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1.
PLoS One ; 15(5): e0231881, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357186

RESUMO

The sequencing and bioinformatics analysis of bacteriophages infecting mycobacteria has yielded a large amount of information on their evolution, including that on their environmental propagation on other genera such as Gordonia, closely related to Mycobacterium. However, little is known on mycobacteriophages cell biology such as the nature of their receptor(s) or their replication cycle. As part of our on-going screening for novel mycobacteriophages, we herein report the isolation and genome bioinformatics analysis of Weirdo19ES, a singleton Siphoviridae temperate mycobacteriophage with a 70.19% GC content. Nucleotide and protein sequence comparison to actinobacteriophage databases revealed that Weirdo19ES shows low homology to Gordonia phage Ruthy and mycobacteriophages falling in clusters Q and G and to singleton DS6A.Weirdo19ES also displays uncommon features such as a very short Lysin A gene (with only one enzymatic domain) and two putative HNH endonucleases. Mycobacterium smegmatis mutants resistant to Weirdo19ES are cross- resistant to I3. In agreement with that phenotype, analysis of cell envelope of those mutants showed that Weirdo19ES shares receptors with the transducing mycobacteriophage I3.This singleton mycobacteriophage adds up to the uncommonness of local mycobacteriophages previously isolated by our group and helps understanding the nature of mycobacteriophage receptors.


Assuntos
Genoma Viral , Glicolipídeos/genética , Micobacteriófagos/genética , Mycobacterium smegmatis/virologia , Composição de Bases , Parede Celular/metabolismo , Análise por Conglomerados , Uso do Códon , Hibridização Genômica Comparativa , Glicolipídeos/deficiência , Micobacteriófagos/classificação , Micobacteriófagos/isolamento & purificação , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/isolamento & purificação , Fenótipo , Filogenia
2.
PLoS One ; 15(5): e0229700, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379829

RESUMO

One of the most important and exclusive characteristics of mycobacteria is their cell wall. Amongst its constituent components are two related families of glycosylated lipids, diphthioceranates and phthiocerol dimycocerosate (PDIM) and its variant phenolic glycolipids (PGL). PGL have been associated with cell wall impermeability, phagocytosis, defence against nitrosative and oxidative stress and, intriguingly, biofilm formation. In bacteria from the Mycobacterium tuberculosis complex (MTBC), the biosynthetic pathway of the phenolphthiocerol moiety of PGL depends upon the expression of several genes encoding type I polyketide synthases (PKS), namely ppsA-E and pks15/1 which constitute the PDIM + PGL locus, and that are highly conserved in PDIM/PGL-producing strains. Consensus has not been achieved regarding the genetic organization of pks15/1 locus and knowledge is lacking on its transcriptional signature. Here we explore publicly available datasets of transcriptome data (RNA-seq) from more than 100 MTBC experiments in 40 growth conditions to outline the transcriptional structure and signature of pks15/1, using a differential expression approach to infer the regulatory patterns involving these and related genes. We show that pks1 expression is highly correlated with fadD22, Rv2949c, lppX, fadD29 and, also, pks6 and pks12, with the first three putatively integrating into a polycistronic structure. We evidence dynamic transcriptional heterogeneity within the genes involved in phenolphtiocerol and phenolic glycolipid production, most exhibiting up-regulation upon acidic pH and antibiotic exposure and down-regulation under hypoxia, dormancy, and low/high iron concentration. We finally propose a model based on transcriptome data in which σD positively regulates pks1, pks15 and fadD22, while σB and σE factors exert negative regulation at an upper level.


Assuntos
Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Glicolipídeos/biossíntese , Glicolipídeos/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Policetídeo Sintases/genética , Transcriptoma , Parede Celular/metabolismo , Simulação por Computador , Redes Reguladoras de Genes , Loci Gênicos , Genoma Bacteriano/genética , Ligases/genética , RNA-Seq , Virulência/genética
3.
Sci Rep ; 10(1): 6583, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313165

RESUMO

Halophytes are increasingly regarded as suitable extractive species and co-products for coastal Integrated Multi-Trophic Aquaculture (IMTA) and studying their lipidome is a valid means towards their economic valorization. Halimione portulacoides (L.) Aellen edible leaves are rich in functional lipids with nutraceutical and pharmaceutical relevance and the present study aimed to investigate the extent to which its lipidome remains unchanged under a range of dissolved inorganic nitrogen (N) and phosphorus (P) concentrations typical of aquaculture effluents. Lipidomics analysis, done by hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry, identified 175 lipid species in the lipid extract of leaves: 140 phospholipids (PLs) and 35 glycolipids (GLs). Plants irrigated with a saline solution with 20-100 mg DIN-N L-1 and 3-15.5 mg DIP-P L-1 under a 1-week hydraulic retention time displayed a relatively stable lipidome. At lower concentrations (6 mg DIN-N L-1 and 0.8 mg DIP-P L-1), plants exhibited less PLs and GLs per unit of leaves dry weight and the GLs fraction of the lipidome changed significantly. This study reveals the importance of analyzing the lipidomic profile of halophytes under different nutritional regimens in order to establish nutrient-limitation thresholds and assure production conditions that deliver a final product with a consistent lipid profile.


