A structure-based mechanism for initiation of AP-3 coated vesicle formation.

Adaptor protein complex 3 (AP-3) mediates cargo sorting from endosomes to lysosomes and lysosome-related organelles. Recently, it was shown that AP-3 is in a constitutively open, active conformation compared to the related AP-1 and AP-2 coat complexes, which are inactive until undergoing large conformational changes upon membrane recruitment. How AP-3 is regulated is therefore an open question. To understand the mechanism of AP-3 membrane recruitment and activation, we reconstituted the core of human AP-3 and determined multiple structures in the soluble and membrane-bound states using electron cryo-microscopy (cryo-EM). Similar to yeast AP-3, human AP-3 is in a constitutively open conformation, with the cargo-binding domain of the µ3 subunit conformationally free. To reconstitute AP-3 activation by the small GTPase Arf1, we used lipid nanodiscs to build Arf1-AP-3 complexes on membranes and determined three structures that show the stepwise conformational changes required for formation of AP-3 coated vesicles. First, membrane-recruitment is driven by one of two predicted Arf1 binding sites on AP-3. In this conformation, AP-3 is flexibly tethered to the membrane and its cargo binding domain remains conformationally dynamic. Second, cargo binding causes AP-3 to adopt a fixed position and rigidifies the complex, which stabilizes binding for a second Arf1 molecule. Finally, binding of the second Arf1 molecule provides the template for AP-3 dimerization, providing a glimpse into the first step of coat polymerization. We propose coat polymerization only occurs after cargo engagement, thereby linking cargo sorting with assembly of higher order coat structures. Additionally, we provide evidence for two amphipathic helices in AP-3, suggesting that AP-3 contributes to membrane deformation during coat assembly. In total, these data provide evidence for the first stages of AP-3 mediated vesicle coat assembly.

Resisting subsidence with a truss Implant: Application of the "Snowshoe" principle for interbody fusion devices.

The primary objective was to compare the subsidence resistance properties of a novel 3D-printed spinal interbody titanium implant versus a predicate polymeric annular cage. We evaluated a 3D-printed spinal interbody fusion device that employs truss-based bio-architectural features to apply the snowshoe principle of line length contact to provide efficient load distribution across the implant/endplate interface as means of resisting implant subsidence. Devices were tested mechanically using synthetic bone blocks of differing densities (osteoporotic to normal) to determine the corresponding resistance to subsidence under compressive load. Statistical analyses were performed to compare the subsidence loads and evaluate the effect of cage length on subsidence resistance. The truss implant demonstrated a marked rectilinear increase in resistance to subsidence associated with increase in the line length contact interface that corresponds with implant length irrespective of subsidence rate or bone density. In blocks simulating osteoporotic bone, comparing the shortest with the longest length truss cage (40 vs. 60 mm), the average compressive load necessary to induce subsidence of the implant increased by 46.4% (383.2 to 561.0 N) and 49.3% (567.4 to 847.2 N) for 1 and 2 mm of subsidence, respectively. In contrast, for annular cages, there was only a modest increase in compressive load when comparing the shortest with the longest length cage at a 1 mm subsidence rate. The Snowshoe truss cages demonstrated substantially more resistance to subsidence than corresponding annular cages. Clinical studies are required to support the biomechanical findings in this work.

Acoustophoresis of a resonant elastic microparticle in a viscous fluid medium.

