Genes Of Known Function Research Articles

Genetic analysis of genotype-specific parameters in the DSSTA-CROPGRO-soybean phenology simulation model via a multi-GWAS method

ContextGenotype-specific parameters (GSP) are the fundamental elements for phenotypic prediction in the DSSAT-CROPGRO soybean phenology simulation model (DCS-PSM). Yet, it is unclear about the quantitative relationship between GSP and genomics, which limits the crop models used for breeding research. ObjectiveExploring the genetic composition of GSP related to four soybean phenological traits in DCS-PSM for providing a basis for whether GSP are genetic coefficient. Highlighting the breeding value of DCS-PSM from a genetic perspective and strengthening its genetic understanding, which could support a further crop model-assisted molecular breeding. MethodsWe conducted a multi-genome-wide association study (GWAS) method by intrgrating the single-locus with multi-locus GWAS models for GSP associated quantitative trait nucleotides (QTN) detection. And the heritability, functional annotation of QTN and genotype-environment interaction (GEI) effect were further analyzed. Results(i). All GSP had high broad-sense heritability, ranging form 88.20–98.80%. (ii). Multi-locus GWAS significantly increased the QTN detection efficiency, the GSP associated QTN detected by multi-GWAS method contributed 22.11–50.25% phenotypic variation explained (PVE) of GSP. Besides, the GSP associated QTN accounted for a high PVE of four phenological traits, ranging from 54.25% to 74.75%. (iii). Most QTN overlapped with the function-known genes which regulate soybean phenology through photoperiod-temperature pathways. (iv). GSP associated QTN could reveal the GEI effect and contributed 1.45–6.27% PVE of soybean phenology. ConclusionsThis study indicated that the genetic factor was the primary source of GSP’s variation, and the multi-GWAS method could be an alternative way for the genetic analysis of GSP. Phenology-related GSP had genetic potential and could be regarded as genetic coefficients and breeding targets.

High-throughput screening of the Saccharomyces cerevisiae genome for 2-amino-3-methylimidazo [4,5-f] quinoline resistance identifies colon cancer-associated genes.

Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred meats and cigarette smoke. However, few eukaryotic resistance genes have been identified. We used Saccharomyces cerevisiae (budding yeast) to identify genes that confer resistance to 2-amino-3-methylimidazo[4,5-f] quinoline (IQ). CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. Deletion libraries expressing human CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 µM IQ for 5 or 10 generations. DNA barcodes were sequenced using the Illumina HiSeq 2500 platform and statistical significance was determined for exactly matched barcodes. We identified 424 ORFs, including 337 genes of known function, in duplicate screens of the "humanized" collection for IQ resistance; resistance was further validated for a select group of 51 genes by growth curves, competitive growth, or trypan blue assays. Screens of the library not expressing human genes identified 143 ORFs conferring resistance to IQ per se. Ribosomal protein and protein modification genes were identified as IQ resistance genes in both the original and "humanized" libraries, while nitrogen metabolism, DNA repair, and growth control genes were also prominent in the "humanized" library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. Among DNA Repair and checkpoint genes, we identified those that function in postreplication repair (RAD18, UBC13, REV7), base excision repair (NTG1), and checkpoint signaling (CHK1, PSY2). These studies underscore the role of ribosomal protein genes in conferring IQ resistance, and illuminate DNA repair pathways for conferring resistance to activated IQ.

Analysis of DNA methylation at birth and in childhood reveals changes associated with season of birth and latitude

