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1.
Int J Food Microbiol ; 317: 108498, 2020 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-31918247

RESUMO

Aspergillus carbonarius consistently produces large amounts of ochratoxin A (OTA), a mycotoxin with nephrotoxic effects on animals and humans. In the present study, we analyzed the transcriptional changes associated to OTA production in three atypical non-ochratoxigenic strains of A. carbonarius. In addition, in vitro interactions between ochratoxigenic strains of A. carbonarius and A. niger and non-ochratoxigenic strains of A. carbonarius and A. tubingensis were studied in order to evaluate their potential for controlling OTA production. RNA-seq analysis revealed that there are 696 differentially expressed genes identified in the three non-OTA-producing strains, including 280 up-regulated and 333 down-regulated genes. A functional and gene ontology enrichment analysis revealed that the processes related to metabolic and oxidation processes, associated with functions such as oxidoreductase and hydrolase activity were down regulated. All the genes related with OTA biosynthesis in A. carbonarius were the most down-regulated genes in non-ochratoxigenic strains. We also showed that these strains possess a deleterious mutation in the AcOTApks gene required for OTA biosynthesis. Moreover, one of these strains gave the best control of OTA production resulting in an OTA reduction of 98-100% in co-inoculation with an ochratoxigenic strain of A. niger and an OTA reduction of 79-89% with an ochratoxigenic strain of A. carbonarius. Results of this study provided novel insights into the knowledge of the OTA biosynthetic pathway in these non-ochratoxigenic wild strains, and showed the biocontrol potential of these strains.


Assuntos
Aspergillus/genética , Aspergillus/metabolismo , Agentes de Controle Biológico/metabolismo , Interações Microbianas/fisiologia , Aspergillus/classificação , Perfilação da Expressão Gênica , Humanos , Hidrolases/metabolismo , Ocratoxinas/biossíntese , Oxirredutases/metabolismo , Vitis/microbiologia
2.
Artigo em Inglês | MEDLINE | ID: mdl-31990641

RESUMO

Condensed phosphates are deliberately added to the fishery and processed marine food products on purpose to increase the weight of products. However, excessive intake overwhelming bodily homoeostasis can result in phosphate toxicity such as mineral and bone disorders, associated with chronic kidney diseases, and cardiovascular events. Rapid analysis for condensed phosphates is required to detect the illegal adulteration of processed marine products. We optimised an analytical method using ion chromatography for the rapid and selective detection of condensed phosphates in various kinds of fishery and processed marine products. We compared the performance of three columns (IonPac AS11, AS11-HC, and AS16) for the detection of condensed phosphates with respect to time of analysis and sensitivity. The IonPac AS11 column exhibited the shortest retention time for the major condensed phosphates (pyro-, tri-, and trimetaphosphate), the highest sensitivity for trimetaphosphate, and good repeatability and precision. Microwave and boiling processing were examined as methods to prevent hydrolysis of condensed phosphates into orthophosphate, which is critical in distinguishing intentionally added condensed phosphates from naturally occurring orthophosphate. Microwave treatment was determined to be the more effective method to suppress hydrolysis of condensed phosphates to orthophosphate. Furthermore, microwave processing could be used for thawing the frozen samples, saving extra effort and time. We confirmed that the method exhibits good recovery (80% or more) and precision (%RSD < 10%) for samples with various matrices. With the method, 14 kinds of fishery and processed marine food products were successfully analysed for condensed phosphates. Especially, we identified that phosphate levels in the processed shrimp and dried shredded squid samples exceeded the maximum allowable levels specified in the CODEX standard. We believe that our method would be useful for the rapid analysis of condensed phosphates in various types of fishery and processed marine food products.


