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1.
Mol Cell ; 83(24): 4509-4523.e11, 2023 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-38134885

RESUMO

The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.


Assuntos
Retículo Endoplasmático , Proteínas , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas/metabolismo , Citosol/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transporte Proteico , Biossíntese de Proteínas
2.
RNA ; 28(1): 76-87, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34706978

RESUMO

Most cellular processes are carried out by protein complexes, but it is still largely unknown how the subunits of lowly expressed complexes find each other in the crowded cellular environment. Here, we will describe a working model where RNA-binding proteins in cytoplasmic condensates act as matchmakers between their bound proteins (called protein targets) and newly translated proteins of their RNA targets to promote their assembly into complexes. Different RNA-binding proteins act as scaffolds for various cytoplasmic condensates with several of them supporting translation. mRNAs and proteins are recruited into the cytoplasmic condensates through binding to specific domains in the RNA-binding proteins. Scaffold RNA-binding proteins have a high valency. In our model, they use homotypic interactions to assemble condensates and they use heterotypic interactions to recruit protein targets into the condensates. We propose that unoccupied binding sites in the scaffold RNA-binding proteins transiently retain recruited and newly translated proteins in the condensates, thus promoting their assembly into complexes. Taken together, we propose that lowly expressed subunits of protein complexes combine information in their mRNAs and proteins to colocalize in the cytoplasm. The efficiency of protein complex assembly is increased by transient entrapment accomplished by multivalent RNA-binding proteins within cytoplasmic condensates.


Assuntos
Condensados Biomoleculares/química , Chaperonas Moleculares/química , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Ribonucleoproteínas/química , Sítios de Ligação , Condensados Biomoleculares/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Eucariotos , Células Eucarióticas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Agregados Proteicos , Ligação Proteica , Biossíntese de Proteínas , Dobramento de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
3.
Proteomics ; 23(12): e2200281, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36843329

RESUMO

Target identification by modification-free proteomic approaches can potentially reveal the pharmacological mechanism of small molecular compounds. By combining the recent solvent-induced protein precipitation (SIP) method with TMT-labeling quantitative proteomics, we propose solvent-induced proteome profiling (SIPP) approach to identify small molecule-protein interactions. The SIPP approach enables to depict denaturation curves of the target protein by varying concentrations of organic solvents to induce unfolding and precipitation of the cellular proteome. By using this approach, we have successfully identified the known targets of market drugs and natural products and extended the proteome information of SIP for target identification.


Assuntos
Proteoma , Proteômica , Solventes , Espectrometria de Massas
4.
Microb Cell Fact ; 22(1): 93, 2023 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-37143012

RESUMO

BACKGROUND: Prolyl endopeptidase from Aspergillus niger (AN-PEP) is a prominent serine proteinase with various potential applications in the food and pharmaceutical industries. However, the availability of efficient and low-cost AN-PEP remains a challenge owing to its low yield and high fermentation cost. RESULTS: Here, AN-PEP was recombinantly expressed in Trichoderma reesei (rAN-PEP) under the control of the cbh1 promoter and its secretion signal. After 4 days of shaking flask cultivation with the model cellulose Avicel PH101 as the sole carbon source, the extracellular prolyl endopeptidase activity reached up to 16.148 U/mL, which is the highest titer reported to date and the secretion of the enzyme is faster in T. reesei than in other eukaryotic expression systems including A. niger and Komagataella phaffii. Most importantly, when cultivated on the low-cost agricultural residue corn cob, the recombinant strain was found to secret a remarkable amount of rAN-PEP (37.125 U/mL) that is twice the activity under the pure cellulose condition. Furthermore, treatment with rAN-PEP during beer brewing lowered the content of gluten below the ELISA kit detection limit (< 10 mg/kg) and thereby, reduced turbidity, which would be beneficial for improving the non-biological stability of beer. CONCLUSION: Our research provides a promising approach for industrial production of AN-PEP and other enzymes (proteins) from renewable lignocellulosic biomass, which provides a new idea with relevant researchers for the utilization of agricultural residues.