BackgroundSeasonal variations in environmental exposures at birth or during gestation are associated with numerous adult traits and health outcomes later in life. Whether DNA methylation (DNAm) plays a role in the molecular mechanisms underlying the associations between birth season and lifelong phenotypes remains unclear.MethodsWe carried out epigenome-wide meta-analyses within the Pregnancy And Childhood Epigenetic Consortium to identify associations of DNAm with birth season, both at differentially methylated probes (DMPs) and regions (DMRs). Associations were examined at two time points: at birth (21 cohorts, N = 9358) and in children aged 1–11 years (12 cohorts, N = 3610). We conducted meta-analyses to assess the impact of latitude on birth season-specific associations at both time points.ResultsWe identified associations between birth season and DNAm (False Discovery Rate-adjusted p values < 0.05) at two CpGs at birth (winter-born) and four in the childhood (summer-born) analyses when compared to children born in autumn. Furthermore, we identified twenty-six differentially methylated regions (DMR) at birth (winter-born: 8, spring-born: 15, summer-born: 3) and thirty-two in childhood (winter-born: 12, spring and summer: 10 each) meta-analyses with few overlapping DMRs between the birth seasons or the two time points. The DMRs were associated with genes of known functions in tumorigenesis, psychiatric/neurological disorders, inflammation, or immunity, amongst others. Latitude-stratified meta-analyses [higher (≥ 50°N), lower (< 50°N, northern hemisphere only)] revealed differences in associations between birth season and DNAm by birth latitude. DMR analysis implicated genes with previously reported links to schizophrenia (LAX1), skin disorders (PSORS1C, LTB4R), and airway inflammation including asthma (LTB4R), present only at birth in the higher latitudes (≥ 50°N).ConclusionsIn this large epigenome-wide meta-analysis study, we provide evidence for (i) associations between DNAm and season of birth that are unique for the seasons of the year (temporal effect) and (ii) latitude-dependent variations in the seasonal associations (spatial effect). DNAm could play a role in the molecular mechanisms underlying the effect of birth season on adult health outcomes.

Sedimentary DNA and pigments show increasing abundance and toxicity of cyanoHABs during the Anthropocene

Abstract Cyanobacterial harmful algal blooms (cyanoHABs) are assumed to be increasing in abundance and toxicity, but comprehensive analysis of change through time is limited, in part, because some key taxa (e.g., Microcystis) leave ambiguous evidence of historical abundance and toxicity. Sedimentary DNA (sedDNA) can allow the reconstruction of the cyanobacteria community as well as the frequency of genes specific to cyanotoxin production, enabling us to determine which taxa are present and their potential for toxin‐production. Using a combination of droplet digital polymerase chain reaction (ddPCR) and high‐throughput sequencing (HTS), we quantified the abundance of cyanobacterial genes of known function and changes in cyanobacteria taxa from sedDNA over the last century in nine lakes along a gradient of lake size, depth and trophic state in Minnesota, U.S.A. Using ddPCR, we quantified genes associated with microcystin toxin‐producing potential (mcyE), total cyanobacteria (CYA, 16S rRNA) and the genus Microcystis (MICR, 16S rRNA). Using HTS on a subset of lakes, we investigated how the abundance of this toxin‐producing gene covaried with the cyanobacteria community composition. We also compared ddPCR and HTS data to fossil pigments, a well‐established palaeolimnological method used to track changes in primary producers over time. Our results showed a significant correlation between MICR and the quantity of mcyE gene and cyanobacterial taxa with known toxin‐production potential. The abundance of both genes likewise increased concomitantly through time. Community analyses of HTS data showed significant change in cyanobacterial communities commencing c. 1950 when major land‐use change in this region led to increased lake productivity, and c. 1990 when Dolichospermum and Microcystis genera increased in abundance, and the subtropical exotic cyanobacteria Raphidiopsis raciborskii and Sphaerospermopsis aphanizomenoides became abundant. Cyanobacteria pigment data reflected these changes only in deeper lakes, suggesting issues related to benthic production or biomarker preservation in shallower systems. This study provides evidence for historical development of increasingly toxic cyanoHABs across a diverse set of lakes and illustrates how sedDNA may help link changes in the cyanobacteria community to the expression of potentially toxic genes.

A nuclear target sequence capture probe set for phylogeny reconstruction of the charismatic plant family Bignoniaceae.

The plant family Bignoniaceae is a conspicuous and charismatic element of the tropical flora. The family has a complex taxonomic history, with substantial changes in the classification of the group during the past two centuries. Recent re-classifications at the tribal and generic levels have been largely possible by the availability of molecular phylogenies reconstructed using Sanger sequencing data. However, our complete understanding of the systematics, evolution, and biogeography of the family remains incomplete, especially due to the low resolution and support of different portions of the Bignoniaceae phylogeny. To overcome these limitations and increase the amount of molecular data available for phylogeny reconstruction within this plant family, we developed a bait kit targeting 762 nuclear genes, including 329 genes selected specifically for the Bignoniaceae; 348 genes obtained from the Angiosperms353 with baits designed specifically for the family; and, 85 low-copy genes of known function. On average, 77.4% of the reads mapped to the targets, and 755 genes were obtained per species. After removing genes with putative paralogs, 677 loci were used for phylogenetic analyses. On-target genes were compared and combined in the Exon-Only dataset, and on-target + off-target regions were combined in the Supercontig dataset. We tested the performance of the bait kit at different taxonomic levels, from family to species-level, using 38 specimens of 36 different species of Bignoniaceae, representing: 1) six (out of eight) tribal level-clades (e.g., Bignonieae, Oroxyleae, Tabebuia Alliance, Paleotropical Clade, Tecomeae, and Jacarandeae), only Tourrettieae and Catalpeae were not sampled; 2) all 20 genera of Bignonieae; 3) seven (out of nine) species of Dolichandra (e.g., D. chodatii, D. cynanchoides, D. dentata, D. hispida, D. quadrivalvis, D. uncata, and D. uniguis-cati), only D. steyermarkii and D. unguiculata were not sampled; and 4) three individuals of Dolichandra unguis-cati. Our data reconstructed a well-supported phylogeny of the Bignoniaceae at different taxonomic scales, opening new perspectives for a comprehensive phylogenetic framework for the family as a whole.

