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Fault-Tolerant Coding for State Machines

Two reliable fault-tolerant coding schemes have been proposed for state machines that are used in field-programmable gate arrays and application-specific integrated circuits to implement sequential logic functions. The schemes apply to strings of bits in state registers, which are typically implemented in practice as assemblies of flip-flop circuits. If a single-event upset (SEU, a radiation-induced change in the bit in one flip-flop) occurs in a state register, the state machine that contains the register could go into an erroneous state or could hang, by which is meant that the machine could remain in undefined states indefinitely. The proposed fault-tolerant coding schemes are intended to prevent the state machine from going into an erroneous or hang state when an SEU occurs. To ensure reliability of the state machine, the coding scheme for bits in the state register must satisfy the following criteria: 1. All possible states are defined. 2. An SEU brings the state machine to a known state. 3. There is no possibility of a hang state. 4. No false state is entered. 5. An SEU exerts no effect on the state machine. Fault-tolerant coding schemes that have been commonly used include binary encoding and "one-hot" encoding. Binary encoding is the simplest state machine encoding and satisfies criteria 1 through 3 if all possible states are defined. Binary encoding is a binary count of the state machine number in sequence; the table represents an eight-state example. In one-hot encoding, N bits are used to represent N states: All except one of the bits in a string are 0, and the position of the 1 in the string represents the state. With proper circuit design, one-hot encoding can satisfy criteria 1 through 4. Unfortunately, the requirement to use N bits to represent N states makes one-hot coding inefficient.

Naegle, Stephanie Taft↗

Toward Synthesis, Analysis, and Certification of Security Protocols

Implemented security protocols are basically pieces of software which are used to (a) authenticate the other communication partners, (b) establish a secure communication channel between them (using insecure communication media), and (c) transfer data between the communication partners in such a way that these data only available to the desired receiver, but not to anyone else. Such an implementation usually consists of the following components: the protocol-engine, which controls in which sequence the messages of the protocol are sent over the network, and which controls the assembly/disassembly and processing (e.g., decryption) of the data. the cryptographic routines to actually encrypt or decrypt the data (using given keys), and t,he interface to the operating system and to the application. For a correct working of such a security protocol, all of these components must work flawlessly. Many formal-methods based techniques for the analysis of a security protocols have been developed. They range from using specific logics (e.g.: BAN-logic [4], or higher order logics [12] to model checking [2] approaches. In each approach, the analysis tries to prove that no (or at least not a modeled intruder) can get access to secret data. Otherwise, a scenario illustrating the &tack may be produced. Despite the seeming simplicity of security protocols ("only" a few messages are sent between the protocol partners in order to ensure a secure communication), many flaws have been detected. Unfortunately, even a perfect protocol engine does not guarantee flawless working of a security protocol, as incidents show. Many break-ins and security vulnerabilities are caused by exploiting errors in the implementation of the protocol engine or the underlying operating system. Attacks using buffer-overflows are a very common class of such attacks. Errors in the implementation of exception or error handling can open up additional vulnerabilities. For example, on a website with a log-in screen: multiple tries with invalid passwords caused the expected error message (too many retries). but let the user nevertheless pass. Finally, security can be compromised by silly implementation bugs or design decisions. In a commercial VPN software, all calls to the encryption routines were incidentally replaced by stubs, probably during factory testing. The product worked nicely. and the error (an open VPN) would have gone undetected, if a team member had not inspected the low-level traffic out of curiosity. Also, the use secret proprietary encryption routines can backfire, because such algorithms often exhibit weaknesses which can be exploited easily (see e.g., DVD encoding). Summarizing, there is large number of possibilities to make errors which can compromise the security of a protocol. In today s world with short time-to-market and the use of security protocols in open and hostile networks for safety-critical applications (e.g., power or air-traffic control), such slips could lead to catastrophic situations. Thus, formal methods and automatic reasoning techniques should not be used just for the formal proof of absence of an attack, but they ought to be used to provide an end-to-end tool-supported framework for security software. With such an approach all required artifacts (code, documentation, test cases) , formal analyses, and reliable certification will be generated automatically, given a single, high level specification. By a combination of program synthesis, formal protocol analysis, certification; and proof-carrying code, this goal is within practical reach, since all the important technologies for such an approach actually exist and only need to be assembled in the right way.