Assuntos
Prolil Oligopeptidases , Trichoderma , Prolil Oligopeptidases/metabolismo , Aspergillus niger/metabolismo , Cerveja , Celulose/metabolismo , Fermentação , Trichoderma/metabolismo
5.
Microb Cell Fact ; 21(1): 76, 2022 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-35525939

RESUMO

BACKGROUND: Ergothioneine (ERG) is a potent histidine-derived antioxidant that confers health-promoting effects. Only certain bacteria and fungi can biosynthesize ERG, but the ERG productivity in natural producers is low. ERG overproduction through genetic engineering represents an efficient and cost-effective manufacturing strategy. RESULTS: Here, we showed that Trichoderma reesei can synthesize ERG during conidiogenesis and hyphal growth. Co-expression of two ERG biosynthesis genes (tregt1 and tregt2) from T. reesei enabled E. coli to generate 70.59 mg/L ERG at the shaking flask level after 48 h of whole-cell biocatalysis, whereas minor amounts of ERG were synthesized by the recombinant E. coli strain bearing only the tregt1 gene. By fed-batch fermentation, the extracellular ERG production reached 4.34 g/L after 143 h of cultivation in a 2-L jar fermenter, which is the highest level of ERG production reported thus far. Similarly, ERG synthesis also occurred in the E. coli strain engineered with the two well-characterized genes from N. crassa and the ERG productivity was up to 4.22 g/L after 143 h of cultivation under the above-mentioned conditions. CONCLUSIONS: Our results showed that the overproduction of ERG in E. coli could be achieved through two-enzymatic steps, demonstrating high efficiency of the fungal ERG biosynthetic pathway. Meanwhile, this work offers a more promising approach for the industrial production of ERG.


Assuntos
Ergotioneína , Vias Biossintéticas , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Histidina/genética , Engenharia Metabólica
6.
BMC Plant Biol ; 19(1): 266, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221095

RESUMO

BACKGROUND: Pogostemon cablin (Blanco) Benth. (Patchouli) is an important aromatic and medicinal plant and widely used in traditional Chinese medicine as well as in the perfume industry. Patchoulol is the primary bioactive component in P. cablin, its biosynthesis has attracted widespread interests. Previous studies have surveyed the putative genes involved in patchoulol biosynthesis using next-generation sequencing method; however, technical limitations generated by short-read sequencing restrict the yield of full-length genes. Additionally, little is known about the expression pattern of genes especially patchoulol biosynthesis related genes in response to methyl jasmonate (MeJA). Our understanding of patchoulol biosynthetic pathway still remained largely incomplete to date. RESULTS: In this study, we analyzed the morphological character and volatile chemical compounds of P. cablin cv. 'Zhanxiang', and 39 volatile chemical components were detected in the patchouli leaf using GC-MS, most of which were sesquiterpenes. Furthermore, high-quality RNA isolated from leaves and stems of P. cablin were used to generate the first full-length transcriptome of P. cablin using PacBio isoform sequencing (Iso-Seq). In total, 9.7 Gb clean data and 82,335 full-length UniTransModels were captured. 102 transcripts were annotated as 16 encoding enzymes involved in patchouli alcohol biosynthesis. Accorded with the uptrend of patchoulol content, the vast majority of genes related to the patchoulol biosynthesis were up-regulated after MeJA treatment, indicating that MeJA led to an increasing synthesis of patchoulol through activating the expression level of genes involved in biosynthesis pathway of patchoulol. Moreover, expression pattern analysis also revealed that transcription factors participated in JA regulation of patchoulol biosynthesis were differentially expressed. CONCLUSIONS: The current study comprehensively reported the morphological specificity, volatile chemical compositions and transcriptome characterization of the Chinese-cultivated P. cablin cv. 'Zhanxiang', these results contribute to our better understanding of the physiological and molecular features of patchouli, especially the molecular mechanism of biosynthesis of patchoulol. Our full-length transcriptome data also provides a valuable genetic resource for further studies in patchouli.


