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1.
Methods Mol Biol ; 2775: 329-347, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38758327

RESUMO

The cell wall of the fungal pathogens Cryptococcus neoformans and C. gattii is critical for cell wall integrity and signaling external threats to the cell, allowing it to adapt and grow in a variety of changing environments. Chitin is a polysaccharide found in the cell walls of fungi that is considered to be essential for fungal survival. Chitosan is a polysaccharide derived from chitin via deacetylation that is also essential for cryptococcal cell wall integrity, fungal pathogenicity, and virulence. Cryptococcus has evolved mechanisms to regulate the amount of chitin and chitosan during growth under laboratory conditions or during mammalian infection. Therefore, levels of chitin and chitosan have been useful phenotypes to define mutant Cryptococcus strains. As a result, we have developed and/or refined various qualitative and quantitative methods for measuring chitin and chitosan. These techniques include those that use fluorescent probes that are known to bind to chitin (e.g., calcofluor white and wheat germ agglutinin), as well as those that preferentially bind to chitosan (e.g., eosin Y and cibacron brilliant red 3B-A). Techniques that enhance the localization and quantification of chitin and chitosan in the cell wall include (i) fluorescence microscopy, (ii) flow cytometry, (iii) and spectrofluorometry. We have also modified two highly selective biochemical methods to measure cellular chitin and chitosan content: the Morgan-Elson and the 3-methyl-2-benzothiazolone hydrazine hydrochloride (MBTH) assays, respectively.


Assuntos
Parede Celular , Quitina , Quitosana , Quitina/metabolismo , Quitina/química , Quitina/análise , Quitosana/química , Quitosana/metabolismo , Parede Celular/metabolismo , Parede Celular/química , Cryptococcus neoformans/metabolismo , Corantes Fluorescentes/química , Cryptococcus/metabolismo , Microscopia de Fluorescência/métodos
2.
Molecules ; 29(9)2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38731405

RESUMO

Chitin, a ubiquitous biopolymer, holds paramount scientific and economic significance. Historically, it has been primarily isolated from marine crustaceans. However, the surge in demand for chitin and the burgeoning interest in biopolymers have necessitated the exploration of alternative sources. Among these methods, the mulberry silkworm (Bombyx mori) has emerged as a particularly intriguing prospect. To isolate chitin from Bombyx mori, a chemical extraction methodology was employed. This process involved a series of meticulously orchestrated steps, including Folch extraction, demineralization, deproteinization, and decolorization. The resultant chitin was subjected to comprehensive analysis utilizing techniques such as attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), 13C nuclear magnetic resonance (NMR) spectroscopy, and wide-angle X-ray scattering (WAXS). The obtained results allow us to conclude that the Bombyx mori represents an attractive alternative source of α-chitin.


Assuntos
Bombyx , Quitina , Bombyx/química , Animais , Quitina/química , Quitina/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X , Espectroscopia de Ressonância Magnética , Morus/química
3.
Carbohydr Polym ; 337: 122149, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38710571

RESUMO

Phytopathogen cell wall polysaccharides have important physiological functions. In this study, we isolated and characterized the alkali-insoluble residue on the inner layers of the Rhizoctonia solani AG1 IA cell wall (RsCW-AIR). Through chemical composition and structural analysis, RsCW-AIR was mainly identified as a complex of chitin/chitosan and glucan (ChCsGC), with glucose and glucosamine were present in a molar ratio of 2.7:1.0. The predominant glycosidic bond linkage of glucan in ChCsGC was ß-1,3-linked Glcp, both the α and ß-polymorphic forms of chitin were presented in it by IR, XRD, and solid-state NMR, and the ChCsGC exhibited a degree of deacetylation measuring 67.08 %. RsCW-AIR pretreatment effectively reduced the incidence of rice sheath blight, and its induced resistance activity in rice was evaluated, such as inducing a reactive oxygen species (ROS) burst, leading to the accumulation of salicylic acid (SA) and the up-regulation of SA-related gene expression. The recognition of RsCW-AIR in rice is partially dependent on CERK1.


