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1.
PLoS One ; 17(9): e0274910, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36126080

RESUMO

It is well known that the presence of comorbidities and age-related health issues may hide biochemical and metabolic features triggered by SARS-CoV-2 infection and other diseases associated to hypoxia, as they are by themselves chronic inflammatory conditions that may potentially disturb metabolic homeostasis and thereby negatively impact on COVID-19 progression. To unveil the metabolic abnormalities inherent to hypoxemia caused by COVID-19, we here applied gas chromatography coupled to mass spectrometry to analyze the main metabolic changes exhibited by a population of male patients less than 50 years of age with mild/moderate and severe COVID-19 without pre-existing comorbidities known to predispose to life-threatening complications from this infection. Several differences in serum levels of particular metabolites between normal controls and patients with COVID-19 as well as between mild/moderate and severe COVID-19 were identified. These included increased glutamic acid and reduced glutamine, cystine, threonic acid, and proline levels. In particular, using the entire metabolomic fingerprint obtained, we observed that glutamine/glutamate metabolism was associated with disease severity as patients in the severe COVID-19 group presented the lowest and higher serum levels of these amino acids, respectively. These data highlight the hypoxia-derived metabolic alterations provoked by SARS-CoV-2 infection in the absence of pre-existing co-morbidities as well as the value of amino acid metabolism in determining reactive oxygen species recycling pathways, which when impaired may lead to increased oxidation of proteins and cell damage. They also provide insights on new supportive therapies for COVID-19 and other disorders that involve altered redox homeostasis and lower oxygen levels that may lead to better outcomes of disease severity.


Assuntos
COVID-19 , Ácido Glutâmico , Aminoácidos/metabolismo , Cistina/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Homeostase , Humanos , Hipóxia , Masculino , Oxirredução , Oxigênio , Prolina/metabolismo , Espécies Reativas de Oxigênio , SARS-CoV-2
2.
Commun Biol ; 5(1): 997, 2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36131087

RESUMO

Hyperprolinemia type II (HPII) is an inborn error of metabolism due to genetic variants in ALDH4A1, leading to a deficiency in Δ-1-pyrroline-5-carboxylate (P5C) dehydrogenase. This leads to an accumulation of toxic levels of P5C, an intermediate in proline catabolism. The accumulating P5C spontaneously reacts with, and inactivates, pyridoxal 5'-phosphate, a crucial cofactor for many enzymatic processes, which is thought to be the pathophysiological mechanism for HPII. Here, we describe the use of a combination of LC-QTOF untargeted metabolomics, NMR spectroscopy and infrared ion spectroscopy (IRIS) to identify and characterize biomarkers for HPII that result of the spontaneous reaction of P5C with malonic acid and acetoacetic acid. We show that these biomarkers can differentiate between HPI, caused by a deficiency of proline oxidase activity, and HPII. The elucidation of their molecular structures yields insights into the disease pathophysiology of HPII.


Assuntos
Prolina Oxidase , Prolina , 1-Pirrolina-5-Carboxilato Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos , Biomarcadores , Fosfatos , Prolina/metabolismo , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Piridoxal , Pirróis
3.
BMC Plant Biol ; 22(1): 453, 2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36131250

