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1.
Microorganisms ; 10(6)2022 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-35744727

RESUMO

Catheter-associated urinary tract infections (CAUTI) are a common clinical concern as they can lead to severe, persistent infections or bacteremia in long-term catheterized patients. This type of CAUTI is difficult to eradicate, as they are caused by multispecies biofilms that may have reduced susceptibility to antibiotics. Many new strategies to tackle CAUTI have been proposed in the past decade, including antibiotic combination treatments, surface modification and probiotic usage. However, those strategies were mainly assessed on mono- or dual-species biofilms that hardly represent the long-term CAUTI cases where, normally, 2-4 or even more species can be involved. We developed a four-species in vitro biofilm model on catheters involving clinical strains of Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca and Proteus mirabilis isolated from indwelling catheters. Interspecies interactions and responses to antibiotics were quantitatively assessed. Collaborative as well as competitive interactions were found among members in our model biofilm and those interactions affected the individual species' abundances upon exposure to antibiotics as mono-, dual- or multispecies biofilms. Our study shows complex interactions between species during the assessment of CAUTI control strategies for biofilms and highlights the necessity of evaluating treatment and control regimes in a multispecies setting.

2.
Crit Rev Microbiol ; 48(3): 283-302, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-34411498

RESUMO

Bacterial biofilms occur in many natural and industrial environments. Besides bacteria, biofilms comprise over 70 wt% water. Water in biofilms occurs as bound- or free-water. Bound-water is adsorbed to bacterial surfaces or biofilm (matrix) structures and possesses different Infra-red and Nuclear-Magnetic-Resonance signatures than free-water. Bound-water is different from intra-cellularly confined-water or water confined within biofilm structures and bacteria are actively involved in building water-filled structures by bacterial swimmers, dispersion or lytic self-sacrifice. Water-filled structures can be transient due to blocking, resulting from bacterial growth, compression or additional matrix formation and are generally referred to as "channels and pores." Channels and pores can be distinguished based on mechanism of formation, function and dimension. Channels allow transport of nutrients, waste-products, signalling molecules and antibiotics through a biofilm provided the cargo does not adsorb to channel walls and channels have a large length/width ratio. Pores serve a storage function for nutrients and dilute waste-products or antimicrobials and thus should have a length/width ratio close to unity. The understanding provided here on the role of water in biofilms, can be employed to artificially engineer by-pass channels or additional pores in industrial and environmental biofilms to increase production yields or enhance antimicrobial penetration in infectious biofilms.


Assuntos
Anti-Infecciosos , Água , Antibacterianos , Bactérias/genética , Biofilmes
3.
mBio ; 10(5)2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31506311

RESUMO

Bacterial adhesion is accompanied by altered gene expression, leading to "emergent" properties of biofilm bacteria that are alien to planktonic ones. With the aim of revealing the role of environmental adhesion forces in emergent biofilm properties, genes in Streptococcus mutans UA159 and a quorum-sensing-deficient mutant were identified that become expressed after adhesion to substratum surfaces. Using atomic force microscopy, adhesion forces of initial S. mutans colonizers on four different substrata were determined and related to gene expression. Adhesion forces upon initial contact were similarly low across different substrata, ranging between 0.2 and 1.2 nN regardless of the strain considered. Bond maturation required up to 21 s, depending on the strain and substratum surface involved, but stationary adhesion forces also were similar in the parent and in the mutant strain. However, stationary adhesion forces were largest on hydrophobic silicone rubber (19 to 20 nN), while being smallest on hydrophilic glass (3 to 4 nN). brpA gene expression in thin (34 to 48 µm) 5-h S. mutans UA159 biofilms was most sensitive to adhesion forces, while expression of gbpB and comDE expressions was weakly sensitive. ftf, gtfB, vicR, and relA expression was insensitive to adhesion forces. In thicker (98 to 151 µm) 24-h biofilms, adhesion-force-induced gene expression and emergent extracellular polymeric substance (EPS) production were limited to the first 20 to 30 µm above a substratum surface. In the quorum-sensing-deficient S. mutans, adhesion-force-controlled gene expression was absent in both 5- and 24-h biofilms. Thus, initial colonizers of substratum surfaces sense adhesion forces that externally trigger emergent biofilm properties over a limited distance above a substratum surface through quorum sensing.IMPORTANCE A new concept in biofilm science is introduced: "adhesion force sensitivity of genes," defining the degree up to which expression of different genes in adhering bacteria is controlled by the environmental adhesion forces they experience. Analysis of gene expression as a function of height in a biofilm showed that the information about the substratum surface to which initially adhering bacteria adhere is passed up to a biofilm height of 20 to 30 µm above a substratum surface, highlighting the importance and limitations of cell-to-cell communication in a biofilm. Bacteria in a biofilm mode of growth, as opposed to planktonic growth, are responsible for the great majority of human infections, predicted to become the number one cause of death in 2050. The concept of adhesion force sensitivity of genes provides better understanding of bacterial adaptation in biofilms, direly needed for the design of improved therapeutic measures that evade the recalcitrance of biofilm bacteria to antimicrobials.