Assuntos
Chenopodiaceae/metabolismo , Lipidômica , Lipídeos/genética , Plantas Tolerantes a Sal/metabolismo , Aquicultura , Chenopodiaceae/genética , Chenopodiaceae/crescimento & desenvolvimento , Glicolipídeos/genética , Glicolipídeos/metabolismo , Humanos , Hidroponia , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/crescimento & desenvolvimento
4.
Yeast ; 37(4): 313-320, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32061177

RESUMO

The Wickerhamiella and Starmerella genera form a clade (W/S clade) that branches close to Yarrowia lipolytica in the Saccharomycotina species tree. It comprises approximately 90 recognized species and 50 putative new species not formally described yet. The large majority of the members of the W/S clade are ecologically associated with flowers and floricolous insects. Many species exhibit unusual metabolic traits, like fructophily and the production of sophorolipids, which are glycolipids that can be used as environmentally friendly biosurfactants. Genomic data have not only firmly established the W/S clade but have also revealed a tumultuous evolution of metabolism marked by losses and gains of important metabolic pathways, among which alcoholic fermentation. Possibly the most surprising finding brought to light by comparative genomics concerned the large number of genes acquired by some species of the W/S clade from bacteria through horizontal gene transfer, many of which were shown to be functional in their new setting. This was facilitated by the genetic tractability of one species in the clade, Starmerella bombicola, which is used for the industrial production of sophorolipids. We suggest that high-density coverage of genome sequencing in this clade, combined with the possibility to conduct molecular genetics experiments in at least one species, has the potential to set the stage for yet more exciting discoveries concerning the evolution of yeast metabolism.


Assuntos
DNA Fúngico/isolamento & purificação , Saccharomycetales/classificação , Saccharomycetales/genética , Animais , DNA Fúngico/genética , Evolução Molecular , Flores/microbiologia , Transferência Genética Horizontal , Glicolipídeos/biossíntese , Glicolipídeos/genética , Insetos/microbiologia , Redes e Vias Metabólicas , Filogenia , Saccharomycetales/isolamento & purificação
5.
Clin Chim Acta ; 501: 27-32, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31770509

RESUMO

Fabry disease (FD [MIM:301500]) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene. Deficient activity of its product, lysosomal enzyme α-galactosidase A (α-Gal A), leads to excessive accumulation of glycosphingolipids in cells of multiple organs. The establishing of the diagnosis is challenge in female patients because of milder clinical manifestation and normal α-Gal A activity. The globotriaosylsphingosine (lysoGb3) is described as a more sensitive diagnostic biomarker for females with pathogenic mutation in the GLA gene. Thus, the aim of this study is to improve the biochemical diagnostic efficiency for FD in females. Here we report the α-Gal A/lysoGb3 ratio as the novel biochemical criteria for diagnosis of female patients with FD, using dried blood spots (DBS) as test samples. It showed 100% sensitivity in distinguishing our group of 35 female patients from control (n = 140). Whereas measurement of α-Gal A and lysoGb3 alone showed 8.6% and 74.4% respectively. A new approach of using the ratio of α-Gal A activity to lysoGb3 concentration in DBS may provide a more accurate screening tool for identification of FD females.


Assuntos
Teste em Amostras de Sangue Seco , Doença de Fabry/sangue , Glicolipídeos/sangue , Esfingolipídeos/sangue , alfa-Galactosidase/sangue , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Criança , Doença de Fabry/diagnóstico , Feminino , Genótipo , Glicolipídeos/genética , Humanos , Pessoa de Meia-Idade , Esfingolipídeos/genética , Adulto Jovem , alfa-Galactosidase/genética
6.
Mar Drugs ; 17(9)2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484443