The mitochondrial genome sequence of Abies alba Mill. reveals a high structural and combinatorial variation

BackgroundPlant mitogenomes vary widely in size and genomic architecture. Although hundreds of plant mitogenomes of angiosperm species have already been sequence-characterized, only a few mitogenomes are available from gymnosperms. Silver fir (Abies alba) is an economically important gymnosperm species that is widely distributed in Europe and occupies a large range of environmental conditions. Reference sequences of the nuclear and chloroplast genome of A. alba are available, however, the mitogenome has not yet been assembled and studied.ResultsHere, we used paired-end Illumina short reads generated from a single haploid megagametophyte in combination with PacBio long reads from high molecular weight DNA of needles to assemble the first mitogenome sequence of A. alba. Assembly and scaffolding resulted in 11 mitogenome scaffolds, with the largest scaffold being 0.25 Mbp long. Two of the scaffolds displayed a potential circular structure supported by PCR. The total size of the A. alba mitogenome was estimated at 1.43 Mbp, similar to the size (1.33 Mbp) of a draft assembly of the Abies firma mitogenome. In total, 53 distinct genes of known function were annotated in the A. alba mitogenome, comprising 41 protein-coding genes, nine tRNA, and three rRNA genes. The proportion of highly repetitive elements (REs) was 0.168. The mitogenome seems to have a complex and dynamic structure featured by high combinatorial variation, which was specifically confirmed by PCR for the contig with the highest mapping coverage. Comparative analysis of all sequenced mitogenomes of gymnosperms revealed a moderate, but significant positive correlation between mitogenome size and proportion of REs.ConclusionsThe A. alba mitogenome provides a basis for new comparative studies and will allow to answer important structural, phylogenetic and other evolutionary questions. Future long-read sequencing with higher coverage of the A. alba mitogenome will be the key to further resolve its physical structure. The observed positive correlation between mitogenome size and proportion of REs will be further validated once available mitogenomes of gymnosperms would become more numerous. To test whether a higher proportion of REs in a mitogenome leads to an increased recombination and higher structural complexity and variability is a prospective avenue for future research.

Genome mining as a biotechnological tool for the discovery of novel biosynthetic genes in lichens.

Natural products (NPs) and their derivatives are a major contributor to modern medicine. Historically, microorganisms such as bacteria and fungi have been instrumental in generating drugs and lead compounds because of the ease of culturing and genetically manipulating them. However, the ever-increasing demand for novel drugs highlights the need to bioprospect previously unexplored taxa for their biosynthetic potential. Next-generation sequencing technologies have expanded the range of organisms that can be explored for their biosynthetic content, as these technologies can provide a glimpse of an organism's entire biosynthetic landscape, without the need for cultivation. The entirety of biosynthetic genes can be compared to the genes of known function to identify the gene clusters potentially coding for novel products. In this study, we mine the genomes of nine lichen-forming fungal species of the genus Umbilicaria for biosynthetic genes, and categorize the biosynthetic gene clusters (BGCs) as "associated product structurally known" or "associated product putatively novel". Although lichen-forming fungi have been suggested to be a rich source of NPs, it is not known how their biosynthetic diversity compares to that of bacteria and non-lichenized fungi. We found that 25%-30% of biosynthetic genes are divergent as compared to the global database of BGCs, which comprises 1,200,000 characterized biosynthetic genes from plants, bacteria, and fungi. Out of 217 BGCs, 43 were highly divergant suggesting that they potentially encode structurally and functionally novel NPs. Clusters encoding the putatively novel metabolic diversity comprise polyketide synthases (30), non-ribosomal peptide synthetases (12), and terpenes (1). Our study emphasizes the utility of genomic data in bioprospecting microorganisms for their biosynthetic potential and in advancing the industrial application of unexplored taxa. We highlight the untapped structural metabolic diversity encoded in the lichenized fungal genomes. To the best of our knowledge, this is the first investigation identifying genes coding for NPs with potentially novel properties in lichenized fungi.