Assuntos
Regulação da Expressão Gênica de Plantas , Pogostemon/genética , Sesquiterpenos/metabolismo , Acetatos , Vias Biossintéticas , Ciclopentanos , Perfilação da Expressão Gênica , Oxilipinas , Transcriptoma
7.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801204

RESUMO

The JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators in the jasmonic acid (JA) signaling pathways of plants, and these proteins have been reported to play key roles in plant secondary metabolism mediated by JA. In this study, we firstly isolated one JAZ from P. cablin, PatJAZ6, which was characterized and revealed based on multiple alignments and a phylogenic tree analysis. The result of subcellular localization indicated that the PatJAZ6 protein was located in the nucleus of plant protoplasts. The expression level of PatJAZ6 was significantly induced by the methyl jasmonate (MeJA). Furthermore, by means of yeast two-hybrid screening, we identified two transcription factors that interact with the PatJAZ6, the PatMYC2b1 and PatMYC2b2. Virus-induced gene silencing (VIGS) of PatJAZ6 caused a decrease in expression abundance, resulting in a significant increase in the accumulation of patchouli alcohol. Moreover, we overexpressed PatJAZ6 in P. cablin, which down-regulated the patchoulol synthase expression, and then suppressed the biosynthesis of patchouli alcohol. The results demonstrate that PatJAZ6 probably acts as a repressor in the regulation of patchouli alcohol biosynthesis, contributed to a model proposed for the potential JA signaling pathway in P. cablin.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Pogostemon/genética , Proteínas Repressoras/genética , Sesquiterpenos/metabolismo , Acetatos/farmacologia , Sequência de Aminoácidos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Inativação Gênica , Isomerases/genética , Isomerases/metabolismo , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Filogenia , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Pogostemon/classificação , Pogostemon/efeitos dos fármacos , Pogostemon/metabolismo , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Técnicas do Sistema de Duplo-Híbrido
8.
Zhongguo Zhong Yao Za Zhi ; 43(12): 2503-2508, 2018 Jun.
Artigo em Zh | MEDLINE | ID: mdl-29950067

RESUMO

To study the SSR loci information and develop molecular markers, a total of 43 683 Unigenes in transcriptome of Andrographis paniculata were used to explore SSR. The distribution frequency of SSR and the basic characteristics of repeat motifs were analyzed using MicroSAtellite software, SSR primers were designed by Primer 3.0 software and then validated by PCR. Moreover, the gene function analysis of SSR Unigene was obtained by Blast. The results showed that 14 135 SSR loci were found in the transcriptome of A. paniculata, which distributed in 9 973 Unigenes with a distribution frequency of 32.36%. Di-nucleotide and Tri-nucleotide repeat were the main types, accounted for 75.54% of all SSRs. The repeat motifs of AT/AT and CCG/CGG were the predominant repeat types of Di-nucleotide and Tri-nucleotide, respectively. A total of 4 740 pairs of SSR primers with the potential to produce polymorphism were designed for maker development. Ten pairs of primers in 20 pairs of randomly picked primers produced fragments with expected molecular size. The gene function of Unigenes containing SSR were mostly related to the basic metabolism function of A. paniculata. The SSR markers in transcriptome of A. paniculata show rich type, strong specificity and high potential of polymorphism, which will benefit the candidate gene mining and marker-assisted breeding.


Assuntos
Andrographis/genética , Repetições de Microssatélites , Transcriptoma , Primers do DNA , Polimorfismo Genético
9.
Biochem Biophys Res Commun ; 459(2): 184-188, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25534854

RESUMO

Trichoderma reesei is thought to be a promising recombinant host for the production and secretion of complex proteins due to its ability to secrete large amounts of proteins. In this study we identified a functional N-acetyl-ß-glucosaminidase (NAGase) gene Nag1 in T. reesei. Nag1, a putative gene encoding a GH 20 family NAGase in T. reesei, was cloned and homologous overexpressed in the T. reesei RutC30ΔU3 with a strong cellobiohydrolase1 gene (cbh1) promoter. Nag1 was secreted in its active form and the highest expression level was around 499.85IU/ml. Nag1 has a molecular mass of 80kDa. The optimum pH and temperature were 4.0 and 60°C, respectively.