RESUMO

BACKGROUND: Alhagi sparsifolia (Camelthorn) is a leguminous shrub species that dominates the Taklimakan desert's salty, hyperarid, and infertile landscapes in northwest China. Although this plant can colonize and spread in very saline soils, how it adapts to saline stress in the seedling stage remains unclear so a pot-based experiment was carried out to evaluate the effects of four different saline stress levels (0, 50, 150, and 300 mM) on the morphological and physio-biochemical responses in A. sparsifolia seedlings. RESULTS: Our results revealed that N-fixing A. sparsifolia has a variety of physio-biochemical anti-saline stress acclimations, including osmotic adjustments, enzymatic mechanisms, and the allocation of metabolic resources. Shoot-root growth and chlorophyll pigments significantly decreased under intermediate and high saline stress. Additionally, increasing levels of saline stress significantly increased Na+ but decreased K+ concentrations in roots and leaves, resulting in a decreased K+/Na+ ratio and leaves accumulated more Na + and K + ions than roots, highlighting their ability to increase cellular osmolarity, favouring water fluxes from soil to leaves. Salt-induced higher lipid peroxidation significantly triggered antioxidant enzymes, both for mass-scavenging (catalase) and cytosolic fine-regulation (superoxide dismutase and peroxidase) of H2O2. Nitrate reductase and glutamine synthetase/glutamate synthase also increased at low and intermediate saline stress levels but decreased under higher stress levels. Soluble proteins and proline rose at all salt levels, whereas soluble sugars increased only at low and medium stress. The results show that when under low-to-intermediate saline stress, seedlings invest more energy in osmotic adjustments but shift their investment towards antioxidant defense mechanisms under high levels of saline stress. CONCLUSIONS: Overall, our results suggest that A. sparsifolia seedlings tolerate low, intermediate, and high salt stress by promoting high antioxidant mechanisms, osmolytes accumulations, and the maintenance of mineral N assimilation. However, a gradual decline in growth with increasing salt levels could be attributed to the diversion of energy from growth to maintain salinity homeostasis and anti-stress oxidative mechanisms.


Assuntos
Antioxidantes , Fabaceae , Antioxidantes/metabolismo , Catalase/metabolismo , Clorofila/metabolismo , Fabaceae/metabolismo , Glutamato Sintase/metabolismo , Glutamato Sintase/farmacologia , Glutamato-Amônia Ligase/metabolismo , Peróxido de Hidrogênio/metabolismo , Íons/metabolismo , Nitrogênio/metabolismo , Prolina/metabolismo , Salinidade , Plântula/metabolismo , Solo , Açúcares/metabolismo , Superóxido Dismutase/metabolismo , Água/metabolismo
4.
Cell Death Dis ; 13(9): 795, 2022 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-36123332

RESUMO

Mammalian WNK kinases (WNKs) are serine/threonine kinases that contain four members, WNK1-4. They function to maintain ion homeostasis and regulate blood pressure in mammals. Recent studies have revealed that the dysregulation of WNKs contributes to tumor growth, metastasis, and angiogenesis through complex mechanisms, especially through phosphorylating kinase substrates SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Here, we review and discuss the relationships between WNKs and several key factors/biological processes in cancer, including ion channels, cation chloride cotransporters, sodium bicarbonate cotransporters, signaling pathways, angiogenesis, autophagy, and non-coding RNAs. In addition, the potential drugs for targeting WNK-SPAK/OSR1 signaling have also been discussed. This review summarizes and discusses knowledge of the roles of WNKs in cancer, which provides a comprehensive reference for future studies.


Assuntos
Bicarbonatos , Neoplasias , Alanina , Animais , Mamíferos , Neoplasias/genética , Neovascularização Patológica , Prolina , Serina , Simportadores de Cloreto de Sódio , Treonina
5.
BMC Plant Biol ; 22(1): 441, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36109698

RESUMO

BACKGROUND: The cationic amino acid transporters (CAT) play indispensable roles in maintaining metabolic functions, such as synthesis of proteins and nitric oxide (NO), biosynthesis of polyamine, and flow of amino acids, by mediating the bidirectional transport of cationic amino acids in plant cells. RESULTS: In this study, we performed a genome-wide and comprehensive study of 79 CAT genes in four species of cotton. Localization of genes revealed that CAT genes reside on the plasma membrane. Seventy-nine CAT genes were grouped into 7 subfamilies by phylogenetic analysis. Structure analysis of genes showed that CAT genes from the same subgroup have similar genetic structure and exon number. RNA-seq and real-time PCR indicated that the expression of most GhCAT genes were induced by salt, drought, cold and heat stresses. Cis-elements analysis of GhCAT promoters showed that the GhCAT genes promoters mainly contained plant hormones responsive elements and abiotic stress elements, which indicated that GhCAT genes may play key roles in response to abiotic stress. Moreover, we also conducted gene interaction network of the GhCAT proteins. Silencing GhCAT10D expression decreased the resistance of cotton to salt stress because of a decrease in the accumulation of NO and proline. CONCLUSION: Our results indicated that CAT genes might be related with salt tolerance in cotton and lay a foundation for further study on the regulation mechanism of CAT genes in cationic amino acids transporting and distribution responsing to abiotic stress.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos , Tolerância ao Sal , Sistemas de Transporte de Aminoácidos Básicos/genética , Aminoácidos/metabolismo , Regulação da Expressão Gênica de Plantas , Genômica , Óxido Nítrico/metabolismo , Filogenia , Reguladores de Crescimento de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poliaminas , Prolina/metabolismo , Tolerância ao Sal/genética
6.
Plant Physiol Biochem ; 189: 83-93, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36058015