Isolation and characterization of two homolog phages infecting Pseudomonas aeruginosa

Bacteriophages (phages) are capable of infecting specific bacteria, and therefore can be used as a biological control agent to control bacteria-induced animal, plant, and human diseases. In this study, two homolog phages (named PPAY and PPAT) that infect Pseudomonas aeruginosa PAO1 were isolated and characterized. The results of the phage plaque assay showed that PPAT plaques were transparent dots, while the PPAY plaques were translucent dots with a halo. Transmission electron microscopy results showed that PPAT (65 nm) and PPAY (60 nm) strains are similar in size and have an icosahedral head and a short tail. Therefore, these belong to the short-tailed phage family Podoviridae. One-step growth curves revealed the latent period of 20 min and burst time of 30 min for PPAT and PPAY. The burst size of PPAT (953 PFUs/infected cell) was higher than that of PPAY (457 PFUs/infected cell). Also, the adsorption rate constant of PPAT (5.97 × 10−7 ml/min) was higher than that of PPAY (1.32 × 10−7 ml/min) at 5 min. Whole-genome sequencing of phages was carried out using the Illumina HiSeq platform. The genomes of PPAT and PPAY have 54,888 and 50,154 bp, respectively. Only 17 of the 352 predicted ORFs of PPAT could be matched to homologous genes of known function. Likewise, among the 351 predicted ORFs of PPAY, only 18 ORFs could be matched to genes of established functions. Homology and evolutionary analysis indicated that PPAT and PPAY are closely related to PA11. The presence of tail fiber proteins in PPAY but not in PPAT may have contributed to the halo effect of its plaque spots. In all, PPAT and PPAY, newly discovered P. aeruginosa phages, showed growth inhibitory effects on bacteria and can be used for research and clinical purposes.

Synthetic biology and opportunities within agricultural crops

AbstractConventional breeding techniques have been integral to the development of many agronomically important traits in numerous crops. The adoption of modern biotechnology approaches further advanced and refined trait development and introduction beyond the scope possible through conventional breeding. However, crop yields continue to be challenged by abiotic and biotic factors that require the development of traits that are more genetically complex than can be addressed through conventional breeding or traditional genetic engineering. Therefore, more advanced trait development approaches are required to maintain and improve yields and production efficiency, especially as climate change accelerates the incidence of biotic and abiotic challenges to food and fibre crops. Synthetic biology (SynBio) encompasses approaches that design and construct new biological elements (e.g., enzymes, genetic circuits, cells) or redesign existing biological systems to build new and improved functions. SynBio ‘upgrades’ the potential of genetic engineering, which involves the transfer of single genes from one organism to another. This technology can enable the introduction of multiple genes in a single transgenic event, either derived from a foreign organism or synthetically generated. It can also enable the assembly of novel genomes from the ground up from a set of standardised genetic parts, which can then be transferred into the target cell or organism. New opportunities to advance breeding applications through exploiting SynBio technology include the introduction of new genes of known function, artificially creating genetic variation, topical applications of small RNAs as pesticides and potentially speeding up the production of new cultivars with elite traits. This review will draw upon case studies to demonstrate the potential application of SynBio to improve crop productivity and resistance to various challenges. Here, we outline specific solutions to challenges including fungal diseases, insect pests, heat and drought stress and nutrient acquisition in a range of important crops using the SynBio toolkit.

Genome Wide Association Study Identifies Candidate Genes Related to the Earlywood Tracheid Properties in Picea crassifolia Kom.