Assuntos
Acetilglucosaminidase/genética , Acetilglucosaminidase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Trichoderma/enzimologia , Trichoderma/genética , Acetilglucosaminidase/química , Sequência de Aminoácidos , Clonagem Molecular , Proteínas Fúngicas/química , Genes Fúngicos , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Dados de Sequência Molecular , Peso Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Temperatura , Regulação para Cima
10.
Biochem Biophys Res Commun ; 460(3): 663-9, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-25817789

RESUMO

Trichoderma reesei (teleomorph Hypocrea jecorina) is an industrially important filamentous fungus for glycoside hydrolases production, with its xylanolytic enzymes widely applied in many areas. However, the molecular mechanisms underlying xylanase expression are still insufficiently understood. In particular, the effect of sugar transporter on the induction of xylanase expression is unclear. In this work, we identified a novel major facilitator transporter TrSTR1 that is capable of transporting xylose by using a xylose utilization system in Saccharomyces cerevisiae. In T. reesei, TrSTR1 is essential for the utilization of d-xylose, l-arabinose, and even their downstream metabolites D-xylitol and L-arabitol. TrSTR1 is also involved in the induction of xylanase expression since both the xylanase activity and extracellular protein concentration in the Tu6△str1 strain were decreased, which further confirmed by a qRT-PCR analysis of the transcript levels of the key transcriptional regulators. Our observations provide new insights into connections between pentose utilization and xylanase production in T. reesei.


Assuntos
Endo-1,4-beta-Xilanases/biossíntese , Pentoses/metabolismo , Trichoderma/metabolismo , Biologia Computacional , Indução Enzimática , Trichoderma/enzimologia
11.
Biotechnol Lett ; 37(10): 2055-62, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26112324

RESUMO

OBJECTIVES: Eukaryotic mitogen-activated protein kinases (MAPKs) play crucial roles in transducing environmental and developmental signals inside the cell and regulating gene expression, however, the roles of MAPKs remain largely unknown in Trichoderma reesei. RESULTS: T. reesei ime2 (TrIme2) encodes an Ime2-like MAPK in T. reesei. The deletion of the TrIme2 gene led to 90% increase in cellulase activity against filter paper during earlier period time of cellulase induction as well as the extracellular protein production. Compared to the parent strain, the transcriptional levels of the three major cellulase genes cbh1,cbh2, egl1 were increased by about 9 times, 4 times, 2 times, respectively, at 8 h after cellulase induction in the ΔTrIme2 mutant. In addition, the disruption of TrIme2 caused over 50% reduction of the transcript levels of cellulase transcriptional regulators cre1 and xyr1. CONCLUSION: TrIme2 functions in regulation of the expression of cellulase gene in T.reesei, and is a good candidate for genetically engineering of T. reesei for higher cellulase production.


Assuntos
Celulase/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Trichoderma/enzimologia , Trichoderma/genética , Fungos/enzimologia , Fungos/genética , Deleção de Genes , Perfilação da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/genética
12.
Acta Radiol ; 56(9): 1100-4, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25168022

RESUMO

BACKGROUND: Differentiation of anomalous systemic artery to the left lower lobe (ASALLL) from pulmonary sequestration (PS) is essential, as ASALLL can be corrected by anastomosis, embolization, or ligation of the anomalous artery. PURPOSE: To compare computed tomography (CT) findings of ASALLL and PS in the left lower lobe (LLL). MATERIALS AND METHODS: This study included 16 patients with ASALLL and 25 patients with PS in LLL confirmed by operative and pathologic findings. RESULTS: Cough and sputum were more common in PS (84% and 60%, respectively) than in ASALLL (25% and 12.5%, respectively) (P < 0.05). Hemoptysis was more common in ASALLL (100%) than in PS (24%) (P < 0.05). The frequency of ground glass opacity (GGO), normal bronchial distribution, dilated left inferior pulmonary veins, and absence of the interlobar artery distal to the origin of the superior segmental artery in LLL differed significantly between ASALLL and PS. Mass was less common in ASALLL (0%) than in PS (88%) (P < 0.01). The mean diameter of the anomalous artery (11.88 ± 1.13 mm) in ASALLL was significantly larger than that (5.96 ± 0.98 mm) in PS (P < 0.01). The presence of anomalous artery arising from thoracic aorta was not different between ASALLL (100%) and PS (72%). CONCLUSION: Radiographic indications of ASALLL differ from those of PS in the LLL. Indications that may suggest ASALLL include an enlarged anomalous systemic artery arising from the thoracic aorta, dilated left inferior pulmonary veins, absence of the interlobar artery distal to the origin of the superior segmental artery, normal bronchial distribution, and GGO in the LLL.