RESUMO

5-Aminolevulinic acid (ALA), an antioxidant existing in plants, has been widely reported to participate in the process of coping with cold stress of plants. In this study, exogenous ALA promoted the growth of tomato plants and alleviated the appearance of purple tomato leaves under low-temperature stress. At the same time, exogenous ALA improved antioxidant enzyme activities, SlSOD gene expression, Fv/Fm, and proline contents and reduced H2O2 contents, SlRBOH gene expression, relative electrical conductivity, and malondialdehyde contents to alleviate the damage caused by low temperature to tomato seedlings. Compared with low-temperature stress, spraying exogenous ALA before low-temperature stress could restore the indicators of photochemical quenching, actual photochemical efficiency, electron transport rate, and nonphotochemical quenching to normal. Exogenous ALA could increase the total contents of the xanthophyll cycle pool, the positive de-epoxidation rate of the xanthophyll cycle and improved the expression levels of key genes in the xanthophyll cycle under low-temperature stress. In addition, we found that exogenous ALA significantly enhanced the absorption of mineral nutrients, promoted the transfer and distribution of mineral nutrients to the leaves, and improved the expression levels of mineral nutrient absorption-related genes, which were all conducive to the improved adaptation of tomato seedlings under low-temperature stress. In summary, the application of exogenous ALA can increase tomato seedlings' tolerance to low-temperature stress by improving the xanthophyll cycle and the ability of the absorption of mineral nutrients in tomato seedlings.


Assuntos
Lycopersicon esculentum , Plântula , Ácido Aminolevulínico/metabolismo , Antioxidantes/metabolismo , Peróxido de Hidrogênio/metabolismo , Lycopersicon esculentum/metabolismo , Malondialdeído/metabolismo , Nutrientes , Fotossíntese , Prolina/metabolismo , Plântula/metabolismo , Estresse Fisiológico , Temperatura , Xantofilas/metabolismo
7.
Plant Physiol Biochem ; 189: 104-114, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36081232

RESUMO

Cadmium (Cd) and lead (Pb) pollution is a major environmental issue affecting plant production. Spermidine (Spd) is involved in plant response to abiotic stress. However, the role and associated mechanism of Spd under Cd + Pb combined stress are poorly understood. The potential protective role of Spd at different concentration on rice (Oryza sativa L.) seedlings exposed to Cd + Pb treatment was investigated by a hydroponic experiment in this study. The results showed that exogenous Spd enhanced the tolerance of rice seedlings to Cd + Pb stress, resulted in an increase in plant height, root length, fresh weight and dry weight of roots and shoots. Further, application of Spd decreased the contents of hydrogen peroxide, superoxide anion, malondialdehyde, and the accumulation of Cd and Pb, and increased the contents of mineral nutrient, carotenoids, chlorophyll, proline, soluble sugar, soluble protein, total phenol, flavonoid, anthocyanin, and antioxidant enzymes activities in roots and shoots of rice seedlings under Cd + Pb stress. Particularly, 0.5 mmol L-1 Spd was the most effective to alleviate the adverse impacts on growth and physiological metabolism of rice seedlings under Cd + Pb stress. Principal component analysis and heat map clustering established correlations between physio-biochemical parameters and further revealed Spd alleviated Cd + Pb damage in rice seedling was associated with inhibition of accumulation and translocation of Cd and Pb, increasing the contents of photosynthetic pigments and mineral nutrient and stimulation of antioxidative response and osmotic adjustment. Overall, our findings provide an important prospect for use of Spd in modulating Cd + Pb tolerance in rice plants. Spd could help to alleviate Cd + Pb damage through inhibition of accumulation and translocation of Cd and Pb and stimulation of oxidant-defense system and osmotic adjustment.