Assuntos
Parede Celular , Quitina , Quitosana , Glucanos , Oryza , Doenças das Plantas , Rhizoctonia , Rhizoctonia/efeitos dos fármacos , Oryza/microbiologia , Oryza/química , Parede Celular/química , Quitosana/química , Quitosana/farmacologia , Quitina/química , Quitina/farmacologia , Glucanos/química , Glucanos/farmacologia , Doenças das Plantas/microbiologia , Resistência à Doença , Espécies Reativas de Oxigênio/metabolismo
4.
J Agric Food Chem ; 72(19): 10794-10804, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38711396

RESUMO

Chitin-degrading enzymes are critical components in regulating the molting process of the Asian corn borer and serve as potential targets for controlling this destructive pest of maize. Here, we used a scaffold-hopping strategy to design a series of efficient naphthylimide insecticides. Among them, compound 8c exhibited potent inhibition of chitinase from OfChi-h and OfChtI at low nanomolar concentrations (IC50 = 1.51 and 9.21 nM, respectively). Molecular docking simulations suggested that 8c binds to chitinase by mimicking the interaction of chitin oligosaccharide substrates with chitinase. At low ppm concentrations, compound 8c performed comparably to commercial insecticides in controlling the highly destructive plant pest, the Asian corn borer. Tests on a wide range of nontarget organisms indicate that compound 8c has very low toxicity. In addition, the effect of inhibitor treatment on the expression of genes associated with the Asian corn borer chitin-degrading enzymes was further investigated by quantitative real-time polymerase chain reaction. In conclusion, our study highlights the potential of 8c as a novel chitinase-targeting insecticide for effective control of the Asian corn borer, providing a promising solution in the quest for sustainable pest management.


Assuntos
Quitina , Quitinases , Proteínas de Insetos , Inseticidas , Simulação de Acoplamento Molecular , Mariposas , Zea mays , Animais , Quitinases/química , Quitinases/genética , Quitinases/metabolismo , Mariposas/enzimologia , Mariposas/efeitos dos fármacos , Mariposas/genética , Quitina/química , Quitina/metabolismo , Inseticidas/química , Inseticidas/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/antagonistas & inibidores , Zea mays/química , Zea mays/parasitologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Desenho de Fármacos , Controle de Insetos , Larva/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Relação Estrutura-Atividade
5.
J Appl Biomater Funct Mater ; 22: 22808000241248887, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38742818

RESUMO

OBJECTIVE: Chitin a natural polymer is abundant in several sources such as shells of crustaceans, mollusks, insects, and fungi. Several possible attempts have been made to recover chitin because of its importance in biomedical applications in various forms such as hydrogel, nanoparticles, nanosheets, nanowires, etc. Among them, deep eutectic solvents have gained much consideration because of their eco-friendly and recyclable nature. However, several factors need to be addressed to obtain a pure form of chitin with a high yield. The development of an innovative system for the production of quality chitin is of prime importance and is still challenging. METHODS: The present study intended to develop a novel and robust approach to investigate chitin purity from various crustacean shell wastes using deep eutectic solvents. This investigation will assist in envisaging the important influencing parameters to obtain a pure form of chitin via a machine learning approach. Different machine learning algorithms have been proposed to model chitin purity by considering the enormous experimental dataset retrieved from previously conducted experiments. Several input variables have been selected to assess chitin purity as the output variable. RESULTS: The statistical criteria of the proposed model have been critically investigated and it was observed that the results indicate XGBoost has the maximum predictive accuracy of 0.95 compared with other selected models. The RMSE and MAE values were also minimal in the XGBoost model. In addition, it revealed better input variables to obtain pure chitin with minimal processing time. CONCLUSION: This study validates that machine learning paves the way for complex problems with substantial datasets and can be an inexpensive and time-saving model for analyzing chitin purity from crustacean shells.


Assuntos
Quitina , Crustáceos , Solventes Eutéticos Profundos , Aprendizado de Máquina , Quitina/química , Quitina/isolamento & purificação , Animais , Crustáceos/química , Solventes Eutéticos Profundos/química , Exoesqueleto/química
6.
Nat Commun ; 15(1): 3733, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740737

RESUMO

Organisms generate shapes across size scales. Whereas patterning and morphogenesis of macroscopic tissues has been extensively studied, the principles underlying the formation of micrometric and submicrometric structures remain largely enigmatic. Individual cells of polychaete annelids, so-called chaetoblasts, are associated with the generation of chitinous bristles of highly stereotypic geometry. Here we show that bristle formation requires a chitin-producing enzyme specifically expressed in the chaetoblasts. Chaetoblasts exhibit dynamic cell surfaces with stereotypical patterns of actin-rich microvilli. These microvilli can be matched with internal and external structures of bristles reconstructed from serial block-face electron micrographs. Individual chitin teeth are deposited by microvilli in an extension-disassembly cycle resembling a biological 3D printer. Consistently, pharmacological interference with actin dynamics leads to defects in tooth formation. Our study reveals that both material and shape of bristles are encoded by the same cell, and that microvilli play a role in micro- to submicrometric sculpting of biomaterials.


