Bacterial motility can govern the dynamics of antibiotic resistance evolution.

Spatial heterogeneity in antibiotic concentrations is thought to accelerate the evolution of antibiotic resistance, but current theory and experiments have overlooked the effect of cell motility on bacterial adaptation. Here, we study bacterial evolution in antibiotic landscapes with a quantitative model where bacteria evolve under the stochastic processes of proliferation, death, mutation and migration. Numerical and analytical results show that cell motility can both accelerate and decelerate bacterial adaptation by affecting the degree of genotypic mixing and ecological competition. Moreover, we find that for sufficiently high rates, cell motility can limit bacterial survival, and we derive conditions for all these regimes. Similar patterns are observed in more complex scenarios, namely where bacteria can bias their motion in chemical gradients (chemotaxis) or switch between motility phenotypes either stochastically or in a density-dependent manner. Overall, our work reveals limits to bacterial adaptation in antibiotic landscapes that are set by cell motility.

Modeling the Batch Sedimentation of Calcium Carbonate Particles in Laboratory Experiments-A Systematic Approach.

The design of continuous thickeners and clarifiers is commonly based on the solid flux theory. Batch sedimentation experiments conducted with solid concentrations still provide useful information for their application. The construction of models for the velocity of settling allows the estimation of the flux of solids throughout time, which can, in turn, be used to find the area of the units required to achieve a given solid concentration in the clarified stream. This paper addresses the numerical treatment of data obtained from batch sedimentation experiments of calcium carbonate particles. We propose a systematic framework to fit a model that is capable of representing the process features that involve (i) the numerical differentiation of data to generate initial estimates for the instantaneous velocity of settling; (ii) the integration of a differential equation to fit the model for the velocity of settling; and (iii) the assessment of the quality of the fit using common statistical indicators. The model used for demonstration has a theoretical basis combined with an empirical component to account for the effect of the particle concentrations and their state of aggregation. The values of the numerical parameters obtained are related to the characteristic dimensions of the aggregates and their mass-length fractal dimensions.

Suicidal chemotaxis in bacteria.

Bacteria commonly live in surface-associated communities where steep gradients of antibiotics and other chemical compounds can occur. While many bacterial species move on surfaces, we know surprisingly little about how such antibiotic gradients affect cell motility. Here, we study the behaviour of the opportunistic pathogen Pseudomonas aeruginosa in stable spatial gradients of several antibiotics by tracking thousands of cells in microfluidic devices as they form biofilms. Unexpectedly, these experiments reveal that bacteria use pili-based ('twitching') motility to navigate towards antibiotics. Our analyses suggest that this behaviour is driven by a general response to the effects of antibiotics on cells. Migrating bacteria reach antibiotic concentrations hundreds of times higher than their minimum inhibitory concentration within hours and remain highly motile. However, isolating cells - using fluid-walled microfluidic devices - reveals that these bacteria are terminal and unable to reproduce. Despite moving towards their death, migrating cells are capable of entering a suicidal program to release bacteriocins that kill other bacteria. This behaviour suggests that the cells are responding to antibiotics as if they come from a competing colony growing nearby, inducing them to invade and attack. As a result, clinical antibiotics have the potential to lure bacteria to their death.

Biofilm Growth under Elastic Confinement.

Bacteria often form surface-bound communities, embedded in a self-produced extracellular matrix, called biofilms. Quantitative studies of bioflim growth have typically focused on unconfined expansion above solid or semisolid surfaces, leading to exponential radial growth. This geometry does not accurately reflect the natural or biomedical contexts in which biofilms grow in confined spaces. Here, we consider one of the simplest confined geometries: a biofilm growing laterally in the space between a solid surface and an overlying elastic sheet. A poroelastic framework is utilized to derive the radial growth rate of the biofilm; it reveals an additional self-similar expansion regime, governed by the Poisson's ratio of the matrix, leading to a finite maximum radius, consistent with our experimental observations of growing Bacillus subtilis biofilms confined by polydimethylsiloxane.

The evolution of strategy in bacterial warfare via the regulation of bacteriocins and antibiotics.

Bacteria inhibit and kill one another with a diverse array of compounds, including bacteriocins and antibiotics. These attacks are highly regulated, but we lack a clear understanding of the evolutionary logic underlying this regulation. Here, we combine a detailed dynamic model of bacterial competition with evolutionary game theory to study the rules of bacterial warfare. We model a large range of possible combat strategies based upon the molecular biology of bacterial regulatory networks. Our model predicts that regulated strategies, which use quorum sensing or stress responses to regulate toxin production, will readily evolve as they outcompete constitutive toxin production. Amongst regulated strategies, we show that a particularly successful strategy is to upregulate toxin production in response to an incoming competitor's toxin, which can be achieved via stress responses that detect cell damage (competition sensing). Mirroring classical game theory, our work suggests a fundamental advantage to reciprocation. However, in contrast to classical results, we argue that reciprocation in bacteria serves not to promote peaceful outcomes but to enable efficient and effective attacks.