This work presents three-dimensional (3D) numerical analysis of acoustic radiation force on an elastic microsphere suspended in a viscous fluid. Acoustophoresis of finite-sized, neutrally buoyant, nearly incompressible soft particles may improve by orders of magnitude and change directions when going through resonant vibrations. These findings offer the potential to manipulate and separate microparticles based on their resonance frequency. This concept has profound implications in cell and microparticle handling, 3D printing, and enrichment in lab-on-chip applications. The existing analytical body of work can predict spheroidal harmonics of an elastic sphere and acoustic radiation force based on monopole and dipole scatter in an ideal fluid. However, little attention is given to the complex interplay of resonant fluid and solid bodies that generate acoustic radiation. The finite element method is used to find resonant modes, damping factors, and acoustic forces of an elastic sphere subject to a standing acoustic wave. Under fundamental spheroidal modes, the radiation force fluctuates significantly around analytical values due to constructive or destructive scatter-incident wave interference. This suggests that for certain materials, relevant to acoustofluidic applications, particle resonances are an important scattering mechanism and design parameter. The 3D model may be applied to any number of particles regardless of geometry or background acoustic field.

Engineered jumpers overcome biological limits via work multiplication.

For centuries, scientists have explored the limits of biological jump height1,2, and for decades, engineers have designed jumping machines3-18 that often mimicked or took inspiration from biological jumpers. Despite these efforts, general analyses are missing that compare the energetics of biological and engineered jumpers across scale. Here we show how biological and engineered jumpers have key differences in their jump energetics. The jump height of a biological jumper is limited by the work its linear motor (muscle) can produce in a single stroke. By contrast, the jump height of an engineered device can be far greater because its ratcheted or rotary motor can 'multiply work' during repeated strokes or rotations. As a consequence of these differences in energy production, biological and engineered jumpers should have divergent designs for maximizing jump height. Following these insights, we created a device that can jump over 30 metres high, to our knowledge far higher than previous engineered jumpers and over an order of magnitude higher than the best biological jumpers. Our work advances the understanding of jumping, shows a new level of performance, and underscores the importance of considering the differences between engineered and biological systems.

Recent progress in acoustic field-assisted 3D-printing of functional composite materials.

Acoustic forces are an attractive pathway to achieve directed assembly for multi-phase materials via additive processes. Programmatic integration of microstructure and structural features during deposition offers opportunities for optimizing printed component performance. We detail recent efforts to integrate acoustic focusing with a direct-ink-write mode of printing to modulate material transport properties (e.g. conductivity). Acoustic field-assisted printing, operating under a multi-node focusing condition, supports deposition of materials with multiple focused lines in a single-pass printed line. Here, we report the demonstration of acoustic focusing in concert with diffusive self-assembly to rapidly assembly and print multiscale, mm-length colloidal solids on a timescale of seconds to minutes. These efforts support the promising capabilities of acoustic field-assisted deposition-based printing to achieve spatial control of printed microstructures with deterministic, long-range ordering across multiple length scales.

Truss implant technology™ for interbody fusion in spinal degenerative disorders: profile of advanced structural design, mechanobiologic and performance characteristics.

Introduction: Interbody fusion devices are customarily used in fusion of the anterior spinal column for treatment of degenerative disc disease. Their traditional role is to reestablish and maintain intervertebral disc height, contain bone graft and provide mechanical support for the spine while osseointegration takes place. Utilizing the principles of mechanobiology, a unique biokinetic interbody fusion device has been developed that employs an advanced structural design to facilitate and actively participate in the fusion consolidation process.Areas covered: This article profiles and characterizes 4WEB Medical's Truss Implant Technology™ which includes a range of 3D-printed titanium spinal interbody implants and non-spinal implants whose design is based on truss structures enabled by advances in additive manufacturing. Four main areas of the implant design and functionality are detailed: bio-architecture, mechanobiologic underpinnings, bioactive surface features, and subsidence resistance. Pre-clinical and clinical examples are provided to describe and specify the bioactive roles and contributions of each design feature.Expert opinion: The distinct and unique combination of features incorporated within the truss cage design results in a biokinetic implant that actively participates in the bone healing cascade and fusion process.

Toward optimal acoustophoretic microparticle manipulation by exploiting asymmetry.