Assuntos
Quitina , Microvilosidades , Microvilosidades/ultraestrutura , Animais , Quitina/metabolismo , Quitina/química , Poliquetos/ultraestrutura , Actinas/metabolismo , Morfogênese
7.
Appl Microbiol Biotechnol ; 108(1): 321, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38709299

RESUMO

Most reduced organic matter entering activated sludge systems is particulate (1-100-µm diameter) or colloidal (0.001-1-µm diameter), yet little is known about colonization of particulate organic matter by activated sludge bacteria. In this study, colonization of biopolymers (chitin, keratin, lignocellulose, lignin, and cellulose) by activated sludge bacteria was compared with colonization of glass beads in the presence and absence of regular nutrient amendment (acetate and ammonia). Scanning electron microscopy and quantitative PCR revealed chitin and cellulose were most readily colonized followed by lignin and lignocellulose, while keratin and glass beads were relatively resistant to colonization. Bacterial community profiles on particles compared to sludge confirmed that specific bacterial phylotypes preferentially colonize different biopolymers. Nitrifying bacteria proved adept at colonizing particles, achieving higher relative abundance on particles compared to bulk sludge. Denitrifying bacteria showed similar or lower relative abundance on particles compared to sludge. KEY POINTS: • Some activated sludge bacteria colonize natural biopolymers more readily than others. • Nitrifying bacteria are overrepresented in natural biopolymer biofilm communities. • Biopolymers in wastewater likely influence activated sludge community composition.


Assuntos
Bactérias , Esgotos , Águas Residuárias , Biopolímeros/metabolismo , Bactérias/metabolismo , Bactérias/genética , Bactérias/classificação , Esgotos/microbiologia , Águas Residuárias/microbiologia , Lignina/metabolismo , Microscopia Eletrônica de Varredura , Celulose/metabolismo , Biofilmes/crescimento & desenvolvimento , Quitina/metabolismo , Nitrificação , Purificação da Água/métodos
8.
Toxins (Basel) ; 16(5)2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38787069

RESUMO

The fungal cell wall serves as the primary interface between fungi and their external environment, providing protection and facilitating interactions with the surroundings. Chitin is a vital structural element in fungal cell wall. Chitin deacetylase (CDA) can transform chitin into chitosan through deacetylation, providing various biological functions across fungal species. Although this modification is widespread in fungi, the biological functions of CDA enzymes in Aspergillus flavus remain largely unexplored. In this study, we aimed to investigate the biofunctions of the CDA family in A. flavus. The A. flavus genome contains six annotated putative chitin deacetylases. We constructed knockout strains targeting each member of the CDA family, including Δcda1, Δcda2, Δcda3, Δcda4, Δcda5, and Δcda6. Functional analyses revealed that the deletion of CDA family members neither significantly affects the chitin content nor exhibits the expected chitin deacetylation function in A. flavus. However, the Δcda6 strain displayed distinct phenotypic characteristics compared to the wild-type (WT), including an increased conidia count, decreased mycelium production, heightened aflatoxin production, and impaired seed colonization. Subcellular localization experiments indicated the cellular localization of CDA6 protein within the cell wall of A. flavus filaments. Moreover, our findings highlight the significance of the CBD1 and CBD2 structural domains in mediating the functional role of the CDA6 protein. Overall, we analyzed the gene functions of CDA family in A. flavus, which contribute to a deeper understanding of the mechanisms underlying aflatoxin contamination and lay the groundwork for potential biocontrol strategies targeting A. flavus.


