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Reliable sample delivery is essential to biological imaging using X-ray Free Electron Lasers (XFELs). Continuous injection using the Gas Dynamic Virtual Nozzle (GDVN) has proven valuable, particularly for time-resolved studies. However, many important aspects of GDVN functionality have yet to be thoroughly understood and/or refined due to fabrication limitations. We report the application of 2-photon polymerization as a form of high-resolution 3D printing to fabricate high-fidelity GDVNs with submicron resolution. This technique allows rapid prototyping of a wide range of different types of nozzles from standard CAD drawings and optimization of crucial dimensions for optimal performance. Three nozzles were tested with pure water to determine general nozzle performance and reproducibility, with nearly reproducible off-axis jetting being the result. X-ray tomography and index matching were successfully used to evaluate the interior nozzle structures and identify the cause of off-axis jetting. Subsequent refinements to fabrication resulted in straight jetting. A performance test of printed nozzles at an XFEL provided high quality femtosecond diffraction patterns.

The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural, and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. In this study, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axoneme's ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ∼2.3 × 10(5) ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights, to our knowledge, into the beating mechanism of flagella and a powerful tool for future studies., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Subcellular localization studies and life cell imaging approaches usually benefit from fusion-reporter proteins, such as enhanced green fluorescent protein (EGFP) and mCherry to the proteins of interest. However, such manipulations have several risks, including protein misfolding, altered protein shuttling, or functional impairment when compared to the wild-type proteins. Here, we demonstrate altered subcellular distribution and function of the pro-inflammatory enzyme procaspase-1 as a result of fusion with the reporter protein mCherry. Our observations are of central importance to further investigations of subcellular behavior and possible protein-protein interactions of naturally occurring genetic variants of human procaspase-1 which have recently been linked to autoinflammatory disorders., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Caspase-1 is a proinflammatory enzyme that is essential in many inflammatory conditions including infectious, autoimmune, and autoinflammatory disorders. The inflammation is mainly mediated by the generation of inflammasomes that activate caspase-1 and subsequently interleukin (IL)-1β and IL-18. In addition, homotypic CARD/CARD interaction of procaspase-1 with RIP2 and thereby activation of the NF-κB pathways may play some role in the inflammation. However, normally, this pathway seems to become downregulated rapidly by the cleavage and excretion of RIP2 by active (pro-)caspase-1. In patients with unexplained recurrent systemic inflammation, CASP1 variants were detected, which often destabilized the caspase-1 dimer interface. Obviously, the resulting decreased or abrogated enzymatic activity and IL-1β production did not prevent the febrile episodes. As an unexpected finding, the inactive procaspase-1 variants significantly enhanced proinflammatory signaling by increasing RIP2 mediated NF-κB activation in an in vitro cell transfection model. A likely reason is the failure of inactive procaspase-1 to cleave bound RIP2 and also to mediate its excretion out of the intracelluar space thereby keeping the RIP2-NF-κB pathway upregulated. Hence, proinflammatory effects of enzymatically inactive procaspase-1 variants may partially explain the inflammatory episodes of the patients.

Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.

We visualize birefringence in microliter sample volumes using a microfluidic dialysis chip optimized for cross polarization microscopy. The chip is composed of two overlapping polydimethylsiloxane (PDMS) channels separated by a commercial cellulose ester membrane. Buffer exchange in the sample chamber is achieved within minutes by dialyzing under continuous reservoir flow. Using fd virus as a birefringent model system, we monitor the fd virus isotropic to liquid crystal phase transition as a function of ionic strength. We show that the reorientation of the fd virus spans a few tens of seconds, indicative of fast ion exchange across the membrane. Complete phase separation reorganization takes minutes to hours as it involves diffusive virus mass transport within the storage chamber.

An emulsion-based serial crystallographic technology has been developed, in which nanolitre-sized droplets of protein solution are encapsulated in oil and stabilized by surfactant. Once the first crystal in a drop is nucleated, the small volume generates a negative feedback mechanism that lowers the supersaturation. This mechanism is exploited to produce one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room-temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 Å, demonstrating the feasibility of high-throughput serial X-ray crystallography using synchrotron radiation.

