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Microfluidic tools and techniques for manipulating fluid droplets have become core to many scientific and technological fields. Despite the plethora of existing approaches to fluidic manipulation, non-Newtonian fluid phenomena are rarely taken advantage of. Here we introduce embedded droplet printing-a system and methods for the generation, trapping, and processing of fluid droplets within yield-stress fluids, materials that exhibit extreme shear thinning. This technique allows for the manipulation of droplets under conditions that are simply unattainable with conventional microfluidic methods, namely the elimination of exterior influences including convection and solid boundaries. Because of this, we believe embedded droplet printing approaches an ideal for the experimentation, processing, or observation of many samples in an "absolutely quiescent" state, while also removing some troublesome aspects of microfluidics including the use of surfactants and the complexity of device manufacturing. We characterize a model material system to understand the process of droplet generation inside yield-stress fluids and develop a nascent set of archetypal operations that can be performed with embedded droplet printing. With these principles and tools, we demonstrate the benefits and versatility of our method, applying it toward the diverse applications of pharmaceutical crystallization, microbatch chemical reactions, and biological assays.
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Marine cyanobacteria are known to produce structurally diverse bioactive specialized metabolites during bloom occurrence. These ecologically active allelochemicals confer chemical defense for the microalgae from competing microbes and herbivores. From a collection of a marine cyanobacterium, cf. Lyngbya sp., a small quantity of a new cyclopropane-containing molecule, benderadiene (2), and lyngbyoic acid (1) were purified and characterized using spectroscopic methods. Using live reporter quorum-sensing (QS) inhibitory assays, based on P. aeruginosa PAO1 lasB-gfp and rhlA-gfp strains, both compounds were found to inhibit QS-regulated gene expression in a dose-dependent manner. In addition to lyngbyoic acid being more active in the PAO1 lasB-gfp biosensor strain (IC50 of 20.4 µM), it displayed anti-biofilm activity when incubated with wild-type P. aeruginosa. The discovery of lyngbyoic acid in relatively high amounts provided insights into its ecological significance as a defensive allelochemical in targeting competing microbes through interference with their QS systems and starting material to produce other related analogs. Similar strategies could be adopted by other marine cyanobacterial strains where the high production of other lipid acids has been reported. Preliminary evidence is provided from the virtual molecular docking of these cyanobacterial free acids at the ligand-binding site of the P. aeruginosa LasR transcriptional protein.
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Cianobactérias , Lyngbya , Lyngbya/metabolismo , Simulação de Acoplamento Molecular , Biofilmes , Percepção de Quorum , Cianobactérias/metabolismo , Ciclopropanos/farmacologia , Pseudomonas aeruginosa/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Fatores de Virulência/genéticaRESUMO
Cells in biofilms sense and interact with their environment through the extracellular matrix. The physicochemical properties of the matrix, particularly at the biofilm-environment interface, determine how cells respond to changing conditions. In this study we describe the application of atomic force microscopy and confocal imaging to probe in situ the mechanical properties of these interfacial regions and to elucidate how key matrix components can contribute to the physical sensing by the cells. We describe how the Young's modulus of microcolonies differs according to the size and morphology of microcolonies, as well as the flow rate. The Young's modulus increased as a function of microcolony diameter, which was correlated with the production of the polysaccharide Psl at later stages of maturation for hemispherical or mushroom shaped microcolonies. The Young's modulus of the periphery of the biofilm colony was however independent of the hydrodynamic shear. The morphology of the microcolonies also influenced interfacial or peripheral stiffness. Microcolonies with a diffuse morphology had a lower Young's modulus than isolated, circular ones and this phenomenon was due to a deficiency of Psl. In this way, changes in the specific polysaccharide components imbue the biofilm with distinct physical properties that may modulate the way in which bacteria perceive or respond to their environment. Further, the physical properties of the polysaccharides are closely linked to the specific architectures formed by the developing biofilm.