Assuntos
Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Streptococcus mutans/genética , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Liases de Carbono-Enxofre/genética , Matriz Extracelular de Substâncias Poliméricas , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Mutação , Percepção de Quorum/genética , Streptococcus mutans/metabolismo
4.
Sci Rep ; 9(1): 9794, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278369

RESUMO

Optical-coherence-tomography (OCT) is a non-destructive tool for biofilm imaging, not requiring staining, and used to measure biofilm thickness and putative comparison of biofilm structure based on signal intensity distributions in OCT-images. Quantitative comparison of biofilm signal intensities in OCT-images, is difficult due to the auto-scaling applied in OCT-instruments to ensure optimal quality of individual images. Here, we developed a method to eliminate the influence of auto-scaling in order to allow quantitative comparison of biofilm densities in different images. Auto- and re-scaled signal intensities could be qualitatively interpreted in line with biofilm characteristics for single and multi-species biofilms of different strains and species (cocci and rod-shaped organisms), demonstrating qualitative validity of auto- and re-scaling analyses. However, specific features of pseudomonas and oral multi-species biofilms were more prominently expressed after re-scaling. Quantitative validation was obtained by relating average auto- and re-scaled signal intensities across biofilm images with volumetric-bacterial-densities in biofilms, independently obtained using enumeration of bacterial numbers per unit biofilm volume. The signal intensities in auto-scaled biofilm images did not significantly relate with volumetric-bacterial-densities, whereas re-scaled intensities in images of biofilms of widely different strains and species increased linearly with independently determined volumetric-bacterial-densities in the biofilms. Herewith, the proposed re-scaling of signal intensity distributions in OCT-images significantly enhances the possibilities of biofilm imaging using OCT.


Assuntos
Bactérias/crescimento & desenvolvimento , Biofilmes , Tomografia de Coerência Óptica , Algoritmos , Biofilmes/crescimento & desenvolvimento , Modelos Teóricos
5.
Appl Environ Microbiol ; 84(1)2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29054874

