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The tellurite toxicity in Haemophilus influenzae and H. parainfluenzae remains unclear. To understand the potential of tellurite as a therapeutic option for these bacteria, we investigated the antimicrobial efficacy of AS101, a tellurium compound, against H. influenzae and H. parainfluenzae and the molecular basis of their differences in AS101 susceptibility. Through broth microdilution, we examined the minimum inhibitory concentration (MIC) of AS101 in 51 H. influenzae and 28 H. parainfluenzae isolates. Whole-genome sequencing was performed on the H. influenzae isolates to identify genetic variations associated with AS101 susceptibility. The MICs of AS101 were ⦠4, 16-32, and ⧠64 µg/mL in 9 (17.6%), 12 (23.5%), and 30 (58.8%) H. influenzae isolates, respectively, whereas ⦠0.5 µg/mL in all H. parainfluenzae isolates, including multidrug-resistant isolates. Time-killing kinetic assay and scanning electron microscopy revealed the in vitro bactericidal activity of AS101 against H. parainfluenzae. Forty variations in nine tellurite resistance-related genes were associated with AS101 susceptibility. Logistic regression, receiver operator characteristic curve analysis, Venn diagram, and protein sequence alignment indicated that Val195Ile substitution in TerC, Ser93Gly in Gor (glutathione reductase), Pro44Ala/Ala50Pro in NapB (nitrate reductase), Val307Leu in TehA (tellurite resistance protein), Cys105Arg in CysK (cysteine synthase), and Thr364Ser in Csd (Cysteine desulfurase) were strongly associated with reduced AS101 susceptibility, whereas Ser155Pro in TehA with increased AS101 susceptibility. In conclusions, the antimicrobial efficacy of AS101 is high against H. parainfluenzae but low against H. influenzae. Genetic variations and corresponding protein changes relevant to AS101 non-susceptibility in H. influenzae were identified.
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Immunoglobulin A1 (IgA1) protease is a critical virulence factor of Haemophilus influenzae that facilitates bacterial mucosal infection. This study investigates the effect of iga gene polymorphism on the enzymatic activity of H. influenzae IgA1 protease. The IgA1 protease activity was examined in the H. influenzae Rd KW20 strain and 51 isolates. Genetic variations in iga and deduced amino acid substitutions affecting IgA1 protease activity were assessed. Machine learning tools and functional complementation assays were used to analyze the effects of identified substitutions on the stability and activity of IgA1 protease, respectively. All 51 isolates exhibited similar iga expression levels. No igaB expression was detected. According to comparisons with the reference Rd KW20 strain, four substitutions in the protease domain, 26 in the nonprotease passenger domain, and two in the ß-barrel domain were associated with the change in IgA1 protease activity. No substitutions in the catalytic site of IgA1 protease were observed. Logistic regression, receiver operating characteristic curves, Venn diagrams, and protein stability analyses revealed that the substitutions Asn352Lys, Pro353Ala, Lys356Asn, Gln916Lys, and Gly917Ser, which were located in the nonactive site of the passenger domain, were associated with decreases in IgA1 protease activity and stability, whereas Asn914Lys was associated with an increase in these events. Functional complementation assays revealed that the Asn914Lys substitution increased IgA1 protease activity in the Rd KW20 strain. This study identified substitutions in the nonactive site of the passenger domain that affect both the activity and stability of H. influenzae IgA1 protease.