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Bactérias/crescimento & desenvolvimento , Biofilmes , Módulo de Elasticidade , Matriz Extracelular/fisiologia , Microscopia de Força AtômicaRESUMO
HU is a protein that plays a role in various bacterial processes including compaction, transcription and replication of the genome. Here, we use atomic force microscopy to study the effect of HU on the stiffness and supercoiling of double-stranded DNA. First, we measured the persistence length, height profile, contour length and bending angle distribution of the DNA-HU complex after different incubation times of HU with linear DNA. We found that the persistence and contour length depend on the incubation time. At high concentrations of HU, DNA molecules first become stiff with a larger value of the persistence length. The persistence length then decreases over time and the molecules regain the flexibility of bare DNA after â¼2 h. Concurrently, the contour length shows a slight increase. Second, we measured the change in topology of closed circular relaxed DNA following binding of HU. Here, we observed that HU induces supercoiling over a similar time span as the measured change in persistence length. Our observations can be rationalized in terms of the formation of a nucleoprotein filament followed by a structural rearrangement of the bound HU on DNA. The rearrangement results in a change in topology, an increase in bending flexibility and an increase in contour length through a decrease in helical pitch of the duplex.
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Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/ultraestrutura , DNA/química , DNA/metabolismo , DNA Circular/ultraestrutura , DNA Super-Helicoidal/ultraestrutura , Microscopia de Força AtômicaRESUMO
YOYO-1 is a green fluorescent dye which is widely used to image single DNA molecules in solution for biophysical studies. However, the question of whether the intercalation of YOYO-1 affects the mechanical properties of DNA is still not clearly answered. Investigators have put forth contradicting data on the changes in persistence length of DNA. Here, we use atomic force microscopy to systematically study the changes in the mechanical properties of DNA due to the intercalation of YOYO-1. We first measured the persistence length, contour length and the bending angle distribution of the DNA-YOYO-1 complex. We find that the persistence length of DNA remains unaffected with the intercalation of YOYO-1. However the contour length increases linearly with about 38% increase at full saturation of 1 YOYO-1 per 4 base pairs of DNA. Next we measured the change in topology of relaxed closed circular DNA after the intercalation of YOYO-1. We find that YOYO-1 introduces supercoiling in closed circular DNA. Our observations indicate that the intercalation of YOYO-1 results in the underwinding of DNA duplex, but does not significantly change the persistence length.
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Benzoxazóis/química , DNA Circular/química , Substâncias Intercalantes/química , Compostos de Quinolínio/química , Fenômenos MecânicosRESUMO
In alignment with widespread interest in carbon neutralization and sustainable practices, we disclose that conjugated polyelectrolyte (CPE) hydrogels are a type of recyclable, electrochemically stable, and environmentally friendly pseudocapacitive material for energy storage applications. By leveraging ionic-electronic coupling in a relatively fluid medium, one finds that hydrogels prepared using a fresh batch of an anionic CPE, namely, Pris-CPE-K, exhibit a specific capacitance of 32.6 ± 6.6 F g-1 in 2 M NaCl and are capable of 80% (26.1 ± 6.5 F g-1) capacitance retention after 100,000 galvanostatic charge-discharge (GCD) cycles at a current density (J) of 10 A g-1. We note that equilibration under a constant potential prior to GCD analysis leads to the K+ counterions in the CPE exchanging with Na+ and, thus, the relevant active material Pris-CPE-Na. It is possible to remove the CPE material from the electrochemical cell via extraction with water and to carry out a simple purification through dialysis to produce a recycled material, namely Re-CPE-Na. The recycling workup has no significant detrimental impact on the electrochemical performance. Specifically, Re-CPE-Na hydrogels display an initial specific capacitance of 26.3 ± 1.2 F g-1 (at 10 A g-1) and retain 77% of the capacitance after a subsequent 100,000 GCD cycles. Characterization by NMR, FTIR, and Raman spectroscopies, together with XPS and GPC measurements, revealed no change in the structure of the backbone or side chains. However, rheological measurements gave evidence of a slight loss in G' and G''. Overall, that CPE hydrogels display recyclability argues in favor of considering them as a novel materials platform for energy storage applications within an economically viable circular recycling strategy.