Assuntos
Aflatoxinas , Amidoidrolases , Aspergillus flavus , Aspergillus flavus/genética , Aspergillus flavus/enzimologia , Aspergillus flavus/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Aflatoxinas/biossíntese , Aflatoxinas/metabolismo , Aflatoxinas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Quitina/metabolismo , Parede Celular/metabolismo
9.
J Appl Microbiol ; 135(5)2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38724455

RESUMO

AIMS: We aimed to investigate the function of an unidentified gene annotated as a PIG-L domain deacetylase (cspld) in Chitiniphilus shinanonensis SAY3. cspld was identified using transposon mutagenesis, followed by negatively selecting a mutant incapable of growing on chitin, a polysaccharide consisting of N-acetyl-d-glucosamine (GlcNAc). We focused on the physiological role of CsPLD protein in chitin utilization. METHODS AND RESULTS: Recombinant CsPLD expressed in Escherichia coli exhibited GlcNAc-6-phosphate deacetylase (GPD) activity, which is involved in the metabolism of amino sugars. However, SAY3 possesses two genes (csnagA1 and csnagA2) in its genome that code for proteins whose primary sequences are homologous to those of typical GPDs. Recombinant CsNagA1 and CsNagA2 also exhibited GPD activity with 23 and 1.6% of catalytic efficiency (kcat/Km), respectively, compared to CsPLD. The gene-disrupted mutant, Δcspld was unable to grow on chitin or GlcNAc, whereas the three mutants, ΔcsnagA1, ΔcsnagA2, and ΔcsnagA1ΔcsnagA2 grew similarly to SAY3. The determination of GPD activity in the crude extracts of each mutant revealed that CsPLD is a major enzyme that accounts for almost all cellular activities. CONCLUSIONS: Deacetylation of GlcNAc-6P catalyzed by CsPLD (but not by typical GPDs) is essential for the assimilation of chitin and its constituent monosaccharide, GlcNAc, as a carbon and energy source in C. shinanonensis.


Assuntos
Quitina , Quitina/metabolismo , Amidoidrolases/metabolismo , Amidoidrolases/genética , Acetilglucosamina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Gammaproteobacteria/genética , Gammaproteobacteria/enzimologia , Gammaproteobacteria/metabolismo
10.
Cell Rep Med ; 5(5): 101560, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38729159

RESUMO

Stimulator of IFN genes (STING) is a promising target for adjuvants utilized in in situ cancer vaccination approaches. However, key barriers remain for clinical translation, including low cellular uptake and accessibility, STING variability necessitating personalized STING agonists, and interferon (IFN)-independent signals that can promote tumor growth. Here, we identify C100, a highly deacetylated chitin-derived polymer (HDCP), as an attractive alternative to conventional STING agonists. C100 promotes potent anti-tumor immune responses, outperforming less deacetylated HDCPs, with therapeutic efficacy dependent on STING and IFN alpha/beta receptor (IFNAR) signaling and CD8+ T cell mediators. Additionally, C100 injection synergizes with systemic checkpoint blockade targeting PD-1. Mechanistically, C100 triggers mitochondrial stress and DNA damage to exclusively activate the IFN arm of the cGAS-STING signaling pathway and elicit sustained IFNAR signaling. Altogether, these results reveal an effective STING- and IFNAR-dependent adjuvant for in situ cancer vaccines with a defined mechanism and distinct properties that overcome common limitations of existing STING therapeutics.


Assuntos
Adjuvantes Imunológicos , Linfócitos T CD8-Positivos , Quitina , Proteínas de Membrana , Camundongos Endogâmicos C57BL , Receptor de Interferon alfa e beta , Transdução de Sinais , Animais , Proteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Receptor de Interferon alfa e beta/genética , Camundongos , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Humanos , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Feminino , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Neoplasias/imunologia , Neoplasias/terapia
11.
Anal Methods ; 16(20): 3278-3286, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38738557

RESUMO

Dextromethorphan (DXM) is a widely utilized central antitussive agent, which is frequently abused by individuals seeking its recreational effect. But DXM overdose can cause some adverse effects, including brain damage, loss of consciousness, and cardiac arrhythmias, and hence its detection is significant. Herein, an electrochemical sensor based on a Cu-coordinated molecularly imprinted polymer (Cu-MIP) was fabricated for its detection. For constructing the sensor, nitrogen-doped carbon nanosheets (CCNs) were prepared through calcining chitin under an argon atmosphere, and molybdenum disulfide (MoS2) was allowed to grow on their surface. Subsequently, the obtained MoS2/CCNs composite was employed to modify a glassy carbon electrode (GCE), and the Cu-MIP was electrodeposited on the electrode in a Cu-1,10-phenanthroline (Cu-Phen) solution containing DXM, where Cu2+ played a role in facilitating electron transfer and binding DXM. Due to the large specific surface area, good electrocatalytic properties and recognition of the resulting composite, the resulting Cu-MIP/MoS2/CCNs/GCE showed high selectivity and sensitivity. Under optimized experimental conditions, the peak current of DXM and its concentration exhibited a good linear relationship over the concentration range of 0.1-100 µM, and the limit of detection (S/N = 3) was 0.02 µM. Furthermore, the electrochemical sensor presented good stability, and it was successfully used for the determination of DXM in pharmaceutical, human serum and urine samples.


