Experimental observation of near-wall effects during the puncture of soft solids.

Performing conventional mechanical characterization techniques on soft materials can be challenging due to issues such as limited sample volumes and clamping difficulties. Deep indentation and puncture is a promising alternative as it is an information-rich measurement with the potential to be performed in a high-throughput manner. Despite its promise, the method lacks standardized protocols, and open questions remain about its possible limitations. Addressing these shortcomings is vital to ensure consistent methodology, measurements, and interpretation across samples and labs. To fill this gap, we examine the role of finite sample dimensions (and by extension, volume) on measured forces to determine the sample geometry needed to perform and unambiguously interpret puncture tests. Through measurements of puncture on a well-characterized elastomer using systematically varied sample dimensions, we show that the apparent mechanical response of a material is in fact sensitive to near-wall effects, and that additional properties, such as the sliding friction coefficient, can only be extracted in the larger dimension case where such effects are negligible.

The living interface between synthetic biology and biomaterial design.

Recent far-reaching advances in synthetic biology have yielded exciting tools for the creation of new materials. Conversely, advances in the fundamental understanding of soft-condensed matter, polymers and biomaterials offer new avenues to extend the reach of synthetic biology. The broad and exciting range of possible applications have substantial implications to address grand challenges in health, biotechnology and sustainability. Despite the potentially transformative impact that lies at the interface of synthetic biology and biomaterials, the two fields have, so far, progressed mostly separately. This Perspective provides a review of recent key advances in these two fields, and a roadmap for collaboration at the interface between the two communities. We highlight the near-term applications of this interface to the development of hierarchically structured biomaterials, from bioinspired building blocks to 'living' materials that sense and respond based on the reciprocal interactions between materials and embedded cells.

Mechanical resilience of the sessile tunicate Botryllus schlosseri.

We demonstrate that the sessile tunicate Botryllus schlosseri is remarkably resilient to applied loads by attaching the animals to an extensile substrate subjected to quasistatic equiradial loads. Animals can withstand radial extension of the substrate to strain values as high as 20% before they spontaneously detach. In the small to moderate strain regime, we found no relationship between the dynamic size of the external vascular bed and the magnitude of applied stretch, despite known force sensitivities of the vascular tissue at the cellular level. We attribute this resilience to the presence and mechanical properties of the tunic, the cellulose-enriched gel-like substance that encases the animal bodies and surrounding vasculature.

Ion channels of cilia: Paramecium as a model.

Holotrichous ciliates, like Paramecium, swim through their aqueous environment by beating their many cilia. They can alter swimming speed and direction, which seems to have mesmerized early microscopists of the 1600s. We know from extensive and elegant physiological studies and generation of mutants that these cells can be considered little swimming neurons because their ciliary beating is under bioelectric control of ion channels in the cilia. This chapter will focus on the ionic control of swimming behavior by ciliary ion channels, primarily in the holotrichous ciliate Paramecium. Voltage-gated and calcium-activated channels for calcium, magnesium, sodium, and potassium are regulated in a closely orchestrated manner that allows cilia to bend and propel the cell forward or backward. Sensory input that generates receptor potentials feeds into the control of this channel activity and allows the cell to turn or speed up. This in turn helps the cell to avoid predators or toxic conditions. While the focus is on P. tetraurelia and P. caudatum, the principles of ciliary ion channel activity and control are easily extendable to other ciliates and protists. The high conservation of channel and ion pump structures also extends the lessons from Paramecium to higher organisms.

Strength of fluid-filled soft composites across the elastofracture length.

Materials that utilize heterogeneous microstructures to control macroscopic mechanical response are ubiquitous in nature. Yet, translating nature's lessons to create synthetic soft solids has remained challenging. This is largely due to the limited synthetic routes available for creating soft composites, particularly with submicron features, as well as uncertainty surrounding the role of such a microstructured secondary phase in determining material behavior. This work leverages recent advances in the development of photocrosslinkable thermogelling nanoemulsions to produce composite hydrogels with a secondary phase assembled at well controlled length scales ranging from tens of nm to tens of µm. Through analysis of the mechanical response of these fluid-filled composite hydrogels, it is found that the size scale of the secondary phase has a profound impact on the strength when at or above the elastofracture length. Moreover, this work shows that mechanical integrity of fluid-filled soft solids can be sensitive to the size scale of the secondary phase.

High-throughput microscopy to determine morphology, microrheology, and phase boundaries applied to phase separating coacervates.

