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Genome Biol ; 23(1): 15, 2022 01 10.
Article En | MEDLINE | ID: mdl-35012625

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) is the cell-entry receptor for SARS-CoV-2. It plays critical roles in both the transmission and the pathogenesis of COVID-19. Comprehensive profiling of ACE2 expression patterns could reveal risk factors of severe COVID-19 illness. While the expression of ACE2 in healthy human tissues has been well characterized, it is not known which diseases and drugs might be associated with ACE2 expression. RESULTS: We develop GENEVA (GENe Expression Variance Analysis), a semi-automated framework for exploring massive amounts of RNA-seq datasets. We apply GENEVA to 286,650 publicly available RNA-seq samples to identify any previously studied experimental conditions that could be directly or indirectly associated with ACE2 expression. We identify multiple drugs, genetic perturbations, and diseases that are associated with the expression of ACE2, including cardiomyopathy, HNF1A overexpression, and drug treatments with RAD140 and itraconazole. Our joint analysis of seven datasets confirms ACE2 upregulation in all cardiomyopathy categories. Using electronic health records data from 3936 COVID-19 patients, we demonstrate that patients with pre-existing cardiomyopathy have an increased mortality risk than age-matched patients with other cardiovascular conditions. GENEVA is applicable to any genes of interest and is freely accessible at . CONCLUSIONS: This study identifies multiple diseases and drugs that are associated with the expression of ACE2. The effect of these conditions should be carefully studied in COVID-19 patients. In particular, our analysis identifies cardiomyopathy patients as a high-risk group, with increased ACE2 expression in the heart and increased mortality after SARS-COV-2 infection.

Angiotensin-Converting Enzyme 2/isolation & purification , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/mortality , Cardiomyopathies/metabolism , Cardiovascular Diseases , Adult , Aged , Angiotensin-Converting Enzyme 2/genetics , Female , Gene Expression , Humans , Male , Middle Aged , Mortality , RNA-Seq , Risk Factors , SARS-CoV-2
Arch Microbiol ; 204(2): 132, 2022 Jan 09.
Article En | MEDLINE | ID: mdl-34999969

Pseudomonas simiae EGD-AQ6 is capable of utilizing chloroaromatic compound i.e., 2-4-D efficiently in its biofilm phenotype. The differential accumulation of intermediate 4-chlorocatechol rates were significant in planktonic and biofilm phenotypes, as well as in the  increased biofilm adapted cell numbers. Interestingly, response surface analysis demonstrated the combined positive effects of 2-4-D degradation and 4-CCA accumulation rates and the gene expression profiles, with significant up-regulation of degradative and biofilm genes, and greater participation of pellicle genes in the biofilm phenotypes than their planktonic counterparts, thereby revealing a phenotype variation. It positively validated the physiological data. Furthermore, the sequence similarity of the 2-4-D catabolic and biofilm-forming proteins (pel ABCDEFG and pga ABCD), which are responsible for building carbohydrate rich extracellular matrix, were significant with the respective organisms. This is the first study, which endorses this strain to be unique in efficient chloro-aromatic degradation through phenotype variation, thereby proving a potential candidate in the improvement of bioremediation technologies.

Gene Expression Regulation, Bacterial , Pseudomonas aeruginosa , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms , Gene Expression , Pseudomonas/genetics , Pseudomonas/metabolism , Pseudomonas aeruginosa/metabolism
BMC Bioinformatics ; 23(1): 24, 2022 Jan 06.
Article En | MEDLINE | ID: mdl-34991441

BACKGROUND: Identifying protein complexes from protein-protein interaction (PPI) networks is a crucial task, and many related algorithms have been developed. Most algorithms usually employ direct neighbors of nodes and ignore resource allocation and second-order neighbors. The effective use of such information is crucial to protein complex detection. RESULT: Based on this observation, we propose a new way by combining node resource allocation and gene expression information to weight protein network (NRAGE-WPN), in which protein complexes are detected based on core-attachment and second-order neighbors. CONCLUSIONS: Through comparison with eleven methods in Yeast and Human PPI network, the experimental results demonstrate that this algorithm not only performs better than other methods on 75% in terms of f-measure+, but also can achieve an ideal overall performance in terms of a composite score consisting of five performance measures. This identification method is simple and can accurately identify more complexes.