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Haemophilus influenzae , Haemophilus influenzae/genética , Haemophilus influenzae/enzimología , Humanos , Sustitución de Aminoácidos , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/química , Inmunoglobulina A/metabolismo , Infecciones por Haemophilus/microbiología , Infecciones por Haemophilus/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/químicaRESUMEN
The relevance of aberrant serum IgG N-glycosylation in liver fibrosis has been identified; however, its causal effect remains unclear. Because hepatic stellate cells (HSCs) contribute substantially to liver fibrosis, we investigated whether and through which mechanisms IgG N-glycosylation affects the fibrogenic properties of HSCs. Analysis of serum IgG1 N-glycome from 151 patients with chronic hepatitis B or liver cirrhosis revealed a positive correlation between Ishak fibrosis grading and IgG1 with agalactosyl N-glycoforms on the crystallizable fragment (Fc). Fc gamma receptor (FcγR) IIIa was observed in cultured human HSCs and HSCs in human liver tissues, and levels of FcγRIIIa in HSCs correlated with the severity of liver fibrosis. Additionally, agalactosyl IgG treatment caused HSCs to have a fibroblast-like morphology, enhanced migration and invasion capabilities, and enhanced expression of the FcγRIIIa downstream tyrosine-protein kinase SYK. Furthermore, agalactosyl IgG treatment increased fibrogenic factors in HSCs, including transforming growth factor (TGF)-ß1, total collagen, platelet-derived growth factor subunit B and its receptors, pro-collagen I-α1, α-smooth muscle actin, and matrix metalloproteinase 9. These effects were more pronounced in HSCs that stably expressed FCGR3A and were reduced in FCGR3A knockout cells. Agalactosyl IgG and TGF-ß1 each increased FCGR3A in HSCs. Furthermore, serum TGF-ß1 concentrations in patients were positively correlated with agalactosyl IgG1 levels and liver fibrosis severity, indicating a positive feedback loop involving agalactosyl IgG, HSC-FcγRIIIa, and TGF-ß1. In conclusion, agalactosyl IgG promotes fibrogenic characteristics in HSCs through FcγRIIIa. © 2024 The Pathological Society of Great Britain and Ireland.
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Células Estrelladas Hepáticas , Inmunoglobulina G , Cirrosis Hepática , Receptores de IgG , Humanos , Receptores de IgG/metabolismo , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Inmunoglobulina G/metabolismo , Inmunoglobulina G/farmacología , Glicosilación , Masculino , Persona de Mediana Edad , Femenino , Movimiento Celular , Hepatitis B Crónica/patología , Hepatitis B Crónica/metabolismo , Transducción de Señal , Quinasa Syk/metabolismo , Adulto , Anciano , Células CultivadasRESUMEN
A decreased chloramphenicol susceptibility in Haemophilus influenzae is commonly caused by the activity of chloramphenicol acetyltransferases (CATs). However, the involvement of membrane proteins in chloramphenicol susceptibility in H. influenzae remains unclear. In this study, chloramphenicol susceptibility testing, whole-genome sequencing, and analyses of membrane-related genes were performed in 51 H. influenzae isolates. Functional complementation assays and structure-based protein analyses were conducted to assess the effect of proteins with sequence substitutions on the minimum inhibitory concentration (MIC) of chloramphenicol in CAT-negative H. influenzae isolates. Six isolates were resistant to chloramphenicol and positive for type A-2 CATs. Of these isolates, A3256 had a similar level of CAT activity but a higher chloramphenicol MIC relative to the other resistant isolates; it also had 163 specific variations in 58 membrane genes. Regarding the CAT-negative isolates, logistic regression and receiver operator characteristic curve analyses revealed that 48T > G (Asn16Lys), 85 C > T (Leu29Phe), and 88 C > A (Leu30Ile) in HI_0898 (emrA), and 86T > G (Phe29Cys) and 141T > A (Ser47Arg) in HI_1177 (artM) were associated with enhanced chloramphenicol susceptibility, whereas 997G > A (Val333Ile) in HI_1612 (hmrM) was associated with reduced chloramphenicol susceptibility. Furthermore, the chloramphenicol MIC was lower in the CAT-negative isolates with EmrA-Leu29Phe/Leu30Ile or ArtM-Ser47Arg substitution and higher in those with HmrM-Val333Ile substitution, relative to their counterparts. The Val333Ile substitution was associated with enhanced HmrM protein stability and flexibility and increased chloramphenicol MICs in CAT-negative H. influenzae isolates. In conclusion, the substitution in H. influenzae multidrug efflux pump HmrM associated with reduced chloramphenicol susceptibility was characterised.