Evolution of composition, rheology, and morphology during phase separation in complex fluids is highly coupled to rheological and mass transport processes within the emerging phases, and understanding this coupling is critical for materials design of multiphase complex fluids. Characterizing these dependencies typically requires careful measurement of a large number of equilibrium and transport properties that are difficult to measure in situ as phase separation proceeds. Here, we propose and demonstrate a high-throughput microscopy platform to achieve simultaneous, in situ mapping of time-evolving morphology and microrheology in phase separating complex fluids over a large compositional space. The method was applied to a canonical example of polyelectrolyte complex coacervation, whereby mixing of oppositely charged species leads to liquid-liquid phase separation into distinct solute-dense and dilute phases. Morphology and rheology were measured simultaneously and kinetically after mixing to track the progression of phase separation. Once equilibrated, the dense phase viscosity was determined to high compositional accuracy using passive probe microrheology, and the results were used to derive empirical relationships between the composition and viscosity. These relationships were inverted to reconstruct the dense phase boundary itself, and further extended to other mixture compositions. The resulting predictions were validated by independent equilibrium compositional measurements. This platform paves the way for rapid screening and formulation of complex fluids and (bio)macromolecular materials, and serves as a critical link between formulation and rheology for multi-phase material discovery.

Influence of Polarity Change and Photophysical Effects on Photosurfactant-Driven Wetting.

Photosurfactants have shown considerable promise for enabling stimuli-responsive control of the properties and motion of fluid interfaces. Recently, a number of photoswitch chemistries have emerged to tailor the photoresponsive properties of photosurfactants. However, systematic studies investigating how photoresponsive surfactant behavior depends on the photochemical and photophysical properties of the switch remain scarce. In this work, we develop synthetic schemes and surfactant designs to produce a well-controlled library of photosurfactants to comparatively assess the behavior of photoswitch chemistry on interfacial behavior. We employ photoinduced spreading of droplets at fluid interfaces as a model for such studies. We show that although photosurfactant response is largely guided by expected trends with changes in polarity of the photoswitch, interfacial behavior also depends nontrivially and sometimes counter-intuitively on the kinetics and mechanisms of photoswitching, particularly at the interface of two solvents, as well as on complex interactions with other surfactants. Understanding these complexities enables the design of new photosurfactant systems and their optimization toward responsive functions including triggered spreading, dewetting, and destabilization of droplets on solid and fluid surfaces.

Non-destructive quantification of anaerobic gut fungi and methanogens in co-culture reveals increased fungal growth rate and changes in metabolic flux relative to mono-culture.

BACKGROUND: Quantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are native to the rumen of large herbivores, where they exist as minority members among a wealth of prokaryotes. AGF have significant biotechnological potential owing to their diverse repertoire of potent lignocellulose-degrading carbohydrate-active enzymes (CAZymes), which indirectly bolsters activity of other rumen microbes through metabolic exchange. While decades of literature suggest that polysaccharide degradation and AGF growth are accelerated in co-culture with prokaryotes, particularly methanogens, methods have not been available to measure concentrations of individual species in co-culture. New methods to disentangle the contributions of AGF and rumen prokaryotes are sorely needed to calculate AGF growth rates and metabolic fluxes to prove this hypothesis and understand its causality for predictable co-culture design. RESULTS: We present a simple, microplate-based method to measure AGF and methanogen concentrations in co-culture based on fluorescence and absorbance spectroscopies. Using samples of < 2% of the co-culture volume, we demonstrate significant increases in AGF growth rate and xylan and glucose degradation rates in co-culture with methanogens relative to mono-culture. Further, we calculate significant differences in AGF metabolic fluxes in co-culture relative to mono-culture, namely increased flux through the energy-generating hydrogenosome organelle. While calculated fluxes highlight uncertainties in AGF primary metabolism that preclude definitive explanations for this shift, our method will enable steady-state fluxomic experiments to probe AGF metabolism in greater detail. CONCLUSIONS: The method we present to measure AGF and methanogen concentrations enables direct growth measurements and calculation of metabolic fluxes in co-culture. These metrics are critical to develop a quantitative understanding of interwoven rumen metabolism, as well as the impact of co-culture on polysaccharide degradation and metabolite production. The framework presented here can inspire new methods to probe systems beyond AGF and methanogens. Simple modifications to the method will likely extend its utility to co-cultures with more than two organisms or those grown on solid substrates to facilitate the design and deployment of microbial communities for bioproduction and beyond.