Protein Interaction Mapping , Protein Interaction Maps , Algorithms , Computational Biology , Gene Expression , Humans , Proteins/genetics , Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
Nat Commun ; 13(1): 142, 2022 01 10.
Article En | MEDLINE | ID: mdl-35013231

The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation.

Cell Lineage/genetics , Epigenesis, Genetic , G-Quadruplexes , Histones/metabolism , Human Embryonic Stem Cells/metabolism , Pluripotent Stem Cells/metabolism , Protein Processing, Post-Translational , Biomarkers/metabolism , Cell Differentiation , Cell Line , DNA/genetics , DNA/metabolism , DNA Methylation , Enhancer Elements, Genetic , Gene Expression , Histones/genetics , Human Embryonic Stem Cells/cytology , Humans , Nanog Homeobox Protein/genetics , Nanog Homeobox Protein/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nestin/genetics , Nestin/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , PAX6 Transcription Factor/genetics , PAX6 Transcription Factor/metabolism , Pluripotent Stem Cells/cytology , Promoter Regions, Genetic , Receptors, Nerve Growth Factor/genetics , Receptors, Nerve Growth Factor/metabolism , Transcription Factor AP-2/genetics , Transcription Factor AP-2/metabolism
Nat Commun ; 13(1): 115, 2022 01 10.
Article En | MEDLINE | ID: mdl-35013254

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.

Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/drug effects , Lipoproteins/chemistry , Membrane Transport Proteins/chemistry , Multidrug Resistance-Associated Proteins/chemistry , Piperazines/pharmacology , Pyridines/pharmacology , Allosteric Regulation/drug effects , Allosteric Site , Anti-Bacterial Agents/chemistry , Bacterial Outer Membrane Proteins/antagonists & inhibitors , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Biological Transport/drug effects , Crystallography, X-Ray , Drug Resistance, Multiple, Bacterial , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression , Lipoproteins/antagonists & inhibitors , Lipoproteins/genetics , Lipoproteins/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Molecular Dynamics Simulation , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Mutation , Oligopeptides/chemistry , Oligopeptides/pharmacology , Oxacillin/chemistry , Oxacillin/pharmacology , Piperazines/chemical synthesis , Promoter Regions, Genetic , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Pyridines/chemical synthesis , Structure-Activity Relationship
Nat Commun ; 13(1): 136, 2022 01 10.
Article En | MEDLINE | ID: mdl-35013263

Emerging research supports that triclosan (TCS), an antimicrobial agent found in thousands of consumer products, exacerbates colitis and colitis-associated colorectal tumorigenesis in animal models. While the intestinal toxicities of TCS require the presence of gut microbiota, the molecular mechanisms involved have not been defined. Here we show that intestinal commensal microbes mediate metabolic activation of TCS in the colon and drive its gut toxicology. Using a range of in vitro, ex vivo, and in vivo approaches, we identify specific microbial ß-glucuronidase (GUS) enzymes involved and pinpoint molecular motifs required to metabolically activate TCS in the gut. Finally, we show that targeted inhibition of bacterial GUS enzymes abolishes the colitis-promoting effects of TCS, supporting an essential role of specific microbial proteins in TCS toxicity. Together, our results define a mechanism by which intestinal microbes contribute to the metabolic activation and gut toxicity of TCS, and highlight the importance of considering the contributions of the gut microbiota in evaluating the toxic potential of environmental chemicals.

Bacterial Proteins/antagonists & inhibitors , Carcinogens/antagonists & inhibitors , Colitis/prevention & control , Colorectal Neoplasms/prevention & control , Glucuronidase/antagonists & inhibitors , Glycoside Hydrolase Inhibitors/pharmacology , Triclosan/antagonists & inhibitors , Animals , Anti-Infective Agents, Local/chemistry , Anti-Infective Agents, Local/metabolism , Anti-Infective Agents, Local/toxicity , Anticarcinogenic Agents/chemistry , Anticarcinogenic Agents/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Biotransformation , Carcinogenesis/drug effects , Carcinogenesis/metabolism , Carcinogens/chemistry , Carcinogens/metabolism , Carcinogens/toxicity , Colitis/chemically induced , Colitis/enzymology , Colitis/microbiology , Colon/drug effects , Colon/microbiology , Colon/pathology , Colorectal Neoplasms/chemically induced , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/microbiology , Gastrointestinal Microbiome/drug effects , Gene Expression , Glucuronidase/chemistry , Glucuronidase/genetics , Glucuronidase/metabolism , Glycoside Hydrolase Inhibitors/chemistry , Humans , Mice , Mice, Inbred C57BL , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Triclosan/chemistry , Triclosan/metabolism , Triclosan/toxicity
Microb Cell Fact ; 21(1): 8, 2022 Jan 10.
Article En | MEDLINE | ID: mdl-35012528