Assuntos
Oryza , Antocianinas/metabolismo , Antioxidantes/metabolismo , Cádmio/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Peróxido de Hidrogênio/metabolismo , Chumbo/metabolismo , Malondialdeído/metabolismo , Oryza/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacologia , Fenóis/metabolismo , Raízes de Plantas/metabolismo , Prolina/metabolismo , Plântula/metabolismo , Espermidina/metabolismo , Espermidina/farmacologia , Açúcares/metabolismo , Superóxidos/metabolismo
8.
Plant Physiol Biochem ; 189: 139-152, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36087439

RESUMO

The role of calcium in fruit ripening has been established, however knowledge regarding the molecular analysis at fruit tissue-level is still lacking. To address this, we examined the impact of foliar-applied calcium (0.5% CaCl2) in the ripening metabolism in skin and flesh tissues of the sweet cherry 'Tragana Edessis' fruit at the harvest stage. Exogenously applied calcium increased endogenous calcium level in flesh tissue and reduced fruit respiration rate and cracking traits. Fruit metabolomic along with transcriptomic analysis unraveled common and tissue-specific metabolic pathways associated with calcium feeding. Treatment with calcium diminished several alcohols (arabitol, sorbitol), sugars (fructose, maltose), acids (glyceric acid, threonic acid) and increased ribose and proline in both fruit tissues. Moreover, numerous primary metabolites, such as proline and galacturonic acid, were differentially accumulated in calcium-exposed tissues. Calcium-affected genes that involved in ubiquitin/ubl conjugation and cell wall biogenesis/degradation were differentially expressed between skin and flesh samples. Notably, skin and flesh tissues shared common calcium-responsive genes and exhibited substantial similarity in their expression patterns. In both tissues, calcium activated gene expression, most strongly those involved in plant-pathogen interaction, plant hormone signaling and MAPK signaling pathway, thus affecting related metabolic processes. By contrast, calcium depressed the expression of genes related to TCA cycle, oxidative phosphorylation, and starch/sucrose metabolism in both tissues. This work established both calcium-driven common and specialized metabolic suites in skin and flesh cherry tissues, demonstrating the utility of this approach to characterize fundamental aspects of calcium in fruit physiology.


Assuntos
Prunus avium , Álcoois/metabolismo , Cálcio/metabolismo , Cloreto de Cálcio , Frutose/metabolismo , Frutas/metabolismo , Ácidos Glicéricos/metabolismo , Maltose/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Prolina/metabolismo , Prunus avium/metabolismo , Ribose/metabolismo , Sorbitol/metabolismo , Amido/metabolismo , Sacarose/metabolismo , Ubiquitinas/metabolismo
10.
Int J Mol Sci ; 23(17)2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-36077020

RESUMO

The NAC (NAM, ATAF1/2, and CUC2) transcription factors are one of the largest families of transcription factors in plants and play an important role in plant development and the response to adversity. In this study, we cloned a new NAC gene, SlNAC10, from the halophyte Suaeda liaotungensis K. The gene has a total length of 1584 bp including a complete ORF of 1107 bp that encodes 369 amino acids. The SlNAC10-GFP fusion protein is located in the nucleus and SlNAC10 has a transcription activation structural domain at the C-terminus. We studied the expression characteristics of SlNAC10 and found that it was highest in the leaves of S. liaotungensis and induced by drought, salt, cold, and abscisic acid (ABA). To analyze the function of SlNAC10 in plants, we obtained SlNAC10 transgenic Arabidopsis. The growth characteristics and physiological indicators of transgenic Arabidopsis were measured under salt and drought stress. The transgenic Arabidopsis showed obvious advantages in the root length and survival rate; chlorophyll fluorescence levels; and the antioxidant enzyme superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and the proline content was higher than that of the wild-type (WT) Arabidopsis, whereas the relative electrolyte leakage and malondialdehyde (MDA) content were lower than those of the wild-type Arabidopsis. We explored the regulatory role of SlNAC10 on proline synthesis-related enzyme genes and found that SlNAC10 binds to the AtP5CS1, AtP5CS2, and AtP5CR promoters and regulates their downstream gene transcription. To sum up, SlNAC10 as a transcription factor improves salt and drought tolerance in plants possibly by regulating proline synthesis.