Safety, Tolerability, and Pharmacokinetics of the Broadly Neutralizing Human Immunodeficiency Virus (HIV)-1 Monoclonal Antibody VRC01 in HIV-Exposed Newborn Infants.

BACKGROUND: Although mother-to-child human immunodeficiency virus (HIV) transmission has dramatically decreased with maternal antiretroviral therapy, breast milk transmission accounts for most of the 180 000 new infant HIV infections annually. Broadly neutralizing antibodies (bNAb) may further reduce transmission. METHODS: A Phase 1 safety and pharmacokinetic study was conducted: a single subcutaneous (SC) dose of 20 or 40 mg/kg (Dose Groups 1 and 2, respectively) of the bNAb VRC01 was administered to HIV-exposed infants soon after birth. Breastfeeding infants (Dose Group 3) received 40 mg/kg SC VRC01 after birth and then 20 mg/kg/dose SC monthly. All infants received appropriate antiretroviral prophylaxis. RESULTS: Forty infants were enrolled (21 in the United States, 19 in Africa). Subcutaneous VRC01 was safe and well tolerated with only mild-to-moderate local reactions, primarily erythema, which rapidly resolved. For multiple-dose infants, local reactions decreased with subsequent injections. VRC01 was rapidly absorbed after administration, with peak concentrations 1-6 days postdose. The 40 mg/kg dose resulted in 13 of 14 infants achieving the serum 50 micrograms (mcg)/mL target at day 28. Dose Group 3 infants maintained concentrations greater than 50 mcg/mL throughout breastfeeding. CONCLUSIONS: Subcutaneous VRC01 as single or multiple doses is safe and well tolerated in very young infants and is suitable for further study to prevent HIV transmission in infants.

Inertial flow focusing: a case study in optimizing cellular trajectory through a microfluidic MEMS device for timing-critical applications.

Although microfluidic micro-electromechanical systems (MEMS) are well suited to investigate the effects of mechanical force on large populations of cells, their high-throughput capabilities cannot be fully leveraged without optimizing the experimental conditions of the fluid and particles flowing through them. Parameters such as flow velocity and particle size are known to affect the trajectories of particles in microfluidic systems and have been studied extensively, but the effects of temperature and buffer viscosity are not as well understood. In this paper, we explored the effects of these parameters on the timing of our own cell-impact device, the µHammer, by first tracking the velocity of polystyrene beads through the device and then visualizing the impact of these beads. Through these assays, we find that the timing of our device is sensitive to changes in the ratio of inertial forces to viscous forces that particles experience while traveling through the device. This sensitivity provides a set of parameters that can serve as a robust framework for optimizing device performance under various experimental conditions, without requiring extensive geometric redesigns. Using these tools, we were able to achieve an effective throughput over 360 beads/s with our device, demonstrating the potential of this framework to improve the consistency of microfluidic systems that rely on precise particle trajectories and timing.

Effects of sea water pH on marine mussel plaque maturation.

Marine mussel plaques are an exceptional model for wet adhesives. Despite advances in understanding their protein composition and strategies for molecular bonding, the process by which these soluble proteins are rapidly processed into load-bearing structures remains poorly understood. Here, we examine the effects of seawater pH on the time evolution of the internal microstructures in plaques harvested from Mytilus californianus. Experimentally, plaques deposited by mussels on glass and acrylic surfaces were collected immediately after foot retraction without plaque separation from the surface, placed into pH-adjusted artificial seawater for varying times, and characterized using scanning electron microscopy and tensile testing. We found a pH dependent transition from a liquid-like state to a porous solid within 30 min for pH ≥ 6.7; these plaques are load-bearing. By contrast, samples maintained at pH 3.0 showed no porosity and no measurable strength. Interestingly, we found cuticle development within 15 min regardless of pH, suggesting that cuticle formation occurs prior to pore assembly. Our results suggest that sea water infusion after deposition by and disengagement of the foot is critical to the rapid formation of internal structures, which in turn plays an important role in the plaques' mechanical performance.

Rapid analysis of cell-generated forces within a multicellular aggregate using microsphere-based traction force microscopy.

We present a new approach to measuring cell-generated forces from the deformations of elastic microspheres embedded within multicellular aggregates. By directly fitting the measured sensor deformation to an analytical model based on experimental observations and invoking linear elasticity, we dramatically reduce the computational complexity of the problem, and directly obtain the full 3D mapping of surface stresses. Our approach imparts extraordinary computational efficiency, allowing tractions to be estimated within minutes and enabling rapid analysis of microsphere-based traction force microscopy data.