Picea crassifolia Kom. is one of the timber and ecological conifers in China and its wood tracheid traits directly affect wood formation and adaptability under harsh environment. Molecular studies on P. crassifolia remain inadequate because relatively few genes have been associated with these traits. To identify markers and candidate genes that can potentially be used for genetic improvement of wood tracheid traits, we examined 106 clones of P. crassifolia, and investigated phenotypic data for 14 wood tracheid traits before specific-locus amplified fragment sequencing (SLAF-seq) was employed to perform a genome wide association study (GWAS). Subsequently, the results were used to screen single nucleotide polymorphism (SNP) loci and candidate genes that exhibited a significant correlation with the studied traits. We developed 4,058,883 SLAF-tags and 12,275,765 SNP loci, and our analyses identified a total of 96 SNP loci that showed significant correlations with three earlywood tracheid traits using a mixed linear model (MLM). Next, candidate genes were screened in the 100 kb zone (50 kb upstream, 50 kb downstream) of each of the SNP loci, whereby 67 candidate genes were obtained in earlywood tracheid traits, including 34 genes of known function and 33 genes of unknown function. We provide the most significant SNP for each trait-locus combination and candidate genes occurring within the GWAS hits. These resources provide a foundation for the development of markers that could be used in wood traits improvement and candidate genes for the development of earlywood tracheid in P. crassifolia.

Preharvest UV-C Hormesis Induces Key Genes Associated With Homeostasis, Growth and Defense in Lettuce Inoculated With Xanthomonas campestris pv. vitians.

Preharvest application of hormetic doses of ultraviolet-C (UV-C) generates beneficial effects in plants. In this study, within 1 week, four UV-C treatments of 0.4 kJ/m2 were applied to 3-week-old lettuce seedlings. The leaves were inoculated with a virulent strain of Xanthomonas campestris pv. vitians (Xcv) 48 h after the last UV-C application. The extent of the disease was tracked over time and a transcriptomic analysis was performed on lettuce leaf samples. Samples of lettuce leaves, from both control and treated groups, were taken at two different times corresponding to T2, 48 h after the last UV-C treatment and T3, 24 h after inoculation (i.e., 72 h after the last UV-C treatment). A significant decrease in disease severity between the UV-C treated lettuce and the control was observed on days 4, 8, and 14 after pathogen inoculation. Data from the transcriptomic study revealed, that in response to the effect of UV-C alone and/or UV-C + Xcv, a total of 3828 genes were differentially regulated with fold change (|log2-FC|) > 1.5 and false discovery rate (FDR) < 0.05. Among these, of the 2270 genes of known function 1556 were upregulated and 714 were downregulated. A total of 10 candidate genes were verified by qPCR and were generally consistent with the transcriptomic results. The differentially expressed genes observed in lettuce under the conditions of the present study were associated with 14 different biological processes in the plant. These genes are involved in a series of metabolic pathways associated with the ability of lettuce treated with hormetic doses of UV-C to resume normal growth and to defend themselves against potential stressors. The results indicate that the hormetic dose of UV-C applied preharvest on lettuce in this study, can be considered as an eustress that does not interfere with the ability of the treated plants to carry on a set of key physiological processes namely: homeostasis, growth and defense.

Integrated Genomic and Bioinformatics Approaches to Identify Molecular Links between Endocrine Disruptors and Adverse Outcomes.

Exposure to Endocrine Disrupting Chemicals (EDC) has been linked with several adverse outcomes. In this review, we examine EDCs that are pervasive in the environment and are of concern in the context of human, animal, and environmental health. We explore the consequences of EDC exposure on aquatic life, terrestrial animals, and humans. We focus on the exploitation of genomics technologies and in particular whole transcriptome sequencing. Genome-wide analyses using RNAseq provides snap shots of cellular, tissue and whole organism transcriptomes under normal physiological and EDC perturbed conditions. A global view of gene expression provides highly valuable information as it uncovers gene families or more specifically, pathways that are affected by EDC exposures, but also reveals those that are unaffected. Hypotheses about genes with unknown functions can also be formed by comparison of their expression levels with genes of known function. Risk assessment strategies leveraging genomic technologies and the development of toxicology databases are explored. Finally, we review how the Adverse Outcome Pathway (AOP) has exploited this high throughput data to provide a framework for toxicology studies.