Assuntos
Arabidopsis , Chenopodiaceae , Arabidopsis/metabolismo , Chenopodiaceae/genética , Chenopodiaceae/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Prolina/metabolismo , Cloreto de Sódio/metabolismo , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
J Inorg Biochem ; 236: 111978, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36063739

RESUMO

Prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), a red-colored microbial pigment, is produced in large quantities by Serratia marcescens KMR-3. This bacterium can grow in a medium with a Cd2+ concentration of 500 mg/L, but it does not produce prodigiosin when the Cd2+ concentration in the medium is higher than 140 mg/L. Therefore, we investigated the mechanisms by which Cd2+ inhibits prodigiosin synthesis. Upon addition of Cd2+ to the medium, the expression of the prodigiosin (pig) gene cluster was significantly downregulated. Simultaneously, genes encoding proteins related to the synthesis of arginine and proline(prodigiosin precursors) were significantly downregulated, while the degradation-related genes were upregulated. Furthermore, PigF, which encodes a key enzyme involved in the synthesis of 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde and PigC, which encodes a key enzyme involved in the last step of prodigiosin synthesis, were downregulated by 80% and 55%, respectively, following Cd2+ treatment. As PigC and PigF are located on the cell membrane and are involved in the final steps of prodigiosin synthesis, the cell membrane might be presumed to be the site of prodigiosin synthesis. The bacterial membrane exhibited different degrees of elongation, folding, fragmentation, and sagging after the addition of Cd2+, while likely destroying the site of prodigiosin synthesis.


Assuntos
Prodigiosina , Serratia marcescens , Animais , Arginina/metabolismo , Cádmio/metabolismo , Feminino , Fator de Crescimento Placentário/metabolismo , Prodigiosina/metabolismo , Prolina , Serratia marcescens/genética , Serratia marcescens/metabolismo
12.
J Plant Physiol ; 277: 153807, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36095952

RESUMO

Annual ryegrass is a widely cultivated forage grass with rapid growth and high productivity. However, drought is one of the abiotic stresses affecting ryegrass growth and quality. In this study, we compared the physiological and transcriptome responses of Chuansi No.1 (drought-tolerant, DT) and Double Barrel (drought-sensitive, DS) under drought stress simulated by PEG-6000 for 7 days. The results showed that Chuansi No. 1 had stronger physiological and biochemical parameters such as root properties, water content, osmotic adjustment ability and antioxidant ability. In addition, RNA-seq was used to elucidate the molecular mechanism of root drought resistance. We identified 8588 differentially expressed genes related to drought tolerance in root, which were mainly enriched in oxidation-reduction process, carbohydrate metabolic process, apoplast, arginine and proline metabolism, and phenylpropanoid biosynthesis pathways. The expression levels of DEGs were consistent with physiological changes of ryegrass under drought stress. We found that genes related to sucrose and starch synthesis, root development, osmotic adjustment, ABA signal regulation and specifically up-regulated transcription factors such as WRKY41, WRKY51, ERF7, ERF109, ERF110, NAC43, NAC68, bHLH162 and bHLH148 in Chuansi No. 1 may be the reason for its higher drought tolerance. This study revealed the underlying physiological and molecular mechanisms of root response to drought stress in ryegrass and provided some new candidate genes for breeding rye drought tolerant varieties.


Assuntos
Secas , Lolium , Antioxidantes , Arginina , Carboidratos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Lolium/genética , Melhoramento Vegetal , Prolina/genética , Amido , Sacarose , Fatores de Transcrição/genética , Água
13.
Viruses ; 14(8)2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-36016300

RESUMO

Direct-acting antivirals (DAAs) have revolutionised the treatment of Hepatitis C virus (HCV), allowing the World Health Organisation (WHO) to set a target of eliminating HCV by 2030. In this study we aimed to investigate glecaprevir and pibrentasvir (GP) treatment outcomes in a cohort of patients with genotype 2a infection. METHODS: Clinical data and plasma samples were collected in NHS Greater Glasgow & Clyde. Next generation whole genome sequencing and replicon assays were carried out at the MRC-University of Glasgow Centre for Virus Research. RESULTS: 132 cases infected with genotype 2a HCV were identified. The SVR rate for this group was 91% (112/123) following treatment with GP. An NS5A polymorphism, L31M, was detected in all cases of g2a infection, and L31M+R353K in individuals that failed treatment. The results showed that R353K was present in 90% of individuals in the Glasgow genotype 2a phylogenetic cluster but in less than 5% of all HCV subtype 2a published sequences. In vitro efficacy of pibrentasvir against sub-genomic replicon constructs containing these mutations showed a 2-fold increase in IC50 compared to wildtype. CONCLUSION: This study describes a cluster of HCV genotype 2a infection associated with a lower-than-expected SVR rate following GP treatment in association with the NS5A mutations L31M+R353K.