RESUMO

Phytoplankton are primary producers in the marine ecosystem, where phosphorus is often a limiting factor of their growth. Hence, they have evolved strategies to recycle phosphorus by replacing membrane phospholipids with phosphorus-free lipids. However, mechanisms for replacement of lipid classes remain poorly understood. To improve our understanding, we performed the lipidomic and transcriptomic profiling analyses of an oleaginous marine microalga Nannochloropsis sp. PJ12 in response to phosphorus depletion (PD) and replenishing. In this study, by using (liquid chromatography couple with tandem mass spectrometry) LC-MS/MS-based lipidomic analysis, we show that membrane phospholipid levels are significantly reduced upon PD, while phosphorus-free betaine lipid levels are increased. However, levels of phosphorus-free photosynthetic galactolipid and sulfolipid are not increased upon PD, consistent with the reduced photosynthetic activity. RNA-seq-based transcriptomic analysis indicates that enzymes involved in phospholipid recycling and phosphorus-free lipid synthesis are upregulated, supporting the lipidomic analysis. Furthermore, enzymes involved in FASII (type II fatty acid synthesis) elongation cycle upon PD are transcriptionally downregulated. EPA (eicosapentaenoic acid) level decrease upon PD is revealed by both GC-MS (gas chromatography coupled with mass spectrometry) and LC-MS/MS-based lipidomic analyses. PD-induced alteration is reversed after phosphorus replenishing. Taken together, our results suggest that the alteration of lipid classes upon environmental change of phosphorus is a result of remodeling rather than de novo synthesis in Nannochloropsis sp. PJ12.


Assuntos
Metabolismo dos Lipídeos/efeitos dos fármacos , Microalgas/efeitos dos fármacos , Fósforo/farmacologia , Transcriptoma/efeitos dos fármacos , Cromatografia Líquida/métodos , Ácidos Graxos/genética , Perfilação da Expressão Gênica/métodos , Glicolipídeos/genética , Metabolismo dos Lipídeos/genética , Lipidômica/métodos , Lipídeos/genética , Microalgas/genética , Fosfolipídeos/genética , Fotossíntese/efeitos dos fármacos , Fotossíntese/genética , Fitoplâncton/efeitos dos fármacos , Fitoplâncton/genética , Espectrometria de Massas em Tandem/métodos , Transcriptoma/genética
7.
J Biol Chem ; 294(40): 14526-14545, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31358619

RESUMO

The avian eggshell is a critical physical barrier, which permits extra-uterine development of the embryo. Its formation involves the fastest known biomineralization process in vertebrates. The eggshell consists of proteins and proteoglycans that interact with the mineral phase to impart its specific microstructure and mechanical properties. In this study, we investigated the role of epidermal growth factor (EGF)-like repeats and discoidin-like domains 3 (EDIL3) and milk fat globule-EGF factor 8 (MFGE8), two glycoproteins that are consistently detected in eggshell proteomes. We verified their common evolutionary history and identified the timing of the duplication event giving rise to these two distinct proteins. Edil3/mfge8 chromosomal locations revealed a nested syntenous relationship with other genes (hapln1/hapln3 and vcan/acan) that are also involved in vertebrate calcification. EDIL3 and MFGE8 proteins possess EGF-like and coagulation factor 5/8 (F5/8C) domains, and their 3D structures predicted that they bind calcium and extracellular vesicles. In chicken, we confirmed the presence of EDIL3 and MFGE8 proteins in eggshell, uterine fluid, and uterus. We observed that only edil3 is overexpressed in tissues in which eggshell mineralization takes place and that this overexpression occurs only at the onset of shell calcification. We therefore propose a model in which EDIL3 and, to a lesser extent, MFGE8 proteins guide vesicles containing amorphous calcium carbonate to the mineralization site. This model was supported by the observation that extracellular vesicles accumulate in uterine fluid during eggshell calcification and that they contain high levels of calcium, carbon, and oxygen that correspond to calcium carbonate.


Assuntos
Antígenos de Superfície/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Casca de Ovo/metabolismo , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Proteínas do Leite/metabolismo , Animais , Antígenos de Superfície/química , Antígenos de Superfície/genética , Biomineralização/genética , Calcificação Fisiológica/genética , Carbonato de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Galinhas/genética , Galinhas/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Glicolipídeos/química , Glicolipídeos/genética , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Gotículas Lipídicas , Proteínas do Leite/química , Proteínas do Leite/genética , Proteoma/genética , Proteômica/métodos , Útero/metabolismo
8.
Food Funct ; 10(7): 4256-4268, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31259333

RESUMO

The composition and functions of milk fat globule membrane (MFGM) proteins are important indicators of the nutritional quality of milk. However, these characteristics of MFGM proteins in donkey milk are unknown at different lactation periods. We characterized and identified MFGM proteins in donkey milk at two lactation periods using label-free proteomics. A total of 947 MFGM proteins were found. There were 902 and 913 MFGM proteins in donkey colostrum and mature milk, respectively. The differentially expressed MFGM proteins were classified into different Gene Ontology annotations. The biological process subgroups containing the most MFGM proteins mainly included cellular process, metabolic process, biological regulation, and regulation of biological processes. Donkey MFGM proteins participated in several Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways at different lactation stages, such as endocytosis, thermogenesis, Alzheimer's disease, cancer, and human papillomavirus infection. The knowledge gained in this study may provide theoretical insights and guidance for the future development of novel infant formulae.