Land use and life history constrain adaptive genetic variation and reduce the capacity for climate change adaptation in turtles

BackgroundRapid anthropogenic climate change will require species to adapt to shifting environmental conditions, with successful adaptation dependent upon current patterns of genetic variation. While landscape genomic approaches allow for exploration of local adaptation in non-model systems, most landscape genomics studies of adaptive capacity are limited to exploratory identification of potentially important functional genes, often without a priori expectations as to the gene functions that may be most important for climate change responses. In this study, we integrated targeted sequencing of genes of known function and genotyping of single-nucleotide polymorphisms to examine spatial, environmental, and species-specific patterns of potential local adaptation in two co-occuring turtle species: the Blanding’s turtle (Emydoidea blandingii) and the snapping turtle (Chelydra serpentina).ResultsWe documented divergent patterns of spatial clustering between neutral and putatively adaptive genetic variation in both species. Environmental associations varied among gene regions and between species, with stronger environmental associations detected for genes involved in stress response and for the more specialized Blanding’s turtle. Land cover appeared to be more important than climate in shaping spatial variation in functional genes, indicating that human landscape alterations may affect adaptive capacity important for climate change responses.ConclusionsOur study provides evidence that responses to climate change will be contingent on species-specific adaptive capacity and past history of exposure to human land cover change.

Identification of genes associated with chapatti quality using transcriptome analysis

Analysis of gene expression in developing grains of 20 diverse wheat genotypes was used to identify genes differentially expressed in genotypes with good chapatti quality. The genes that were identified as potentially playing an important role in chapatti quality included both annotated genes and genes of unknown function. Genes of known function differentially expressed in wheat genotypes with good chapatti quality included those associated with cell wall polysaccharide biosynthesis (upregulated) and protein synthesis (upregulation of many gliadins and down regulation of many LMW glutenins and other storage proteins). The expression of many other genes was associated with chapatti quality. The wbm gene that was upregulated in good bread wheats was not highly expressed in wheats known for good chapatti quality. This confirms that in wheat genotypes, the expression of some of the transcripts associated with good bread and chapatti quality characteristics are negatively correlated. This RNAseq analysis provides wheat breeders with an insight into the genes found to be associated with good chapatti quality. The large differences in the quality requirements for bread and chapatti make it difficult for breeders to breed wheat for both good bread and chapatti quality in the same genotype.