Assuntos
Hepatite C Crônica , Hepatite C , Abuso de Substâncias por Via Intravenosa , Ácidos Aminoisobutíricos , Antivirais/uso terapêutico , Benzimidazóis , Ciclopropanos , Combinação de Medicamentos , Genótipo , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Humanos , Lactamas Macrocíclicas , Leucina/análogos & derivados , Filogenia , Prolina/análogos & derivados , Prolina/genética , Pirrolidinas , Quinoxalinas , Escócia/epidemiologia , Sulfonamidas
15.
Microb Cell Fact ; 21(1): 159, 2022 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-35953819

RESUMO

BACKGROUND: In recent years, there has been a growing demand for microbial production of trans-4-hydroxy-L-proline (t4Hyp), which is a value-added amino acid and has been widely used in the fields of medicine, food, and cosmetics. In this study, a multivariate modular metabolic engineering approach was used to remove the bottleneck in the synthesis pathway of t4Hyp. RESULTS: Escherichia coli t4Hyp synthesis was performed using two modules: a α-ketoglutarate (α-KG) synthesis module (K module) and L-proline synthesis with hydroxylation module (H module). First, α-KG attrition was reduced, and then, L-proline consumption was inhibited. Subsequently, to improve the contribution to proline synthesis with hydroxylation, optimization of gene overexpression, promotor, copy number, and the fusion system was performed. Finally, optimization of the H and K modules was performed in combination to balance metabolic flow. Using the final module H1K4 in a shaking flask culture, 8.80 g/L t4Hyp was produced, which was threefold higher than that produced by the W0 strain. CONCLUSIONS: These strategies demonstrate that a microbial cell factory can be systematically optimized by modular engineering for efficient production of t4Hyp.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hidroxiprolina , Ácidos Cetoglutáricos/metabolismo , Engenharia Metabólica , Prolina/metabolismo
16.
Int J Mol Sci ; 23(15)2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35955930

RESUMO

Calcium-dependent protein kinases (CDPK) are implicated in signaling transduction in eukaryotic organisms. It is largely unknown whether StCDPK28 plays a role in the response to water deficiency and osmotic stress in potato plants (Solanum tuberosum L.). Potato cv. Zihuabai was cultivated under natural, moderate, and severe water deficiency conditions; to induce osmotic stress, potato plants were treated with 10% or 20% PEG. StCDPK28-overexpression and StCDPK28-knockdown plants were constructed. StCDPKs were evaluated by qRT-PCR. The subcellular location of the StCDPK28 protein was observed with confocal scanning laser microscopy. Phenotypic changes were indicated by photosynthetic activity, the contents of H2O2, MDA and proline, and the activities of CAT, SOD and POD. Results showed water deficiency and osmotic stress altered StCDPK expression patterns. StCDPK28 exhibited a membrane, cytosolic and nuclear localization. Water deficiency and osmotic stress induced StCDPK28 upregulation. Photosynthetic activity was enhanced by StCDPK28 overexpression, while decreased by StCDPK2 knockdown under water deficiency and osmotic stress. StCDPK28 overexpression decreased H2O2 and MDA, and increased proline, while StCDPK28 knockdown showed reverse results, compared with the wild type, in response to water deficiency and osmotic stress. StCDPK28 overexpression increased the activities of CAT, SOD and POD, while StCDPK28-knockdown plants indicated the reverse trend under water deficiency and osmotic stress conditions. Regulation of StCDPK28 expression could be a promising approach to improve the tolerance ability of potato plants in response to drought or high salt media.