Schumann, Johann↗

Light Energy Transduction in Green Sulfur Bacteria

Green sulfur bacteria (GSB) are exquisitely adapted for growth at extraordinarily low light intensities. They are important primary producers of biomass in many anoxic environments, and they contribute significantly to the biogeochemical cycling of carbon, nitrogen, and sulfur on Earth. Green bacteria more generally share the property of using chlorosomes for light-harvesting, and these unusual organelles have many unique features. These include the presence of a monolayer lipid-protein envelope, self-assembling bacteriochlorophylls (BChl) in which pigment-pigment interactions predominate, and the presence of redox components ([2Fe-2S] ferredoxins and quinones) that play a role in regulating excitation energy transfer to the type-1 homodimeric reaction centers. The reaction centers of GSB are related to Photosystem I of cyanobacteria and higher plants but also exhibit several unique structural and functional features. The long-term objectives of this research program are to understand the structure, functions, and biogenesis of the chlorosomes, reaction centers, and electron transport chains that carry out the photochemical transduction of light energy into chemical energy in the model green sulfur bacterium, Chlorobaculum (formerly Chlorobium) tepidum. Over a period of 26 years, we characterized chlorosomes in detail. We identified the proteins in the chlorosome envelopes of diverse organisms, identified the nearest neighbors of those proteins, and characterized the chlorosomes of mutant strains lacking one to five of these proteins. After the genome sequence of Cba. tepidum became available, we identified all genes encoding enzymes for BChl a , BChl c/d/e/ƒ , and Chl a biosynthesis Cba. tepidum . We additionally identified all genes encoding enzymes for carotenoid biosynthesis. By constructing a bchQRU mutant strain that produces [Et, Me]-BChl d , together with collaborators who are specialists in solid-state NMR and cryo-electron microscopy, we solved the structure of the BChls in chlorosomes. Using similar methods and chlorosomes containing [Et, Me]-BChl c , we then showed that alternative structures were possible using the same syn-anti BChl dimer. The structures were refined by introducing spectroscopic data from single-chlorosome measurements. Over the course of this project, we sequenced the genomes of approximately twenty GSB strains, which provided important information for comparative analyses. Through analyses of metagenomic data from Mushroom and Octopus Springs in Yellowstone National Park, we identified a novel chlorophototroph belonging to the phylum Acidobacteriota. We successfully isolated an axenic culture of this organism. We characterized the photosynthetic apparatus of this bacterium, named Chloracidobacterium thermophilum, in significant detail, in particular its type-1, homodimeric reaction centers. Surprisingly, these reaction centers contain three types of Chls, BChl a , Chl a , and Zn-BChl a' . We showed that a dimer of Zn-BChl a' is the primary donor and Chl a the primary acceptor of electrons in this reaction center by using advanced spectroscopic techniques. We isolated eight additional strains of Chloracidobacterium spp. from Mushroom Spring and Rupite hot springs in Bulgaria. Comparative genomes showed that these represent three species, Cab. thermophilum , Cab. aggregatum , and Cab. validum . By applying comparative genomics, genetic, biochemical biophysical and physiological approaches to study green bacteria, we produced a wealth of new information about the remarkable light-harvesting and energy transduction capabilities of these poorly characterized microorganisms.