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Microbial electrosynthesis-using renewable electricity to stimulate microbial metabolism-holds the promise of sustainable chemical production. A key limitation hindering performance is slow electron-transfer rates at biotic-abiotic interfaces. Here a new n-type conjugated polyelectrolyte is rationally designed and synthesized and its use is demonstrated as a soft conductive material to encapsulate electroactive bacteria Shewanella oneidensis MR-1. The self-assembled 3D living biocomposite amplifies current uptake from the electrode ≈674-fold over controls with the same initial number of cells, thereby enabling continuous synthesis of succinate from fumarate. Such functionality is a result of the increased number of bacterial cells having intimate electronic communication with the electrode and a higher current uptake per cell. This is underpinned by the molecular design of the polymer to have an n-dopable conjugated backbone for facile reduction by the electrode and zwitterionic side chains for compatibility with aqueous media. Moreover, direct arylation polycondensation is employed instead of the traditional Stille polymerization to avoid non-biocompatible tin by-products. By demonstrating synergy between living cells with n-type organic semiconductor materials, these results provide new strategies for improving the performance of bioelectrosynthesis technologies.
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Fontes de Energia Bioelétrica , Elétrons , Fontes de Energia Bioelétrica/microbiologia , Eletricidade , Eletrodos , Transporte de Elétrons , PolieletrólitosRESUMO
BPI-inducible protein A (BipA), a highly conserved paralog of the well-known translational GTPases LepA and EF-G, has been implicated in bacterial motility, cold shock, stress response, biofilm formation, and virulence. BipA binds to the aminoacyl-(A) site of the bacterial ribosome and establishes contacts with the functionally important regions of both subunits, implying a specific role relevant to the ribosome, such as functioning in ribosome biogenesis and/or conditional protein translation. When cultured at suboptimal temperatures, the Escherichia coli bipA genomic deletion strain (ΔbipA) exhibits defects in growth, swimming motility, and ribosome assembly, which can be complemented by a plasmid-borne bipA supplementation or suppressed by the genomic rluC deletion. Based on the growth curve, soft agar swimming assay, and sucrose gradient sedimentation analysis, mutation of the catalytic residue His78 rendered plasmid-borne bipA unable to complement its deletion phenotypes. Interestingly, truncation of the C-terminal loop of BipA exacerbates the aforementioned phenotypes, demonstrating the involvement of BipA in ribosome assembly or its function. Furthermore, tandem mass tag-mass spectrometry analysis of the ΔbipA strain proteome revealed upregulations of a number of proteins (e.g., DeaD, RNase R, CspA, RpoS, and ObgE) implicated in ribosome biogenesis and RNA metabolism, and these proteins were restored to wild-type levels by plasmid-borne bipA supplementation or the genomic rluC deletion, implying BipA involvement in RNA metabolism and ribosome biogenesis. We have also determined that BipA interacts with ribosome 50S precursor (pre-50S), suggesting its role in 50S maturation and ribosome biogenesis. Taken together, BipA demonstrates the characteristics of a bona fide 50S assembly factor in ribosome biogenesis.
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The flow properties of DNA are important for understanding cell division and, indirectly, cancer therapy. DNA topology controlling enzymes such as topoisomerase II are thought to play an essential role. We report experiments showing how double-strand passage facilitated by topoisomerase II controls DNA rheology. For this purpose, we have measured the elastic storage and viscous loss moduli of a model system comprising bacteriophage λ-DNA and human topoisomerase IIα using video tracking of the Brownian motion of colloidal probe particles. We found that the rheology is critically dependent on the formation of temporal entanglements among the DNA molecules with a relaxation time of â¼1 s. We observed that topoisomerase II effectively removes these entanglements and transforms the solution from an elastic physical gel to a viscous fluid depending on the consumption of ATP. A second aspect of this study is the effect of the generic topoisomerase II inhibitor adenylyl-imidodiphosphate (AMP-PNP). In mixtures of AMP-PNP and ATP, the double-strand passage reaction gets blocked and progressively fewer entanglements are relaxed. A total replacement of ATP by AMP-PNP results in a temporal increase in elasticity at higher frequencies, but no transition to an elastic gel with fixed cross-links.