BACKGROUND: Phenylpropanoids represent a diverse class of industrially important secondary metabolites, synthesized in plants from phenylalanine and tyrosine. Cyanobacteria have a great potential for sustainable production of phenylpropanoids directly from CO2, due to their photosynthetic lifestyle with a fast growth compared to plants and the ease of generating genetically engineered strains. This study focuses on photosynthetic production of the starting compounds of the phenylpropanoid pathway, trans-cinnamic acid and p-coumaric acid, in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). RESULTS: A selected set of phenylalanine ammonia lyase (PAL) enzymes from different organisms was overexpressed in Synechocystis, and the productivities of the resulting strains compared. To further improve the titer of target compounds, we evaluated the use of stronger expression cassettes for increasing PAL protein levels, as well as knock-out of the laccase gene slr1573, as this was previously reported to prevent degradation of the target compounds in the cell. Finally, to investigate the effect of growth conditions on the production of trans-cinnamic and p-coumaric acids from Synechocystis, cultivation conditions promoting rapid, high density growth were tested. Comparing the different PALs, the highest specific titer was achieved for the strain AtC, expressing PAL from Arabidopsis thaliana. A subsequent increase of protein level did not improve the productivity. Production of target compounds in strains where the slr1573 laccase had been knocked out was found to be lower compared to strains with wild type background, and the Δslr1573 strains exhibited a strong phenotype of slower growth rate and lower pigment content. Application of a high-density cultivation system for the growth of production strains allowed reaching the highest total titers of trans-cinnamic and p-coumaric acids reported so far, at around 0.8 and 0.4 g L-1, respectively, after 4 days. CONCLUSIONS: Production of trans-cinnamic acid, unlike that of p-coumaric acid, is not limited by the protein level of heterologously expressed PAL in Synechocystis. High density cultivation led to higher titres of both products, while knocking out slr1573 did not have a positive effect on production. This work contributes to capability of exploiting the primary metabolism of cyanobacteria for sustainable production of plant phenylpropanoids.

Cinnamates/metabolism , Coumaric Acids/metabolism , Metabolic Engineering , Phenylalanine Ammonia-Lyase/biosynthesis , Phenylalanine Ammonia-Lyase/genetics , Synechocystis/metabolism , Arabidopsis/enzymology , Arabidopsis/genetics , Gene Expression , Phenylalanine Ammonia-Lyase/metabolism , Photosynthesis , Synechocystis/genetics , Synechocystis/growth & development
J Exp Med ; 219(1)2022 01 03.
Article En | MEDLINE | ID: mdl-34817548

Transcription factors (TFs) regulate cell fates, and their expression must be tightly regulated. Autoregulation is assumed to regulate many TFs' own expression to control cell fates. Here, we manipulate and quantify the (auto)regulation of PU.1, a TF controlling hematopoietic stem and progenitor cells (HSPCs), and correlate it to their future fates. We generate transgenic mice allowing both inducible activation of PU.1 and noninvasive quantification of endogenous PU.1 protein expression. The quantified HSPC PU.1 dynamics show that PU.1 up-regulation occurs as a consequence of hematopoietic differentiation independently of direct fast autoregulation. In contrast, inflammatory signaling induces fast PU.1 up-regulation, which does not require PU.1 expression or its binding to its own autoregulatory enhancer. However, the increased PU.1 levels induced by inflammatory signaling cannot be sustained via autoregulation after removal of the signaling stimulus. We conclude that PU.1 overexpression induces HSC differentiation before PU.1 up-regulation, only later generating cell types with intrinsically higher PU.1.