Assuntos
Fosfatos/análise , Animais , Cromatografia por Troca Iônica , Pesqueiros , Hidrolases/metabolismo , Limite de Detecção , Micro-Ondas , Alimentos Marinhos , Sensibilidade e Especificidade , Extração em Fase Sólida
3.
Ecotoxicol Environ Saf ; 190: 110148, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31911388

RESUMO

Phthalate esters have raised public concerns owing to their effects on the environment and human health. We identified a novel phthalate-degrading hydrolase, EstJ6, from a metagenomic library using function-driven screening. Phylogenetic analysis indicated that EstJ6 is a member of family IV esterases. EstJ6 hydrolyzed various dialkyl and monoalkyl phthalate esters, and exhibited high hydrolytic activity (128 U/mg) toward dibutyl phthalate at 40 °C and pH 7.5. EstJ6 hydrolyzed not only common phthalate esters with simple side chains but also diethylhexyl phthalate and monoethylhexyl phthalate, which have complex and long side chains. Site-directed mutagenesis indicated that the catalytic triad residues of EstJ6 consists of Ser146, Glu240, and His270. EstJ6 is therefore a promising biodegradation enzyme, and our study illustrates the advantages of a metagenomic approach in identifying enzyme-coding genes for agricultural, food, and biotechnological applications.


Assuntos
Biodegradação Ambiental , Hidrolases/metabolismo , Ácidos Ftálicos/metabolismo , Dibutilftalato/metabolismo , Dietilexilftalato/metabolismo , Esterases/metabolismo , Ésteres/química , Biblioteca Gênica , Hidrolases/genética , Hidrólise , Metagenoma , Filogenia , Solo
4.
Rev Soc Bras Med Trop ; 53: e20190336, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31994664

RESUMO

INTRODUCTION: Candida parapsilosis complex species differ from each other with regard to their prevalence and virulence. METHODS: The hydrolytic enzyme activity, biofilm production, and adhesion to epithelial cells were analyzed in 87 C. parapsilosis complex strains. RESULTS: Among the studied isolates, 97.7%, 63.2%, and 82.8% exhibited very strong proteinase, esterase, and hemolysin activity, respectively. All the C. parapsilosis complex isolates produced biofilms and presented an average adherence of 96.0 yeasts/100 epithelial cells. CONCLUSIONS: Our results show that Candida parapsilosis complex isolates showed different levels of enzyme activity, biofilm production, and adhesion to epithelial cells.


Assuntos
Candida parapsilosis/patogenicidade , Fatores de Virulência/análise , Biofilmes/crescimento & desenvolvimento , Candida parapsilosis/classificação , Candida parapsilosis/enzimologia , Candida parapsilosis/isolamento & purificação , Adesão Celular , Humanos , Hidrolases/biossíntese , Técnicas de Tipagem Micológica
5.
Appl Microbiol Biotechnol ; 104(5): 2067-2077, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31932896

RESUMO

Halohydrin dehalogenases (HHDHs) have attracted much attention due to their ability to synthesize enantiomerically enriched epoxides and ß-haloalcohols. However, most of the HHDHs exhibit low enantioselectivity. Here, a HHDH from the alphaproteobacteria isolate 46_93_T64 (AbHHDH), which shows only poor enantioselectivity in the catalytic resolution of rac-PGE (E = 9.9), has been subjected to protein engineering to enhance its enantioselectivity. Eight mutants (R89K, R89Y, V137I, P178A, N179Q, N179L, F187L, F187A) showed better enantioselectivity than the wild type. The best single mutant N179L (E = 93.0) showed a remarkable 9.4-fold increase in the enantioselectivity. Then, the single mutations were combined to produce the double, triple, quadruple, and quintuple mutants. Among the combinational mutants, the best variant (R89Y/N179L) showed an increased E value of up to 48. The E values of the variants N179L and R89Y/N179L for other epoxides 2-7 were 12.2 to > 200, which showed great improvement compared to 1.2 to 10.5 for the wild type. Using the variant N179L, enantiopure (R)-PGE with > 99% ee could be readily prepared, affording a high yield and a high concentration.


