibility and reliability of gene expression data. Results The correlation coefficient alone is not sufficient to assess equality among sample replicates but when coupled with slope and scatter plots expression data equality can be better assessed. Narrow-intervals of scatter plots should be shown as a tool to inspect the actual level of noise within the data. Here we propose a method to examine expression data reproducibility, which is based on the ratios of both the means and the standard deviations for the inter-treatment expression ratios of genes. In addition, we introduce a fold-change threshold with an inter-replicate occurrence likelihood lower than 5% to perform analysis even when reproducibility is not acceptable. There is no possibility to find a perfect correlation between transcript and protein levels even when there is not any post-transcriptional regulatory mechanism. We therefore propose an adjustment for protein abundance with that of transcript abundance based on open reading frame length. Conclusions Here, we introduce a very efficient reproducibility approach. Our method detects very small changes in large datasets which was not possible through regular correlation analysis. We also introduce a correction on protein quantities which allows us to examine the post-transcriptional regulatory effects with a higher accuracy

Identification of circular RNAs from the parental genes involved in multiple aspects of cellular metabolism in barley

RNA circularization made by head-to-tail back-splicing events is involved in the regulation of gene expression from transcriptional to post-translational levels. By exploiting RNA-Seq data and down-stream analysis, we shed light on the importance of circular RNAs in plants. The results introduce circular RNAs as novel interactors in the regulation of gene expression in plants and imply the comprehensiveness of this regulatory pathway by identifying circular RNAs for a diverse set of genes. These genes are involved in several aspects of cellular metabolism as hormonal signaling, intracellular protein sorting, carbohydrate metabolism and cell-wall biogenesis, respiration, amino acid biosynthesis, transcription and translation, and protein ubiquitination. Additionally, these parental loci of circular RNAs, from both nuclear and mitochondrial genomes, encode for different transcript classes including protein coding transcripts, microRNA, rRNA, and long non-coding/microprotein coding RNAs. The results shed light on the mitochondrial exonic circular RNAs and imply the importance of circular RNAs for regulation of mitochondrial genes. Importantly, we introduce circular RNAs in barley and elucidate their cellular-level alterations across tissues and in response to micronutrients iron and zinc. In further support of circular RNAs' functional roles in plants, we report several cases where fluctuations of circRNAs do not correlate with the levels of their parental-loci encoded linear transcripts.Keywords: circular RNAs, coding and non-coding transcripts, leaves, seeds, transfer cells, micronutrients, mitochondri

The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in <i>Sorghum bicolor</i> contains its co-expressed vacuolar MATE transporter

Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters

Coupling engineering of Saccharomyces cerevisiae with medium optimization for the production of ergothioneine

Ergothioneine (ERG) is a naturally occurring, exogenous antioxidant that is nonetheless abundant in the human body. It has been shown both to reduce oxidative damage and to be involved in several diseases in vivo1,2. Therefore, ergothioneine is poised to take a place in the dietary supplement industry. Here we describe the engineering of the yeast Saccharomyces cerevisiae and subsequent medium optimization to produce ergothioneine by fermentation. After integrating combinations of biosynthetic pathways from different organisms, we screened yeast strains for their production of ERG. Next, the highest producing strain was engineered with ergothioneine transporters, and its amino acid metabolism was altered by knock-out of Tor1 or Yih1. The bottleneck for ergothioneine production was determined by integration of a second copy of the pathway enzymes. We also optimized the media composition for production of ergothioneine using yeast S. cerevisiae. Following these manipulations, we obtained a titer of 630 mg/l in fed-batch cultivation in bioreactors. This work shows that with further engineering of the strain, current chemical synthesis of ergothioneine could be replaced with a sustainable alternative. 1. Cheah, I. K. & Halliwell, B. Ergothioneine; antioxidant potential, physiological function and role in disease. Biochim. Biophys. Acta - Mol. Basis Dis. 1822, 784–793 (2012). 2. Halliwell, B., Cheah, I. K. & Tang, R. M. Y. Ergothioneine - a diet-derived antioxidant with therapeutic potential. FEBS Lett. (2018). doi:10.1002/1873-3468.1312