Assuntos
Colostro/química , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Leite/química , Proteômica , Animais , Cromatografia Líquida , Equidae , Feminino , Ontologia Genética , Glicolipídeos/genética , Glicoproteínas/genética , Gotículas Lipídicas , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Gravidez , Domínios e Motivos de Interação entre Proteínas , Espectrometria de Massas em Tandem
9.
J Bacteriol ; 201(19)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31308071

RESUMO

Pseudomonas aeruginosa is among the many bacteria that swarm, where groups of cells coordinate to move over surfaces. It has been challenging to determine the behavior of single cells within these high-cell-density swarms. To track individual cells within P. aeruginosa swarms, we imaged a fluorescently labeled subset of the larger population. Single cells at the advancing swarm edge varied in their motility dynamics as a function of time. From these data, we delineated four phases of early swarming prior to the formation of the tendril fractals characteristic of P. aeruginosa swarming by collectively considering both micro- and macroscale data. We determined that the period of greatest single-cell motility does not coincide with the period of greatest collective swarm expansion. We also noted that flagellar, rhamnolipid, and type IV pilus motility mutants exhibit substantially less single-cell motility than the wild type.IMPORTANCE Numerous bacteria exhibit coordinated swarming motion over surfaces. It is often challenging to assess the behavior of single cells within swarming communities due to the limitations of identifying, tracking, and analyzing the traits of swarming cells over time. Here, we show that the behavior of Pseudomonas aeruginosa swarming cells can vary substantially in the earliest phases of swarming. This is important to establish that dynamic behaviors should not be assumed to be constant over long periods when predicting and simulating the actions of swarming bacteria.


Assuntos
Mutação , Pseudomonas aeruginosa/fisiologia , Análise de Célula Única/métodos , Rastreamento de Células , Fímbrias Bacterianas/genética , Flagelos/genética , Fluorescência , Glicolipídeos/genética , Microscopia de Fluorescência , Movimento , Pseudomonas aeruginosa/genética
10.
Intern Med ; 58(4): 603-607, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30333391

RESUMO

Anderson-Fabry disease (AFD) is a rare X-linked disorder caused by deficient activity of the lysosomal enzyme α-galactosidase A (α-GAL A). We herein report 10 cases of AFD in 5 families (3 men and 7 women) that were found to have a specific common mutation in R301Q [G-to-A transition in exon 6 (codon 301) resulting in the replacement of a glutamine with an arginine residue]. We evaluated their clinical characteristics, residual enzymatic activity, and plasma concentrations of globotriaosylsphingosine (Lyso-Gb3). Although all 10 cases had cardiac and renal manifestations in common, their clinical manifestations were markedly divergent despite the same genetic abnormality.


Assuntos
Doença de Fabry/genética , Doença de Fabry/fisiopatologia , Glicolipídeos/genética , Mutação , Esfingolipídeos/genética , alfa-Galactosidase/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Adulto Jovem
11.
Genet Med ; 21(1): 44-52, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29543226

RESUMO

PURPOSE: Plasma globotriaosylsphingosine (lyso-Gb3) is a promising secondary screening biomarker for Fabry disease. Here, we examined its applicability as a primary screening biomarker for classic and late-onset Fabry disease in males and females. METHODS: Between 1 July 2014 and 31 December 2015, we screened 2,359 patients (1,324 males) referred from 168 Japanese specialty clinics (cardiology, nephrology, neurology, and pediatrics), based on clinical symptoms suggestive of Fabry disease. We used the plasma lyso-Gb3 concentration, α-galactosidase A (α-Gal A) activity, and analysis of the α-Gal A gene (GLA) for primary and secondary screens, respectively. RESULTS: Of 8 males with elevated lyso-Gb3 levels (≥2.0 ng ml-1) and low α-Gal A activity (≤4.0 nmol h-1 ml-1), 7 presented a GLA mutation (2 classic and 5 late-onset). Of 14 females with elevated lyso-Gb3, 7 displayed low α-Gal A activity (5 with GLA mutations; 4 classic and 1 late-onset) and 7 exhibited normal α-Gal A activity (1 with a classic GLA mutation and 3 with genetic variants of uncertain significance). CONCLUSION: Plasma lyso-Gb3 is a potential primary screening biomarker for classic and late-onset Fabry disease probands.