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Sustitución de Aminoácidos , Antibacterianos , Proteínas Bacterianas , Cloranfenicol O-Acetiltransferasa , Cloranfenicol , Haemophilus influenzae , Pruebas de Sensibilidad Microbiana , Cloranfenicol/farmacología , Haemophilus influenzae/genética , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/metabolismo , Haemophilus influenzae/aislamiento & purificación , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Resistencia al Cloranfenicol/genética , Humanos , Infecciones por Haemophilus/microbiología , Secuenciación Completa del GenomaRESUMEN
Ammonium (NH4+) serves as a vital nitrogen source for plants, but it can turn toxic when it accumulates in excessive amounts. Toxicity is aggravated under hypoxic/anaerobic conditions, e.g., during flooding or submergence, due to a lower assimilation capacity. AMT1; 1 mediates NH4+ uptake into roots. Under conditions of oxygen-deficiency, i.e., submergence, the CBL-interacting protein kinase OsCIPK15 has been shown to trigger SnRK1A signaling, promoting starch mobilization, thereby the increasing availability of ATP, reduction equivalents and acceptors for NH4+ assimilation in rice. Our previous study in Arabidopsis demonstrates that AtCIPK15 phosphorylates AMT1; 1 whose activity is under allosteric feedback control by phosphorylation of T460 in the cytosolic C-terminus. Here we show that submergence cause higher NH4+ accumulation in wild-type, plant but not of nitrate, nor in a quadruple amt knock-out mutant. In addition, submergence triggers rapid accumulation of AtAMT1;1 and AtCIPK15 transcripts as well as AMT1 phosphorylation. Significantly, cipk15 knock-out mutants do not exhibit an increase in AMT1 phosphorylation; however, they do display heightened sensitivity to submergence. These findings suggest that CIPK15 suppresses AMT activity, resulting in decreased NH4+ accumulation during submergence, a period when NH4+ assimilation capacity may be impaired.
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BACKGROUND: Rifampin is a potent chemoprophylactic antibiotic for Haemophilus influenzae infection, and the resistance rate in H. influenzae is low. In this study, we assessed rifampin resistance-related genetic variations in H. influenzae. METHODS: Rifampin susceptibility testing and whole-genome sequencing were performed in 51 H. influenzae isolates. Variations associated with rifampin resistance were identified using Fisher's exact tests. Functional assays were performed to evaluate the effect of RpoB substitutions on rifampin susceptibility. RESULTS: Using the genome of the Rd KW20 H. influenzae strain as the reference, we detected 40 genetic variations in rpoB, which resulted in 39 deduced amino acid substitutions among the isolates. Isolate A0586 was resistant to rifampin, with a minimum inhibitory concentration (MIC) = 8 µg/mL. Phylogenetic analyses revealed that the RpoB sequence of isolate A0586 was distinct from other isolates. Five substitutions, including H526N located in cluster I and L623F, R628C, L645F, and L672F in the region between clusters II and III, were unique to isolate A0586. In two rifampin-susceptible H. influenzae isolates, RpoB-H526N alone and in combination with RpoB-L672F increased the MICs of rifampin to 4 and 8 µg/mL, respectively. RpoB-L672F did not affect cell growth and transcription in H. influenzae isolates. No amino acid substitutions in the AcrAB-TolC efflux pump or outer membrane proteins were found to be associated with rifampin resistance in H. influenzae. CONCLUSIONS: Our findings indicate that L672F substitution in the region between RpoB clusters II and III has an aggravating effect on rifampin resistance in H. influenzae.
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Infecciones por Haemophilus , Rifampin , Humanos , Rifampin/farmacología , Haemophilus influenzae , Sustitución de Aminoácidos , Filogenia , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Infecciones por Haemophilus/microbiología , Pruebas de Sensibilidad Microbiana , MutaciónRESUMEN
Nitrate (NO3-) is an essential element and nutrient for plants and animals. Despite extensive studies on the regulation of nitrate uptake and downstream responses in various cells, our knowledge of the distribution of nitrogen forms in different root cell types and their cellular compartments is still limited. Previous physiological models have relied on in vitro biochemistry and metabolite level analysis, which limits the ability to differentiate between cell types and compartments. Here, to address this, we report a nuclear-localized, genetically encoded fluorescent biosensor, which we named nlsNitraMeter3.0, for the quantitative visualization of nitrate concentration and distribution at the cellular level in Arabidopsis thaliana. This biosensor was specifically designed for nitrate measurements, not nitrite. Through genetic engineering to create and select sensors using yeast, Xenopus oocyte, and Arabidopsis expression systems, we developed a reversible and highly specific nitrate sensor. This method, combined with fluorescence imaging systems such as confocal microscopy, allows for the understanding and monitoring of nitrate transporter activity in plant root cells in a minimally invasive manner. Furthermore, this approach enables the functional analysis of nitrate transporters and the measurement of nitrate distribution in plants, providing a valuable tool for plant biology research. In summary, we provide a protocol for sensor development and a biosensor that can be used to monitor nitrate levels in plants. Key features This protocol builds upon the concept of FRET biosensors for in vivo visualization of spatiotemporal nitrate levels at a cellular resolution. Nitrate levels can be quantified utilizing the biosensor in conjunction with either a plate reader or a fluorescence microscope.