Assuntos
Carbono , Cobre , Dextrometorfano , Dissulfetos , Técnicas Eletroquímicas , Polímeros Molecularmente Impressos , Molibdênio , Molibdênio/química , Dissulfetos/química , Dextrometorfano/análise , Dextrometorfano/química , Dextrometorfano/urina , Cobre/química , Técnicas Eletroquímicas/métodos , Carbono/química , Polímeros Molecularmente Impressos/química , Quitina/química , Humanos , Limite de Detecção , Eletrodos , Antitussígenos/química , Antitussígenos/análise , Antitussígenos/urina
12.
Microb Cell Fact ; 23(1): 126, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38698402

RESUMO

BACKGROUND: Hydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to  evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by  response surface methodology (RSM) which delvs  into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers. RESULTS: In this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number "OR643436". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts. CONCLUSIONS: The recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.


Assuntos
Biodegradação Ambiental , Quitinases , Quitosana , Oligossacarídeos , Proteínas Recombinantes , Quitinases/metabolismo , Quitinases/genética , Oligossacarídeos/metabolismo , Animais , Quitosana/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/biossíntese , Quitina/metabolismo , Hidrocarbonetos/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Crustáceos/metabolismo , Emulsificantes/metabolismo , Emulsificantes/química
13.
Carbohydr Polym ; 337: 122165, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38710577

RESUMO

This research intended to remove residual protein from chitin with proteases in deep eutectic solvents (DESs). The activities of some proteases in several DESs, including choline chloride/p-toluenesulfonic acid, betaine/glycerol (Bet/G), choline chloride/malic acid, choline chloride/lactic acid, and choline chloride/urea, which are capable of dissolving chitin, were tested, and only in Bet/G some proteases were found to be active, with subtilisin A, ficin, and bromelain showing higher activity than other proteases. However, the latter two proteases caused degradation of chitin molecules. Further investigation revealed that subtilisin A in Bet/G did not exhibit "pH memory", which is a universal characteristic displayed by enzymes dispersed in organic phases, and the catalytic characteristics of subtilisin A in Bet/G differed significantly from those in aqueous phase. The conditions for protein removal from chitin by subtilisin A in Bet/G were determined: Chitin dissolved in Bet/G with 0.5 % subtilisin A (442.0 U/mg, based on the mass of chitin) was hydrolyzed at 45 °C for 30 min. The residual protein content in chitin decreased from 5.75 % ± 0.10 % to 1.01 % ± 0.12 %, improving protein removal by 57.20 % compared with protein removal obtained by Bet/G alone. The crystallinity and deacetylation degrees of chitin remained unchanged after the treatment.


Assuntos
Betaína , Quitina , Solventes Eutéticos Profundos , Glicerol , Quitina/química , Betaína/química , Glicerol/química , Solventes Eutéticos Profundos/química , Hidrólise , Subtilisina/metabolismo , Subtilisina/química , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/química , Colina/química
14.
World J Gastroenterol ; 30(16): 2258-2271, 2024 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-38690023