59 BASIC BIOLOGICAL SCIENCES↗

A Comparison of Functional Models for Use in the Function-Failure Design Method

When failure analysis and prevention, guided by historical design knowledge, are coupled with product design at its conception, shorter design cycles are possible. By decreasing the design time of a product in this manner, design costs are reduced and the product will better suit the customer s needs. Prior work indicates that similar failure modes occur with products (or components) with similar functionality. To capitalize on this finding, a knowledge base of historical failure information linked to functionality is assembled for use by designers. One possible use for this knowledge base is within the Elemental Function-Failure Design Method (EFDM). This design methodology and failure analysis tool begins at conceptual design and keeps the designer cognizant of failures that are likely to occur based on the product s functionality. The EFDM offers potential improvement over current failure analysis methods, such as FMEA, FMECA, and Fault Tree Analysis, because it can be implemented hand in hand with other conceptual design steps and carried throughout a product s design cycle. These other failure analysis methods can only truly be effective after a physical design has been completed. The EFDM however is only as good as the knowledge base that it draws from, and therefore it is of utmost importance to develop a knowledge base that will be suitable for use across a wide spectrum of products. One fundamental question that arises in using the EFDM is: At what level of detail should functional descriptions of components be encoded? This paper explores two approaches to populating a knowledge base with actual failure occurrence information from Bell 206 helicopters. Functional models expressed at various levels of detail are investigated to determine the necessary detail for an applicable knowledge base that can be used by designers in both new designs as well as redesigns. High level and more detailed functional descriptions are derived for each failed component based on NTSB accident reports. To best record this data, standardized functional and failure mode vocabularies are used. Two separate function-failure knowledge bases are then created aid compared. Results indicate that encoding failure data using more detailed functional models allows for a more robust knowledge base. Interestingly however, when applying the EFDM, high level descriptions continue to produce useful results when using the knowledge base generated from the detailed functional models.

Stock, Michael E.↗

The role of CenKR in the coordination of Rhodobacter sphaeroides cell elongation and division

ABSTRACT Cell elongation and division are essential aspects of the bacterial life cycle that must be coordinated for viability and replication. The impact of misregulation of these processes is not well understood as these systems are often not amenable to traditional genetic manipulation. Recently, we reported on the CenKR two-component system (TCS) in the Gram-negative bacterium Rhodobacter sphaeroides that is genetically tractable, widely conserved in α-proteobacteria, and directly regulates the expression of components crucial for cell elongation and division, including genes encoding subunit of the Tol-Pal complex. In this work, we show that overexpression of cenK results in cell filamentation and chaining. Using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), we generated high-resolution two-dimensional (2D) images and three-dimensional (3D) volumes of the cell envelope and division septum of wild-type cells and a cenK overexpression strain finding that these morphological changes stem from defects in outer membrane (OM) and peptidoglycan (PG) constriction. By monitoring the localization of Pal, PG biosynthesis, and the bacterial cytoskeletal proteins MreB and FtsZ, we developed a model for how increased CenKR activity leads to changes in cell elongation and division. This model predicts that increased CenKR activity decreases the mobility of Pal, delaying OM constriction, and ultimately disrupting the midcell positioning of MreB and FtsZ and interfering with the spatial regulation of PG synthesis and remodeling. IMPORTANCE By coordinating cell elongation and division, bacteria maintain their shape, support critical envelope functions, and orchestrate division. Regulatory and assembly systems have been implicated in these processes in some well-studied Gram-negative bacteria. However, we lack information on these processes and their conservation across the bacterial phylogeny. In R. sphaeroides and other α-proteobacteria, CenKR is an essential two-component system (TCS) that regulates the expression of genes known or predicted to function in cell envelope biosynthesis, elongation, and/or division. Here, we leverage unique features of CenKR to understand how increasing its activity impacts cell elongation/division and use antibiotics to identify how modulating the activity of this TCS leads to changes in cell morphology. Our results provide new insight into how CenKR activity controls the structure and function of the bacterial envelope, the localization of cell elongation and division machinery, and cellular processes in organisms with importance in health, host-microbe interactions, and biotechnology.