Clinical Research on Antioxidant-Based Modalities in 2021

Being updated biweekly till the end of the year, this Education &amp; Resources web page lists major clinical studies on antioxidant-based modalities or related entities in disease intervention and health promotion, which have been published in highly influential journals during 2021. It should be noted that this is not intended to be a complete list, but is rather to focus on rigorously designed and well conducted high-profile randomized controlled trials (RCTs) whose findings were reported in medical or bioscience journals of the highest impact. For more comprehensive information on antioxidant-based clinical trials, the reader may refer to the ClinicalTrials.gov (https://clinicaltrials.gov), the largest clinical trials database, run by the US National Library of Medicine, that holds registrations from over 368,000 trials from 219 countries. 2021 LIST IN REVERSE CHRONOLOGICAL ORDER Xu et al. Edaravone dexborneol versus edaravone alone for the treatment of acute ischemic stroke: a phase III, randomized, double-blind, comparative trial. Stroke 2021 Mar; 52(3):772-780. doi: https://dx.doi.org/10.1161/STROKEAHA.120.031197. Key finding: Edaravone dexborneol (a combination of edaravone and borneol) was superior to edaravone alone in improving the clinical outcomes of the acute ischemic stroke patients. Note: Edaravone, a synthetic free radical scavenger, was approved by the US FDA in 2017 for treating amyotrophic lateral sclerosis (ALS). Borneol is a phytochemical with diverse biological activities including antioxidative and anti-inflammatory effects. Kim et al. Reactive oxygen species scavenger in acute intracerebral hemorrhage patients: a multicenter, randomized controlled trial. Stroke 2021 Feb 25; doi: https://doi.org/10.1161/STROKEAHA.120.032266. Key finding: Giving N-acetylcysteine 2000 mg/day and selenium 1600 µg/day, intravenously, for 14 days significantly improved the clinical outcomes in the acute intracerebral hemorrhage patients. Note: N-Acetylcysteine is a precursor of glutathione (GSH). Selenium acts as an antioxidant element due, at least partly, to its essentialness for the function of various selenoproteins, including selenium-dependent glutathione peroxidases (GPx). Kalstad et al. Effects of n-3 fatty acid supplements in elderly patients after myocardial infarction: a randomized, controlled trial. Circulation 2021 Feb 9; 143(6):528-539. doi: https://dx.doi.org/10.1161/CIRCULATIONAHA.120.052209. Key finding: Null Note: n-3 Fatty acids, also known as omega-3 fatty acids, possess potent antioxidative and anti-inflammatory activities. Lynch et al. Safety and efficacy of omaveloxolone in Friedreich ataxia (MOXIe Study). Ann Neurol 2021 Feb; 89(2):212-225. doi: https://dx.doi.org/10.1002/ana.25934. Key finding: Omaveloxolone significantly improved neurological function compared to placebo and is well tolerated. Note: Omaveloxolone, a synthetic oleanane triterpenoid, is an activator of Nrf2, the chief regulator of cellular antioxidant and other cytoprotective genes. Meir et al. Effect of green-Mediterranean diet on intrahepatic fat: the DIRECT PLUS randomised controlled trial. Gut 2021 Jan 18; gutjnl-2020-323106. doi: https://dx.doi.org/10.1136/gutjnl-2020-323106. Key finding: Green-Mediterranean diet enriched with green plants and polyphenols improved non-alcoholic fatty liver disease (NAFLD). Note: Mediterranean diet and green plants (green tea) and nuts are rich in antioxidants and anti-inflammatory compounds and possess many health benefits, especially cardiovascular protection. However, the exact contribution of antioxidant components to the health benefits of Mediterranean diet remains to be established. Rinott et al. Effects of diet-modulated autologous fecal microbiota transplantation on weight regain. Gastroenterology 2021 Jan; 160(1):158-173.e10. doi: https://dx.doi.org/10.1053/j.gastro.2020.08.041. Key finding: Polyphenol-enriched diet-modulated autologous fecal microbiota transplantation attenuated weight regain and preserved glycemic control. Note: Dietary polyphenols possess antioxidative, anti-inflammatory, and many other biological activities. Zheng et al. Plasma vitamin C and type 2 diabetes: genome-wide association study and Mendelian randomization analysis in European populations. Diabetes Care 2021 Jan; 44(1):98-106. doi: https://dx.doi.org/10.2337/dc20-1328. Key finding: Null; no evidence to support an efficacy of vitamin C supplement in type 2 diabetes prevention. Note: Vitamin C is a multitasking compound; it is an antioxidant, but also possesses many other biological functions. According to Dr Davey Smith, Mendelian randomization is a method of using measured variation in genes of known function to examine the causal effect of a modifiable exposure on disease in observational studies (from the US CDC website: https://cdc.gov). A positive finding in a Mendelian randomization study provides strong evidence for a causal relationship. Luo et al. Diet-derived circulating antioxidants and risk of coronary heart disease: a Mendelian randomization study. J Am Coll Cardiol 2021 Jan 5; 77(1):45-54. doi: https://dx.doi.org/10.1016/j.jacc.2020.10.048. Key finding: Null Note: According to Dr Davey Smith, Mendelian randomization is a method of using measured variation in genes of known function to examine the causal effect of a modifiable exposure on disease in observational studies (from the US CDC website: https://cdc.gov). A positive finding in a Mendelian randomization study provides strong evidence for a causal relationship.

Clinical Research on Antioxidant-Based Modalities in 2020

Clinical Research on Antioxidant-Based Modalities in 2020

CRISPR-Cas9 editing of non-coding genomic loci as a means of controlling gene expression in the sea urchin

We seek to manipulate gene function here through CRISPR-Cas9 editing of cis-regulatory sequences, rather than the more typical mutation of coding regions. This approach would minimize secondary effects of cellular responses to nonsense mediated decay pathways or to mutant protein products by premature stops. This strategy also allows for reducing gene activity in cases where a complete gene knockout would result in lethality, and it can be applied to the rapid identification of key regulatory sites essential for gene expression. We tested this strategy here with genes of known function as a proof of concept, and then applied it to examine the upstream genomic region of the germline gene Nanos2 in the sea urchin, Strongylocentrotus purpuratus. We first used CRISPR-Cas9 to target established genomic cis-regulatory regions of the skeletogenic cell transcription factor, Alx1, and the TGF-β signaling ligand, Nodal, which produce obvious developmental defects when altered in sea urchin embryos. Importantly, mutation of cis-activator sites (Alx1) and cis-repressor sites (Nodal) result in the predicted decreased and increased transcriptional output, respectively. Upon identification of efficient gRNAs by genomic mutations, we then used the same validated gRNAs to target a deadCas9-VP64 transcriptional activator to increase Nodal transcription directly. Finally, we paired these new methodologies with a more traditional, GFP reporter construct approach to further our understanding of the transcriptional regulation of Nanos2, a key gene required for germ cell identity in S. purpuratus. With a series of reporter assays, upstream Cas9-promoter targeted mutagenesis, coupled with qPCR and in situ RNA hybridization, we concluded that the promoter of Nanos2 drives strong mRNA expression in the sea urchin embryo, indicating that its primordial germ cell (PGC)-specific restriction may rely instead on post-transcriptional regulation. Overall, we present a proof-of-principle tool-kit of Cas9-mediated manipulations of promoter regions that should be applicable in most cells and embryos for which CRISPR-Cas9 is employed.