Cell Differentiation/genetics , Hematopoietic Stem Cells/metabolism , Homeostasis/genetics , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Up-Regulation/genetics , Animals , Cells, Cultured , Gene Expression , Male , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence/methods , Proto-Oncogene Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Time-Lapse Imaging/methods , Trans-Activators/metabolism
Methods Mol Biol ; 2401: 79-100, 2022.
Article En | MEDLINE | ID: mdl-34902124

DNA microarray data preprocessing is of utmost importance in the analytical path starting from the experimental design and leading to a reliable biological interpretation. In fact, when all relevant aspects regarding the experimental plan have been considered, the following steps from data quality check to differential analysis will lead to robust, trustworthy results. In this chapter, all the relevant aspects and considerations about microarray preprocessing will be discussed. Preprocessing steps are organized in an orderly manner, from experimental design to quality check and batch effect removal, including the most common visualization methods. Furthermore, we will discuss data representation and differential testing methods with a focus on the most common microarray technologies, such as gene expression and DNA methylation.

Research Design , DNA Methylation , Gene Expression , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis
Methods Mol Biol ; 2401: 187-194, 2022.
Article En | MEDLINE | ID: mdl-34902129

Gene expression profiling is a useful way to measure the activity of genes in molecular biology and, because of its effectiveness, researchers have released thousands of gene expression datasets publicly in online databases and repositories, such as Gene Expression Omnibus (GEO). To read and analyze gene expression data, the computational biology community has developed several tools and platforms, including Bioconductor, an R open-source platform of software packages that can be used to analyze these data. Despite the usefulness of Bioconductor and of its packages, it is still difficult to read gene expression data from GEO, and to assign gene symbols to the probesets of datasets. To alleviate this problem, we introduce here a new R software package, geneExpressionFromGEO, which provides to the users the possibility to easily download gene expression data from GEO and to easily associate gene symbols to probesets. In this short chapter, we describe the assets of our software package, and we report an example of its usage. We believe that geneExpressionFromGEO can be very useful for the R community of bioinformaticians working on gene expression data.

Gene Expression , Computational Biology , Gene Expression Profiling , Reading , Software
Methods Mol Biol ; 2401: 249-261, 2022.
Article En | MEDLINE | ID: mdl-34902133

Microarrays are experimental methods that can provide information about gene expression and SNP data that hold great potential for new understanding, driving advances in functional genomics and clinical and molecular biology. Cluster analysis is used to analyze data that are not a priori to contain any specific subgroup. The goal is to use the data itself to recognize meaningful and informative subgroups. Also, cluster analysis helps data reduction purposes, exposes hidden patterns, and generates hypotheses regarding the relationship between genes and phenotypes. This chapter outlines a collection of cluster methods suitable for the analysis of microarray data sets.

Microarray Analysis , Algorithms , Cluster Analysis , Gene Expression , Gene Expression Profiling , Genomics , Oligonucleotide Array Sequence Analysis
Food Chem ; 371: 131050, 2022 Mar 01.
Article En | MEDLINE | ID: mdl-34537615

Vitamin B3, derived primarily from plant sources, is an essential nutrient for humans. Torreya grandis is rich in vitamin B3, however, the mechanism underlying the biosynthesis and regulation of vitamin B3 in T. grandis remains unclear. A systematic transcriptomic investigation was thus conducted to identify the gene expression pattern of vitamin B3 biosynthesis in 10 T. grandis cultivars. The findings suggest that biosynthesis occurs mainly via the aspartate pathway. Expression and correlation analyses indicate that aspartate oxidase (AOX) and quinolinate synthase (QS) may play important roles in vitamin B3 accumulation. Furthermore, co-expression network and ethephon treatments indicate that the ethylene response factor (ERF) may be involved in the regulation of vitamin B3 biosynthesis in T. grandis nuts. Our findings not only help to elucidate the biosynthesis of vitamin B3, but also provide valuable resource material for future genomic research and molecular-assisted breeding to develop genotypes with higher vitamin B3 levels.