Live Respiratory Syncytial Virus (RSV) Vaccine Candidate Containing Stabilized Temperature-Sensitivity Mutations Is Highly Attenuated in RSV-Seronegative Infants and Children.

Background: Respiratory syncytial virus (RSV) is the most important viral cause of severe respiratory illness in young children and lacks a vaccine. RSV cold-passage/stabilized 2 (RSVcps2) is a modification of a previously evaluated vaccine candidate in which 2 major attenuating mutations have been stabilized against deattenuation. Methods: RSV-seronegative 6-24-month-old children received an intranasal dose of 105.3 plaque-forming units (PFU) of RSVcps2 (n = 34) or placebo (n = 16) (International Maternal Pediatric Adolescent AIDS Clinical Trials protocol P1114 and companion protocol CIR285). RSV serum neutralizing antibody titers before and 56 days after vaccination, vaccine virus infectivity (defined as vaccine virus shedding detectable in nasal wash and/or a ≥4-fold rise in serum antibodies), reactogenicity, and genetic stability were assessed. During the following RSV transmission season, participants were monitored for respiratory illness, with serum antibody titers measured before and after the season. Results: A total of 85% of vaccinees were infected with RSVcps2 (median peak titer, 0.5 log10 PFU/mL by culture and 2.9 log10 copies/mL by polymerase chain reaction analysis); 77% shed vaccine virus, and 59% developed a ≥4-fold rise in RSV-serum neutralizing antibody titers. Respiratory tract and/or febrile illness occurred at the same rate (50%) in the vaccine and placebo groups. Deattenuation was not detected at either of 2 stabilized mutation sites. Conclusions: RSVcps2 was well tolerated and moderately immunogenic and had increased genetic stability in 6-24-month-old RSV-seronegative children. Clinical Trials Registration: NCT01852266 and NCT01968083.

Live-Attenuated Respiratory Syncytial Virus Vaccine Candidate With Deletion of RNA Synthesis Regulatory Protein M2-2 is Highly Immunogenic in Children.

Background: Live respiratory syncytial virus (RSV) candidate vaccine LIDΔM2-2 is attenuated by deletion of the RSV RNA regulatory protein M2-2, resulting in upregulated viral gene transcription and antigen expression but reduced RNA replication. Methods: RSV-seronegative children ages 6-24 months received a single intranasal dose of 105 plaque forming units (PFU) of LIDΔM2-2 (n = 20) or placebo (n = 9) (NCT02237209, NCT02040831). RSV serum antibodies, vaccine infectivity, and reactogenicity were assessed. During the following RSV season, participants were monitored for respiratory illness and pre- and post-RSV season serum antibodies. Results: Vaccine virus was shed by 95% of vaccinees (median peak titers of 3.8 log10 PFU/mL by quantitative culture and 6.3 log10 copies/mL by PCR); 90% had ≥4-fold rise in serum neutralizing antibodies. Respiratory symptoms and fever were common in vaccine (95%) and placebo (78%). One vaccinee had grade 2 rhonchi concurrent with vaccine shedding, rhinovirus, and enterovirus. Eight of 19 vaccinees versus 2 of 9 placebo recipients had substantially increased RSV antibody titers after the RSV season without medically attended RSV disease, indicating anamnestic vaccine responses to wild-type RSV without significant illness. Conclusion: LIDΔM2-2 had excellent infectivity and immunogenicity, encouraging further study of vaccine candidates attenuated by M2-2 deletion. Clinical Trials Registration: NCT02237209, NCT02040831.

Influence of multi-cycle loading on the structure and mechanics of marine mussel plaques.

The proteinaceous byssal plaque-thread structures created by marine mussels exhibit extraordinary load-bearing capability. Although the nanoscopic protein interactions that support interfacial adhesion are increasingly understood, major mechanistic questions about how mussel plaques maintain toughness on supramolecular scales remain unanswered. This study explores the mechanical properties of whole mussel plaques subjected to repetitive loading cycles, with varied recovery times. Mechanical measurements were complemented with scanning electron microscopy to investigate strain-induced structural changes after yield. Multicyclic loading of plaques decreases their low-strain stiffness and introduces irreversible, strain-dependent plastic damage within the plaque microstructure. However, strain history does not compromise critical strength or maximum extension compared with plaques monotonically loaded to failure. These results suggest that a multiplicity of force transfer mechanisms between the thread and plaque-substrate interface allow the plaque-thread structure to accommodate a wide range of extensions as it continues to bear load. This improved understanding of the mussel system at micron-to-millimeter lengthscales offers strategies for including similar fail-safe mechanisms in the design of soft, tough and resilient synthetic structures.