RESUMO

BACKGROUND: Irritable bowel syndrome (IBS) is one of the most frequent and debilitating conditions leading to gastroenterological referrals. However, recommended treatments remain limited, yielding only limited therapeutic gains. Chitin-glucan (CG) is a novel dietary prebiotic classically used in humans at a dosage of 1.5-3.0 g/d and is considered a safe food ingredient by the European Food Safety Authority. To provide an alternative approach to managing patients with IBS, we performed preclinical molecular, cellular, and animal studies to evaluate the role of chitin-glucan in the main pathophysiological mechanisms involved in IBS. AIM: To evaluate the roles of CG in visceral analgesia, intestinal inflammation, barrier function, and to develop computational molecular models. METHODS: Visceral pain was recorded through colorectal distension (CRD) in a model of long-lasting colon hypersensitivity induced by an intra-rectal administration of TNBS [15 milligrams (mg)/kilogram (kg)] in 33 Sprague-Dawley rats. Intracolonic pressure was regularly assessed during the 9 wk-experiment (weeks 0, 3, 5, and 7) in animals receiving CG (n = 14) at a human equivalent dose (HED) of 1.5 g/d or 3.0 g/d and compared to negative control (tap water, n = 11) and positive control (phloroglucinol at 1.5 g/d HED, n = 8) groups. The anti-inflammatory effect of CG was evaluated using clinical and histological scores in 30 C57bl6 male mice with colitis induced by dextran sodium sulfate (DSS) administered in their drinking water during 14 d. HT-29 cells under basal conditions and after stimulation with lipopolysaccharide (LPS) were treated with CG to evaluate changes in pathways related to analgesia (µ-opioid receptor (MOR), cannabinoid receptor 2 (CB2), peroxisome proliferator-activated receptor alpha, inflammation [interleukin (IL)-10, IL-1b, and IL-8] and barrier function [mucin 2-5AC, claudin-2, zonula occludens (ZO)-1, ZO-2] using the real-time PCR method. Molecular modelling of CG, LPS, lipoteichoic acid (LTA), and phospholipomannan (PLM) was developed, and the ability of CG to chelate microbial pathogenic lipids was evaluated by docking and molecular dynamics simulations. Data were expressed as the mean ± SEM. RESULTS: Daily CG orally-administered to rats or mice was well tolerated without including diarrhea, visceral hypersensitivity, or inflammation, as evaluated at histological and molecular levels. In a model of CRD, CG at a dosage of 3 g/d HED significantly decreased visceral pain perception by 14% after 2 wk of administration (P < 0.01) and reduced inflammation intensity by 50%, resulting in complete regeneration of the colonic mucosa in mice with DSS-induced colitis. To better reproduce the characteristics of visceral pain in patients with IBS, we then measured the therapeutic impact of CG in rats with TNBS-induced inflammation to long-lasting visceral hypersensitivity. CG at a dosage of 1.5 g/d HED decreased visceral pain perception by 20% five weeks after colitis induction (P < 0.01). When the CG dosage was increased to 3.0 g/d HED, this analgesic effect surpassed that of the spasmolytic agent phloroglucinol, manifesting more rapidly within 3 wk and leading to a 50% inhibition of pain perception (P < 0.0001). The underlying molecular mechanisms contributing to these analgesic and anti-inflammatory effects of CG involved, at least in part, a significant induction of MOR, CB2 receptor, and IL-10, as well as a significant decrease in pro-inflammatory cytokines IL-1b and IL-8. CG also significantly upregulated barrier-related genes including muc5AC, claudin-2, and ZO-2. Molecular modelling of CG revealed a new property of the molecule as a chelator of microbial pathogenic lipids, sequestering gram-negative LPS and gram-positive LTA bacterial toxins, as well as PLM in fungi at the lowesr energy conformations. CONCLUSION: CG decreased visceral perception and intestinal inflammation through master gene regulation and direct binding of microbial products, suggesting that CG may constitute a new therapeutic strategy for patients with IBS or IBS-like symptoms.


Assuntos
Quitina , Colo , Modelos Animais de Doenças , Glucanos , Síndrome do Intestino Irritável , Ratos Sprague-Dawley , Dor Visceral , Animais , Síndrome do Intestino Irritável/tratamento farmacológico , Síndrome do Intestino Irritável/fisiopatologia , Masculino , Humanos , Colo/efeitos dos fármacos , Colo/patologia , Ratos , Dor Visceral/tratamento farmacológico , Dor Visceral/fisiopatologia , Dor Visceral/metabolismo , Dor Visceral/etiologia , Quitina/farmacologia , Glucanos/farmacologia , Glucanos/administração & dosagem , Camundongos , Prebióticos/administração & dosagem , Ácido Trinitrobenzenossulfônico/toxicidade , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Mucosa Intestinal/metabolismo , Colite/tratamento farmacológico , Colite/induzido quimicamente , Colite/fisiopatologia , Colite/patologia , Células HT29
15.
Curr Biol ; 34(8): 1705-1717.e6, 2024 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-38574729