59 BASIC BIOLOGICAL SCIENCES↗

T-Slide Linear Actuators

T-slide linear actuators use gear bearing differential epicyclical transmissions (GBDETs) to directly drive a linear rack, which, in turn, performs the actuation. Conventional systems use a rotary power source in conjunction with a nut and screw to provide linear motion. Non-back-drive properties of GBDETs make the new actuator more direct and simpler. Versions of this approach will serve as a long-stroke, ultra-precision, position actuator for NASA science instruments, and as a rugged, linear actuator for NASA deployment duties. The T slide can operate effectively in the presence of side forces and torques. Versions of the actuator can perform ultra-precision positioning. A basic T-slide actuator is a long-stroke, rack-and-pinion linear actuator that, typically, consists of a T-slide, several idlers, a transmission to drive the slide (powered by an electric motor) and a housing that holds the entire assembly. The actuator is driven by gear action on its top surface, and is guided and constrained by gear-bearing idlers on its other two parallel surfaces. The geometry, implemented with gear-bearing technology, is particularly effective. An electronic motor operating through a GBDET can directly drive the T slide against large loads, as a rack and pinion linear actuator, with no break and no danger of back driving. The actuator drives the slide into position and stops. The slide holes position with power off and no brake, regardless of load. With the T slide configuration, this GBDET has an entire T-gear surface on which to operate. The GB idlers coupling the other two T slide parallel surfaces to their housing counterpart surfaces provide constraints in five degrees-of-freedom and rolling friction in the direction of actuation. Multiple GB idlers provide roller bearing strength sufficient to support efficient, rolling friction movement, even in the presence of large, resisting forces. T-slide actuators can be controlled using the combination of an off-the-shelf, electric servomotor, a motor angle resolution sensor (typically an encoder or resolver), and microprocessor-based intelligent software. In applications requiring precision positioning, it may be necessary to add strain gauges to the T-slide housing. Existing sensory- interactive motion control art will work for T slides. For open-loop positioning, a stepping motor emulation technique can be used.

Vranish, John↗

Entropy production fluctuations encode collective behavior in active matter

We derive a general lower bound on distributions of entropy production in interacting active matter systems. The bound is tight in the limit that interparticle correlations are small and short-ranged, which we explore in four canonical active matter models. In all models studied, the bound is weak where collective fluctuations result in long-ranged correlations, which subsequently links the locations of phase transitions to enhanced entropy production fluctuations. We develop a theory for the onset of enhanced fluctuations and relate it to specific phase transitions in active Brownian particles. Furthermore, we also derive optimal control forces that realize the dynamics necessary to tune dissipation and manipulate the system between phases. In so doing, we uncover a general relationship between entropy production and pattern formation in active matter, as well as ways of controlling it.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

Major Revisions in Arthropod Phylogeny Through Improved Supermatrix, With Support for Two Possible Waves of Land Invasion by Chelicerates

Deep phylogeny involving arthropod lineages is difficult to recover because the erosion of phylogenetic signals over time leads to unreliable multiple sequence alignment (MSA) and subsequent phylogenetic reconstruction. One way to alleviate the problem is to assemble a large number of gene sequences to compensate for the weakness in each individual gene. Such an approach has led to many robustly supported but contradictory phylogenies. A close examination shows that the supermatrix approach often suffers from two shortcomings. The first is that MSA is rarely checked for reliability and, as will be illustrated, can be poor. The second is that, to alleviate the problem of homoplasy at the third codon position of protein-coding genes due to convergent evolution of nucleotide frequencies, phylogeneticists may remove or degenerate the third codon position but may do it improperly and introduce new biases. We performed extensive reanalysis of one of such “big data” sets to highlight these two problems, and demonstrated the power and benefits of correcting or alleviating these problems. Our results support a new group with Xiphosura and Arachnopulmonata (Tetrapulmonata + Scorpiones) as sister taxa. This favors a new hypothesis in which the ancestor of Xiphosura and the extinct Eurypterida (sea scorpions, of which many later forms lived in brackish or freshwater) returned to the sea after the initial chelicerate invasion of land. Our phylogeny is supported even with the original data but processed with a new “principled” codon degeneration. We also show that removing the 1673 codon sites with both AGN and UCN codons (encoding serine) in our alignment can partially reconcile discrepancies between nucleotide-based and AA-based tree, partly because two sequences, one with AGN and the other with UCN, would be identical at the amino acid level but quite different at the nucleotide level.