Assuntos
Solanum tuberosum , Secas , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Pressão Osmótica , Fotossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Prolina/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Solanum tuberosum/metabolismo , Estresse Fisiológico , Superóxido Dismutase/metabolismo , Água/metabolismo
17.
Molecules ; 27(15)2022 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-35956767

RESUMO

Vibrational spectroscopic techniques, i.e., attenuated total reflectance infrared (ATR-IR), near infrared spectroscopy (NIRS) and Raman spectroscopy (RS), coupled with Partial Least Squares Regression (PLSR), were evaluated as cost-effective label-free and reagent-free tools to monitor water content in Levulinic Acid/L-Proline (LALP) (2:1, mol/mol) Natural Deep Eutectic Solvent (NADES). ATR-IR delivered the best outcome of Root Mean Squared Error (RMSE) of Cross-Validation (CV) = 0.27% added water concentration, RMSE of Prediction (P) = 0.27% added water concentration and mean % relative error = 2.59%. Two NIRS instruments (benchtop and handheld) were also compared during the study, respectively yielding RMSECV = 0.35% added water concentration, RMSEP = 0.56% added water concentration and mean % relative error = 5.13% added water concentration, and RMECV = 0.36% added water concentration, RMSEP = 0.68% added water concentration and mean % relative error = 6.23%. RS analysis performed in quartz cuvettes enabled accurate water quantification with RMECV = 0.43% added water concentration, RMSEP = 0.67% added water concentration and mean % relative error = 6.75%. While the vibrational spectroscopic techniques studied have shown high performance in relation to reliable determination of water concentration, their accuracy is most likely related to their sensitivity to detect the LALP compounds in the NADES. For instance, whereas ATR-IR spectra display strong features from water, Levulinic Acid and L-Proline that contribute to the PLSR predictive models constructed, NIRS and RS spectra are respectively dominated by either water or LALP compounds, representing partial molecular information and moderate accuracy compared to ATR-IR. However, while ATR-IR instruments are common in chemistry and physics laboratories, making the technique readily transferable to water quantification in NADES, Raman spectroscopy offers promising potential for future development for in situ, sample withdrawal-free analysis for high throughput and online monitoring.


Assuntos
Solventes Eutéticos Profundos , Água , Análise dos Mínimos Quadrados , Prolina , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos
18.
Sci Rep ; 12(1): 13634, 2022 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-35948609

RESUMO

The epidermal differentiation complex (EDC) is a cluster of genes encoding components of the skin barrier in terrestrial vertebrates. EDC genes can be categorized as S100 fused-type protein (SFTP) genes such as filaggrin, which contain two coding exons, and single-coding-exon EDC (SEDC) genes such as loricrin. SFTPs are known to be present in amniotes (mammals, reptiles and birds) and amphibians, whereas SEDCs have not yet been reported in amphibians. Here, we show that caecilians (Amphibia: Gymnophiona) have both SFTP and SEDC genes. Two to four SEDC genes were identified in the genomes of Rhinatrema bivittatum, Microcaecilia unicolor and Geotrypetes seraphini. Comparative analysis of tissue transcriptomes indicated predominant expression of SEDC genes in the skin of caecilians. The proteins encoded by caecilian SEDC genes resemble human SEDC proteins, such as involucrin and small proline-rich proteins, with regard to low sequence complexity and high contents of proline, glutamine and lysine. Our data reveal diversification of EDC genes in amphibians and suggest that SEDC-type skin barrier genes have originated either in a common ancestor of tetrapods followed by loss in Batrachia (frogs and salamanders) or, by convergent evolution, in caecilians and amniotes.


Assuntos
Anfíbios , Répteis , Anfíbios/genética , Animais , Epiderme , Humanos , Mamíferos , Filogenia , Prolina/genética , Répteis/genética , Proteínas S100/genética
19.
Proc Natl Acad Sci U S A ; 119(35): e2110105119, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35994646