RESUMO

Plants establish symbiotic associations with arbuscular mycorrhizal fungi (AMF) to facilitate nutrient uptake, particularly in nutrient-limited conditions. This partnership is rooted in the plant's ability to recognize fungal signaling molecules, such as chitooligosaccharides (chitin) and lipo-chitooligosaccharides. In the legume Medicago truncatula, chitooligosaccharides trigger both symbiotic and immune responses via the same lysin-motif-receptor-like kinases (LysM-RLKs), notably CERK1 and LYR4. The nature of plant-fungal engagement is opposite according to the outcomes of immunity or symbiosis signaling, and as such, discrimination is necessary, which is challenged by the dual roles of CERK1/LYR4 in both processes. Here, we describe a LysM-RLK, LYK8, that is functionally redundant with CERK1 for mycorrhizal colonization but is not involved in chitooligosaccharides-induced immunity. Genetic mutation of both LYK8 and CERK1 blocks chitooligosaccharides-triggered symbiosis signaling, as well as mycorrhizal colonization, but shows no further impact on immunity signaling triggered by chitooligosaccharides, compared with the mutation of CERK1 alone. LYK8 interacts with CERK1 and forms a receptor complex that appears essential for chitooligosaccharides activation of symbiosis signaling, with the lyk8/cerk1 double mutant recapitulating the impact of mutations in the symbiosis signaling pathway. We conclude that this novel receptor complex allows chitooligosaccharides activation specifically of symbiosis signaling and helps the plant to differentiate between activation of these opposing signaling processes.


Assuntos
Quitina , Quitosana , Medicago truncatula , Micorrizas , Proteínas de Plantas , Simbiose , Micorrizas/fisiologia , Quitina/metabolismo , Medicago truncatula/microbiologia , Medicago truncatula/metabolismo , Medicago truncatula/imunologia , Medicago truncatula/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Imunidade Vegetal , Oligossacarídeos/metabolismo , Raízes de Plantas/microbiologia , Raízes de Plantas/metabolismo
16.
Int J Biol Macromol ; 268(Pt 2): 131731, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38649081

RESUMO

In this work we have characterized and compared chitin sourced from exoskeleton of Tenebrio molitor larvae fed with polystyrene or plastic kitchen wrap combined with bran in the ratio 1: 1 with chitin sourced from larvae exoskeleton fed only with bran. Analysis of the frass by ATR-FTIR showed very similar spectra and confirmed degradation of the plastic feed components, while ATR-FTIR analysis of the exoskeleton verified the absence of any plastic residue. Deproteinization followed by demineralization produced 6.78-5.29 % chitin, showing that plastic (polystyrene or plastic kitchen wrap) in the larvae diet resulted in heavier insect exoskeleton, but yielded slightly less chitin, with the lowest value obtained for plastic kitchen wrap in the insect diet. The deacetylation degree of 98.17-98.61 % was determined from measured ATR-FTIR spectra. XRD analysis confirmed the presence of α-chitin with a crystallinity index of 66.5-62 % and crystallite size 4-5 nm. Thermogravimetric analysis showed similar degradation curves for all chitin samples, with two degradation steps. These results show that chitin sourced from exoskeleton of T. molitor larvae fed with plastic (polystyrene or plastic kitchen wrap) and contributing to significant biodegradation of major polluting materials can be a feasible and alternative source of chitin, further promoting a bio-circular economy.


Assuntos
Quitina , Poliestirenos , Tenebrio , Animais , Tenebrio/química , Quitina/química , Poliestirenos/química , Plásticos/química , Exoesqueleto/química , Larva , Espectroscopia de Infravermelho com Transformada de Fourier
17.
Int J Biol Macromol ; 268(Pt 2): 131787, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38657939

RESUMO

Chitin oligosaccharides (CTOS) possess potential applications in food, medicine, and agriculture. However, lower mass transfer and catalytic efficiency are the main kinetic limitations for the production of CTOS from shrimp shell waste (SSW) and crystalline chitin. Chemical or physical methods are usually used for pretreatment to improve chitinase hydrolysis efficiency, but this is not eco-friendly and cost-effective. To address this challenge, a chitinase nanoreactor with the liquid-solid system (BcChiA1@ZIF-8) was manufactured to boost the one-step degradation of SSW and crystalline chitin. Compared with free enzyme, the catalytic efficiency of BcChiA1@ZIF-8 on colloidal chitin was significantly improved to 142 %. SSW and crystalline chitin can be directly degraded by BcChiA1@ZIF-8 without any pretreatments. The yield of N, N'-diacetylchitobiose [(GlcNAc)2] from SSW and N-acetyl-D-glucosamine (GlcNAc) from crystalline chitin was 2 times and 3.1 times than that of free enzyme, respectively. The reason was that BcChiA1@ZIF-8 with a liquid-solid system enlarged the interface area, increased the collision frequency between enzyme and substrate, and improved the large-substrates binding activity of chitinase. Moreover, the biphasic system exhibited excellent stability, and the design showed universal applicability. This strategy provided novel guidance for other polysaccharide biosynthesis and the conversion of environmental waste into carbohydrates.