The performance of a micro-acousto-fluidic device designed for microparticle trapping is simulated using a three-dimensional (3D) numerical model. It is demonstrated by numerical simulations that geometrically asymmetric architecture and actuation can increase the acoustic radiation forces in a liquid-filled cavity by almost 2 orders of magnitude when setting up a standing pressure half wave in a microfluidic chamber. Similarly, experiments with silicon-glass devices show a noticeable improvement in acoustophoresis of 20-µm silica beads in water when asymmetric devices are used. Microparticle acoustophoresis has an extensive array of applications in applied science fields ranging from life sciences to 3D printing. A more efficient and powerful particle manipulation system can boost the overall effectiveness of an acoustofluidic device. The numerical simulations are developed in the COMSOL Multiphysics® software package (COMSOL AB, Stockholm, Sweden). By monitoring the modes and magnitudes of simulated acoustophoretic fields in a relatively wide range of ultrasonic frequencies, a map of device performance is obtained. 3D resonant acoustophoretic fields are identified to quantify the improved performance of the chips with an asymmetric layout. Four different device designs are analyzed experimentally, and particle tracking experimental data qualitatively supports the numerical results.

Subsidence of Additively-Manufactured Cages in Foam Substrates: Effect of Contact Topology.

Subsidence of implants into bone is a major source of morbidity. The underlying mechanics of the phenomenon are not clear, but are likely related to interactions between contact stresses and the underlying porous trabecular bone structure. To gain insight into these interactions, we studied the penetration of three-dimensional (3D)-printed indenters with systematically varying geometries into Sawbones® foam substrates and isolated the effects of contact geometry from those of overall contact size and area. When size, contact area, and indented material stiffness and strength are controlled for, we show that resistance to penetration is in fact a function of topology only. Indenters with greater line contact lengths support higher subsidence loads in compression. These results have direct implications for the design of implants to resist subsidence into bone.

Role of Zika Virus Envelope Protein Domain III as a Target of Human Neutralizing Antibodies.

Zika virus (ZIKV) is a flavivirus that is structurally highly similar to the related viruses, dengue virus (DENV), West Nile virus, and yellow fever virus. ZIKV causes an acute infection that often results in mild symptoms but that can cause severe disease in rare instances. Following infection, individuals mount an adaptive immune response, composed of antibodies (Abs) that target the envelope (E) glycoprotein of ZIKV, which covers the surface of the virus. Groups have studied monoclonal antibodies and polyclonal immune sera isolated from individuals who recovered from natural ZIKV infections. Some of these antibodies bind to domain III of E (EDIII), but the functional importance of these antibodies is unknown. In this study, we aimed to determine if EDIII is a major target of the potent serum neutralizing antibodies present in people after ZIKV infection. By generating a chimeric virus containing ZIKV EDIII in a DENV4 virus backbone, our data show a minor role of EDIII-targeting antibodies in human polyclonal neutralization. These results reveal that while monoclonal antibody (MAb) studies are informative in identifying individual antibody epitopes, they can overestimate the importance of epitopes contained within EDIII as targets of serum neutralizing antibodies. Additionally, these results argue that the major target of human ZIKV neutralizing antibodies resides elsewhere in E; however, further studies are needed to assess the epitope specificity of the neutralizing response at the population level. Identification of the major epitopes on the envelope of ZIKV recognized by serum neutralizing antibodies is critical for understanding protective immunity following natural infection and for guiding the design and evaluation of vaccines.IMPORTANCE Zika virus is a flavivirus that was recently introduced to Latin America, where it caused a massive epidemic. Individuals infected with ZIKV generate an immune response composed of antibodies which bind to the envelope (E) protein. These anti-E antibodies are critical in protecting individuals from subsequent infection. Multiple groups have found that many ZIKV antibodies bind to domain III of E (EDIII), suggesting that this region is an important target of neutralizing antibodies. Here, we generated a chimeric virus containing ZIKV EDIII in a dengue virus backbone to measure ZIKV EDIII-specific antibody responses. We found that while polyclonal ZIKV immune serum contains antibodies targeting EDIII, they constitute only a small fraction of the total population of antibodies that neutralize ZIKV. Further studies are needed to define the main targets on the viral envelope recognized by human neutralizing antibodies, which is critical for guiding the development of ZIKV vaccines.