Recent advances on open fluidic systems for biomedical applications: A review.

Microfluidics has become an important tool to engineer microenvironments with high precision, comprising devices and methods for controlling and manipulating fluids at the submillimeter scale. A specific branch of microfluidics comprises open fluidic systems, which is mainly characterized by displaying a higher air/liquid interface when compared with traditional closed-channel setups. The use of open channel systems has enabled the design of singular architectures in devices that are simple to fabricate and to clean. Enhanced functionality and accessibility for liquid handling are additional advantages inputted to technologies based on open fluidics. While benchmarked against closed fluidics approaches, the use of directly accessible channels decreases the risk of clogging and bubble-driven flow perturbation. In this review, we discuss the advantages of open fluidics systems when compared to their closed fluidics counterparts. Platforms are analyzed in two separated groups based on different confinement principles: wall-based physical confinement and wettability-contrast confinement. The physical confinement group comprises both open and traditional microfluidics; examples based on open channels with rectangular and triangular cross-section, suspended microfluidics, and the use of narrow edge of a solid surface for fluid confinement are addressed. The second group covers (super)hydrophilic/(super)hydrophobic patterned surfaces, and examples based on polymer-, textile- and paper-based microfluidic devices are explored. The technologies described in this review are critically discussed concerning devices' performance and versatility, manufacturing techniques and fluid transport/manipulation methods. A gather-up of recent biomedical applications of open fluidics devices is also presented.

Bioactive Hydrogel Marbles.

Liquid marbles represented a significant advance in the manipulation of fluids as they used particle films to confine liquid drops, creating a robust and durable soft solid. We exploit this technology to engineering a bioactive hydrogel marble (BHM). Specifically, pristine bioactive glass nanoparticles were chemically tuned to produce biocompatible hydrophobic bioactive glass nanoparticles (H-BGNPs) that shielded a gelatin-based bead. The designed BHM shell promoted the growth of a bone-like apatite layer upon immersion in a physiological environment. The fabrication process allowed the efficient incorporation of drugs and cells into the engineered structure. The BHM provided a simultaneously controlled release of distinct encapsulated therapeutic model molecules. Moreover, the BHM sustained cell encapsulation in a 3D environment as demonstrated by an excellent in vitro stability and cytocompatibility. The engineered structures also showed potential to regulate a pre-osteoblastic cell line into osteogenic commitment. Overall, these hierarchical nanostructured and functional marbles revealed a high potential for future applications in bone tissue engineering.

Open Fluidics: A Cell Culture Flow System Developed Over Wettability Contrast-Based Chips.

Biological tissues are recurrently exposed to several dynamic mechanical forces that influence cell behavior. On this work, the focus is on the shear stress forces induced by fluid flow. The study of flow-induced effects on cells leads to important advances in cardiovascular, cancer, stem cell, and bone biology understanding. These studies are performed using cell culture flow (CCF) systems, mainly parallel plate flow chambers (PPFC), and microfluidic systems. Here, it is proposed an original CCF system based on the open fluidics concept. The system is developed using a planar superhydrophobic platform with hydrophilic paths. The paths work as channels to drive cell culture medium flows without using walls for liquid confinement. The liquid streams are controlled just based on the wettability contrast. To validate the concept, the effect of the shear stress stimulus in the osteogenic differentiation of C2C12 myoblast cells is studied. Combining bone morphogenic protein (specifically BMP-2) stimulation with this mechanical stimulus, a synergistic effect is found on osteoblast differentiation. This effect is confirmed by the enhancement of alkaline phosphatase activity, a well-known early marker of osteogenic differentiation. The suggested CCF system combines characteristics and advantages of both the PPFC and microfluidic systems.

The Potential of Liquid Marbles for Biomedical Applications: A Critical Review.