Assuntos
Exoesqueleto , Quitina , Quitinases , Oligossacarídeos , Quitina/química , Quitina/metabolismo , Animais , Quitinases/metabolismo , Quitinases/química , Oligossacarídeos/química , Exoesqueleto/química , Hidrólise , Reatores Biológicos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Crustáceos , Cinética , Resíduos , Penaeidae/enzimologia
18.
Nat Genet ; 56(5): 970-981, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38654131

RESUMO

Barnacles are the only sessile lineages among crustaceans, and their sessile life begins with the settlement of swimming larvae (cyprids) and the formation of protective shells. These processes are crucial for adaptation to a sessile lifestyle, but the underlying molecular mechanisms remain poorly understood. While investigating these mechanisms in the acorn barnacle, Amphibalanus amphitrite, we discovered a new gene, bcs-6, which is involved in the energy metabolism of cyprid settlement and originated from a transposon by acquiring the promoter and cis-regulatory element. Unlike mollusks, the barnacle shell comprises alternate layers of chitin and calcite and requires another new gene, bsf, which generates silk-like fibers that efficiently bind chitin and aggregate calcite in the aquatic environment. Our findings highlight the importance of exploring new genes in unique adaptative scenarios, and the results will provide important insights into gene origin and material development.


Assuntos
Thoracica , Animais , Thoracica/genética , Adaptação Fisiológica/genética , Larva/genética , Quitina/metabolismo , Filogenia , Carbonato de Cálcio , Elementos de DNA Transponíveis/genética , Metabolismo Energético/genética , Evolução Molecular
19.
Int J Biol Macromol ; 268(Pt 1): 131815, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38670192

RESUMO

We report on the extraction of ß-chitin from pens (or Gladius) of Uroteuthis edulis, a squid species prevalent in the Pacific coastal regions of East Asia. In particular, we employ cryogenic mechanical grinding (or cryomilling) as a pre-treatment process for the raw squid pens. We show that the cryomilling step enables an effective pulverization of the raw materials, which facilitates the removal of protein residues allowing the extraction of high-purity ß-chitin with a high acetylation degree (∼97 %) and crystallinity (∼82 %). We also demonstrate that the Uroteuthis edulis extract ß-chitin affords a free-standing film with excellent optical transmittance and mechanical properties.


Assuntos
Quitina , Decapodiformes , Quitina/química , Quitina/isolamento & purificação , Decapodiformes/química , Animais , Acetilação
20.
Int J Biol Macromol ; 266(Pt 2): 131314, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38569995

RESUMO

Breeding of the black soldier fly is carried out to produce proteins. It is accompanied by releases during the life cycle of this insect. This work is a study of the valorization of these rejects through the production of chitins and chitosans with controlled characteristics. An extraction process is developed with an order of treatments and reaction conditions that provide chitins with high contents. These contents increase as the stages of the life cycle progress and drop for the adult. However, the exuviae chitins present organic impurities which will be eliminated at the N-deacetylation reaction for pupe and after a purification treatment for chitosan from larval stages. All these chitins have an α structure although certain physicochemical characteristics of the larval exuviae chitins are close to those presented by γ chitin. The observed shifts are linked to the effect of impurities rather than to a difference in structure. N-deacetylation of chitins makes possible the valorization of all rejects by the production of pure chitosans with high yields which retain a porous structure for the exuviae and fibrous for the adult which allow complementary applications. These chitosans are highly to completely deacetylated and their molar masses can vary depending on the process and life stage.


Assuntos
Quitina , Quitosana , Quitosana/química , Animais , Quitina/química , Dípteros/metabolismo , Dípteros/química , Dípteros/crescimento & desenvolvimento , Acetilação , Larva , Cruzamento , Estágios do Ciclo de Vida , Fenômenos Químicos
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