Assuntos
Sequestro Broncopulmonar/diagnóstico por imagem , Artéria Pulmonar/anormalidades , Tomografia Computadorizada por Raios X/métodos , Adulto , Sequestro Broncopulmonar/cirurgia , Meios de Contraste , Feminino , Humanos , Iohexol/análogos & derivados , Masculino , Pneumonectomia , Veias Pulmonares/anormalidades
13.
Biochem Biophys Res Commun ; 443(3): 899-904, 2014 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-24361891

RESUMO

Bacillus subtilis is a facultative anaerobic Gram-positive non-pathogenic bacterium that includes members displaying hemolytic activity. To identify the genes responsible for hemolysis, a random mariner-based transposon insertion mutant library of B. subtilis 168 was constructed. More than 20,000 colonies were screened for the hypohemolytic phenotype on blood agar plates. One mutant showed significantly less pronounced hemolytic phenotype than the wild type. DNA sequencing and Southern blot analysis showed this mutant has a single transposable element inserted into the open reading frame (ORF) of the spoVG gene; complementation of the spoVG-disrupted mutant with a wild-type copy restored its hemolytic phenotype. It was therefore concluded that the spoVG gene, which plays a role in regulating asymmetric septation during sporulation in B. subtilis, is involved in hemolysis by B. subtilis.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Hemólise , Animais , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Mutagênese Insercional/genética , Mutação/genética , Fenótipo , Plasmídeos/metabolismo , Coelhos
14.
Quant Imaging Med Surg ; 14(2): 1616-1635, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38415168

RESUMO

Background: The high-definition standard (HD-standard) scan mode has been proven to display stents better than the standard (STND) scan mode but with more image noise. Deep learning image reconstruction (DLIR) is capable of reducing image noise. This study examined the impact of HD-standard scan mode with DLIR algorithms on stent and coronary artery image quality in coronary computed tomography angiography (CCTA) via a comparison with conventional STND scan mode and adaptive statistical iterative reconstruction-Veo (ASIR-V) algorithms. Methods: The data of 121 patients who underwent HD-standard mode scans (group A: N=47, with coronary stent) or STND mode scans (group B: N=74, without coronary stent) were retrospectively collected. All images were reconstructed with ASIR-V at a level of 50% (ASIR-V50%) and a level of 80% (ASIR-V80%) and with DLIR at medium (DLIR-M) and high (DLIR-H) levels. The noise, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), artifact index (AI), and in-stent diameter were measured as objective evaluation parameters. Subjective assessment involved a 5-point scale for overall image quality, image noise, stent appearance, stent artifacts, vascular sharpness, and diagnostic confidence. Diagnostic confidence was evaluated based on the presence or absence of significant stenosis (≥50% lumen reduction). Both subjective and objective evaluations were conducted by two radiologists independently, with kappa and intraclass correlation statistics being used to test the interobserver agreement. Results: There were 76 evaluable stents in group A, and the DLIR-H algorithm significantly outperformed other algorithms, demonstrating the lowest noise (41.6±7.1/41.3±7.2) and AI (32.4±8.9/31.2±10.1), the highest SNR (14.6±3.5/15.0±3.5) and CNR (13.6±3.8/13.9±3.8), and the largest in-stent diameter (2.18±0.61/2.19±0.61) in representing true stent diameter (all P values <0.01), as well as the highest score in each subjective evaluation parameter. In group B, a total of 296 coronary arteries were evaluated, and the DLIR-H algorithm provided the best objective image quality, with statistically superior noise, SNR, and CNR compared with the other algorithms (all P values <0.05). Moreover, the HD-standard mode scan with DLIR provided better image quality and a lower radiation dose than did the STND mode scan with ASIR-V (P<0.01). Conclusions: HD-standard scan mode with DLIR-H improves image quality of both stents and coronary arteries on CCTA under a lower radiation dose.