Liquid marbles (LM) are freestanding droplets covered by micro/nanoparticles with hydrophobic/hydrophilic properties, which can be manipulated as a soft solid. The phenomenon that generates these soft structures is regarded as a different method to generate a superhydrophobic behavior in the liquid/solid interface without modifying the surface. Several applications for the LM have been reported in very different fields, however the developments for biomedical applications are very recent. At first, the LM properties are reviewed, namely shell structure, LM shape, evaporation, floatability and robustness. The different strategies for LM manipulation are also described, which make use of magnetic, electrostatic and gravitational forces, ultraviolet and infrared radiation, and approaches that induce LM self-propulsion. Then, very distinctive applications for LM in the biomedical field are presented, namely for diagnostic assays, cell culture, drug screening and cryopreservation of mammalian cells. Finally, a critical outlook about the unexplored potential of LM for biomedical applications is presented, suggesting possible advances on this emergent scientific area.

The evolution of siderophore production as a competitive trait.

Microbes have the potential to be highly cooperative organisms. The archetype of microbial cooperation is often considered to be the secretion of siderophores, molecules scavenging iron, where cooperation is threatened by "cheater" genotypes that use siderophores without making them. Here, we show that this view neglects a key piece of biology: siderophores are imported by specific receptors that constrain their use by competing strains. We study the effect of this specificity in an ecoevolutionary model, in which we vary siderophore sharing among strains, and compare fully shared siderophores with private siderophores. We show that privatizing siderophores fundamentally alters their evolution. Rather than a canonical cooperative good, siderophores become a competitive trait used to pillage iron from other strains. We also study the physiological regulation of siderophores using in silico long-term evolution. Although shared siderophores evolve to be downregulated in the presence of a competitor, as expected for a cooperative trait, privatized siderophores evolve to be upregulated. We evaluate these predictions using published experimental work, which suggests that some siderophores are upregulated in response to competition akin to competitive traits like antibiotics. Although siderophores can act as a cooperative good for single genotypes, we argue that their role in competition is fundamental to understanding their biology.

Single-cell twitching chemotaxis in developing biofilms.

Bacteria form surface-attached communities, known as biofilms, which are central to bacterial biology and how they affect us. Although surface-attached bacteria often experience strong chemical gradients, it remains unclear whether single cells can effectively perform chemotaxis on surfaces. Here we use microfluidic chemical gradients and massively parallel automated tracking to study the behavior of the pathogen Pseudomonas aeruginosa during early biofilm development. We show that individual cells can efficiently move toward chemoattractants using pili-based "twitching" motility and the Chp chemosensory system. Moreover, we discovered the behavioral mechanism underlying this surface chemotaxis: Cells reverse direction more frequently when moving away from chemoattractant sources. These corrective maneuvers are triggered rapidly, typically before a wayward cell has ventured a fraction of a micron. Our work shows that single bacteria can direct their motion with submicron precision and reveals the hidden potential for chemotaxis within bacterial biofilms.

Stimulus electrodiagnosis and motor and functional evaluations during ulnar nerve recovery

BACKGROUND: Distal ulnar nerve injury leads to impairment of hand function due to motor and sensorial changes. Stimulus electrodiagnosis (SE) is a method of assessing and monitoring the development of this type of injury. OBJECTIVE: To identify the most sensitive electrodiagnostic parameters to evaluate ulnar nerve recovery and to correlate these parameters (Rheobase, Chronaxie, and Accommodation) with motor function evaluations. METHOD: A prospective cohort study of ten patients submitted to ulnar neurorrhaphy and evaluated using electrodiagnosis and motor assessment at two moments of neural recovery. A functional evaluation using the DASH questionnaire (Disability of the Arm, Shoulder, and Hand) was conducted at the end to establish the functional status of the upper limb. RESULTS: There was significant reduction only in the Chronaxie values in relation to time of injury and side (with and without lesion), as well as significant correlation of Chronaxie with the motor domain score. CONCLUSION: Chronaxie was the most sensitive SE parameter for detecting differences in neuromuscular responses during the ulnar nerve recovery process and it was the only parameter correlated with the motor assessment.

Model-based optimal design of experiments - semidefinite and nonlinear programming formulations.

We use mathematical programming tools, such as Semidefinite Programming (SDP) and Nonlinear Programming (NLP)-based formulations to find optimal designs for models used in chemistry and chemical engineering. In particular, we employ local design-based setups in linear models and a Bayesian setup in nonlinear models to find optimal designs. In the latter case, Gaussian Quadrature Formulas (GQFs) are used to evaluate the optimality criterion averaged over the prior distribution for the model parameters. Mathematical programming techniques are then applied to solve the optimization problems. Because such methods require the design space be discretized, we also evaluate the impact of the discretization scheme on the generated design. We demonstrate the techniques for finding D-, A- and E-optimal designs using design problems in biochemical engineering and show the method can also be directly applied to tackle additional issues, such as heteroscedasticity in the model. Our results show that the NLP formulation produces highly efficient D-optimal designs but is computationally less efficient than that required for the SDP formulation. The efficiencies of the generated designs from the two methods are generally very close and so we recommend the SDP formulation in practice.