Assuntos
Proteínas de Bactérias/metabolismo , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Hidrolases/metabolismo , Alphaproteobacteria/enzimologia , Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Hidrolases/química , Hidrolases/genética , Hidrolases/isolamento & purificação , Cinética , Modelos Moleculares , Mutação , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por Substrato
6.
Sci Total Environ ; 709: 135897, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-31887512

RESUMO

The danger of zearalenone (ZEN) as an endocrine disruptor to humans and the environment has aroused increasing attention. In this study, we implemented the quantum mechanics/molecular mechanics (QM/MM) method to investigate the degradation mechanism of ZEN hydrolase (RmZHD) toward ZEN at the atomic level. The degradation process involves two concerted reaction pathways, where the active site contains a Ser-His-Glu triplet as a proton donor. With the Boltzmann-weighted average potential barriers of 18.1 and 21.5 kcal/mol, the process undergoes proton transfer and nucleophilic-substituted ring opening to form a hydroxyl product. Non-covalent interaction analyses elucidated hydrogen bonding between key amino acids with ZEN. The electrostatic influence analysis of 16 amino acids proposes residues Asp34 and His128 as the possible mutation target for future mutation design of enzyme RmZHD. An in-depth investigation of the protein environment of RmZHD can improve the bioremediation efficiency of endocrine disrupting chemicals.


Assuntos
Zearalenona/metabolismo , Ligações de Hidrogênio , Hidrolases , Simulação de Dinâmica Molecular , Prótons , Zearalenona/química
7.
Chemosphere ; 238: 124594, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31445334

RESUMO

Enhancing the biodegradation efficiency of atrazine, a kind of commonly applied herbicide, has been attracted much more concern. Here, Zn2+ which has long been considered essential in adjusting cell physiological status was selected to investigate its role on the biodegradation of atrazine by Arthrobacter sp. DNS10 as well as the transmembrane transport of atrazine during the biodegradation period. The results of gas chromatography showed that the atrazine removal percentages (initial concentration was 100 mg L-1) in 0.05 mM Zn2+ and 1.0 mM Zn2+ treatments were 94.42% and 86.02% respectively at 48 h, while there was also 66.43% of atrazine left in the treatment without exogenous Zn2+ existence. The expression of atrazine chlorohydrolase gene trzN in the strain DNS10 cultured with 0.05 mM and 1.0 mM Zn2+ was 7.30- and 4.67- times respectively compared with that of the non-zinc treatment. In addition, the flow cytometry test suggests that 0.05 mM of Zn2+ could better adjust the membrane permeability of strain DNS10, meanwhile, the amount of atrazine accumulation in the strain DNS10 co-cultured with this level Zn2+ was 2.21 times of that of the strain without Zn2+. This study may facilitate a better understanding of the mechanisms that exogenous Zn2+ enhances the biodegradation of atrazine by Arthrobacter sp. DNS10.


Assuntos
Arthrobacter/metabolismo , Atrazina/análise , Herbicidas/análise , Hidrolases/biossíntese , Zinco/farmacologia , Biodegradação Ambiental/efeitos dos fármacos , Transporte Biológico/fisiologia , Hidrolases/genética , Permeabilidade/efeitos dos fármacos , Microbiologia do Solo
8.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140302, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678195

RESUMO

Bacteriophage endolysins have the potential to be a long-term antibacterial replacement for antibiotics. The exogenous application of endolysins on some bacteria results in rapid cell lysis. The prospects for endolysins are furthered by the ability to engineer them; novel endolysins can be developed with optimised stability, specificity, and lytic function. But the success of endolysin engineering and application requires a comprehensive understanding of the relationship between the enzymes biochemical, biophysical and bacteriolytic properties. Here, we examine their catalytic mechanisms, opportunities for developing novel endolysins, and highlight areas where a better understanding would support their long-term success as antibacterial agents.


Assuntos
Antibacterianos/química , Bacteriófagos/enzimologia , Hidrolases/química , Catálise , Engenharia de Proteínas
9.
Chin J Physiol ; 62(6): 273-278, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31793464