Bridging functional nanocomposites to robust macroscale devices.

At the intersection of the outwardly disparate fields of nanoparticle science and three-dimensional printing lies the promise of revolutionary new "nanocomposite" materials. Emergent phenomena deriving from the nanoscale constituents pave the way for a new class of transformative materials with encoded functionality amplified by new couplings between electrical, optical, transport, and mechanical properties. We provide an overview of key scientific advances that empower the development of such materials: nanoparticle synthesis and assembly, multiscale assembly and patterning, and mechanical characterization to assess stability. The focus is on recent illustrations of approaches that bridge these fields, facilitate the design of ordered nanocomposites, and offer clear pathways to device integration. We conclude by highlighting the remaining scientific challenges, including the critical need for assembly-compatible particle-fluid systems that ultimately yield mechanically robust materials. The role of domain boundaries and/or defects emerges as an important open question to address, with recent advances in fabrication setting the stage for future work in this area.

In situ characterization of low-viscosity direct ink writing: Stability, wetting, and rotational flows.

HYPOTHESIS: Direct ink writing (DIW) of composites can be coupled with magnetic, electric, or acoustic fields to control spatial variations of microstructure that enhance performance. The use of such external fields often requires inks with lower viscosities than conventional DIW, which presents new challenges with regards to maintaining the stability of printed lines and targeted microstructures. In-situ monitoring of the print bead, combined with slot-die models, can be used to predict printing modality and guide printing protocols for low viscosity inks. EXPERIMENTS: Using videos of the nozzle-substrate gap, we systematically study how ink composition, stand-off distance, and nozzle and substrate surface coatings influence the printing process. We establish in-situ digital image analysis techniques to evaluate filament stability, nozzle wetting, and rotational flows in low viscosity composite inks. FINDINGS: Variations in the fluid-substrate contact line position and angle can be used to evaluate stability on-the-fly, and lubrication theory can be used to predict filament-to-droplet transitions. To limit nozzle wetting and disruption of microstructures established in the nozzle, it is necessary to use low flow to stage speed ratios, in regimes close to those associated with instabilities. However, low viscosities, small stand-off distances, and functionalized nozzles can improve the print line's resilience against instabilities.

An Immunocompetent Mouse Model of Zika Virus Infection.

Progress toward understanding Zika virus (ZIKV) pathogenesis is hindered by lack of immunocompetent small animal models, in part because ZIKV fails to effectively antagonize Stat2-dependent interferon (IFN) responses in mice. To address this limitation, we first passaged an African ZIKV strain (ZIKV-Dak-41525) through Rag1-/- mice to obtain a mouse-adapted virus (ZIKV-Dak-MA) that was more virulent than ZIKV-Dak-41525 in mice treated with an anti-Ifnar1 antibody. A G18R substitution in NS4B was the genetic basis for the increased replication, and resulted in decreased IFN-ß production, diminished IFN-stimulated gene expression, and the greater brain infection observed with ZIKV-Dak-MA. To generate a fully immunocompetent mouse model of ZIKV infection, human STAT2 was introduced into the mouse Stat2 locus (hSTAT2 KI). Subcutaneous inoculation of pregnant hSTAT2 KI mice with ZIKV-Dak-MA resulted in spread to the placenta and fetal brain. An immunocompetent mouse model of ZIKV infection may prove valuable for evaluating countermeasures to limit disease.

Transplantation of a quaternary structure neutralizing antibody epitope from dengue virus serotype 3 into serotype 4.