15.
Antibiotics (Basel) ; 13(3)2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38534713

RESUMO

Antimicrobial peptides (AMPs) hold promise as alternatives to combat bacterial infections, addressing the urgent global threat of antibiotic resistance. COG1410, a synthetic peptide derived from apolipoprotein E, has exhibited potent antimicrobial properties against various bacterial strains, including Mycobacterium smegmatis. However, our study reveals a previously unknown resistance mechanism developed by M. smegmatis against COG1410 involving ClpC. Upon subjecting M. smegmatis to serial passages in the presence of sub-MIC COG1410, resistance emerged. The comparative genomic analysis identified a point mutation in ClpC (S437P), situated within its middle domain, which led to high resistance to COG1410 without compromising bacterial fitness. Complementation of ClpC in mutant restored bacterial sensitivity. In-depth analyses, including transcriptomic profiling and in vitro assays, uncovered that COG1410 interferes with ClpC at both transcriptional and functional levels. COG1410 not only stimulated the ATPase activity of ClpC but also enhanced the proteolytic activity of Clp protease. SPR analysis confirmed that COG1410 directly binds with ClpC. Surprisingly, the identified S437P mutation did not impact their binding affinity. This study sheds light on a unique resistance mechanism against AMPs in mycobacteria, highlighting the pivotal role of ClpC in this process. Unraveling the interplay between COG1410 and ClpC enriches our understanding of AMP-bacterial interactions, offering potential insights for developing innovative strategies to combat antibiotic resistance.

16.
Front Microbiol ; 15: 1397830, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38784808

RESUMO

The rise of antimicrobial resistance in ESKAPEE pathogens poses significant clinical challenges, especially in polymicrobial infections. Bacteriophage-derived endolysins offer promise in combating this crisis, but face practical hurdles. Our study focuses on engineering endolysins from a Klebsiella pneumoniae phage, fusing them with ApoE23 and COG133 peptides. We assessed the resulting chimeric proteins' bactericidal activity against ESKAPEE pathogens in vitro. ApoE23-Kp84B (CHU-1) reduced over 3 log units of CFU for A. baumannii, E. faecalis, K. pneumoniae within 1 h, while COG133-Kp84B (CHU-2) showed significant efficacy against S. aureus. COG133-L1-Kp84B, with a GS linker insertion in CHU-2, exhibited outstanding bactericidal activity against E. cloacae and P. aeruginosa. Scanning electron microscopy revealed alterations in bacterial morphology after treatment with engineered endolysins. Notably, CHU-1 demonstrated promising anti-biofilm and anti-persister cell activity against A. baumannii and E. faecalis but had limited efficacy in a bacteremia mouse model of their coinfection. Our findings advance the field of endolysin engineering, facilitating the customization of these proteins to target specific bacterial pathogens. This approach holds promise for the development of personalized therapies tailored to combat ESKAPEE infections effectively.

17.
Int J Antimicrob Agents ; 64(2): 107220, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38810939

RESUMO

Phage therapy offers a promising approach to combat the growing threat of antimicrobial resistance. Yet, key questions remain regarding dosage, administration routes, combination therapy, and the causes of therapeutic failure. In this study, we focused on a novel lytic phage, ФAb4B, which specifically targeted the Acinetobacter baumannii strains with KL160 capsular polysaccharide, including the pan-drug resistant A. baumannii YQ4. ФAb4B exhibited the ability to effectively inhibit biofilm formation and eradicate mature biofilms independently of dosage. Additionally, it demonstrated a wide spectrum of antibiotic-phage synergy and did not show any cytotoxic or haemolytic effects. Continuous phage injections, both intraperitoneally and intravenously over 7 d, showed no acute toxicity in vivo. Importantly, phage therapy significantly improved neutrophil counts, outperforming ciprofloxacin. However, excessive phage injections suppressed neutrophil levels. The combinatorial treatment of phage-ciprofloxacin rescued 91% of the mice, a superior outcome compared to phage alone (67%). The efficacy of the combinatorial treatment was independent of phage dosage. Notably, prophylactic administration of the combinatorial regimen provided no protection, but even when combined with a delayed therapeutic regimen, it saved all the mice. Bacterial resistance to the phage was not a contributing factor to treatment failure. Our preclinical study systematically describes the lytic phage's effectiveness in both in vitro and in vivo settings, filling in crucial details about phage treatment against bacteriemia caused by A. baumannii, which will provide a robust foundation for the future of phage therapy.