59 BASIC BIOLOGICAL SCIENCES↗

Functional in vitro diversity of an intrinsically disordered plant protein during freeze–thawing is encoded by its structural plasticity

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix–helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.

59 BASIC BIOLOGICAL SCIENCES↗

Photon Counting Using Edge-Detection Algorithm

New applications such as high-datarate, photon-starved, free-space optical communications require photon counting at flux rates into gigaphoton-per-second regimes coupled with subnanosecond timing accuracy. Current single-photon detectors that are capable of handling such operating conditions are designed in an array format and produce output pulses that span multiple sample times. In order to discern one pulse from another and not to overcount the number of incoming photons, a detection algorithm must be applied to the sampled detector output pulses. As flux rates increase, the ability to implement such a detection algorithm becomes difficult within a digital processor that may reside within a field-programmable gate array (FPGA). Systems have been developed and implemented to both characterize gigahertz bandwidth single-photon detectors, as well as process photon count signals at rates into gigaphotons per second in order to implement communications links at SCPPM (serial concatenated pulse position modulation) encoded data rates exceeding 100 megabits per second with efficiencies greater than two bits per detected photon. A hardware edge-detection algorithm and corresponding signal combining and deserialization hardware were developed to meet these requirements at sample rates up to 10 GHz. The photon discriminator deserializer hardware board accepts four inputs, which allows for the ability to take inputs from a quadphoton counting detector, to support requirements for optical tracking with a reduced number of hardware components. The four inputs are hardware leading-edge detected independently. After leading-edge detection, the resultant samples are ORed together prior to deserialization. The deserialization is performed to reduce the rate at which data is passed to a digital signal processor, perhaps residing within an FPGA. The hardware implements four separate analog inputs that are connected through RF connectors. Each analog input is fed to a high-speed 1-bit comparator, which digitizes the input referenced to an adjustable threshold value. This results in four independent serial sample streams of binary 1s and 0s, which are ORed together at rates up to 10 GHz. This single serial stream is then deserialized by a factor of 16 to create 16 signal lines at a rate of 622.5 MHz or lower for input to a high-speed digital processor assembly. The new design and corresponding hardware can be employed with a quad-photon counting detector capable of handling photon rates on the order of multi-gigaphotons per second, whereas prior art was only capable of handling a single input at 1/4 the flux rate. Additionally, the hardware edge-detection algorithm has provided the ability to process 3-10 higher photon flux rates than previously possible by removing the limitation that photoncounting detector output pulses on multiple channels being ORed not overlap. Now, only the leading edges of the pulses are required to not overlap. This new photon counting digitizer hardware architecture supports a universal front end for an optical communications receiver operating at data rates from kilobits to over one gigabit per second to meet increased mission data volume requirements.

Gin, Jonathan W.↗

Preliminary results on noncollocated torque control of space robot actuators

In the Space Station era, more operations will be performed robotically in space in the areas of servicing, assembly, and experiment tending among others. These robots may have various sets of requirements for accuracy, speed, and force generation, but there will be design constraints such as size, mass, and power dissipation limits. For actuation, a leading motor candidate is a dc brushless type, and there are numerous potential drive trains each with its own advantages and disadvantages. This experiment uses a harmonic drive and addresses some inherent limitations, namely its backdriveability and low frequency structural resonances. These effects are controlled and diminished by instrumenting the actuator system with a torque transducer on the output shaft. This noncollocated loop is closed to ensure that the commanded torque is accurately delivered to the manipulator link. The actuator system is modelled and its essential parameters identified. The nonlinear model for simulations will include inertias, gearing, stiction, flexibility, and the effects of output load variations. A linear model is extracted and used for designing the noncollocated torque and position feedback loops. These loops are simulated with the structural frequency encountered in the testbed system. Simulation results are given for various commands in position. The use of torque feedback is demonstrated to yield superior performance in settling time and positioning accuracy. An experimental setup being finished consists of a bench mounted motor and harmonic drive actuator system. A torque transducer and two position encoders, each with sufficient resolution and bandwidth, will provide sensory information. Parameters of the physical system are being identified and matched to analytical predictions. Initial feedback control laws will be incorporated in the bench test equipment and various experiments run to validate the designs. The status of these experiments is given.