RESUMO

The viscoelasticity of a biofilm's EPS (extracellular polymeric substance) matrix conveys protection against mechanical challenges, but adaptive responses of biofilm inhabitants to produce EPS are not well known. Here, we compare the responses of a biofilm of an EPS-producing (ATCC 12600) and a non-EPS producing (5298) Staphylococcus aureus strain to fluid shear and mechanical challenge. Confocal laser scanning microscopy confirmed absence of calcofluor-white-stainable EPS in biofilms of S. aureus 5298. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy combined with tribometry indicated that polysaccharide production per bacterium in the initial adhering layer was higher during growth at high shear than at low shear and that this increased EPS production extended to entire biofilms, as indicated by tribometrically measured coefficients of friction (CoF). CoF of biofilms grown under high fluid shear were higher than those when grown under low shear, likely due to wash-off polysaccharides. Measurement of a biofilm's CoF implies application of mechanical pressure that yielded an immediate increase in the polysaccharide band area of S. aureus ATCC 12600 biofilms due to their compression. Compression decreased after relief of pressure to the level observed prior to mechanical pressure. For biofilms grown under high shear, this coincided with a higher percent whiteness in optical coherence tomography-images indicative of water outflow, returning back into the biofilm during stress relaxation. Biofilms grown under low shear, however, were stimulated during tribometry to produce EPS, also after relief of stress. Knowledge of factors that govern EPS production and water flow in biofilms will allow better control of biofilms under mechanical challenge and better understanding of the barrier properties of biofilms against antimicrobial penetration.IMPORTANCE Adaptive responses of biofilm inhabitants in nature to environmental challenges such as fluid shear and mechanical pressure often involve EPS production with the aim of protecting biofilm inhabitants. EPS can assist biofilm bacteria in remaining attached or can impede antimicrobial penetration. The TriboChemist is a recently introduced instrument, allowing the study of initially adhering bacteria to a germanium crystal using ATR-FTIR spectroscopy, while simultaneously allowing measurement of the coefficient of friction of a biofilm, which serves as an indicator of the EPS content of a biofilm. EPS production can be stimulated by both fluid shear during growth and mechanical pressure, while increased EPS production can continue after pressure relaxation of the biofilm. Since EPS is pivotal in the protection of biofilm inhabitants against mechanical and chemical challenges, knowledge of the factors that make biofilm inhabitants decide to produce EPS, as provided in this study, is important for the development of biofilm control measures.


Assuntos
Matriz Extracelular de Substâncias Poliméricas/fisiologia , Staphylococcus aureus/fisiologia , Hidrodinâmica , Microscopia Confocal , Pressão , Espectroscopia de Infravermelho com Transformada de Fourier
7.
J R Soc Interface ; 13(123)2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27733700

RESUMO

Water-based lubrication provides cheap and environmentally friendly lubrication and, although hydrophilic surfaces are preferred in water-based lubrication, often lubricating surfaces do not retain water molecules during shear. We show here that hydrophilic (42° water contact angle) quartz surfaces facilitate water-based lubrication to the same extent as more hydrophobic Si crystal surfaces (61°), while lubrication by hydrophilic Ge crystal surfaces (44°) is best. Thus surface hydrophilicity is not sufficient for water-based lubrication. Surface-thermodynamic analyses demonstrated that all surfaces, regardless of their water-based lubrication, were predominantly electron donating, implying water binding with their hydrogen groups. X-ray photoelectron spectroscopy showed that Ge crystal surfaces providing optimal lubrication consisted of a mixture of -O and =O functionalities, while Si crystal and quartz surfaces solely possessed -O functionalities. Comparison of infrared absorption bands of the crystals in water indicated fewer bound-water layers on hydrophilic Ge than on hydrophobic Si crystal surfaces, while absorption bands for free water on the Ge crystal surface indicated a much more pronounced presence of structured, free-water clusters near the Ge crystal than near Si crystal surfaces. Accordingly, we conclude that the presence of structured, free-water clusters is essential for water-based lubrication. The prevalence of structured water clusters can be regulated by adjusting the ratio between surface electron-donating and electron-accepting groups and between -O and =O functionalities.