18.
EMBO Rep ; 12(3): 244-51, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21331095

RESUMO

Two copies of each core histone exist in every nucleosome; however, it is not known whether both histones within a nucleosome are required to be symmetrically methylated at the same lysine residues. We report that for most lysine methylation states, wild-type histones paired with mutant, unmethylatable histones in mononucleosomes have comparable methylation levels to bulk histones. Our results indicate that symmetrical histone methylation is not required on a global scale. However, wild-type H4 histones paired with unmethylatable H4K20R histones showed reduced levels of H4K20me2 and H4K20me3, suggesting that some fractions of these modifications might exist symmetrically, and enzymes mediating these modifications might, to some extent, favour nucleosome substrates with premethylated H4K20.


Assuntos
Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Linhagem Celular , Células HeLa , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Lisina/química , Espectrometria de Massas , Metilação
19.
Heliyon ; 9(6): e16590, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37292283

RESUMO

As nodes in Social Internet of Things (SIoT) become more intelligent, malicious information occurs more frequently and spreads more widely. This problem can severely affect the trustworthiness of services and applications in SIoT. Methods to effectively control malicious information spreading in SIoT are essential and necessary. Reputation mechanism provides a powerful tool to tackle this challenge. In this paper, we propose a reputation-based mechanism to activate the self-purification capacity of the SIoT network by balancing information conflicts triggered by reporters and supporters. In order to find the best rewarding and punishment strategy, a bilateral cumulative-prospect-based evolutionary game model of SIoT network information conflict is constructed. Using local stability analysis and numerical simulation, the evolutionary trends of the proposed game model under different theoretical application scenarios are analyzed. The findings indicate that the basic income and deposit of both sides, the popularity of information as well as the importance of the conformity effect all have a significant impact on the system's steady state and evolutionary path. The specific conditions that both participating sides of the game tend to treat conflicts relatively rationally are analyzed. Dynamic evolution analysis and sensitivity analysis of selected parameters show that basic income is positively related to smart object's feedback strategies, while deposit is negatively related to that. While weight of conformity effect or the information popularity goes up, the rising of feedback probability is observed. Based on the above results, suggestions on dynamic reward and punishment strategies are given. The proposed model is a helpful attempt to model the evolution of information spreading in SIoT networks, with the ability to simulate several well-known regularities of message dissemination. Proposed model and suggested quantitative strategies can be helpful to build feasible malicious information control facilities in SIoT networks.

20.
bioRxiv ; 2023 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-37961296

RESUMO

It is currently not known that mRNAs fulfill structural roles in the cytoplasm. Here, we report the FXR1 network, an mRNA-protein (mRNP) network present throughout the cytoplasm: FXR1 packages exceptionally long mRNAs that serve as an underlying network scaffold and concentrate FXR1 molecules, which have multiple protein binding sites. The proximity of FXR1 molecules makes the FXR1 network a hub for transient interactions of proteins lacking RNA-binding domains. We show that the FXR1 network is necessary for RhoA signaling-induced actomyosin reorganization to provide spatial proximity between kinases and their substrates. A point mutation in FXR1, which is found in its FMR1 homolog and causes Fragile X syndrome, disrupts the network. FXR1 network disruption prevents actomyosin remodeling-an essential and ubiquitous process for the regulation of cell shape, migration, and synaptic function. These findings uncover a structural role for cytoplasmic mRNA and show how the FXR1 RNA-binding protein as part of the FXR1 network acts as organizer of signaling reactions.

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