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BACKGROUND: Non-typeable Haemophilus influenzae (NTHi) has become the major cause of invasive H. influenzae diseases in the post-H. influenzae type b vaccine era. The emergence of multidrug-resistant (MDR) NTHi is a growing public health problem. Herein, we investigated the molecular basis of MDR in NTHi. The isolated NTHi were subjected to antimicrobial susceptibility testing for 12 agents. Whole genome and plasmid sequencing were conducted and analyzed to identify significant genetic variations and plasmid-encoded genes conferred antibiotic resistance. RESULTS: Thirteen (50%) MDR NTHi isolates were obtained; of these, 92.3% were non-susceptible to ampicillin, 30.8% to amoxicillin-clavulanate, 61.5% to cefuroxime, 61.5% to ciprofloxacin/levofloxacin, 92.3% to trimethoprim-sulfamethoxazole, 30.8% to tetracycline, and 7.7% to azithromycin. Eight ampicillin-resistant isolates were ß-lactamase positive; of these, 6 carried blaTEM-1 and 2 carried blaROB-1, whereas 4 were ß-lactamase negative. Genetic variations in mrdA, mepA, and pbpG were correlated with amoxicillin-clavulanate non-susceptibility, whereas variations in ftsI and lpoA conferred cefuroxime resistance. Five variations in gyrA, 2 in gyrB, 3 in parC, 1 in parE, and 1 in the parC-parE intergenic region were associated with levofloxacin/ciprofloxacin non-susceptibility. Among these genes, 8 variations were linked to high-level levofloxacin resistance. Six variations in folA were associated with trimethoprim-sulfamethoxazole resistance. Plasmid-bearing tet(B) and mef(A) genes were responsible for tetracycline and azithromycin resistance in 4 and 1 MDR isolates, respectively. CONCLUSIONS: This study clarified the molecular epidemiology of MDR in NTHi. This can benefit the monitoring of drug resistance trends in NTHi and the adequate medical management of patients with NTHi infection.
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Infecciones por Haemophilus , Haemophilus influenzae , Humanos , Haemophilus influenzae/genética , Cefuroxima/farmacología , Levofloxacino/farmacología , Combinación Trimetoprim y Sulfametoxazol/farmacología , Azitromicina , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Ampicilina , Infecciones por Haemophilus/tratamiento farmacológico , Combinación Amoxicilina-Clavulanato de Potasio , Tetraciclina , Ciprofloxacina , beta-Lactamasas/genéticaRESUMEN
BACKGROUND: Cefepime and aztreonam are highly efficacious against H. influenzae, and resistant strains are rare. In this study, we isolated cefepime- and aztreonam-nonsusceptible H. influenzae strains and addressed the molecular basis of their resistance to cefepime and aztreonam. METHODS: Two hundred and 28 specimens containing H. influenzae were screened, of which 32 isolates were enrolled and applied to antimicrobial susceptibility testing and whole-genome sequencing. Genetic variations that were detected in all nonsusceptible isolates with statistical significance by Fisher's exact tests were identified as cefepime or aztreonam nonsusceptibility related. Functional complementation assays were conducted to assess the in vitro effects of proteins with sequence substitutions on drug susceptibility. RESULTS: Three H. influenzae isolates were nonsusceptible to cefepime, one of which was also nonsusceptible to aztreonam. Genes encoding TEM, SHV and CTX-M extended-spectrum ß-lactamases were not detected in the cefepime- and aztreonam-nonsusceptible isolates. Five genetic variations in four genes and 10 genetic variations in five genes were associated with cefepime and aztreonam nonsusceptibility, respectively. Phylogenetic analyses revealed that changes in FtsI were correlated strongly with the MIC of cefepime and moderately with aztreonam. FtsI Thr532Ser-Tyr557His cosubstitution linked to cefepime nonsusceptibility and Asn305Lys-Ser385Asn-Glu416Asp cosubstitution to aztreonam nonsusceptibility. Functional complementation assays revealed that these cosubstitutions increased MICs of cefepime and aztreonam in susceptible H. influenzae isolates, respectively. CONCLUSIONS: Genetic variations relevant to resistant phenotypes of cefepime and aztreonam nonsusceptibility in H. influenzae were identified. Moreover, the effects of FtsI cosubstitutions on increasing MICs of cefepime and aztreonam in H. influenzae were demonstrated.