Identification and characterisation of two high-affinity glucose transporters from the spoilage yeast Brettanomyces bruxellensis

The yeast Brettanomyces bruxellensis (syn. Dekkera bruxellensis) is an emerging and undesirable contaminant in industrial low-sugar ethanol fermentations that employ the yeast Saccharomyces cerevisiae. High-affinity glucose import in B. bruxellensis has been proposed to be the mechanism by which this yeast can outcompete S. cerevisiae. The present study describes the characterization of two B. bruxellensis genes (BHT1 and BHT3) believed to encode putative high-affinity glucose transporters. In vitro-generated transcripts of both genes as well as the S. cerevisiae HXT7 high-affinity glucose transporter were injected into Xenopus laevis oocytes and subsequent glucose uptake rates were assayed using 14C-labelled glucose. At 0.1 mM glucose, Bht1p was shown to transport glucose five times faster than Hxt7p. pH affected the rate of glucose transport by Bht1p and Bht3p, indicating an active glucose transport mechanism that involves proton symport. These results suggest a possible role for BHT1 and BHT3 in the competitive ability of B. bruxellensis

Genome evolutionary dynamics meets functional genomics: A case story on the identification of slc25a44

Gene clusters are becoming promising tools for gene identification. The study reveals the purposive genomic distribution of genes toward higher inheritance rates of intact metabolic pathways/phenotypes and, thereby, higher fitness. The co-localization of co-expressed, co-interacting, and functionally related genes was found as genome-wide trends in humans, mouse, golden eagle, rice fish, Drosophila, peanut, and Arabidopsis. As anticipated, the analyses verified the co-segregation of co-localized events. A negative correlation was notable between the likelihood of co-localization events and the inter-loci distances. The evolution of genomic blocks was also found convergent and uniform along the chromosomal arms. Calling a genomic block responsible for adjacent metabolic reactions is therefore recommended for identification of candidate genes and interpretation of cellular functions. As a case story, a function in the metabolism of energy and secondary metabolites was proposed for Slc25A44, based on its genomic local information. Slc25A44 was further characterized as an essential housekeeping gene which has been under evolutionary purifying pressure and belongs to the phylogenetic ETC-clade of SLC25s. Pathway enrichment mapped the Slc25A44s to the energy metabolism. The expression of peanut and human Slc25A44s in oocytes and Saccharomyces cerevisiae strains confirmed the transport of common precursors for secondary metabolites and ubiquinone. These results suggest that SLC25A44 is a mitochondrion-ER-nucleus zone transporter with biotechnological applications. Finally, a conserved three-amino acid signature on the cytosolic face of transport cavity was found important for rational engineering of SLC25s

The Expression and Polymorphism of Entry Machinery for COVID-19 in Human: Juxtaposing Population Groups, Gender, and Different Tissues

(1) Background: Combating viral disease outbreaks has doubtlessly been one of the major public health challenges for the 21st century. (2) Methods: The host entry machinery required for COVID-19 (SARS-CoV-2) infection was examined for the gene expression profiles and polymorphism. (3) Results: Lung, kidney, small intestine, and salivary glands were among the tissues which expressed the entry machinery coding genes Ace2, Tmprss2, CtsB, and CtsL. The genes had no significant expression changes between males and females. The four human population groups of Europeans, Africans, Asians, and Americans had specific and also a common pool of rare variants for the X-linked locus of ACE2 receptor. Several specific and common ACE2 variants including S19P, I21T/V, E23K, A25T, K26R, T27A, E35D/K, E37K, Y50F, N51D/S, M62V, N64K, K68E, F72V, E75G, M82I, T92I, Q102P, G220S, H239Q, G326E, E329G, G352V, D355N, H378R, Q388L, P389H, E467K, H505R, R514G/*, and Y515C were of the utmost importance to the viral entry and infection. The variants of S19P, I21T, K26R, T27A, E37K, N51D, N64K, K68E, F72V, M82I, G326E, H378R, Q388L, and P389H also had significant differences in frequencies among the population groups. Most interestingly, the analyses revealed that more than half of the variants can exist in males, i.e., as hemizygous. (4) Conclusions: The rare variants of human ACE2 seem to be one of the determinant factors associated with fitness in the battle against SARS viruses. The hemizygous viral-entry booster variants of ACE2 describe the higher SARS-CoV-2 mortality rate in males. This is also supported by the lack of gender bias for the gene expression profiles of entry machinery. A personalized medicine strategy is conceived for isolating high-risk individuals in epidemic circumstances