Niacinamide , Nuts , Taxaceae , Ethylenes , Gene Expression , Gene Expression Regulation, Plant , Humans , Nuts/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Vitamins
Hum Genet ; 141(1): 31-47, 2022 Jan.
Article En | MEDLINE | ID: mdl-34907472

The discovery of introns over four decades ago revealed a new vision of genes and their interrupted arrangement. Throughout the years, it has appeared that introns play essential roles in the regulation of gene expression. Unique processing of excised introns through the formation of lariats suggests a widespread role for these molecules in the structure and function of cells. In addition to rapid destruction, these lariats may linger on in the nucleus or may even be exported to the cytoplasm, where they remain stable circular RNAs (circRNAs). Alternative splicing (AS) is a source of diversity in mature transcripts harboring retained introns (RI-mRNAs). Such RNAs may contain one or more entire retained intron(s) (RIs), but they may also have intron fragments resulting from sequential excision of smaller subfragments via recursive splicing (RS), which is characteristic of long introns. There are many potential fates of RI-mRNAs, including their downregulation via nuclear and cytoplasmic surveillance systems and the generation of new protein isoforms with potentially different functions. Various reports have linked the presence of such unprocessed transcripts in mammals to important roles in normal development and in disease-related conditions. In certain human neurological-neuromuscular disorders, including myotonic dystrophy type 2 (DM2), frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS) and Duchenne muscular dystrophy (DMD), peculiar processing of long introns has been identified and is associated with their pathogenic effects. In this review, we discuss different mechanisms involved in the processing of introns during AS and the functions of these large sections of the genome in our biology.

Alternative Splicing , Disease/genetics , Gene Expression , Introns , RNA, Circular/physiology , RNA, Messenger/physiology , Amyotrophic Lateral Sclerosis/genetics , Animals , Cell Nucleus/genetics , Frontotemporal Dementia/genetics , Humans , Mammals/genetics , Muscular Dystrophy, Duchenne/genetics , Myotonic Dystrophy/genetics
Sci Total Environ ; 810: 152260, 2022 Mar 01.
Article En | MEDLINE | ID: mdl-34896498

Weedy rice grows competitively with cultivated rice and significantly diminishes rice grain production worldwide. The different effects of Cu-based nanomaterials on the production of weedy and cultivated rice, especially the grain qualities are not known. Grains were collected from weedy and cultivated rice grown for four months in field soil amended with nanoscale CuO (nCuO), bulk CuO (bCuO), and copper sulfate (CuSO4) at 0, 75, 150, 300, and 600 mg Cu/kg soil. Cu translocation, essential element accumulation, yield, sugar, starch, protein content, and the expression of auxin associated genes in grains were determined. The grains of weedy and cultivated rice were differentially impacted by CuO-based compounds. At ≥300 mg/kg, nCuO and bCuO treated rice had no grain production. Treatment at 75 mg/kg significantly decreased grain yield as compared to control with the order: bCuO (by 88.7%) > CuSO4 (by 47.2%) ~ nCuO (by 38.3% only in cultivated rice); at the same dose, the Cu grain content was: nCuO ~ CuSO4 > bCuO > control. In weedy grains, K, Mg, Zn, and Ca contents were decreased by 75 and 150 mg/kg nCuO by up to 47.4%, 34.3%, 37.6%, and 60.0%, but no such decreases were noted in cultivated rice, and Fe content was increased by up to 88.6%, and 53.2%. In rice spikes, nCuO increased Mg, Ca, Fe, and Zn levels by up to 118.1%, 202.6%, 133.8%, and 103.9%, respectively. Nanoscale CuO at 75 and 150 mg/kg upregulated the transcription of an auxin associated gene by 5.22- and 1.38-fold, respectively, in grains of weedy and cultivated rice. The biodistribution of Cu-based compounds in harvested grain was determined by two-photon microscopy. These findings demonstrate a cultivar-specific and concentration-dependent response of rice to nCuO. A potential use of nCuO at 75 and 150 mg/kg in cultivar-dependent delivery system was suggested based on enhanced grain nutritional quality, although the yield was compromised. This knowledge, at the physiological and molecular level, provides valuable information for the future use of Cu-based nanomaterials in sustainable agriculture.

Nanoparticles , Oryza , Copper/toxicity , Gene Expression , Indoleacetic Acids , Nanoparticles/toxicity , Nutritive Value , Oryza/genetics , Tissue Distribution
Methods Mol Biol ; 2377: 69-88, 2022.
Article En | MEDLINE | ID: mdl-34709611

Studying life-threatening fungal pathogens such as Candida albicans is of critical importance, yet progress can be hindered by challenges associated with manipulating these pathogens genetically. CRISPR-based technologies have significantly improved our ability to manipulate the genomes of countless organisms, including fungal pathogens such as C. albicans. CRISPR interference (CRISPRi) is a modified variation of CRISPR technology that enables the targeted genetic repression of specific genes of interest and can be used as a technique for studying essential genes. We recently developed tools to enable CRISPRi in C. albicans and the repression of essential genes in this fungus. Here, we describe a protocol for CRISPRi in C. albicans, including the design of the single-guide RNAs (sgRNAs) for targeting essential genes, the high-efficiency cloning of sgRNAs into C. albicans-optimized CRISPRi plasmids, transformation into fungal strains, and testing to monitor the repression capabilities of these constructs. Together, this protocol will illuminate efficient strategies for targeted genetic repression of essential genes in C. albicans using a novel CRISPRi platform.