RESUMO

The aim of the study was to assess the effect of one session of high-intensity laser therapy (HILT) on the levels of selected oxidative stress parameters, lysosomal hydrolases, and anti-inflammatory serine protease inhibitor in the peripheral blood of amateur athletes with torn or pulled tendons of the ankle or the knee joint. The group of injured athletes comprised 16 males and females aged 16.3 ± 1.3 years, while the control group of 14 healthy, noninjured amateur athletes of both sexes (controls; age 17.4 ± 4.6 years). Material for the study was peripheral blood taken at three study time points: Immediately before, 30 min after, and 24 h after HILT intervention. In plasma and erythrocytes, thiobarbituric acid reactive substances (TBARSpl and TBARSer, respectively) were determined. In erythrocytes, the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were measured. In serum, the activity of acid phosphatase (AcP), arylsulfatase (ASA), cathepsin D (CTS D), and α1-antitrypsin (AAT) were determined. Among oxidative stress parameters, only the CAT activity significantly decreased 24 h after HILT compared to measurement 30 min after the treatment in the injured individuals (P < 0.01), while the GPx activity in that group was meaningfully higher 30 min after HILT compared to controls (P < 0.05). Thirty min after the intervention, the activities of AcP and ASA were lower in the injured participants compared to the uninjured ones (P < 0.01 and P < 0.05, respectively). The CTS D activity was lower 30 min and 24 h after HILT in both groups (P < 0.001) and did not differ significantly between them (P > 0.05). Moreover, the study showed statistically significant linear relationships between the TBARSer concentration and the SOD activity before HILT in the healthy participants (r = -0.6, P = 0.021) and 24 h after HILT in the injured ones (r = 0.6, P = 0.025). In the noninjured athletes before HILT, the CTS D activity linearly correlated with the AAT activity (r = -0.70, P = 0.005), and 30 min after the treatment, with the AcP activity (r = 0.5, P = 0.041). 24 h after the HILT intervention, the CTS D and AcP activities were also correlated in the injured athletes (r = 0.8, P = 0.002). The study suggests that one HILT intervention does not significantly influence the redox equilibrium but stabilizes lysosomal membranes.


Assuntos
Terapia a Laser , Estresse Oxidativo , Adolescente , Antioxidantes , Atletas , Catalase , Feminino , Glutationa Peroxidase , Humanos , Hidrolases , Lisossomos , Masculino , Inibidores de Proteases , Superóxido Dismutase
10.
Science ; 366(6469): 1072-1073, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31780543
11.
Acta Crystallogr D Struct Biol ; 75(Pt 11): 995-1002, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31692473

RESUMO

Atrazine is an s-triazine-based herbicide that is used in many countries around the world in many millions of tons per year. A small number of organisms, such as Pseudomonas sp. strain ADP, have evolved to use this modified s-triazine as a food source, and the various genes required to metabolize atrazine can be found on a single plasmid. The atomic structures of seven of the eight proteins involved in the breakdown of atrazine by Pseudomonas sp. strain ADP have been determined by X-ray crystallography, but the structures of the proteins required by the cell to import atrazine for use as an energy source are still lacking. The structure of AtzT, a periplasmic binding protein that may be involved in the transport of a derivative of atrazine, 2-hydroxyatrazine, into the cell for mineralization, has now been determined. The structure was determined by SAD phasing using an ethylmercury phosphate derivative that diffracted X-rays to beyond 1.9 Šresolution. `Native' (guanine-bound) and 2-hydroxyatrazine-bound structures were also determined to high resolution (1.67 and 1.65 Å, respectively), showing that 2-hydroxyatrazine binds in a similar way to the purportedly native ligand. Structural similarities led to the belief that it may be possible to evolve AtzT from a purine-binding protein to a protein that can bind and detect atrazine in the environment.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Cristalografia por Raios X/métodos , Hidrolases/química , Proteínas Periplásmicas de Ligação/química , Atrazina/análogos & derivados , Atrazina/metabolismo , Estrutura Terciária de Proteína , Pseudomonas/metabolismo
12.
Nat Chem Biol ; 15(11): 1120-1128, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31636435

RESUMO

Characterizing the adaptive landscapes that encompass the emergence of novel enzyme functions can provide molecular insights into both enzymatic and evolutionary mechanisms. Here, we combine ancestral protein reconstruction with biochemical, structural and mutational analyses to characterize the functional evolution of methyl-parathion hydrolase (MPH), an organophosphate-degrading enzyme. We identify five mutations that are necessary and sufficient for the evolution of MPH from an ancestral dihydrocoumarin hydrolase. In-depth analyses of the adaptive landscapes encompassing this evolutionary transition revealed that the mutations form a complex interaction network, defined in part by higher-order epistasis, that constrained the adaptive pathways available. By also characterizing the adaptive landscapes in terms of their functional activities towards three additional organophosphate substrates, we reveal that subtle differences in the polarity of the substrate substituents drastically alter the network of epistatic interactions. Our work suggests that the mutations function collectively to enable substrate recognition via subtle structural repositioning.