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Aztreonam , Haemophilus influenzae , Cefepima/farmacología , Aztreonam/farmacología , Filogenia , beta-Lactamasas/metabolismo , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacologíaRESUMEN
Gut microbiota play a key role in health maintenance and disease pathogenesis in animals. Dietary phytochemicals are crucial factors shaping gut bacteria. Here, we investigated the function and mechanism of a phytogenic formulation, EUBIO-BPSG (BP), in laying hens. We found that BP dose-dependently improved health and egg production in 54-week-old hens. Furthermore, BP was correlated with increased fecal Lactobacillus, decreased Escherichia coli and Salmonella enterica, and reduced antibiotic resistance (AR) and antibiotic resistance genes (ARG) in chicken stools. The 16S rDNA data showed that BP increased seven genera of probiotics and reduced 13 genera of pathogens in chicken feces. In vitro co-culture experiments showed that BP at 4 µg/mL and above promoted growth of L. reuteri while large 100- and 200-fold higher doses suppressed growth of E. coli and S. enterica, respectively. Mechanistic studies indicated that L. reuteri and its supernatants antagonized growth of E. coli and S. enterica but not vice-versa. Five short-chain fatty acids and derivatives (SCFA) produced from L. reuteri directly killed both pathogens via membrane destruction. Furthermore, BP inhibited conjugation and recombination of ARG via interference with conjugation machinery and integrase activity in E. coli. Collectively, this work suggests that BP promotes host health and reproductive performance in laying hens through regulation of gut microbiota through increasing probiotics and decreasing pathogens and spreading ARG.
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Nitrate (NO3-) uptake and distribution are critical to plant life. Although the upstream regulation of NO3- uptake and downstream responses to NO3- in a variety of cells have been well studied, it is still not possible to directly visualize the spatial and temporal distribution of NO3- with high resolution at the cellular level. Here, we report a nuclear-localized, genetically encoded fluorescent biosensor, which we named NitraMeter3.0, for the quantitative visualization of NO3- distribution in Arabidopsis thaliana. This biosensor tracked the spatiotemporal distribution of NO3- along the primary root axis and disruptions by genetic mutation of transport (low NO3- uptake) and assimilation (high NO3- accumulation). The developed biosensor effectively monitors NO3- concentrations at the cellular level in real time and spatiotemporal changes during the plant life cycle.
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Proteínas de Arabidopsis , Arabidopsis , Técnicas Biosensibles , Nitratos , Raíces de Plantas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genéticaRESUMEN
The NRT1/PTR FAMILY (NPF) in Arabidopsis (Arabidopsis thaliana) plays a major role as a nitrate transporter. The first nitrate transporter activity biosensor NiTrac1 converted the dual-affinity nitrate transceptor NPF6.3 into fluorescence activity sensors. To test whether this approach is transferable to other members of this family, screening for genetically encoded fluorescence transport activity sensor was performed with the member of the NPF family in Arabidopsis. In this study, NPF1.3, an uncharacterized member of NPF in Arabidopsis, was converted into a transporter activity biosensor NiTrac-NPF1.3 that responds specifically to nitrate. The emission ratio change of NiTrac-NPF1.3 triggered by the addition of nitrate reveals the important function of NPF1.3 in nitrate transport in Arabidopsis. A functional analysis of Xenopus laevis oocytes confirmed that NPF1.3 plays a role as a nitrate transporter. This new technology is applicable in plant and medical research.
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Arabidopsis , Técnicas Biosensibles , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Nitratos , Proteínas de PlantasRESUMEN
The understanding of signaling and metabolic processes in multicellular organisms requires knowledge of the spatial dynamics of small molecules and the activities of enzymes, transporters, and other proteins in vivo, as well as biophysical parameters inside cells and across tissues. The cellular distribution of receptors, ligands, and activation state must be integrated with information about the cellular distribution of metabolites in relation to metabolic fluxes and signaling dynamics in order to achieve the promise of in vivo biochemistry. Genetically encoded sensors are engineered fluorescent proteins that have been developed for a wide range of small molecules, such as ions and metabolites, or to report biophysical processes, such as transmembrane voltage or tension. First steps have been taken to monitor the activity of transporters in vivo. Advancements in imaging technologies and specimen handling and stimulation have enabled researchers in plant sciences to implement sensor technologies in intact plants. Here, we provide a brief history of the development of genetically encoded sensors and an overview of the types of sensors available for quantifying and visualizing ion and metabolite distribution and dynamics. We further discuss the pros and cons of specific sensor designs, imaging systems, and sample manipulations, provide advice on the choice of technology, and give an outlook into future developments.