Tilley, Scott W.↗

3D domain swapping in the TIM barrel of the α subunit of Streptococcus pneumoniae tryptophan synthase

Tryptophan synthase catalyzes the last two steps of tryptophan biosynthesis in plants, fungi and bacteria. It consists of two protein chains, designated α and β, encoded by trpA and trpB genes, that function as an αββα complex. Structural and functional features of tryptophan synthase have been extensively studied, explaining the roles of individual residues in the two active sites in catalysis and allosteric regulation. TrpA serves as a model for protein-folding studies. In 1969, Jackson and Yanofsky observed that the typically monomeric TrpA forms a small population of dimers. Dimerization was postulated to take place through an exchange of structural elements of the monomeric chains, a phenomenon later termed 3D domain swapping. The structural details of the TrpA dimer have remained unknown. Here, the crystal structure of the Streptococcus pneumoniae TrpA homodimer is reported, demonstrating 3D domain swapping in a TIM-barrel fold for the first time. The N-terminal domain comprising the H0–S1–H1–S2 elements is exchanged, while the hinge region corresponds to loop L2 linking strand S2 to helix H2′. The structural elements S2 and L2 carry the catalytic residues Glu52 and Asp63. As the S2 element is part of the swapped domain, the architecture of the catalytic apparatus in the dimer is recreated from two protein chains. The homodimer interface overlaps with the α–β interface of the tryptophan synthase αββα heterotetramer, suggesting that the 3D domain-swapped dimer cannot form a complex with the β subunit. In the crystal, the dimers assemble into a decamer comprising two pentameric rings.

3D domain swapping↗

Biallelic variants in SNUPN cause a limb girdle muscular dystrophy with myofibrillar-like features

Abstract Alterations in RNA-splicing are a molecular hallmark of several neurological diseases, including muscular dystrophies, where mutations in genes involved in RNA metabolism or characterized by alterations in RNA splicing have been described. Here, we present five patients from two unrelated families with a limb-girdle muscular dystrophy (LGMD) phenotype carrying a biallelic variant in SNUPN gene. Snurportin-1, the protein encoded by SNUPN, plays an important role in the nuclear transport of small nuclear ribonucleoproteins (snRNPs), essential components of the spliceosome. We combine deep phenotyping, including clinical features, histopathology and muscle MRI, with functional studies in patient-derived cells and muscle biopsies to demonstrate that variants in SNUPN are the cause of a new type of LGMD according to current definition. Moreover, an in vivo model in Drosophila melanogaster further supports the relevance of Snurportin-1 in muscle. SNUPN patients show a similar phenotype characterized by proximal weakness starting in childhood, restrictive respiratory dysfunction and prominent contractures, although inter-individual variability in terms of severity even in individuals from the same family was found. Muscle biopsy showed myofibrillar-like features consisting of myotilin deposits and Z-disc disorganization. MRI showed predominant impairment of paravertebral, vasti, sartorius, gracilis, peroneal and medial gastrocnemius muscles. Conservation and structural analyses of Snurportin-1 p.Ile309Ser variant suggest an effect in nuclear-cytosol snRNP trafficking. In patient-derived fibroblasts and muscle, cytoplasmic accumulation of snRNP components is observed, while total expression of Snurportin-1 and snRNPs remains unchanged, which demonstrates a functional impact of SNUPN variant in snRNP metabolism. Furthermore, RNA-splicing analysis in patients’ muscle showed widespread splicing deregulation, in particular in genes relevant for muscle development and splicing factors that participate in the early steps of spliceosome assembly. In conclusion, we report that SNUPN variants are a new cause of limb girdle muscular dystrophy with specific clinical, histopathological and imaging features, supporting SNUPN as a new gene to be included in genetic testing of myopathies. These results further support the relevance of splicing-related proteins in muscle disorders.