Simple peptide coacervates adapted for rapid pressure-sensitive wet adhesion.

We report here that a dense liquid formed by spontaneous condensation, also known as simple coacervation, of a single mussel foot protein-3S-mimicking peptide exhibits properties critical for underwater adhesion. A structurally homogeneous coacervate is deposited on underwater surfaces as micrometer-thick layers, and, after compression, displays orders of magnitude higher underwater adhesion at 2 N m-1 than that reported from thin films of the most adhesive mussel-foot-derived peptides or their synthetic mimics. The increase in adhesion efficiency does not require nor rely on post-deposition curing or chemical processing, but rather represents an intrinsic physical property of the single-component coacervate. Its wet adhesive and rheological properties correlate with significant dehydration, tight peptide packing and restriction in peptide mobility. We suggest that such dense coacervate liquids represent an essential adaptation for the initial priming stages of mussel adhesive deposition, and provide a hitherto untapped design principle for synthetic underwater adhesives.

The Short- to Medium-Term Predictive Validity of Static and Dynamic Risk-of-Violence Measures in Medium- to Low-Secure Forensic and Civil Inpatients.

The prediction and subsequent management of aggression by psychiatric inpatients is a crucial role of the mental health professional. This retrospective cohort study examines the predictive validity of 10 static and dynamic risk-of-violence measures and subscales in 37 forensic and 37 civil inpatients residing in a medium- to-low security psychiatric facility for a period of up to 6 months. Retrospective file records were sourced to conduct an AUC analysis of the ROC curve for short- and medium-term follow-up periods. The hypothesis that dynamic measures would be better predictors than static measures over the short term was supported. Albeit to a lesser extent, dynamic measures were still better predictors than static measures over the medium term. This result was seen in both civil and forensic groups. Three previously untested measures were found to predict aggression within the sample. It is recommended that mental health services employ the use of dynamic measures when making short-term risk-of-violence predictions for civil and/or forensic inpatients.

Molecular control of stress transmission in the microtubule cytoskeleton.

In this article, we will summarize recent progress in understanding the mechanical origins of rigidity, strength, resiliency and stress transmission in the MT cytoskeleton using reconstituted networks formed from purified components. We focus on the role of network architecture, crosslinker compliance and dynamics, and molecular determinants of single filament elasticity, while highlighting open questions and future directions for this work.

Improved calibration of the nonlinear regime of a single-beam gradient optical trap.

We report an improved method for calibrating the nonlinear region of a single-beam gradient optical trap. Through analysis of the position fluctuations of a trapped object that is displaced from the trap center by controlled flow we measure the local trap stiffness in both the linear and nonlinear regimes without knowledge of the magnitude of the applied external forces. This approach requires only knowledge of the system temperature, and is especially useful for measurements involving trapped objects of unknown size, or objects in a fluid of unknown viscosity.

Voltage-gated calcium channels of Paramecium cilia.

Paramecium cells swim by beating their cilia, and make turns by transiently reversing their power stroke. Reversal is caused by Ca2+ entering the cilium through voltage-gated Ca2+ (CaV) channels that are found exclusively in the cilia. As ciliary Ca2+ levels return to normal, the cell pivots and swims forward in a new direction. Thus, the activation of the CaV channels causes cells to make a turn in their swimming paths. For 45 years, the physiological characteristics of the Paramecium ciliary CaV channels have been known, but the proteins were not identified until recently, when the P. tetraurelia ciliary membrane proteome was determined. Three CaVα1 subunits that were identified among the proteins were cloned and confirmed to be expressed in the cilia. We demonstrate using RNA interference that these channels function as the ciliary CaV channels that are responsible for the reversal of ciliary beating. Furthermore, we show that Pawn (pw) mutants of Paramecium that cannot swim backward for lack of CaV channel activity do not express any of the three CaV1 channels in their ciliary membrane, until they are rescued from the mutant phenotype by expression of the wild-type PW gene. These results reinforce the correlation of the three CaV channels with backward swimming through ciliary reversal. The PwB protein, found in endoplasmic reticulum fractions, co-immunoprecipitates with the CaV1c channel and perhaps functions in trafficking. The PwA protein does not appear to have an interaction with the channel proteins but affects their appearance in the cilia.