Stimulus electrodiagnosis and motor and functional evaluations during ulnar nerve recovery.

BACKGROUND: Distal ulnar nerve injury leads to impairment of hand function due to motor and sensorial changes. Stimulus electrodiagnosis (SE) is a method of assessing and monitoring the development of this type of injury. OBJECTIVE: To identify the most sensitive electrodiagnostic parameters to evaluate ulnar nerve recovery and to correlate these parameters (Rheobase, Chronaxie, and Accommodation) with motor function evaluations. METHOD: A prospective cohort study of ten patients submitted to ulnar neurorrhaphy and evaluated using electrodiagnosis and motor assessment at two moments of neural recovery. A functional evaluation using the DASH questionnaire (Disability of the Arm, Shoulder, and Hand) was conducted at the end to establish the functional status of the upper limb. RESULTS: There was significant reduction only in the Chronaxie values in relation to time of injury and side (with and without lesion), as well as significant correlation of Chronaxie with the motor domain score. CONCLUSION: Chronaxie was the most sensitive SE parameter for detecting differences in neuromuscular responses during the ulnar nerve recovery process and it was the only parameter correlated with the motor assessment.

Fabrication and characterization of Eri silk fibers-based sponges for biomedical application.

Cocoon-derived semi-domesticated Eri silk fibers still lack exploitation for tissue engineering applications due to their poor solubility using conventional methods. The present work explores the ability to process cocoon fibers of non-mulberry Eri silk (Samia/Philosamia ricini) into sponges through a green approach using ionic liquid (IL)--1-buthyl-imidazolium acetate as a solvent. The formation of ß-sheet structures during Eri silk/IL gelation was acquired by exposing the Eri silk/IL gels to a saturated atmosphere composed of two different solvents: (i) isopropanol/ethanol (physical stabilization) and (ii) genipin, a natural crosslinker, dissolved in ethanol (chemical crosslinking). The sponges were then obtained by freeze-drying. This approach promotes the formation of both stable and ordered non-crosslinked Eri silk fibroin matrices. Moreover, genipin-crosslinked silk fibroin sponges presenting high height recovery capacity after compression, high swelling degree and suitable mechanical properties for tissue engineering applications were produced. The incorporation of a model drug--ibuprofen--and the corresponding release study from the loaded sponges demonstrated the potential of using these matrices as effective drug delivery systems. The assessment of the biological performance of ATDC5 chondrocyte-like cells in contact with the developed sponges showed the promotion of cell adhesion and proliferation, as well as extracellular matrix production within 2 weeks of culture. Sponges' intrinsic properties and biological findings open up their potential use for biomedical applications. STATEMENT OF SIGNIFICANCE: This work addresses the preparation and characterization of non-mulberry cocoon-derived Eri silk sponges. The insolubility of cocoons-derived non-mulberry silkworms impairs their processability and applications in the healthcare field. We used a green approach with ionic liquids to overcome the lack solubility of such silk fibers. The formation of beta-sheet structures into Eri-based sponges was physically and chemically induced. The sponges were obtained by freeze-drying. The developed structures exhibited flexibility to adapt and recover their shapes upon application and subsequent removal of load, high swelling degree, ability to load an anti-inflammatory drug and to promote its sustained release. They promoted in vitro cellular adhesion, proliferation and extracellular matrix production of a chondrocyte-like cell line, opening up their potential application for biomedical applications.

Fucoidan Hydrogels Photo-Cross-Linked with Visible Radiation As Matrices for Cell Culture.

Algae are abundant sources of bioactive components with extensive therapeutic properties, receiving much interest in recent years. The research on marine brown algae, namely one of its polysaccharide-fucoidan, has increased exponentially. Fucoidan is a sulfated cell-wall polysaccharide with several reported biological properties including anticancer, antivirus, anticoagulant, antioxidant and anti-inflammatory effects. In this study, fucoidan was functionalized by grafting methacrylic groups in the chain backbone, photo-cross-linkable under visible light to obtain biodegradable structures for tissue engineering. The functionalization reaction was carried out by concentrations (8 and 12%) of methacrylic anhydride (MA). The modified fucoidan (MFu) was characterized by FTIR and 1HNMR spectroscopy to confirm the functionalization. Further, modified fucoidan was photo-cross-linked under visible light and using superhydrophobic surfaces, to obtain spherical particles with controlled geometries benefiting from the high repellence of the surfaces. When using higher concentrations of MA, the particles were observed to exhibit a smaller average diameter. Moreover, the behavior of L929 mouse fibroblast-like cells was evaluated when cultured in contact with photo-cross-linked particles was investigated, being observed up to 14 days in culture. The photo-cross-linking of MFu under visible light enables thus the formation of particles here suggested as potentially relevant in a wide range of biomedical applications.