Assuntos
Biomarcadores/sangue , Doença de Fabry/sangue , Testes Genéticos , Glicolipídeos/sangue , Esfingolipídeos/sangue , Idoso , Doença de Fabry/genética , Doença de Fabry/patologia , Feminino , Galactosidases/sangue , Galactosidases/genética , Glicolipídeos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Seleção de Pacientes , Fatores de Risco , Esfingolipídeos/genética
12.
Biosci Rep ; 38(6)2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30487163

RESUMO

Tuberculosis caused by Mycobacterium tuberculosis is currently one of the leading causes of death from an infectious agent. The main difficulties encountered in eradicating this bacteria are mainly related to (i) a very complex lipid composition of the bacillus cell wall, (ii) its ability to hide from the immune system inside the granulomas, and (iii) the increasing number of resistant strains. In this context, we were interested in the Rv0646c (lipGMTB ) gene located upstream to the mmaA cluster which is described as being crucial for the production of cell wall components and required for the bacilli adaptation and survival in mouse macrophages. Using biochemical experiments combined with the construction of deletion and overexpression mutant strains in Mycobacterium smegmatis, we found that LipGMTB is a cytoplasmic membrane-associated enzyme that displays both phospholipase and thioesterase activities. Overproduction of LipGMTB decreases the glycopeptidolipids (GPL) level concomitantly to an increase in phosphatidylinositol (PI) which is the precursor of the PI mannoside (PIM), an essential lipid component of the bacterial cell wall. Conversely, deletion of the lipGMS gene in M. smegmatis leads to an overproduction of GPL, and subsequently decreases the strain susceptibility to various antibiotics. All these findings demonstrate that LipG is involved in cell envelope biosynthesis/remodeling, and consequently this enzyme may thus play an important role in mycobacterial physiology.


Assuntos
Parede Celular/enzimologia , Glicopeptídeos/genética , Fosfolipases/genética , Tuberculose/microbiologia , Animais , Antibacterianos/farmacologia , Parede Celular/química , Glicolipídeos/química , Glicolipídeos/genética , Glicopeptídeos/química , Humanos , Macrófagos/enzimologia , Camundongos , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/patogenicidade , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Fosfolipases/química , Tuberculose/enzimologia
13.
Int J Mol Sci ; 19(12)2018 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-30477121

RESUMO

Anderson-Fabry disease (FD) is a rare, progressive, multisystem storage disorder caused by the partial or total deficit of the lysosomal enzyme α-galactosidase A (α-Gal A). It is an X-linked, lysosomal enzymopathy due to mutations in the galactosidase alpha gene (GLA), encoding the α-Gal A. To date, more than 900 mutations in this gene have been described. In our laboratories, the study of genetic and enzymatic alterations related to FD was performed in about 17,000 subjects with a symptomatology referable to this disorder. The accumulation of globotriaosylsphingosine (LysoGb3) was determined in blood of positives. Exonic mutations in the GLA gene were detected in 471 patients (207 Probands and 264 relatives): 71.6% of mutations were associated with the classic phenotype, 19.8% were associated with the late-onset phenotype, and 8.6% of genetic variants were of unknown significance (GVUS). The accumulation of LysoGb3 was found in all male patients with a mutation responsible for classic or late-onset FD. LysoGb3 levels were consistent with the type of mutations and the symptomatology of patients. α-Gal A activity in these patients is absent or dramatically reduced. In recent years, confusion about the pathogenicity of some mutations led to an association between non-causative mutations and FD. Our study shows that the identification of FD patients is possible by associating clinical history, GLA gene analysis, α-Gal A assay, and blood accumulation of LysoGB3. In our experience, LysoGB3 can be considered a reliable marker, which is very useful to confirm the diagnosis of Fabry disease.