CRISPR-Cas Systems , Candida albicans , CRISPR-Cas Systems/genetics , Candida albicans/genetics , Gene Expression , Genes, Essential , RNA, Guide/genetics
Chem Biol Interact ; 352: 109782, 2022 Jan 25.
Article En | MEDLINE | ID: mdl-34932954

Indole derivatives from various plants are known to have health benefits because of their anti-cancer, anti-oxidant, anti-inflammatory, and anti-tubercular effects. However, their effects on adipogenesis have not been fully elucidated yet. Herein, we show that a newly synthesized indole derivative, CF3-allylated indole, [(E)-1-(pyrimidin- 2-yl)-2-(4,4,4- trifluorobut-2-enyl)-1H-indole], effectively inhibits adipogenesis. We found that CF3-allylated indole inhibited lipid accumulation and suppressed the expression of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator activated receptor γ (PPARγ) in 3T3-L1 cells. The inhibitory effect of CF3-allylated indole primarily occurred at the early phase of adipocyte differentiation by increasing intracellular cyclic adenosine monophosphate (cAMP) levels and enhancing protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) signaling. Conversely, depletion of PKA or treatment with a protein kinase A inhibitor (H89) reversed such inhibitory effects of CF3-allylated indole on adipogenesis and PPARγ expression. These results suggest that CF3-allylated indole inhibits early stages of adipogenesis by increasing phosphorylation of PKA/AMPK, leading to decreased expression of adipogenic genes in 3T3-L1 cells. These results indicate that CF3-allylated indole has potential for controlling initial adipocyte differentiation in metabolic disorders such as obesity.

Adipogenesis/drug effects , Indoles/pharmacology , 3T3-L1 Cells , AMP-Activated Protein Kinases/metabolism , Adipocytes/cytology , Adipocytes/drug effects , Adipocytes/metabolism , Adipogenesis/genetics , Adipogenesis/physiology , Animals , Anti-Obesity Agents/chemistry , Anti-Obesity Agents/pharmacology , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression/drug effects , Indoles/chemistry , Lipid Metabolism/drug effects , Mice , Obesity/drug therapy , Phosphorylation , Signal Transduction/drug effects
J Card Surg ; 37(1): 138-147, 2022 Jan.
Article En | MEDLINE | ID: mdl-34713498

BACKGROUND: Whether perioperative glycemic control is associated with neurocognitive decline (NCD) after cardiac surgery was examined. METHODS: Thirty patients undergoing cardiac surgery utilizing cardiopulmonary bypass (CPB) were screened for NCD preoperatively and on postoperative day 4 (POD4). Indices of glucose control were examined. Serum cytokine levels were measured and human transcriptome analysis was performed on blood samples. Neurocognitive data are presented as a change from baseline to POD4 in a score standardized with respect to age and gender. RESULTS: A decline in neurocognitive function was identified in 73% (22/30) of patients on POD4. There was no difference in neurocognitive function between patients with elevated HbA1c levels preoperatively (p = .973) or elevated fasting blood glucose levels the morning of surgery (>126 mg/dl, p = .910), or a higher maximum blood glucose levels during CPB (>180 mg/dl, p = .252), or higher average glucose levels during CPB (>160 mg/dl, p = .639). Patients with postoperative leukocytosis (WBC ≥ 10.5) had more NCD when compared to their baseline function (p = .03). Patients with elevated IL-8 levels at 6 h postoperatively had a significant decline in NCD at POD4 (p = .04). Human transcriptome analysis demonstrated unique and differential patterns of gene expression in patients depending on the presence of DM and NCD. CONCLUSIONS: Perioperative glycemic control does not have an effect on NCD soon after cardiac surgery. The profile of gene expression was altered in patients with NCD with or without diabetes.