The Expression and Polymorphism of Entry Machinery for COVID-19 in Human: Juxtaposing Population Groups, Gender, and Different Tissues

BACKGROUND: Combating viral disease outbreaks has doubtlessly been one of the major public health challenges for the 21st century. METHODS: Methods: The host entry machinery required for COVID-19 (SARS-CoV-2) infection was examined for the gene expression profiles and polymorphism. RESULTS: Lung, kidney, small intestine, and salivary glands were among the tissues which expressed the entry machinery coding genes Ace2, Tmprss2, CtsB, and CtsL. The genes had no significant expression changes between males and females. The four human population groups of Europeans, Africans, Asians, and Americans had specific and also a common pool of rare variants for the X-linked locus of ACE2 receptor. Several specific and common ACE2 variants including S19P, I21T/V, E23K, A25T, K26R, T27A, E35D/K, E37K, Y50F, N51D/S, M62V, N64K, K68E, F72V, E75G, M82I, T92I, Q102P, G220S, H239Q, G326E, E329G, G352V, D355N, H378R, Q388L, P389H, E467K, H505R, R514G/*, and Y515C were of the utmost importance to the viral entry and infection. The variants of S19P, I21T, K26R, T27A, E37K, N51D, N64K, K68E, F72V, M82I, G326E, H378R, Q388L, and P389H also had significant differences in frequencies among the population groups. Most interestingly, the analyses revealed that more than half of the variants can exist in males, i.e., as hemizygous. CONCLUSIONS: The rare variants of human ACE2 seem to be one of the determinant factors associated with fitness in the battle against SARS viruses. The hemizygous viral-entry booster variants of ACE2 describe the higher SARS-CoV-2 mortality rate in males. This is also supported by the lack of gender bias for the gene expression profiles of entry machinery. A personalized medicine strategy is conceived for isolating high-risk individuals in epidemic circumstances

Energetic evolution of cellular Transportomes

Abstract Background Transporter proteins mediate the translocation of substances across the membranes of living cells. Many transport processes are energetically expensive and the cells use 20 to 60% of their energy to power the transportomes. We hypothesized that there may be an evolutionary selection pressure for lower energy transporters. Results We performed a genome-wide analysis of the compositional reshaping of the transportomes across the kingdoms of bacteria, archaea, and eukarya. We found that the share of ABC transporters is much higher in bacteria and archaea (ca. 27% of the transportome) than in primitive eukaryotes (13%), algae and plants (10%) and in fungi and animals (5–6%). This decrease is compensated by an increased occurrence of secondary transporters and ion channels. The share of ion channels is particularly high in animals (ca. 30% of the transportome) and algae and plants with (ca. 13%), when compared to bacteria and archaea with only 6–7%. Therefore, our results show a move to a preference for the low-energy-demanding transporters (ion channels and carriers) over the more energy-costly transporter classes (ATP-dependent families, and ABCs in particular) as part of the transition from prokaryotes to eukaryotes. The transportome analysis also indicated seven bacterial species, including Neorickettsia risticii and Neorickettsia sennetsu, as likely origins of the mitochondrion in eukaryotes, based on the phylogenetically restricted presence therein of clear homologues of modern mitochondrial solute carriers. Conclusions The results indicate that the transportomes of eukaryotes evolved strongly towards a higher energetic efficiency, as ATP-dependent transporters diminished and secondary transporters and ion channels proliferated. These changes have likely been important in the development of tissues performing energetically costly cellular functions