Assuntos
Doença de Fabry/genética , Glicolipídeos/genética , Mutação , Esfingolipídeos/genética , alfa-Galactosidase/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Substituição de Aminoácidos , Biomarcadores , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Fenótipo , Adulto Jovem
14.
Artigo em Inglês | MEDLINE | ID: mdl-30041907

RESUMO

INTRODUCTION: The only known non-pharmacological means to alter long chain polyunsaturated fatty acid (LCPUFA) abundance in mammalian tissue is by altering substrate fatty acid ratios. Alternative mRNA splicing is increasingly recognized as a modulator of protein structure and function. Here we report identification of a novel alternative transcript (AT) of fatty acid desaturase 2 (FADS2) that inhibits production of omega-3 but not omega-6 LCPUFA, discovered during study of ATs in human milk fat globules (MFG). METHODS: Human breastmilk collected from a single donor was used to isolate MFG. An mRNA-sequencing library was constructed from the total RNA isolated from the MFG. The constructed library was sequenced using an Illumina HiSeq instrument operating in high output mode. Expression levels of evolutionary conserved FADSAT were measured using cDNA from MFG by semi-quantitative RT-PCR assay. RESULTS: RNA sequencing revealed >15,000 transcripts, including moderate expression of the FADS2 classical transcript (CS). A novel FADS2 alternative transcript (FADS2AT2) with 386 amino acids was discovered. When FADS2AT2 was transiently transfected into MCF7 cells stably expressing FADS2, delta-6 desaturation (D6D) of alpha-linolenic acid 18:3n-3 → 18:4n-3 was suppressed as were downstream products 20:4n-3 and 20:5n-3. In contrast, no significant effect on D6D of linoleic acid 18:2n-6 → 18:3n-6 or downstream products was observed. FADS2, FADS2AT1 and 5 out of 8 known FADS3AT were expressed in MFG. FADS1, FADS3AT3, and FADS3AT5 are undetectable. CONCLUSION: The novel, noncatalytic FADS2AT2 regulates FADS2CS-mediated Δ6-desaturation of omega-3 but not omega-6 PUFA biosynthesis. This spliced isoform mediated interaction is the first molecular mechanism by which desaturation of one PUFA family but not the other is modulated.


Assuntos
Ácidos Graxos Dessaturases/metabolismo , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Leite Humano/enzimologia , Ácido alfa-Linoleico/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Ômega-6/genética , Ácidos Graxos Ômega-6/metabolismo , Glicolipídeos/genética , Glicoproteínas/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Gotículas Lipídicas , Células MCF-7 , Ácido alfa-Linoleico/genética
15.
Appl Microbiol Biotechnol ; 102(19): 8537-8549, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29992435

RESUMO

This study aimed to identify and characterise biosurfactant compounds produced by bacteria associated with a marine eukaryotic phytoplankton bloom. One strain, designated MCTG214(3b1), was isolated by enrichment with polycyclic aromatic hydrocarbons and based on 16S rDNA, and gyrB sequencing was found to belong to the genus Pseudomonas, however not related to P. aeruginosa. Cell-free supernatant samples of strain MCTG214(3b1) at stationary phase showed significant reductions in surface tension. HPLC-MS and NMR analysis of these samples indicated the presence of five different rhamnolipid (RL) congeners. Di-rhamnolipids accounted for 87% relative abundance and all congeners possessed fatty acid moieties consisting of 8-12 carbons. PCR screening of strain MCTG214(3b1) DNA revealed homologues to the P. aeruginosa RL synthesis genes rhlA and rhlB; however, no rhlC homologue was identified. Using the Galleria mellonella larvae model, strain MCTG214(3b1) was demonstrated to be far less pathogenic than P. aeruginosa. This study identifies for the first time a significantly high level of synthesis of short chain di-rhamnolipids by a non-pathogenic marine Pseudomonas species. We postulate that RL synthesis in Pseudomonas sp. MCTG214(3b1) is carried out by enzymes expressed from rhlA/B homologues similar to those of P. aeruginosa; however, a lack of rhlC potentially indicates the presence of a second novel rhamnosyltransferase responsible for the di-rhamnolipid congeners identified by HPLC-MS.


Assuntos
Proteínas de Bactérias/metabolismo , Glicolipídeos/biossíntese , Glicolipídeos/química , Pseudomonas/metabolismo , Água do Mar/microbiologia , Tensoativos/metabolismo , Animais , Proteínas de Bactérias/genética , DNA Girase/genética , Glicolipídeos/genética , Pseudomonas/química , Pseudomonas/classificação , Pseudomonas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
16.
Appl Microbiol Biotechnol ; 102(16): 6877-6884, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29926140

RESUMO

Mannosylerythritol lipids (MELs) are a type of glycolipid biosurfactant produced by basidiomycetous yeasts, most notably those belonging to the genera Pseudozyma and Ustilago. Mannosylerythritol lipids are environmentally friendly and possess many unique functions, such as gene delivery, bio-activation, and human skin repair, and thus have potential applications in cosmetic, pharmaceutical, agriculture, food, and environmental industries. However, MELs will require overcoming same issues related to the commercialization, e.g., expansion of the structure and function variety and cost reduction. In the past decade, various studies have attempted to tailor production of targeted MELs in order to expand the utility of these biosurfactants. Moreover, the rapid development of genomic sequencing techniques will enhance our ability to modify MEL producers. In this review, we focus on current research into the tailored production of MELs, including conventional and advanced approaches.