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DNA Topoisomerases Tipo II/metabolismo , DNA Viral/metabolismo , Movimento/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia , Monofosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/metabolismo , Adenilil Imidodifosfato/farmacologia , Bacteriófago lambda , DNA Viral/química , Dimaprit/análogos & derivados , Dimaprit/metabolismo , Módulo de Elasticidade/efeitos dos fármacos , Humanos , Reologia , Inibidores da Topoisomerase II/metabolismo , Viscosidade/efeitos dos fármacosRESUMO
Weak acids such as acetic acid and N-acetyl cysteine (NAC) at pH less than their pKa can effectively eradicate biofilms due to their ability to penetrate the biofilm matrix and the cell membrane. However, the optimum conditions for their activity against drug resistant strains, and safety, need to be understood for their application to treat infections or to inactivate biofilms on hard surfaces. Here, we investigate the efficacy and optimum conditions at which weak acids can eradicate biofilms. We compared the efficacy of various mono and triprotic weak acids such as N-acetyl cysteine (NAC), acetic acid, formic acid and citric acid, in eradicating biofilms. We found that monoprotic weak acids/acid drugs can kill mucoid P. aeruginosa mucA biofilm bacteria provided the pH is less than their pKa, demonstrating that the extracellular biofilm matrix does not protect the bacteria from the activity of the weak acids. Triprotic acids, such as citric acid, kill biofilm bacteria at pH â< âpKa1. However, at a pH between pKa1 and pKa2, citric acid is effective in killing the bacteria at the core of biofilm microcolonies but does not kill the bacteria on the periphery. The efficacy of a monoprotic weak acid (NAC) and triprotic weak acid (citric acid) were tested on biofilms formed by Klebsiella pneumoniae KP1, Pseudomonas putida OUS82, Staphylococcus aureus 15981, P. aeruginosa DK1-NH57388A, a mucoid cystic fibrosis isolate and P. aeruginosa PA_D25, an antibiotic resistant strain. We showed that weak acids have a broad spectrum of activity against a wide range of bacteria, including antibiotic resistant bacteria. Further, we showed that a weak acid drug, NAC, can kill bacteria without being toxic to human cells, if its pH is maintained close to its pKa. Thus weak acids/weak acid drugs target antibiotic resistant bacteria and eradicate the persister cells in biofilms which are tolerant to other conventional methods of biofilm eradication.
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The viscoelastic moduli of lambda-phage DNA through the entanglement transition were obtained with particle tracking microrheology. With increasing frequency, the viscous loss modulus first increases, then levels off, and eventually increases again. Concurrently, the elastic storage modulus monotonously increases and eventually levels off to a constant high frequency plateau value. Once the DNA molecules become entangled at about ten times the overlap concentration, the elastic storage modulus becomes larger than the viscous loss modulus in an intermediate frequency range. The number of entanglements per chain is obtained from the plateau value of the elasticity modulus. The longest, global relaxation time pertaining to the motion of the DNA molecules is obtained from the low shear viscosity as well as from the lowest crossover frequency of the viscous loss and elastic storage moduli. The concentration dependencies of the low shear viscosity, the number of entanglements per chain, and the relaxation time agree with the relevant scaling laws for reptation dynamics of entangled polyelectrolytes in an excess of simple, low molecular weight salt with screened electrostatic interactions.
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Bacteriófago lambda/genética , DNA Viral/química , Substâncias Viscoelásticas/química , Soluções Tampão , Ácido Edético/química , Reologia , SoluçõesRESUMO
Topically administered ocular drug delivery systems typically face severe bioavailability challenges because of the natural protective mechanisms of eyes. The rational design of drug delivery systems that are able to persist on corneal surfaces for sustained drug release is critical to tackle this problem. In this study, we fabricated monodisperse chitosan-coated PLGA microparticles with tailored diameters from 5 to 120 µm by capillary microfluidic techniques and conducted detailed investigations of their mucoadhesion to artificial mucin-coated substrates. AFM force spectroscopy revealed strong instant adhesion to mucins, whereas the adhesion force, rupture length, and adhesion energy were positively correlated to the particle diameter and contact time. Particle detachment tests under shear flow in a microfluidic mucin-coated flow cell were in accord with the AFM measurements and revealed that microparticles smaller than 25 µm exhibited strong persistence in the flow cell, withstanding high shear rates up to 28,750 s-1 which are equivalent to the harshest in vivo ocular conditions. A simple scaling analysis connects the AFM and detachment tests, and reveals the existence of a threshold diameter below which mucoadhesion performance essentially saturates-an important insight in managing the opposing design criteria of enhanced mucoadhesion and slow, sustained drug delivery. Our findings thus pave the way for the rational design of mucoadhesive microparticulate ocular drug delivery systems that are capable of enhancing the bioavailability of topically applied drugs to eyes, as well as to other tissues whose epithelial surfaces contain mucosae.