Assuntos
Lubrificantes/química , Modelos Químicos , Quartzo/química , Dióxido de Silício/química , Água/química , Interações Hidrofóbicas e Hidrofílicas
8.
Materials (Basel) ; 9(9)2016 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-28773886

RESUMO

Fatigue properties and cracking behavior of cold-drawn commercially pure aluminum wires (CPAWs) widely used as the overhead transmission conductors were investigated. It was found that the fracture surface of the CPAWs shows an obvious four-stage fracture characteristic, i.e., crack initiation, planar crack propagation, 45°-inclined crack propagation and final rapid fracture. The crack growth mechanisms for the CPAWs were found quite different from those for the conventional coarse-grained materials. The cracks in the CPAWs firstly grow along the grain boundaries (Stage I crack growth), and then grow along the plane of maximum shear stress during the last stage of cycling (Stage II crack growth), leading to the distinctive fracture surfaces, i.e., the granular surface in the planar crack propagation region and the coarse fatigue striations in the 45°-inclined crack propagation region. The grain boundary migration was observed in the fatigued CPAWs. The increase in fatigue load enhances the dislocation recovery, increases the grain boundary migration rate, and thus promotes the occurrence of softening and damage localization up to the final failure.

9.
Acta Pharm Sin B ; 5(6): 577-82, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26713273

RESUMO

This paper reports investigations into the preparation and characterization of surface molecularly imprinted nanoparticles (SMINs) designed to adhere to Helicobacter pylori (H. pylori). Imprinted nanoparticles were prepared by the inverse microemulsion polymerization method. A fraction of Lpp20, an outer membrane protein of H. pylori known as NQA, was chosen as template and modified with myristic acid to facilitate its localization on the surface of the nanoparticles. The interaction between these SMINs with the template NQA were evaluated using surface plasmon resonance (SPR), change in zeta potential and fluorescence polarization (FP). The results were highly consistent in demonstrating a preferential recognition of the template NQA for SMINs compared with the control nanoparticles. In vitro experiments also indicate that such SMINs are able to adhere to H. pylori and may be useful for H. pylori eradication.

10.
Int J Pharm ; 496(2): 1006-14, 2015 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-26524712

RESUMO

This paper reports studies on preparation and evaluation of amoxicillin loaded dual molecularly imprinted nanoparticles (Amo/Dual-MIPs) designed for anti-H. pylori therapy. Both MNQA and AmoNa were chosen as templates to prepare Dual-MIPs using inverse microemulsion polymerization method. NQA was modified with myristic acid (MNQA) to become amphiphilic and assist in leaving NQA cavities on the surface of Dual-MIPs for H. pylori adhesion. AmoNa was applied to produce imprinting sites in Dual-MIPs for rebinding AmoNa to exert its anti-H. pylori effect. Batch rebinding test demonstrated a preferential rebinding effect of NQA toward the Dual-MIPs. In vivofluorescence imaging showed the prolonged residence time of Dual-MIPs in H. pylori infected mice stomachs after intragastric administration of nanoparticles.In vivo H. pylori clearance tests indicated Amo/Dual-MIPs had a better aniti-H. pylori effect than amoxicillin powder did. In conclusion, Amo/Dual-MIPs may provide an alternative drug delivery strategy for anti-H. pylori therapy.


Assuntos
Amoxicilina/administração & dosagem , Antibacterianos/administração & dosagem , Helicobacter pylori/efeitos dos fármacos , Impressão Molecular , Nanopartículas/administração & dosagem , Amoxicilina/química , Amoxicilina/farmacologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Solubilidade
11.
Yao Xue Xue Bao ; 50(1): 15-20, 2015 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-25924469

RESUMO

Molecular imprinting technique (MIT) involves the synthesis of polymer in the presence of a template to produce complementary binding sites in terms of its size, shape and functional group orientation. Such kind of polymer possesses specific recognition ability towards its template molecule. Despite the rapid development of MIT over the years, the majority of the template molecules that have been studied are small molecules, while molecular imprinting of proteins remains a significant yet challenging task due to their large size, structural flexibility and complex conformation. This review, we summarized the research findings over the past years, and discussed the nano-reinforcing materials used to prepare molecular imprinting of proteins and the perspective of these nano-reinforcing materials.