Assuntos
Epistasia Genética , Hidrolases/metabolismo , Metil Paration/metabolismo , Xenobióticos/metabolismo
13.
Biofouling ; 35(8): 883-899, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31663364

RESUMO

Biofilm growth is a significant source of contamination in the food industry. Enzymes are considered green countermeasures against biofilm formation in the food industry owing to their biodegradability and low toxicity. In this study, the synergistic effect of enzymes was studied against biofilm cleaning from hard surfaces. A mixed-microbial sample was sourced from a meat packaging line and biofilms were grown under high shear conditions on stainless steel and polyethylene surfaces. A model cleaning-in-place (CIP) parallel-plate flow chamber was used for firstly, the enzymatic cleaning and secondly, a disinfection step. The cleaning effectiveness was evaluated in response to different formulations containing non-foaming commercial surfactants among with amylase, protease and lipase at neutral pH. The formulation combining all three enzymes was the most effective, showing a synergy essential for the deformation of biofilm structure and consequently better disinfection of both material surfaces.


Assuntos
Biofilmes/crescimento & desenvolvimento , Detergentes/química , Desinfecção/métodos , Indústria de Processamento de Alimentos/normas , Hidrolases/química , Saneamento/métodos , Incrustação Biológica/prevenção & controle , Modelos Teóricos , Plásticos , Aço Inoxidável , Propriedades de Superfície
14.
BMC Genomics ; 20(1): 722, 2019 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-31585525

RESUMO

BACKGROUND: Nicotiana benthamiana is an important model organism of the Solanaceae (Nightshade) family. Several draft assemblies of the N. benthamiana genome have been generated, but many of the gene-models in these draft assemblies appear incorrect. RESULTS: Here we present an improved proteome based on the Niben1.0.1 draft genome assembly guided by gene models from other Nicotiana species. Due to the fragmented nature of the Niben1.0.1 draft genome, many protein-encoding genes are missing or partial. We complement these missing proteins by similarly annotating other draft genome assemblies. This approach overcomes problems caused by mis-annotated exon-intron boundaries and mis-assigned short read transcripts to homeologs in polyploid genomes. With an estimated 98.1% completeness; only 53,411 protein-encoding genes; and improved protein lengths and functional annotations, this new predicted proteome is better in assigning spectra than the preceding proteome annotations. This dataset is more sensitive and accurate in proteomics applications, clarifying the detection by activity-based proteomics of proteins that were previously predicted to be inactive. Phylogenetic analysis of the subtilase family of hydrolases reveal inactivation of likely homeologs, associated with a contraction of the functional genome in this alloploid plant species. Finally, we use this new proteome annotation to characterize the extracellular proteome as compared to a total leaf proteome, which highlights the enrichment of hydrolases in the apoplast. CONCLUSIONS: This proteome annotation provides the community working with Nicotiana benthamiana with an important new resource for functional proteomics.


Assuntos
Hidrolases/metabolismo , Proteômica/métodos , Tabaco/genética , Genoma de Planta , Anotação de Sequência Molecular , Filogenia , Ploidias , Homologia de Sequência , Tabaco/metabolismo
15.
Enzyme Microb Technol ; 131: 109432, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615671