The proinflammatory enzyme caspase-1 plays an important role in the innate immune system and is involved in a variety of inflammatory conditions. Rare naturally occurring human variants of the caspase-1 gene (CASP1) lead to different protein expression and structure and to decreased or absent enzymatic activity. Paradoxically, a significant number of patients with such variants suffer from febrile episodes despite decreased IL-1β production and secretion. In this study, we investigate how variant (pro)caspase-1 can possibly contribute to inflammation. In a transfection model, such variant procaspase-1 binds receptor interacting protein kinase 2 (RIP2) via Caspase activation and recruitment domain (CARD)/CARD interaction and thereby activates NF-κB, whereas wild-type procaspase-1 reduces intracellular RIP2 levels by enzymatic cleavage and release into the supernatant. We approach the protein interactions by coimmunoprecipitation and confocal microscopy and show that NF-κB activation is inhibited by anti-RIP2-short hairpin RNA and by the expression of a RIP2 CARD-only protein. In conclusion, variant procaspase-1 binds RIP2 and thereby activates NF-κB. This pathway could possibly contribute to proinflammatory signaling.

Inflammasomes are multiprotein complexes that serve as activating platforms for the enzyme caspase-1 in response to various danger signals. Active caspase-1 can cleave the precursors of the pro-inflammatory cytokines IL-1β and IL-18 and thereby activate them. Deregulation of this cascade caused by mutations in genes coding for inflammasomal components and their interaction partners can lead to severe disease. This review summarizes the contribution of deregulated inflammasomes to the field of autoinflammatory syndromes. In addition, it gives insight into currently available mouse models that are used to study and characterize the role of the inflammasome components in the pathophysiology of these diseases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Caspase-1 (Interleukin-1 Converting Enzyme, ICE) is a proinflammatory enzyme that plays pivotal roles in innate immunity and many inflammatory conditions such as periodic fever syndromes and gout. Inflammation is often mediated by enzymatic activation of interleukin (IL)-1β and IL-18. We detected seven naturally occurring human CASP1 variants with different effects on protein structure, expression, and enzymatic activity. Most mutations destabilized the caspase-1 dimer interface as revealed by crystal structure analysis and homology modeling followed by molecular dynamics simulations. All variants demonstrated decreased or absent enzymatic and IL-1β releasing activity in vitro, in a cell transfection model, and as low as 25% of normal ex vivo in a whole blood assay of samples taken from subjects with variant CASP1, a subset of whom suffered from unclassified autoinflammation. We conclude that decreased enzymatic activity of caspase-1 is compatible with normal life and does not prevent moderate and severe autoinflammation., (© 2012 Wiley Periodicals, Inc.)

Microfluidic devices can be molded easily from PDMS using soft lithography. However, the softness of the resulting microchannels makes it difficult to photolithographically pattern their surface properties, as is needed for applications such as double emulsification. We introduce a new patterning method for PDMS devices, using integrated oxygen reservoirs fabricated simultaneously with the microfluidic channels, which serve as "chemo-masks". Oxygen diffuses through the PDMS to the nearby channel segments and there inhibits functional polymer growth; by placement of the chemo-masks, we thus control the polymerization pattern. This patterning method is simple, scalable, and compatible with a variety of surface chemistries.

Objective: We present a formal approach and methodology for the analysis and generation of user interfaces, with special emphasis on human-automation interaction., Background: A conceptual approach for modeling, analyzing, and verifying the information content of user interfaces is discussed., Methods: The proposed methodology is based on two criteria: First, the interface must be correct--that is, given the interface indications and all related information (user manuals, training material, etc.), the user must be able to successfully perform the specified tasks. Second, the interface and related information must be succinct--that is, the amount of information (mode indications, mode buttons, parameter settings, etc.) presented to the user must be reduced (abstracted) to the minimum necessary., Results: A step-by-step procedure for generating the information content of the interface that is both correct and succinct is presented and then explained and illustrated via two examples., Conclusions: Every user interface is an abstract description of the underlying system. The correspondence between the abstracted information presented to the user and the underlying behavior of a given machine can be analyzed and addressed formally., Applications: The procedure for generating the information content of user interfaces can be automated, and a software tool for its implementation has been developed. Potential application areas include adaptive interface systems and customized/personalized interfaces.

This paper discusses a formal and rigorous approach to the analysis of operator interaction with machines. It addresses the acute problem of detecting design errors in human-machine interaction and focuses on verifying the correctness of the interaction in complex and automated control systems. The paper describes a systematic methodology for evaluating whether the interface provides the necessary information about the machine to enable the operator to perform a specified task successfully and unambiguously. It also addresses the adequacy of information provided to the user via training material (e.g., user manual) about the machine's behavior. The essentials of the methodology, which can be automated and applied to the verification of large systems, are illustrated by several examples and through a case study of pilot interaction with an autopilot aboard a modern commercial aircraft. The expected application of this methodology is an augmentation and enhancement, by formal verification, of human-automation interfaces.