Iruzubieta, Pablo (ORCID:0000000303316222)↗

Unraveling the mechanism of recognition of the 3’ splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3’). We found that the PUF60–AdML3’ dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60’s two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3’ revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF60 2 :AdML3’) stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3’ splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3’ splice sites.

59 BASIC BIOLOGICAL SCIENCES↗

Scale-free, programmable design of morphable chain loops of kilobots and colloidal motors

Micron-scale robots require systems that can morph into arbitrary target configurations controlled by external agents such as heat, light, electricity, and chemical environment. Achieving this behavior using conventional approaches is challenging because the available materials at these scales are not programmable like their macroscopic counterparts. To overcome this challenge, we propose a design strategy to make a robotic machine that is both programmable and compatible with colloidal-scale physics. Our strategy uses motors in the form of active colloidal particles that constantly propel forward. We sequence these motors end-to-end in a closed chain forming a two-dimensional loop that folds under its mechanical constraints. We encode the target loop shape and its motion by regulating six design parameters, each scale-invariant and achievable at the colloidal scale. We demonstrate the plausibility of our design strategy using centimeter-scale robots called kilobots . We use Brownian dynamics simulation to explore the large design space beyond that possible with kilobots, and present an analytical theory to aid the design process. Multiple loops can also be fused together to achieve several complex shapes and robotic behaviors, demonstrated by folding a letter shape “M,” a dynamic gripper , and a dynamic pacman . The material-agnostic, scale-free, and programmable nature of our design enables building a variety of reconfigurable and autonomous robots at both colloidal scales and macroscales.

36 MATERIALS SCIENCE↗

Small Ion Channel Linking Molecular Simulations and Electrophysiology

Ion channels are pore-forming protein assemblies that mediate the transport of small ions across cell membranes. Otherwise, membrane bilayers would be almost impermeable to ions incapable to traverse the low dielectric constant, hydrophobic membrane core. Ion channels are ubiquitous to all life forms. In humans and other higher organisms they play the central role in conducting nerve impulses, cardiac functions, muscle contraction and apoptosis. On the other extreme of biological complexity, viral ion channels (viroporins) influence many stages of the virus infection cycle either through regulating virus replication, such as entry, assembly and release or modulating the electrochemical balance in the subcellular compartments of host cells. Ion channels were crucial components of protocells. Their emergence facilitated adaptation of nascent life to different environmental conditions. The earliest ion channels must have been much simpler than most of their modern ancestors. Viral channels are among only a few naturally occurring models to study the structure, function and evolution of primordial channels. Experimental studies of these properties are difficult and often unreliable. In principle, computational methods, and molecular dynamics (MD) simulations in particular, can aid in providing information about both the structure and the function of ion channels. However, MD suffers from its own problems, such as inability to access sufficiently long time scales or limited accuracy of force fields. It is, therefore, essential to determine the reliability of MD simulations. We propose to do so on the basis of two criteria. One is channel stability on time scales that extend for several microseconds or longer. The other is the ability to reproduce the measured ionic conductance as a function of applied voltage. If both the stability and the calculated ionic conductance are satisfactory it will greatly increase our confidence that the structure and the function of a channel are described sufficiently accurately. To our knowledge, long time scale stability (approx.10 micro-sec) and the correct electrophysiology have been shown so far for only one channel - the synthetic LS3 hexamer). In this presentation, this approach will be discussed in application to two viral channels - Vpu, encoded by the HIV-1 genome and p7 of hepatitis C.