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Biología , Técnicas Biosensibles/métodos , Proteínas Luminiscentes/genética , Plantas/genética , Transporte Biológico , Colorantes Fluorescentes , Iones/metabolismo , Imagen Molecular , Transducción de SeñalRESUMEN
BACKGROUND: Ammonium (NH4+), a key nitrogen form, becomes toxic when it accumulates to high levels. Ammonium transporters (AMTs) are the key transporters responsible for NH4+ uptake. AMT activity is under allosteric feedback control, mediated by phosphorylation of a threonine in the cytosolic C-terminus (CCT). However, the kinases responsible for the NH4+-triggered phosphorylation remain unknown. RESULTS: In this study, a functional screen identified protein kinase CBL-Interacting Protein Kinase15 (CIPK15) as a negative regulator of AMT1;1 activity. CIPK15 was able to interact with several AMT1 paralogs at the plasma membrane. Analysis of AmTryoshka, an NH4+ transporter activity sensor for AMT1;3 in yeast, and a two-electrode-voltage-clamp (TEVC) of AMT1;1 in Xenopus oocytes showed that CIPK15 inhibits AMT activity. CIPK15 transcript levels increased when seedlings were exposed to elevated NH4+ levels. Notably, cipk15 knockout mutants showed higher 15NH4+ uptake and accumulated higher amounts of NH4+ compared to the wild-type. Consistently, cipk15 was hypersensitive to both NH4+ and methylammonium but not nitrate (NO3-). CONCLUSION: Taken together, our data indicate that feedback inhibition of AMT1 activity is mediated by the protein kinase CIPK15 via phosphorylation of residues in the CCT to reduce NH4+-accumulation.
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Compuestos de Amonio/metabolismo , Arabidopsis/fisiología , Proteínas de Transporte de Catión/genética , Retroalimentación Fisiológica , Proteínas de Plantas/genética , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Transporte de Catión/metabolismo , Fosforilación , Proteínas de Plantas/metabolismoRESUMEN
Although mass spectrometry-based plasma proteomics enables sensitive and large-scale discovery and validation of biomarkers for various diseases, its integrative application to hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) is not well investigated. Therefore, we analyzed albumin- and immunoglobulin G-depleted plasma samples from 148 and 60 patients with HCC and CCA, respectively, using liquid chromatography-tandem mass spectrometry. The algorithm used to measure the content of each protein was the percentage of exponentially modified protein abundance index. From 5320 proteins assayed in plasma, 53 and 25 biomarker candidates were identified for HCC and CCA, respectively. The abundance of six and two HCC markers particularly protruded in stage II and III, respectively, whereas plasma serine protease inhibitor was the sole marker the level of which steadily decreased with CCA progression. From a prognostic facet, we showed candidate markers and their cutoff levels for evaluating probability of tumor recurrence and patient survival period. Combination Kaplan-Meier models showed that HCC stage III or IV and both the content of alpha-2-HS-glycoprotein and apolipoprotein CIII <0.2% exhibited the poorest post-surgical recurrence-free and overall survivals. Furthermore, the content of afamin ≥0.2% played a significant role on the poor prognosis in patients with CCA. Our findings, taken together, characterized novel plasma biomarker signatures in dissecting tumor stages and post-surgical outcomes of HCC and CCA.