A metabolic and physiological design study of Pseudomonas putida KT2440 capable of anaerobic respiration

BackgroundPseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and thus interesting microbial chassis for biotechnological applications. However, its obligate aerobic nature hampers production of oxygen sensitive products and drives up costs in large scale fermentation. The inability to perform anaerobic fermentation has been attributed to insufficient ATP production and an inability to produce pyrimidines under these conditions. Addressing these bottlenecks enabled growth under micro-oxic conditions but does not lead to growth or survival under anoxic conditions.ResultsHere, a data-driven approach was used to develop a rational design for a P. putida KT2440 derivative strain capable of anaerobic respiration. To come to the design, data derived from a genome comparison of 1628 Pseudomonas strains was combined with genome-scale metabolic modelling simulations and a transcriptome dataset of 47 samples representing 14 environmental conditions from the facultative anaerobe Pseudomonas aeruginosa.ConclusionsThe results indicate that the implementation of anaerobic respiration in P. putida KT2440 would require at least 49 additional genes of known function, at least 8 genes encoding proteins of unknown function, and 3 externally added vitamins.

The Lipid A from the Lipopolysaccharide of the Phototrophic Bacterium Rhodomicrobium vannielii ATCC 17100 Revisited.

The structure of lipid A from lipopolysaccharide (LPS) of Rhodomicrobium vannielii ATCC 17100 (Rv) a phototrophic, budding bacterium was re-investigated using high-resolution mass spectrometry, NMR, and chemical degradation protocols. It was found that the (GlcpN)-disaccharide lipid A backbone was substituted by a GalpA residue that was connected to C-1 of proximal GlcpN. Some of this GalpA residue was β-eliminated by alkaline de-acylation, which indicated the possibility of the presence of another so far unidentified substituent at C-4 in non-stoichiometric amounts. One Manp residue substituted C-4′ of distal GlcpN. The lipid A backbone was acylated by 16:0(3-OH) at C-2 of proximal GlcpN, and by 16:0(3-OH), i17:0(3-OH), or 18:0(3-OH) at C-2′ of distal GlcpN. Two acyloxy-acyl moieties that were mainly formed by 14:0(3-O-14:0) and 16:0(3-O-22:1) occupied the distal GlcpN of lipid A. Genes that were possibly involved in the modification of Rv lipid A were compared with bacterial genes of known function. The biological activity was tested at the model of human mononuclear cells (MNC), showing that Rv lipid A alone does not significantly stimulate MNC. At low concentrations of toxic Escherichia coli O111:B4 LPS, pre-incubation with Rv lipid A resulted in a substantial reduction of activity, but, when higher concentrations of E. coli LPS were used, the stimulatory effect was increased.

Discovery of Three Toxic Proteins of Klebsiella Phage fHe-Kpn01.

The lytic phage, fHe-Kpn01 was isolated from sewage water using an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae as a host. The genome is 43,329 bp in size and contains direct terminal repeats of 222 bp. The genome contains 56 predicted genes, of which proteomics analysis detected 29 different proteins in purified phage particles. Comparison of fHe-Kpn01 to other phages, both morphologically and genetically, indicated that the phage belongs to the family Podoviridae and genus Drulisvirus. Because fHe-Kpn01 is strictly lytic and does not carry any known resistance or virulence genes, it is suitable for phage therapy. It has, however, a narrow host range since it infected only three of the 72 tested K. pneumoniae strains, two of which were of capsule type KL62. After annotation of the predicted genes based on the similarity to genes of known function and proteomics results on the virion-associated proteins, 22 gene products remained annotated as hypothetical proteins of unknown function (HPUF). These fHe-Kpn01 HPUFs were screened for their toxicity in Escherichia coli. Three of the HPUFs, encoded by the genes g10, g22, and g38, were confirmed to be toxic.