Pohorille, Andrzej↗

Validating Phasing and Geometry of Large Focal Plane Arrays

The Kepler Mission is designed to survey our region of the Milky Way galaxy to discover hundreds of Earth-sized and smaller planets in or near the habitable zone. The Kepler photometer is an array of 42 CCDs (charge-coupled devices) in the focal plane of a 95-cm Schmidt camera onboard the Kepler spacecraft. Each 50x25-mm CCD has 2,200 x 1,024 pixels. The CCDs accumulate photons and are read out every six seconds to prevent saturation. The data is integrated for 30 minutes, and then the pixel data is transferred to onboard storage. The data is subsequently encoded and transmitted to the ground. During End-to-End Information System (EEIS) testing of the Kepler Mission System (KMS), there was a need to verify that the pixels requested by the science team operationally were correctly collected, encoded, compressed, stored, and transmitted by the FS, and subsequently received, decoded, uncompressed, and displayed by the Ground Segment (GS) without the outputs of any CCD modules being flipped, mirrored, or otherwise corrupted during the extensive FS and GS processing. This would normally be done by projecting an image on the focal plane array (FPA), collecting the data in a flight-like way, and making a comparison between the original data and the data reconstructed by the science data system. Projecting a focused image onto the FPA through the telescope would normally involve using a collimator suspended over the telescope opening. There were several problems with this approach: the collimation equipment is elaborate and expensive; as conceived, it could only illuminate a limited section of the FPA (.25 percent) during a given test; the telescope cover would have to be deployed during testing to allow the image to be projected into the telescope; the equipment was bulky and difficult to situate in temperature-controlled environments; and given all the above, test setup, execution, and repeatability were significant concerns. Instead of using this complicated approach of projecting an optical image on the FPA, the Kepler project developed a method using known defect features in the CCDs to verify proper collection and reassembly of the pixels, thereby avoiding the costs and risks of the optical projection approach. The CCDs composing the Kepler FPA, as all CCDs, had minor defects. At ambient temperature, some pixels look far brighter than they should. These ghot h pixels have a higher rate of charge leakage than the others due to manufacturing variations. They are usually stable over time, and appear at temperatures above 5 oC. The hot pixels on the Kepler FPA were mapped before photometer assembly during module testing. Selected hot pixels were used as target gstars h for the purposes of EEIS testing. gDead h pixels are permanently off, producing a permanently black pixel. These can also be used if there is some illumination of the FPA. During EEIS testing, Dark Current Full Frame Images (FFIs) taken at room temperature were used to create the hot pixel maps for all 84 Kepler photometer CCD channels. Data from two separate nights were used to create two hot pixel maps per channel, which were cross-correlated to remove cosmic ray events which appear to be hot pixels. These hot pixel maps obtained during EEIS testing were compared to the maps made during module testing to verify that the end-to-end data flow was correct.

Standley, Shaun P.↗

Symmetry-Breaking Dendrimer Synthons in Colloidal Crystal Engineering with DNA

Breaking symmetry in colloidal crystals is challenging due to the inherent chemical and structural isotropy of many nanoscale building blocks. If a non-particle component could be used to anisotropically encode such building blocks with orthogonal recognition properties, one could expand the scope of structural and compositional possibilities of colloidal crystals beyond what is thus far possible with purely particle-based systems. In this work, we report the synthesis and characterization of novel DNA dendrimers that function as symmetry-breaking synthons, capable of programming anisotropic and orthogonal interactions within colloidal crystals. When the DNA dendrimers have identical sticky ends, they hybridize with DNA-functionalized nanoparticles to yield three distinct colloidal crystals, dictated by dendrimer size, including a structure not previously reported in the field of colloidal crystal engineering, Si 2 Sr. When used as symmetry-breaking synthons (when the sticky ends deliberately consist of orthogonal sequences), the synthesis of binary and ternary colloidal alloys with structures that can only be realized through directional interactions is possible. Furthermore, by modulating the extent of shape anisotropy within the DNA dendrimers, the local distribution of the nanoparticles within the crystals can be directed.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