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Neoplasias de los Conductos Biliares/patología , Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/patología , Colangiocarcinoma/patología , Neoplasias Hepáticas/patología , Adulto , Anciano , Anciano de 80 o más Años , Apolipoproteína C-III/sangre , Conductos Biliares Intrahepáticos/patología , Proteínas Portadoras/sangre , Femenino , Glicoproteínas/sangre , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Pronóstico , Proteoma/análisis , Inhibidores de Serina Proteinasa/sangre , Albúmina Sérica Humana , alfa-2-Glicoproteína-HS/análisisRESUMEN
BACKGROUND: Concern about Haemophilus influenzae infection has been increasing over recent decades. Given the emergence of H. influenzae with severe drug resistance, we assessed the prevalence of as well as risk factors and potential therapies for extensively drug-resistant (XDR) H. influenzae infection in Taiwan. RESULTS: In total, 2091 H. influenzae isolates with disk diffusion-based antibiotic susceptibility testing from 2007 to 2018 were enrolled. H. influenzae strains resistant to ampicillin, chloramphenicol, levofloxacin, and trimethoprim-sulfamethoxazole tended to be isolated from patient wards (â§41%), whereas those resistant to amoxicillin-clavulanate, cefotaxime, and cefuroxime were more likely to be isolated from intensive care units (approximately 50%). XDR H. influenzae was first identified in 2007, and its incidence did not significantly change thereafter. Overall prevalence of single, multiple, and extensively drug-resistant H. influenzae over 2007-2018 was 21.5% (n = 450), 26.6% (n = 557), and 2.5% (n = 52), respectively. A stepwise logistic regression analysis revealed that blood culture (odds ratio: 4.069, 95% confidence intervals: 1.339-12.365, P = 0.013) was an independent risk factor for XDR H. influenzae infection. No nosocomial transmission of XDR H. influenzae observed. Antibiotic susceptibility testing results demonstrated that cefotaxime was effective against 78.8% (n = 41) of the XDR strains. CONCLUSIONS: The presence of XDR H. influenzae strains was identified in Taiwan, and cefotaxime was efficacious against most of these strains.
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Antibacterianos/farmacología , Cefotaxima/farmacología , Infección Hospitalaria/microbiología , Farmacorresistencia Bacteriana , Infecciones por Haemophilus/epidemiología , Haemophilus influenzae/clasificación , Ampicilina/farmacología , Antibacterianos/uso terapéutico , Cefotaxima/uso terapéutico , Cefuroxima/farmacología , Cloranfenicol/farmacología , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/epidemiología , Femenino , Infecciones por Haemophilus/tratamiento farmacológico , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/aislamiento & purificación , Humanos , Incidencia , Unidades de Cuidados Intensivos , Levofloxacino/farmacología , Modelos Logísticos , Masculino , Pruebas de Sensibilidad Microbiana , Taiwán/epidemiología , Combinación Trimetoprim y Sulfametoxazol/farmacologíaRESUMEN
As debate rumbles on about whether anti-hepatitis B virus (HBV) nucleos(t)ide analogue treatments modulate host immune system during end-stage liver diseases, we studied effects of two potent anti-HBV agents, telbivudine or entecavir, on humoral immune activities including cytokine secretion, immunoglobulin production, and IgG-Fc agalactosylation, which is known to induce proinflammatory responses, in liver cirrhosis. Serum IgG-Fc N-glycan structures in patients with HBV-related liver cirrhosis, who had received either telbivudine treatment or entecavir treatment for at least 48 weeks were analyzed using liquid chromatography tandem-mass spectrometry. Levels of cytokines and each immunoglobulin isotype were measured using enzyme-linked immunosorbent assays. Results showed that 48 weeks of entecavir treatment caused HBV DNA loss, alanine aminotransferase normalization, and an amelioration of hypergammaglobulinemia in cirrhotic patients; however, telbivudine treatment, though possessing similar efficacies on HBV suppression and an improvement in liver inflammation to entecavir treatment, did not mitigate IgG-related hypergammaglobulinemia. Levels of IgG and transforming growth factor (TGF)-ß1 in sera of the cirrhotic patients before and during treatment were positively correlated. In vitro assays revealed that telbivudine treatment induced TGF-ß1 expression in human macrophagic cells. Moreover, recombinant TGF-ß1 treatment stimulated cell proliferation and IgG overproduction in human IgG-producing B cell lines. Finally, we found that telbivudine treatment enhanced the proportion of serum IgG-Fc agalactosylation in cirrhotic patients, which was associated with enhanced levels of TGF-ß1 and IgG. In conclusion, telbivudine therapy was associated with TGF-ß1 hyperactivity, IgG-related hypergammaglobulinemia, and IgG-Fc agalactosylation in HBV-related liver cirrhosis.