Assuntos
Basidiomycota/genética , Basidiomycota/metabolismo , Glicolipídeos/biossíntese , Glicolipídeos/genética , Ustilago/genética , Ustilago/metabolismo , Cosméticos , Tensoativos
17.
PLoS Negl Trop Dis ; 12(6): e0006532, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29953440

RESUMO

Mycobacterium leprae (M. leprae) is a human pathogen and the causative agent for leprosy, a chronic disease characterized by lesions of the skin and peripheral nerve damage. Zoonotic transmission of M. leprae to humans by nine-banded armadillos (Dasypus novemcinctus) has been shown to occur in the southern United States, mainly in Texas, Louisiana, and Florida. Nine-banded armadillos are also common in South America, and residents living in some areas in Brazil hunt and kill armadillos as a dietary source of protein. This study examines the extent of M. leprae infection in wild armadillos and whether these New World mammals may be a natural reservoir for leprosy transmission in Brazil, similar to the situation in the southern states of the U.S. The presence of the M. leprae-specific repetitive sequence RLEP was detected by PCR amplification in purified DNA extracted from armadillo spleen and liver tissue samples. A positive RLEP signal was confirmed in 62% of the armadillos (10/16), indicating high rates of infection with M. leprae. Immunohistochemistry of sections of infected armadillo spleens revealed mycobacterial DNA and cell wall constituents in situ detected by SYBR Gold and auramine/rhodamine staining techniques, respectively. The M. leprae-specific antigen, phenolic glycolipid I (PGL-I) was detected in spleen sections using a rabbit polyclonal antibody specific for PGL-I. Anti-PGL-I titers were assessed by ELISA in sera from 146 inhabitants of Belterra, a hyperendemic city located in western Pará state in Brazil. A positive anti-PGL-I titer is a known biomarker for M. leprae infection in both humans and armadillos. Individuals who consumed armadillo meat most frequently (more than once per month) showed a significantly higher anti-PGL-I titer than those who did not eat or ate less frequently than once per month. Armadillos infected with M. leprae represent a potential environmental reservoir. Consequently, people who hunt, kill, or process or eat armadillo meat are at a higher risk for infection with M. leprae from these animals.


Assuntos
Antígenos de Bactérias/imunologia , Tatus/microbiologia , Reservatórios de Doenças/microbiologia , Glicolipídeos/imunologia , Hanseníase/transmissão , Carne/microbiologia , Mycobacterium leprae/isolamento & purificação , Adulto , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Brasil/epidemiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Glicolipídeos/genética , Glicolipídeos/isolamento & purificação , Humanos , Hanseníase/epidemiologia , Hanseníase/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Reação em Cadeia da Polimerase , Coelhos , Risco , Baço/microbiologia , Adulto Jovem , Zoonoses
18.
Methods Mol Biol ; 1772: 95-123, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29754224

RESUMO

In this chapter, a step-by-step approach on how to transform non-conventional yeasts or fungi into platform organisms is described. The non-conventional glycolipid producing yeast Starmerella bombicola (and in some cases also Pseudohyphozyma bogoriensis) is used as a case study. And more specifically how to engineer it toward production of new-to-nature glycolipids like bola sophorolipids. When starting genetic engineering efforts for non-lab strains, one should start at the very basis: identifying selection markers and possibly developing auxotrophic strains. Once this is done, the quest for the development of an optimal transformation method can be started. After optimization thereof, knock-out and knock-in strains can be generated based upon the specific strategy/aim. Sometimes this can lead to unexpected, but yet very interesting findings. To fully and efficiently expand the potential as a production platform of these yeast strains, a range of additional molecular tools are required. A well-equipped molecular toolbox should contain a set of characterized promotors, terminators, and defined genomic landing paths. The availability of an episomal system greatly facilitates engineering and screening efforts, but also offers the possibility of developing more advanced engineering techniques such as Crispr-Cas. InBio.be is a world leading pioneer to do this for the yeast S. bombicola and combined, these efforts will result in the commercialization of new types of glycolipids in the next few years.