Cardiac Surgical Procedures , Glycemic Control , Cardiopulmonary Bypass , Gene Expression , Humans
J Microbiol ; 60(1): 63-69, 2022 Jan.
Article En | MEDLINE | ID: mdl-34964943

In a previous study, a putative 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD) was highly expressed in a mutant strain of Pyropia yezoensis, which exhibited an improved growth rate compared to its wild strain. To investigate the functional role of the putative ACMSD (Pyacmsd) of P. yezoensis, the putative Pyacmsd was cloned and expressed in Chlamydomonas reinhardtii. Recombinant C. reinhardtii cells with Pyacmsd (Cr_Pyacmsd) exhibited enhanced tolerance compared to control C. reinhardtii cells (Cr_control) under nitrogen starvation. Notably, Cr_Pyacmsd cells showed accumulation of lipids in nitrogen-enriched conditions. These results demonstrate the role of Pyacmsd in the generation of acetyl-coenzyme A. Thus, it can be used to enhance the production of biofuel using microalgae such as C. reinhardtii and increase the tolerance of other biological systems to nitrogen-deficient conditions.

Carboxy-Lyases/genetics , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/metabolism , Gene Expression , Nitrogen/metabolism , Rhodophyta/enzymology , Carboxy-Lyases/metabolism , Cloning, Molecular , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodophyta/genetics
Biochim Biophys Acta Gen Subj ; 1866(1): 130046, 2022 01.
Article En | MEDLINE | ID: mdl-34743989

BACKGROUND: Mucin-type O-glycosylation is one of the most abundant types of O-glycosylation and plays important roles in various human carcinomas, including breast cancer. A large family of polypeptide N-acetyl-α-galactosaminyltransferases (GALNTs) initiate and define sites of mucin-type O-glycosylation. However, the specific mechanisms underlying GALNT8 expression and its roles in tumorigenesis remain poorly characterized. METHODS: GALNT8 expression was assessed in 140 breast cancer patients. Immunofluorescence, immunoprecipitation, lectin blot and quantitative real-time PCR were used to investigate the expression of GALNT8 and its role in regulating estrogen receptor α (ERα) via bone morphogenetic protein (BMP) signaling. RESULTS: The expression of GALNT8 was associated with breast cancer patient survival. GALNT8 downregulation was associated with a reduction in ERα levels, while GALNT8 overexpression elevated the transcription and protein levels of ERα and suppressed colony formation, suggesting an important role of GALNT8 in cancer cell proliferation. Conversely, GALNT8 knockdown led to the inhibition of BMP/SMAD/RUNX2 axis, which decreased ERα transcription. Further analysis suggested that BMP receptor 1A (BMPR1A) was O-GalNAcylated. Sites mutation of BMPR1A indicated that Thr137 and Ser37/Ser39/Ser44/Thr49 of BMPR1A were the main O-glycosylation sites. Although we cannot exclude the indirect effect of GALNT8, our results demonstrated that the expression of GALNT8 and O-glycosylation of BMPR1A play key roles in regulating the activity of BMP/SMAD/RUNX2 signaling and ERα expression. CONCLUSION: These findings suggest that GALNT8 expression and abnormal O-GalNAcylation of BMPR1A increase ERα expression and suppress breast cancer cell proliferation by modulating the BMP signaling pathway. GENERAL SIGNIFICANCE: Our results identify the involvement of GALNT8 in regulating ERα expression.

Bone Morphogenetic Protein Receptors, Type I/metabolism , Estrogen Receptor alpha/metabolism , N-Acetylgalactosaminyltransferases/genetics , Bone Morphogenetic Protein 1/metabolism , Bone Morphogenetic Protein Receptors/genetics , Bone Morphogenetic Protein Receptors/metabolism , Bone Morphogenetic Protein Receptors, Type I/genetics , Breast Neoplasms/metabolism , Carcinogenesis/metabolism , Cell Line, Tumor , Cell Proliferation , Core Binding Factor Alpha 1 Subunit , Databases, Genetic , Estrogen Receptor alpha/genetics , Estrogens/metabolism , Female , Gene Expression/genetics , Glycosylation , Humans , Mucin-1 , N-Acetylgalactosaminyltransferases/metabolism , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Signal Transduction , Transcriptome/genetics