RESUMO

The potential anticancer activity of arginine deiminase (ADI) via deimination of l-arginine into citrulline has been extensively verified against various arginine-auxotrophic tumors, however, the higher antigenicity, structural instability and in vivo proteolysis are the major challenges that limit this enzyme from further clinical implementation. Since, this clinically applied enzyme was derived from Mycobacterium spp, thus, searching for ADI from eukaryotic microbes "especially thermophilic fungi" could have a novel biochemical, conformational and catalytic properties. Aspergillus nidulans ADI was purified with 5.3 folds, with molecular subunit structure 48 kDa and entire molecular mass 120 kDa, ensuring its homotrimeric identity. The peptide fingerprinting analysis revealing the domain Glu95-Gly96-Gly97 as the conserved active site of A. nidulans ADI, with higher proximity to Mycobacterium ADI clade IV. In an endeavor to fortify the structural stability and anticancer activity of A. nidulans ADI, the enzyme was chemically modified with dextran. The optimal activity of Dextran-ADI conjugates was determined at 0.08:20 M ratio of ADI: Dextran, with an overall increase to ADI molecular subunit mass to ˜100 kDa. ADI was conjugated with dextran via the ε-amino groups interaction of surface lysine residues of ADI. The resistance of Dextran-ADI conjugate to proteolysis had been increased by 2.5 folds to proteinase K and trypsin, suggesting the shielding of >50% of ADI surface proteolytic recognition sites. The native and Dextran-ADI conjugates have the same optimum reaction temperature (37 °C), reaction pH and pH stability (7.0-8.0) with dependency on K+ ions as a cofactor. Dextran-ADI conjugates exhibited a higher thermal stability by ˜ 2 folds for all the tested temperatures, ensuring the acquired structural and catalytic stability upon dextran conjugation. Dextran conjugation slightly protect the reactive amino and thiols groups of surface amino acids of ADI from amino acids suicide inhibitors. The affinity of ADI was increased by 5.3 folds to free L-arginine with a dramatic reduction in citrullination of peptidylarginine residues upon dextran conjugation. The anticancer activity of ADI to breast (MCF-7), liver (HepG-2) and colon (HCT8, HT29, DLD1 and LS174 T) cancer cell lines was increased by 1.7 folds with dextran conjugation in vitro. Pharmacokinetically, the half-life time of ADI was increased by 1.7 folds upon dextran conjugation, in vivo. From the biochemical and hematological parameters, ADIs had no signs of toxicity to the experimental animals. In addition to the dramatic reduction of L-arginine in serum, citrulline level was increased by 2.5 folds upon dextran conjugation of ADI. This is first report exploring thermostable ADI from thermophilic A. nidulans with robust structural stability, catalytic efficiency and proteolytic resistance.


Assuntos
Antineoplásicos/química , Antineoplásicos/metabolismo , Aspergillus nidulans/enzimologia , Dextranos/metabolismo , Estabilidade Enzimática , Hidrolases/química , Hidrolases/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Arginina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Citrulina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidrolases/farmacocinética , Hidrolases/farmacologia , Cinética , Camundongos , Peso Molecular , Multimerização Proteica , Proteólise , Temperatura Ambiente
16.
Neurology ; 93(20): e1873-e1880, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31624089

RESUMO

OBJECTIVE: To define the clinicopathologic features of amphiphysin-immunoglobulin G (IgG)-mediated neuropathy. METHODS: Patients examined at our institution from January 1, 1995, to September 30, 2018, with amphiphysin-IgG by indirect immunofluorescence and Western blot, were reviewed. Their phenotypes were compared to cases of coexisting collapsin response-mediator protein-5 (CRMP5)-IgG or anti-neuronal nuclear antibody type 1 (ANNA1-IgG) and CRMP5-IgG autoimmunity. Improvement in modified Rankin Scale (mRS) (≥1) on follow-up was considered a favorable outcome. Amphiphysin RNA expression was assessed in healthy nerves. RESULTS: Fifty-three amphiphysin-IgG-positive cases were identified. Of 33 (60%) patients with neuropathy, 21 had amphiphysin-IgG alone, and 12 had coexisting autoantibodies (ANNA1-IgG, n = 8; CRMP5-IgG, n = 2; ANNA1-IgG and CRMP5-IgG, n = 2). The neuropathies in isolated amphiphysin-IgG autoimmunity included polyradiculoneuropathy (62%), diffuse sensory neuronopathy (35%), and facial neuropathy with gastroparesis (3%). Among these, pain (80%), breast cancer (63%), and CNS (57%) involvements commonly coexisted, and neuropathy frequently prompted breast cancer diagnosis (76%). Stiff-person spectrum disorder was the most common CNS accompaniment (45%). Nerve biopsies showed axonal loss (n = 6/6), subperineurial edema (n = 4/6), and CD4 predominant inflammation (n = 2/6). Median mRS score at last follow-up was 3.5; 58% of patients were immunotherapy-responsive. Patients with amphiphysin-IgG alone had more favorable immunotherapy response than patients with CRMP5-IgG polyneuropathy (n = 45) (44% vs 16%, p = 0.028, odds ratio 4.2, 95% confidence interval 1.1 to 15.5). Only 1/9 (11%) patients with amphiphysin-IgG with coexisting CRMP5-IgG or ANNA1-IgG had immunotherapy response. RNA amphiphysin expression occurred at low levels in nerve. CONCLUSION: Amphiphysin-IgG autoimmune neuropathy has a recognizable phenotype, is frequently immune responsive, and can prompt early diagnosis of breast cancer.


Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes do Sistema Nervoso/imunologia , Proteínas do Tecido Nervoso/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antinucleares/imunologia , Doenças Autoimunes do Sistema Nervoso/epidemiologia , Doenças Autoimunes do Sistema Nervoso/patologia , Doenças Autoimunes do Sistema Nervoso/fisiopatologia , Biópsia , Neoplasias da Mama/epidemiologia , Comorbidade , Doenças do Nervo Facial/epidemiologia , Doenças do Nervo Facial/imunologia , Doenças do Nervo Facial/patologia , Doenças do Nervo Facial/fisiopatologia , Feminino , Humanos , Hidrolases/imunologia , Imunoglobulina G/imunologia , Masculino , Proteínas Associadas aos Microtúbulos/imunologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Dor , Nervos Periféricos/imunologia , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Polirradiculoneuropatia/epidemiologia , Polirradiculoneuropatia/imunologia , Polirradiculoneuropatia/patologia , Polirradiculoneuropatia/fisiopatologia , Rigidez Muscular Espasmódica/epidemiologia , Síndrome
17.
Microb Cell Fact ; 18(1): 171, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601227

RESUMO

BACKGROUND: The biological degradation of plastics is a promising method to counter the increasing pollution of our planet with artificial polymers and to develop eco-friendly recycling strategies. Polyethylene terephthalate (PET) is a thermoplast industrially produced from fossil feedstocks since the 1940s, nowadays prevalently used in bottle packaging and textiles. Although established industrial processes for PET recycling exist, large amounts of PET still end up in the environment-a significant portion thereof in the world's oceans. In 2016, Ideonella sakaiensis, a bacterium possessing the ability to degrade PET and use the degradation products as a sole carbon source for growth, was isolated. I. sakaiensis expresses a key enzyme responsible for the breakdown of PET into monomers: PETase. This hydrolase might possess huge potential for the development of biological PET degradation and recycling processes as well as bioremediation approaches of environmental plastic waste. RESULTS: Using the photosynthetic microalga Phaeodactylum tricornutum as a chassis we generated a microbial cell factory capable of producing and secreting an engineered version of PETase into the surrounding culture medium. Initial degradation experiments using culture supernatant at 30 °C showed that PETase possessed activity against PET and the copolymer polyethylene terephthalate glycol (PETG) with an approximately 80-fold higher turnover of low crystallinity PETG compared to bottle PET. Moreover, we show that diatom produced PETase was active against industrially shredded PET in a saltwater-based environment even at mesophilic temperatures (21 °C). The products resulting from the degradation of the PET substrate were mainly terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalic acid (MHET) estimated to be formed in the micromolar range under the selected reaction conditions. CONCLUSION: We provide a promising and eco-friendly solution for biological decomposition of PET waste in a saltwater-based environment by using a eukaryotic microalga instead of a bacterium as a model system. Our results show that via synthetic biology the diatom P. tricornutum indeed could be converted into a valuable chassis for biological PET degradation. Overall, this proof of principle study demonstrates the potential of the diatom system for future biotechnological applications in biological PET degradation especially for bioremediation approaches of PET polluted seawater.