Assuntos
Glicolipídeos/genética , Leveduras/genética , Engenharia Genética/métodos , Regiões Promotoras Genéticas/genética , Regiões Terminadoras Genéticas/genética
19.
Mol Ecol ; 27(13): 2871-2883, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29772096

RESUMO

Positive selection acting on Toll-like receptors (TLRs) has been recently investigated to reveal evolutionary mechanisms of host-pathogen molecular co-adaptation. Much of this research, however, has focused mainly on the identification of sites predicted to be under positive selection, bringing little insight into the functional differences and similarities among species and a limited understanding of convergent evolution in the innate immune molecules. In this study, we provide evidence of phenotypic variability in the avian TLR4 ligand-binding region (LBR), the direct interface between host and pathogen molecular structures. We show that 55 passerine species vary substantially in the distribution of electrostatic potential on the surface of the receptor, and based on these distinct patterns, we identified four species clusters. Seven of the 34 evolutionarily nonconservative and positively selected residues correspond topologically to sites previously identified as being important for lipopolysaccharide, lipid IVa or MD-2 binding. Five of these positions codetermine the identity of the charge clusters. Groups of species that host-related communities of pathogens were predicted to cluster based on their TLR4 LBR charge. Despite some evidence for convergence among taxa, there were no clear associations between the TLR4 LBR charge distribution and any of the general ecological characteristics compared (migration, latitudinal distribution and diet). Closely related species, however, mostly belonged to the same surface charge cluster indicating that phylogenetic constraints are key determinants shaping TLR4 adaptive evolution. Our results suggest that host innate immune evolution is consistent with Fahrenholz's rule on the cospeciation of hosts and their parasites.


Assuntos
Evolução Molecular , Interações Hospedeiro-Patógeno/genética , Seleção Genética , Receptor 4 Toll-Like/genética , Animais , Aves/genética , Aves/parasitologia , Glicolipídeos/química , Glicolipídeos/genética , Imunidade Inata/genética , Ligantes , Lipídeo A/análogos & derivados , Lipídeo A/química , Lipídeo A/genética , Lipopolissacarídeos/química , Lipopolissacarídeos/genética , Antígeno 96 de Linfócito/química , Antígeno 96 de Linfócito/genética , Microbiota/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Seleção Genética/genética , Análise de Sequência de DNA , Eletricidade Estática , Receptor 4 Toll-Like/química
20.
Cell Physiol Biochem ; 47(1): 378-389, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29794418

RESUMO

BACKGROUND/AIMS: The adverse effects of obesity on male fertility have been widely reported. In recent years, the relationship between the differential expression of proteins and long non-coding RNAs with male reproductive disease has been reported. However, the exact mechanism in underlying obesity-induced decreased male fertility remains unclear. METHODS: We used isobaric tags for relative and absolute quantification to identify differential protein expression patterns in the testis of rats fed a high-fat diet and normal diet. A microarray-based gene expression analysis protocol was used to compare the differences in long non-coding RNAs in high-fat diet-fed and normal diet-fed rats. Five obviously upregulated or downregulated proteins were examined using western blot to verify the accuracy of their expression. Then, we carried out functional enrichment analysis of the differentially expressed proteins using gene ontology and pathway analysis. Finally, the metabolic Gene Ontology terms and pathways involved in the differential metabolites were analyzed using the MetaboAnalyst 2.0 software to explore the co-expression relationship between long non-coding RNAs and proteins. RESULTS: We found 107 proteins and 263 long non-coding RNAs differentially expressed between rats fed a high-fat diet and normal diet. The Gene Ontology term enrichment analysis showed that the protein function most highly enriched was related to negative regulation of reproductive processes. We also found five Gene Ontology terms and two metabolic pathways upregulated or downregulated for both proteins and long non-coding RNAs. CONCLUSION: The study revealed different expression levels for both proteins and long non-coding RNAs and showed that the function and metabolic pathways of differently expressed proteins were related to reproductive processes. The Gene Ontology terms and metabolic pathways upregulated or downregulated in both proteins and long non-coding RNAs may provide new candidates to explore the mechanisms of obesity-induced male infertility for both protein and epigenetic pathways.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Perfilação da Expressão Gênica , Obesidade/etiologia , Obesidade/genética , Testículo/metabolismo , Animais , Peso Corporal , Ontologia Genética , Glicolipídeos/genética , Glicolipídeos/metabolismo , Masculino , Redes e Vias Metabólicas , Obesidade/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteômica , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ratos , Ratos Sprague-Dawley , Sêmen/metabolismo , Testículo/ultraestrutura
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