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Antivirales/uso terapéutico , Hepatitis B Crónica/tratamiento farmacológico , Hipergammaglobulinemia/patología , Inmunoglobulina G/sangre , Cirrosis Hepática/patología , Telbivudina/uso terapéutico , Factor de Crecimiento Transformador beta1/sangre , Adulto , Anciano , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Proliferación Celular/efectos de los fármacos , Citocinas/sangre , Femenino , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/virología , Humanos , Hipergammaglobulinemia/complicaciones , Cirrosis Hepática/etiología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Estudios Retrospectivos , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Developmental processes that control root system architecture are critical for soil exploration by plants, allowing for uptake of water and nutrients. Conversion of the auxin precursor indole-3-butyric acid (IBA) to active auxin (indole-3-acetic acid; IAA) modulates lateral root formation. However, mechanisms governing IBA-to-IAA conversion have yet to be elucidated. We identified TRANSPORTER OF IBA1 (TOB1) as a vacuolar IBA transporter that limits lateral root formation. Moreover, TOB1, which is transcriptionally regulated by the phytohormone cytokinin, is necessary for the ability of cytokinin to exert inhibitory effects on lateral root production. The increased production of lateral roots in tob1 mutants, TOB1 transport of IBA into the vacuole, and cytokinin-regulated TOB1 expression provide a mechanism linking cytokinin signaling and IBA contribution to the auxin pool to tune root system architecture.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Proteínas de Arabidopsis/metabolismo , Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , Indoles/metabolismo , Raíces de Plantas/metabolismo , Arabidopsis , Membranas Intracelulares/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Vacuolas/metabolismoRESUMEN
BACKGROUND: The nursing process system (NPS) is used to establish the nursing process involving assessment, diagnosis, planning, intervention, and evaluation in solving the health problems of patients. OBJECTIVES: The factors influencing the use of the NPS by nurses were analyzed based on user satisfaction and technology acceptance within the 3Q (service quality, information quality, and system quality) model. METHODS: In this cross-sectional quantitative study, the valid responses of 222 nurses to a questionnaire were obtained; these nurses worked at eight hospitals affiliated with public organizations in Taiwan. Structural equation modeling was used to analyze information quality, system quality, service quality, user satisfaction, perceived usefulness, perceived ease of use, perceived enjoyment, behavioral attitude, and intention after the nurses had used the NPS system for more than 1 month. RESULTS: Information quality, service quality, and system quality influenced user satisfaction. User satisfaction affected perceived usefulness, perceived ease of use, and perceived enjoyment and had the highest explanatory power (R2 = 0.75). Furthermore, perceived usefulness, perceived ease of use, and perceived enjoyment influenced behavioral attitude and intention to use the system. The proposed model explained 53% of the variance in the intention to use the NPS. CONCLUSIONS: The relationships between the variables of the 3Q model were successfully used to examine the intention of nurses toward using the NPS. Using the findings of this study, designers and programmers can comprehensively understand the perceptions of nurses and further improve the performance of the NPS.
Asunto(s)
Sistemas de Información en Hospital , Proceso de Enfermería , Satisfacción Personal , Femenino , Humanos , Masculino , Modelos TeóricosRESUMEN
Hepatobiliary cancer is the third leading cause of cancer death worldwide. Appropriate markers for early diagnosis, monitoring of disease progression, and prediction of postsurgical outcome are still lacking. As the majority of circulating N-glycoproteins are originated from the hepatobiliary system, we sought to explore new markers by assessing the dynamics of N-glycoproteome in plasma samples from patients with hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), or combined HCC and CCA (cHCC-CCA). Using a mass spectrometry-based quantitative proteomic approach, we found that 57 of 5358 identified plasma proteins were differentially expressed in hepatobiliary cancers. The levels of four essential proteins, including complement C3 and apolipoprotein C-III in HCC, galectin-3-binding protein in CCA, and 72 kDa inositol polyphosphate 5-phosphatase in cHCC-CCA, were highly correlated with tumor stage, tumor grade, recurrence-free survival, and overall survival. Postproteomic site-specific N-glycan analyses showed that human complement C3 bears high-mannose and hybrid glycoforms rather than complex glycoforms at Asn85. The abundance of complement C3 with mannose-5 or mannose-6 glycoform at Asn85 was associated with HCC tumor grade. Furthermore, stepwise Cox regression analyses revealed that HCC patients with a hybrid glycoform at Asn85 of complement C3 had a lower postsurgery tumor recurrence rate or mortality rate than those with a low amount of complement C3 protein. In conclusion, our data show that particular plasma N-glycoproteins with specific N-glycan compositions could be potential noninvasive markers to evaluate oncological status and prognosis of hepatobiliary cancers.