Assuntos
Burkholderiales/metabolismo , Hidrolases/metabolismo , Microalgas/metabolismo , Polietilenotereftalatos/metabolismo , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Biologia Marinha , Microbiologia da Água
18.
Int J Mol Sci ; 20(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561555

RESUMO

The application of purified enzymes as well as whole-cell biocatalysts in synthetic organic chemistry is becoming more and more popular, and both academia and industry are keen on finding and developing novel enzymes capable of performing otherwise impossible or challenging reactions. The diverse genus Rhodococcus offers a multitude of promising enzymes, which therefore makes it one of the key bacterial hosts in many areas of research. This review focused on the broad utilization potential of the genus Rhodococcus in organic chemistry, thereby particularly highlighting the specific enzyme classes exploited and the reactions they catalyze. Additionally, close attention was paid to the substrate scope that each enzyme class covers. Overall, a comprehensive overview of the applicability of the genus Rhodococcus is provided, which puts this versatile microorganism in the spotlight of further research.


Assuntos
Técnicas de Química Sintética , Rhodococcus/metabolismo , Biocatálise , Catálise , Hidrolases/metabolismo , Redes e Vias Metabólicas , Nitrilos/metabolismo , Oxirredução , Oximas/metabolismo , Compostos de Enxofre
19.
Nat Commun ; 10(1): 4239, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31534136

RESUMO

One of the grand challenges in chemistry is the construction of functional out-of-equilibrium networks, which are typical of living cells. Building such a system from molecular components requires control over the formation and degradation of the interacting chemicals and homeostasis of the internal physical-chemical conditions. The provision and consumption of ATP lies at the heart of this challenge. Here we report the in vitro construction of a pathway in vesicles for sustained ATP production that is maintained away from equilibrium by control of energy dissipation. We maintain a constant level of ATP with varying load on the system. The pathway enables us to control the transmembrane fluxes of osmolytes and to demonstrate basic physicochemical homeostasis. Our work demonstrates metabolic energy conservation and cell volume regulatory mechanisms in a cell-like system at a level of complexity minimally needed for life.


Assuntos
Trifosfato de Adenosina/metabolismo , Células Artificiais/metabolismo , Metabolismo Energético/fisiologia , Redes e Vias Metabólicas/fisiologia , Trifosfato de Adenosina/biossíntese , Arginina/metabolismo , Proteínas de Transporte/metabolismo , Citrulina/metabolismo , Hidrolases/metabolismo , Lactococcus lactis/genética , Ornitina/metabolismo , Ornitina Carbamoiltransferase/metabolismo , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo
20.
Extremophiles ; 23(6): 793-808, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31555903

RESUMO

Anaerobic cultivable microbial communities in thermal springs producing hydrolytic enzymes were studied. Thermal water samples from seven thermal springs located in the Andean volcanic belt, in the eastern and central mountain ranges of the Colombian Andes were used as inocula for the growth and isolation of thermophilic microorganisms using substrates such as starch, gelatin, xylan, cellulose, Tween 80, olive oil, peptone and casamino acids. These springs differed in temperature (50-70 °C) and pH (6.5-7.5). The predominant ion in eastern mountain range thermal springs was sulphate, whereas that in central mountain range springs was bicarbonate. A total of 40 anaerobic thermophilic bacterial strains that belonged to the genera Thermoanaerobacter, Caloramator, Anoxybacillus, Caloranaerobacter, Desulfomicrobium, Geotoga, Hydrogenophilus, Desulfacinum and Thermoanaerobacterium were isolated. To investigate the metabolic potential of these isolates, selected strains were analysed for enzymatic activities to identify strains than can produce hydrolytic enzymes. We demonstrated that these thermal springs contained diverse microbial populations of anaerobic thermophilic comprising different metabolic groups of bacteria including strains belonging to the genera Thermoanaerobacter, Caloramator, Anoxybacillus, Caloranaerobacter, Desulfomicrobium, Geotoga, Hydrogenophilus, Desulfacinum and Thermoanaerobacterium with amylases, proteases, lipases, esterases, xylanases and pectinases; therefore, the strains represent a promising source of enzymes with biotechnological potential.


Assuntos
Bactérias Anaeróbias/enzimologia , Proteínas de Bactérias/química , Fontes Termais/microbiologia , Temperatura Alta , Hidrolases/química , Microbiota , Microbiologia da Água , Bactérias Anaeróbias/classificação , Proteínas de Bactérias/metabolismo , Colômbia , Concentração de Íons de Hidrogênio , Hidrolases/metabolismo , Filogenia
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