Biofilm Formation As a Response to Ecological Competition.

Bacteria form dense surface-associated communities known as biofilms that are central to their persistence and how they affect us. Biofilm formation is commonly viewed as a cooperative enterprise, where strains and species work together for a common goal. Here we explore an alternative model: biofilm formation is a response to ecological competition. We co-cultured a diverse collection of natural isolates of the opportunistic pathogen Pseudomonas aeruginosa and studied the effect on biofilm formation. We show that strain mixing reliably increases biofilm formation compared to unmixed conditions. Importantly, strain mixing leads to strong competition: one strain dominates and largely excludes the other from the biofilm. Furthermore, we show that pyocins, narrow-spectrum antibiotics made by other P. aeruginosa strains, can stimulate biofilm formation by increasing the attachment of cells. Side-by-side comparisons using microfluidic assays suggest that the increase in biofilm occurs due to a general response to cellular damage: a comparable biofilm response occurs for pyocins that disrupt membranes as for commercial antibiotics that damage DNA, inhibit protein synthesis or transcription. Our data show that bacteria increase biofilm formation in response to ecological competition that is detected by antibiotic stress. This is inconsistent with the idea that sub-lethal concentrations of antibiotics are cooperative signals that coordinate microbial communities, as is often concluded. Instead, our work is consistent with competition sensing where low-levels of antibiotics are used to detect and respond to the competing genotypes that produce them.

Chitosan-alginate multilayered films with gradients of physicochemical cues.

Tissues presenting continuous variations of properties in one direction have inspired the development of functional graded materials. In this work, we developed a new facile method for the development of continuous gradients in chitosan (CHIT) and alginate (ALG) polyelectrolyte multilayers (PEMs) obtained layer-by-layer based on the gradual dipping of CHIT/ALG coated glass slides in genipin solution. Stiffness gradients were produced in the cm scale by varying the reaction time with genipin. Quartz crystal microbalance, colorimetric measurements, trypan blue assay, attenuated total reflection-Fourier transform infrared spectroscopy, swelling ability, water contact angle and dynamic mechanical analysis (DMA) were used to find suitable conditions for the stiffness gradient. The PEMs can be successfully built up and cross-linked with genipin to yield surfaces with uniform physicochemical properties or with gradients of different physicochemical properties. It was found that a large reduction in the hydrophobic nature of the CHIT/ALG PEMs could be produced with higher cross-linking reaction times, regardless of the decrease in their swelling ability. Moreover, the mechanical properties were evaluated using an innovative and non-conventional DMA to monitor the cross-linking reaction in situ. The results confirm an enhancement on the tensile storage modulus with increasing reaction times from 60 to 140 MPa. In another original DMA testing protocol the local compression storage modulus was also measured directly on the films along the stiffness gradient, with results consistent with the tensile tests obtained on the freestanding membranes with different cross-linking degrees. The in vitro biological performance demonstrates that L929 adhered and spread more in the stiffer regions. This work demonstrates the versatility and feasibility of the LbL methodology to generate functional biomimetic surfaces with tuned mechanical and physicochemical properties, which hold great promise for the study of cell-substrate interactions.

Liquid marbles for high-throughput biological screening of anchorage-dependent cells.

Stable liquid marbles (LM) are produced by coating liquid droplets with a hydrophobic powder. The used hydrophobic powder is produced by fluorosi-lanization of diatomaceous earth, used before to produce superhydrophobic structures. Here, the use of LM is proposed for high-throughput drug screening on anchorage-dependent cells. To provide the required cell adhesion sites inside the liquid environment of LM, surface-modified poly(l-lactic acid) microparticles are used. A simple method that takes advantage from LM appealing features is presented, such as the ability to inject liquid on LM without disrupting (self-healing ability), and to monitor color changes inside of LM. After promoting cell adhesion, a cytotoxic screening test is performed as a proof of concept. Fe(3+) is used as a model cytotoxic agent and is injected on LM. After incubation, AlamarBlue reagent is injected and used to assess the presence of viable cells, by monitoring color change from blue to red. Color intensity is measured by image processing and the analysis of pictures takes using an ordinary digital camera. The proposed method is fully validated in counterpoint to an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo​xymethoxyphenyl)-2-(4-sulfophenyl)-2H-te​trazolium) colorimetric assay, a well-known method used for the cytotoxicity assessment.