Assuntos
Impressão Molecular , Nanoestruturas/química , Proteínas/química , Sítios de Ligação , Conformação Molecular , Polímeros/química
12.
Elife ; 42015 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-25738228

RESUMO

Huntington's disease (HD) represents an important model for neurodegenerative disorders and proteinopathies. It is mainly caused by cytotoxicity of the mutant huntingtin protein (Htt) with an expanded polyQ stretch. While Htt is ubiquitously expressed, HD is characterized by selective neurodegeneration of the striatum. Here we report a striatal-enriched orphan G protein-coupled receptor(GPCR) Gpr52 as a stabilizer of Htt in vitro and in vivo. Gpr52 modulates Htt via cAMP-dependent but PKA independent mechanisms. Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum. Finally, reducing Gpr52 suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models. Thus, our discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies.


Assuntos
Corpo Estriado/metabolismo , Doença de Huntington/metabolismo , Íntrons , Receptores Acoplados a Proteínas G/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Células Cultivadas , Corpo Estriado/citologia , Modelos Animais de Doenças , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Feminino , Humanos , Doença de Huntington/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Atividade Motora/genética , Atividade Motora/fisiologia , Mutação , Desempenho Psicomotor/fisiologia , Interferência de RNA , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética
13.
J Huntingtons Dis ; 3(3): 253-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25300329

RESUMO

High-throughput measurement of huntingtin (Htt) levels is useful for Huntington's disease research. For example, identification of genetic or chemical modifiers that reduce Htt levels by high-throughput screening provides promising strategy for HD drug discovery. In the human cells, high-throughput measurement of Htt levels has been established based on the Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) technology, using the 2B7/MW1 antibody pair. Unfortunately, application of this assay in the mouse cells has been problematic due to discrepancies between TR-FRET signals and Western-blots, possibly caused by non-specific antibody binding. Here we report TR-FRET assays that are able to detect endogenous Htt levels of the mouse striatal cell line (STHdh).


Assuntos
Modelos Animais de Doenças , Transferência Ressonante de Energia de Fluorescência/métodos , Imunoensaio/métodos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Animais , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Proteína Huntingtina , Proteínas do Tecido Nervoso/análise , Proteínas Nucleares/análise
14.
Mol Pharm ; 11(10): 3261-8, 2014 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-24673510

RESUMO

The blood-brain barrier (BBB) prevents most drugs from reaching the site of central nervous system (CNS) diseases, intensively confining the therapeutic efficiency. Angiopep-2 (here termed (L)Angiopep), which is a 19-mer peptide derived from human Kunitz domain, can trigger transcytosis and traverse the BBB by recognizing low density lipoprotein-related protein 1 (LRP-1) expressed on the brain capillary endothelial cells. Various enzymes in the blood and the BBB, however, present multiple metabolic barriers to peptide-inspired brain-targeted drug delivery. Here we designed a retro-inverso isomer of (L)Angiopep, termed (D)Angiopep, to inspire brain-targeted drug delivery. Both (D)Angiopep and (L)Angiopep displayed high uptake capacity in LRP-1 overexpressed cells, including bEnd.3 and U87 cells. (D)Angiopep demonstrated lower uptake efficiency in both cell lines than did (L)Angiopep, suggestive of lower binding affinity to LRP-1 of the d-peptide. (D)Angiopep was resistant to proteolysis in fresh rat blood serum, while more than 85% of (L)Angiopep disappeared within 2 h. Endocytosed (D)Angiopep and (L)Angiopep were found to be colocalized with lysosomal compartments of bEnd.3 cells, indicating that susceptibility to proteolysis of (L)Angiopep in the BBB may further attenuate its transcytosis efficiency. In vivo, (D)Angiopep modified PEG-DSPE micelles displayed high distribution in normal brain and intracranial glioblastoma. Due to the expression of LRP-1 on the BBB and glioblastoma cells, proteolytically stable (D)Angiopep holds much potential for designing two-order brain tumor targeted delivery systems.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Peptídeos/química , Animais , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Micelas , Ratos
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