Dengue vaccine trials have revealed deficits in our understanding of the mechanisms of protective immunity, demonstrating a need to measure epitope-specific antibody responses against each DENV serotype. HmAb 5J7 binds to a complex, 3-monomer spanning quaternary epitope in the DENV3 envelope (E) protein, but it is unclear whether all interactions are needed for neutralization. Structure guided design and reverse genetics were used to sequentially transplant larger portions of the DENV3-specific 5J7 mAb epitope into dengue virus serotype 4 (DENV4). We observed complete binding and neutralization only when the entire 3 monomer spanning epitope was transplanted into DENV4, providing empirical proof that cooperative monomer-hmAb 5J7 interactions maximize activity. The rDENV4/3 virus containing the most expanded 5J7 epitope was also significantly more sensitive than WT DENV4 to neutralization by DENV3 primary immune sera. We conclude that the hinge-spanning region of the 5J7 quaternary epitope is a target for serotype-specific neutralizing antibodies after DENV3 infection.

Multilevel fluidic flow control in a rotationally-driven polyester film microdevice created using laser print, cut and laminate.

This paper presents a simple and cost-effective polyester toner microchip fabricated with laser print and cut lithography (PCL) to use with a battery-powered centrifugal platform for fluid handling. The combination of the PCL microfluidic disc and centrifugal platform: (1) allows parallel aliquoting of two different reagents of four different volumes ranging from nL to µL with an accuracy comparable to a piston-driven air pipette; (2) incorporates a reciprocating mixing unit driven by a surface-tension pump for further dilution of reagents, and (3) is amenable to larger scale integration of assay multiplexing (including all valves and mixers) without substantially increasing fabrication cost and time. For a proof of principle, a 10 min colorimetric assay for the quantitation of the protein level in the human blood plasma samples is demonstrated on chip with a limit of detection of ∼5 mg mL(-1) and coefficient of variance of ∼7%.

Simultaneous metering and dispensing of multiple reagents on a passively controlled microdevice solely by finger pressing.

In this work, we report a novel design of a passively controlled, finger-driven microfluidic circuit for the metering and delivery (MaD) of a liquid reagent. The proposed design modularized the fluidic circuit for a single reagent's MaD so that it can be multiplexed conveniently for the MaD of an arbitrary number of reagents solely by finger pressing. The microdevice has comparable accuracy with pipettes and we have demonstrated its applicability in the preparation of biochemical assays. The proposed design of the modularized, structurally "stackable" fluidic circuit provides a reference in designing future single-pressure-source-driven, passively controlled multi-liquid handling microfluidic platforms.

Detachment of compliant films adhered to stiff substrates via van der Waals interactions: role of frictional sliding during peeling.

The remarkable ability of some plants and animals to cling strongly to substrates despite relatively weak interfacial bonds has important implications for the development of synthetic adhesives. Here, we examine the origins of large detachment forces using a thin elastomer tape adhered to a glass slide via van der Waals interactions, which serves as a model system for geckos, mussels and ivy. The forces required for peeling of the tape are shown to be a strong function of the angle of peeling, which is a consequence of frictional sliding at the edge of attachment that serves to dissipate energy that would otherwise drive detachment. Experiments and theory demonstrate that proper accounting for frictional sliding leads to an inferred work of adhesion of only approximately 0.5 J m(-2) (defined for purely normal separations) for all load orientations. This starkly contrasts with the interface energies inferred using conventional interface fracture models that assume pure sticking behaviour, which are considerably larger and shown to depend not only on the mode-mixity, but also on the magnitude of the mode-I stress intensity factor. The implications for developing frameworks to predict detachment forces in the presence of interface sliding are briefly discussed.

Flow switching in microfluidic networks using passive features and frequency tuning.