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Selective permeability of a biofilm matrix to some drugs has resulted in the development of drug tolerant bacteria. Here we studied the efficacy of a weak organic acid drug, N-acetyl-L-cysteine (NAC), on the eradication of biofilms formed by the mucoid strain of Pseudomonas aeruginosa and investigated the commonality of this drug with that of acetic acid. We showed that NAC and acetic acid at pH < pKa can penetrate the matrix and eventually kill 100% of the bacteria embedded in the biofilm. Once the bacteria are killed, the microcolonies swell in size and passively shed bacteria, suggesting that the bacteria act as crosslinkers within the extracellular matrix. Despite shedding of the bacteria, the remnant matrix remains intact and behaves as a pH-responsive hydrogel. These studies not only have implications for drug design but also offer a route to generate robust soft matter materials.
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Ácido Acético/farmacologia , Acetilcisteína/farmacologia , Biofilmes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Polissacarídeos BacterianosRESUMO
An approach to automatically detect bacteria division with temporal models is presented. To understand how bacteria migrate and proliferate to form complex multicellular behaviours such as biofilms, it is desirable to track individual bacteria and detect cell division events. Unlike eukaryotic cells, prokaryotic cells such as bacteria lack distinctive features, causing bacteria division difficult to detect in a single image frame. Furthermore, bacteria may detach, migrate close to other bacteria and may orientate themselves at an angle to the horizontal plane. Our system trains a hidden conditional random field (HCRF) model from tracked and aligned bacteria division sequences. The HCRF model classifies a set of image frames as division or otherwise. The performance of our HCRF model is compared with a Hidden Markov Model (HMM). The results show that a HCRF classifier outperforms a HMM classifier. From 2D bright field microscopy data, it is a challenge to separate individual bacteria and associate observations to tracks. Automatic detection of sequences with bacteria division will improve tracking accuracy.
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Algoritmos , Divisão Celular , Processamento de Imagem Assistida por Computador , Microscopia/métodos , Pseudomonas aeruginosa/citologia , Cadeias de Markov , MovimentoRESUMO
Biofilms are densely populated communities of microbial cells protected and held together by a matrix of extracellular polymeric substances. The structure and rheological properties of the matrix at the microscale influence the retention and transport of molecules and cells in the biofilm, thereby dictating population and community behavior. Despite its importance, quantitative descriptions of the matrix microstructure and microrheology are limited. Here, particle-tracking microrheology in combination with genetic approaches was used to spatially and temporally study the rheological contributions of the major exopolysaccharides Pel and Psl in Pseudomonas aeruginosa biofilms. Psl increased the elasticity and effective cross-linking within the matrix, which strengthened its scaffold and appeared to facilitate the formation of microcolonies. Conversely, Pel reduced effective cross-linking within the matrix. Without Psl, the matrix becomes more viscous, which facilitates biofilm spreading. The wild-type biofilm decreased in effective cross-linking over time, which would be advantageous for the spreading and colonization of new surfaces. This suggests that there are regulatory mechanisms to control production of the exopolysaccharides that serve to remodel the matrix of developing biofilms. The exopolysaccharides were also found to have profound effects on the spatial organization and integration of P. aeruginosa in a mixed-species biofilm model of P. aeruginosa-Staphylococcus aureus. Pel was required for close association of the two species in mixed-species microcolonies. In contrast, Psl was important for P. aeruginosa to form single-species biofilms on top of S. aureus biofilms. Our results demonstrate that Pel and Psl have distinct physical properties and functional roles during biofilm formation. Importance: Most bacteria grow as biofilms in the environment or in association with eukaryotic hosts. Removal of biofilms that form on surfaces is a challenge in clinical and industrial settings. One of the defining features of a biofilm is its extracellular matrix. The matrix has a heterogeneous structure and is formed from a secretion of various biopolymers, including proteins, extracellular DNA, and polysaccharides. It is generally known to interact with biofilm cells, thus affecting cell physiology and cell-cell communication. Despite the fact that the matrix may comprise up to 90% of the biofilm dry weight, how the matrix properties affect biofilm structure, maturation, and interspecies interactions remain largely unexplored. This study reveals that bacteria can use specific extracellular polymers to modulate the physical properties of their microenvironment. This in turn impacts biofilm structure, differentiation, and interspecies interactions.