RESUMO

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for neutralizing antibodies (NAbs). The S protein trimer is anchored in the virion membrane in its prefusion (preS) but metastable form. The preS protein has been stabilized by introducing two or six proline substitutions, to generate stabilized, soluble 2P or HexaPro (6P) preS proteins. Currently, it is not known which form is the most immunogenic. Here, we generated recombinant vesicular stomatitis virus (rVSV) expressing preS-2P, preS-HexaPro, and native full-length S, and compared their immunogenicity in mice and hamsters. The rVSV-preS-HexaPro produced and secreted significantly more preS protein compared to rVSV-preS-2P. Importantly, rVSV-preS-HexaPro triggered significantly more preS-specific serum IgG antibody than rVSV-preS-2P in both mice and hamsters. Antibodies induced by preS-HexaPro neutralized the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.617.2 variants approximately two to four times better than those induced by preS-2P. Furthermore, preS-HexaPro induced a more robust Th1-biased cellular immune response than preS-2P. A single dose (104 pfu) immunization with rVSV-preS-HexaPro and rVSV-preS-2P provided complete protection against challenge with mouse-adapted SARS-CoV-2 and B.1.617.2 variant, whereas rVSV-S only conferred partial protection. When the immunization dose was lowered to 103 pfu, rVSV-preS-HexaPro induced two- to sixfold higher antibody responses than rVSV-preS-2P in hamsters. In addition, rVSV-preS-HexaPro conferred 70% protection against lung infection whereas only 30% protection was observed in the rVSV-preS-2P. Collectively, our data demonstrate that both preS-2P and preS-HexaPro are highly efficacious but preS-HexaPro is more immunogenic and protective, highlighting the advantages of using preS-HexaPro in the next generation of SARS-CoV-2 vaccines.


Assuntos
Prolina , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Desenvolvimento de Vacinas , Estomatite Vesicular , Vacinas Virais , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/genética , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Vacinas contra COVID-19/imunologia , Cricetinae , Humanos , Camundongos , Prolina/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Estomatite Vesicular/imunologia , Estomatite Vesicular/prevenção & controle , Estomatite Vesicular/virologia , Vesiculovirus/imunologia , Proteínas Virais/imunologia , Vacinas Virais/imunologia
20.
N Engl J Med ; 387(9): 790-798, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36001529

RESUMO

BACKGROUND: The oral protease inhibitor nirmatrelvir has shown substantial efficacy in high-risk, unvaccinated patients infected with the B.1.617.2 (delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data regarding the effectiveness of nirmatrelvir in preventing severe coronavirus disease 2019 (Covid-19) outcomes from the B.1.1.529 (omicron) variant are limited. METHODS: We obtained data for all members of Clalit Health Services who were 40 years of age or older at the start of the study period and were assessed as being eligible to receive nirmatrelvir therapy during the omicron surge. A Cox proportional-hazards regression model with time-dependent covariates was used to estimate the association of nirmatrelvir treatment with hospitalization and death due to Covid-19, with adjustment for sociodemographic factors, coexisting conditions, and previous SARS-CoV-2 immunity status. RESULTS: A total of 109,254 patients met the eligibility criteria, of whom 3902 (4%) received nirmatrelvir during the study period. Among patients 65 years of age or older, the rate of hospitalization due to Covid-19 was 14.7 cases per 100,000 person-days among treated patients as compared with 58.9 cases per 100,000 person-days among untreated patients (adjusted hazard ratio, 0.27; 95% confidence interval [CI], 0.15 to 0.49). The adjusted hazard ratio for death due to Covid-19 was 0.21 (95% CI, 0.05 to 0.82). Among patients 40 to 64 years of age, the rate of hospitalization due to Covid-19 was 15.2 cases per 100,000 person-days among treated patients and 15.8 cases per 100,000 person-days among untreated patients (adjusted hazard ratio, 0.74; 95% CI, 0.35 to 1.58). The adjusted hazard ratio for death due to Covid-19 was 1.32 (95% CI, 0.16 to 10.75). CONCLUSIONS: Among patients 65 years of age or older, the rates of hospitalization and death due to Covid-19 were significantly lower among those who received nirmatrelvir than among those who did not. No evidence of benefit was found in younger adults.


Assuntos
Antivirais , COVID-19 , Lactamas , Leucina , Nitrilas , Prolina , Adulto , Idoso , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/virologia , Hospitalização , Humanos , Lactamas/uso terapêutico , Leucina/uso terapêutico , Pessoa de Meia-Idade , Nitrilas/uso terapêutico , Prolina/uso terapêutico , SARS-CoV-2 , Resultado do Tratamento
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