Manipulating fluids in microchips remains a persistent challenge in the development of inexpensive and portable point-of-care diagnostic tools. Flow in microfluidic chips can be controlled via frequency tuning, wherein the excitation frequency of a pressure source is matched with the characteristic frequencies of network branches. The characteristic frequencies of each branch arise from coupling between fluid in the channels and passive deformable features, and can be programmed by adjusting the dimensions and stiffness of the features. In contrast to quasi-static 'on-off' valves, such networks require only a single active element and relatively small dynamic displacements. To achieve effective flow switching between different pathways in the chip, well-separated peak frequencies and narrow bandwidths are required (such that branches are independently addressable). This paper illustrates that high selectivity can be achieved in fluidic networks that exploit fluidic inertia, with flow driven selectively at peak frequencies between ~1-100 Hz with bandwidths less than ~25% of the peak frequency. Precise frequency-based flow switching between two on-chip microchannels is demonstrated. A simple theoretical framework is presented that predicts the characteristic frequencies in terms of feature properties, thus facilitating the design of networks with specific activation frequencies. The approach provides a clear pathway to simplification and miniaturization of flow-control hardware for microchips with several fluidic domains.

Rapid patterning of 'tunable' hydrophobic valves on disposable microchips by laser printer lithography.

We recently defined a method for fabricating multilayer microdevices using poly(ethylene terephthalate) transparency film and printer toner, and showed these could be successfully applied to DNA extraction and amplification (Duarte et al., Anal. Chem. 2011, 83, 5182-5189). Here, we advance the functionality of these microdevices with flow control enabled by hydrophobic valves patterned using laser printer lithography. Laser printer patterning of toner within the microchannel induces a dramatic change in surface hydrophobicity (change in contact angle of DI water from 51° to 111°) with good reproducibility. Moreover, the hydrophobicity of the surface can be controlled by altering the density of the patterned toner via varying the gray-scale setting on the laser printer, which consequently tunes the valve's burst pressure. Toner density provided a larger burst pressure bandwidth (158 ± 18 Pa to 573 ± 16 Pa) than could be achieved by varying channel geometry (492 ± 18 Pa to 573 ± 16 Pa). Finally, we used a series of tuned toner valves (with varied gray-scale) for passive valve-based fluidic transfer in a predictable manner through the architecture of a rotating PeT microdevice. While an elementary demonstration, this presents the possibility for simplistic and cost-effective microdevices with valved fluid flow control to be fabricated using nothing more than a laser printer, a laser cutter and a laminator.

Crystal structure of human methyl-binding domain IV glycosylase bound to abasic DNA.

The mammalian repair protein MBD4 (methyl-CpG-binding domain IV) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC), and downstream base excision repair proteins restore a G·C pair. MBD4 is also implicated in active DNA demethylation by initiating base excision repair of G·T mispairs generated by a deaminase enzyme. The question of how mismatch glycosylases attain specificity for excising thymine from G·T, but not A·T, pairs remains largely unresolved. Here, we report a crystal structure of the glycosylase domain of human MBD4 (residues 427-580) bound to DNA containing an abasic nucleotide paired with guanine, providing a glimpse of the enzyme-product complex. The mismatched guanine remains intrahelical, nestled into a recognition pocket. MBD4 provides selective interactions with the mismatched guanine (N1H, N2H(2)) that are not compatible with adenine, which likely confer mismatch specificity. The structure reveals no interactions that would be expected to provide the MBD4 glycosylase domain with specificity for acting at CpG sites. Accordingly, we find modest 1.5- to 2.7-fold reductions in G·T activity upon altering the CpG context. In contrast, 37- to 580-fold effects were observed previously for thymine DNA glycosylase. These findings suggest that specificity of MBD4 for acting at CpG sites depends largely on its methyl-CpG-binding domain, which binds preferably to G·T mispairs in a methylated CpG site. MBD4 glycosylase cannot excise 5-formylcytosine (fC) or 5-carboxylcytosine (caC), intermediates in a Tet (ten eleven translocation)-initiated DNA demethylation pathway. Our structure suggests that MBD4 does not provide the electrostatic interactions needed to excise these oxidized forms of mC.

New detection modality for label-free quantification of DNA in biological samples via superparamagnetic bead aggregation.

Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, enabling label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without prior DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques.