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Scaffold proteins link signaling molecules into linear pathways by physically assembling them into complexes. Scaffolds may also have a higher-order role as signal-processing hubs, serving as the target of feedback loops that optimize signaling amplitude and timing. We demonstrate that the Ste5 scaffold protein can be used as a platform to systematically reshape output of the yeast mating MAP kinase pathway. We constructed synthetic positive- and negative-feedback loops by dynamically regulating recruitment of pathway modulators to an artificial binding site on Ste5. These engineered circuits yielded diverse behaviors: ultrasensitive dose response, accelerated or delayed response times, and tunable adaptation. Protein scaffolds provide a flexible platform for reprogramming cellular responses and could be exploited to engineer cells with novel therapeutic and biotechnological functions.

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.

Although the β-rich self-assemblies are a major structural class for polypeptides and the focus of intense research, little is known about their atomic structures and dynamics due to their insoluble and noncrystalline nature. We developed a protein engineering strategy that captures a self-assembly segment in a water-soluble molecule. A predefined number of self-assembling peptide units are linked, and the β-sheet ends are capped to prevent aggregation, which yields a mono-dispersed soluble protein. We tested this strategy by using Borrelia outer surface protein (OspA) whose single-layer β-sheet located between two globular domains consists of two β-hairpin units and thus can be considered as a prototype of self-assembly. We constructed self-assembly mimics of different sizes and determined their atomic structures using x-ray crystallography and NMR spectroscopy. Highly regular β-sheet geometries were maintained in these structures, and peptide units had a nearly identical conformation, supporting the concept that a peptide in the regular β-geometry is primed for self-assembly. However, we found small but significant differences in the relative orientation between adjacent peptide units in terms of β-sheet twist and bend, suggesting their inherent flexibility. Modeling shows how this conformational diversity, when propagated over a large number of peptide units, can lead to a substantial degree of nanoscale polymorphism of self-assemblies.

Many cells can sense and respond to time-varying stimuli, selectively triggering changes in cell fate only in response to inputs of a particular duration or frequency. A common motif in dynamically controlled cells is a dual-timescale regulatory network: although long-term fate decisions are ultimately controlled by a slow-timescale switch (e.g., gene expression), input signals are first processed by a fast-timescale signaling layer, which is hypothesized to filter what dynamic information is efficiently relayed downstream. Directly testing the design principles of how dual-timescale circuits control dynamic sensing, however, has been challenging, because most synthetic biology methods have focused solely on rewiring transcriptional circuits, which operate at a single slow timescale. Here, we report the development of a modular approach for flexibly engineering phosphorylation circuits using designed phospho-regulon motifs. By then linking rapid phospho-feedback with slower downstream transcription-based bistable switches, we can construct synthetic dual-timescale circuits in yeast in which the triggering dynamics and the end-state properties of the ON state can be selectively tuned. These phospho-regulon tools thus open up the possibility to engineer cells with customized dynamical control.

The energy landscape of a protein is defined as a hyper-dimensional surface constituted by all possible conformations with associated free energies, where the global minimum corresponds to the native structure, and local minima and maxima respectively to folding intermediates and transition states. The energy landscape determines a protein's folding and dynamic behaviors, and thus function. My research focuses on understanding general properties of the energy landscape and their sequence determinants. I used Borrelia OspA as a model system. This protein contains two globular domains connected with a unique single-layer β-sheet, providing a relatively simplified system to study complex folding. First, I developed a method based on amide hydrogen exchange to determine both thermodynamics and kinetics for the formation of multiple folding intermediates. With this method, I dissected OspA into five folding units, identified and characterized four folding intermediates, capturing the global features of the OspA energy landscape. Second, I applied systematic mutagenesis coupled with a novel Brønsted-like analysis to quantify conformational heterogeneity in the energy landscape. This method also addressed the inherent ambiguity of the Φ-value analysis, the most commonly used approach to characterize folding transition states. I resolved a previously identified, apparently single intermediate into two distinct species, and demonstrated that fractional Φ-values, which are often observed in Φ-value analysis, more likely result from conformational heterogeneity than from partial structure formation as generally presumed.

Designing a multifunctional separator with abundant ion migration paths is crucial for tuning the ion transport in rocking‐chair‐type batteries. Herein, a polydopamine‐functionalized PVDF (PVDF@PDA) nanofibrous membrane is designed to serve as a separator for aqueous zinc‐ion batteries (AZIBs). The functional groups (OH and NH) in PDA facilitate the formation of ZnO and ZnN coordination bonds with Zn ions, homogenizing the Zn‐ion flux and thus enabling dendrite‐free Zn deposition. Moreover, the PVDF@PDA separator effectively inhibits the shuttling of V‐species through the formation of VO coordination bonds. As a result, the Zn/NH4V4O10 battery with the PVDF@PDA separator exhibits enhanced cycling stability (92.3% after 1000 cycles at 5 A g−1) and rate capability compared to that using a glass fiber separator. This work provides a new avenue to design functionalized separators for high‐performance AZIBs. A polydopamine‐functionalized PVDF (PVDF@PDA) nanofibrous membrane is designed as a separator for aqueous zinc‐ion batteries. The PVDF@PDA separator homogenizes the Zn‐ion flux distribution to achieve the dendrite‐free Zn deposition via the metal‐PDA coordination chemistry. Moreover, the PVDF@PDA separator inhibits the shuttle of V‐species. Benefiting from the separator, the Zn/NH4V4O10 full cell retains 92.3% capacity after 1000 cycles at 5 A g−1.

To obtain high joint strength, friction stir lap welding (FSLW) was used to join 6061-T6 aluminum alloy and Ti-6Al-4V alloy using low rotating speeds. The effect of tool rotating speed on the microstructure and lap shear failure loads of the joints was mainly discussed. Results showed that voids were easily observed at the lap interface when small penetration was used. The void was eliminated by decreasing the rotating speed. The hook acted as a “lock” during the lap shear test. At the inner side of hook, metallurgical bonding was formed due to the formation of intermetallic compounds. The joint failed through Al alloy and showed higher lap shear failure loads than other joints when the rotating speed of 300 rpm was used.

Hook and cold lap are the key factors which can affect the friction stir lap welding (FSLW) joint performance. Pin length has also an important effect on the morphologies of the hook and cold lap. Joints of AA7075 and AA2024 which are frequently used in aircraft structural elements mainly adopt lap joint configuration. The effect of pin length on dissimilar AA7075 and AA2024 lap joints has not been reported. Current paper intends to provide some further insights on this subject by discussing the relations among pin length, welding speed, and the corresponding tensile shear strength. It is interesting to find that the joint with high strength can be obtained when the harder material is placed as the upper sheet.

Gravitational lensing distorts post-dark age Universe A measure of the star-forming activity that took place during the epoch of reionization — when the Universe was emerging from a dark age and neutral hydrogen was being reionized — can be obtained from the number counts of high-redshift galaxy candidates at redshifts z >∼7. Gravitational lensing by galaxies between these distant objects and us can complicate matters, however. A new theoretical modelling study suggests that gravitational lensing is likely to dominate the observed properties of galaxies with redshifts of z >∼12, where the instrumental limiting magnitude is expected to be brighter than the characteristic magnitude of the galaxy sample. This factor could alter number counts by an order of magnitude. Future surveys will therefore need to account for a significant gravitational lensing bias in high-redshift galaxy samples. The observed number counts of high-redshift galaxy candidates have been used to build up a statistical description of star-forming activity at redshift of about z>7. Here it is reported that gravitational lensing is likely to dominate the observed properties of galaxies with redshifts of about z>12, when the instrumental limiting magnitude is expected to be brighter than the characteristic magnitude of the galaxy sample. The number counts could be modified by an order of magnitude. Future surveys will need to be designed to account for a significant gravitational lensing bias in high-redshift galaxy samples. The observed number counts of high-redshift galaxy candidates 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 have been used to build up a statistical description of star-forming activity at redshift z  ≳ 7, when galaxies reionized the Universe 1 , 2 , 9 , 10 . Standard models 11 predict that a high incidence of gravitational lensing will probably distort measurements of flux and number of these earliest galaxies. The raw probability of this happening has been estimated to be ∼0.5 per cent (refs 11 , 12 ), but can be larger owing to observational biases. Here we report that gravitational lensing is likely to dominate the observed properties of galaxies with redshifts of z  ≳ 12, when the instrumental limiting magnitude is expected to be brighter than the characteristic magnitude of the galaxy sample. The number counts could be modified by an order of magnitude, with most galaxies being part of multiply imaged systems, located less than 1 arcsec from brighter foreground galaxies at z  ≈ 2. This lens-induced association of high-redshift and foreground galaxies has perhaps already been observed among a sample of galaxy candidates identified at z  ≈ 10.6. Future surveys will need to be designed to account for a significant gravitational lensing bias in high-redshift galaxy samples.

Background. A Sabin strain–based inactivated poliomyelitis vaccine (Sabin-IPV) is the rational option for completely eradicating poliovirus transmission. The neutralizing capacity of Sabin-IPV immune serum to different strains of poliovirus is a key indicator of the clinical protective efficacy of this vaccine. Methods. Sera collected from 500 infants enrolled in a randomized, blinded, positive control, phase 2 clinical trial were randomly divided into 5 groups: Groups A, B, and C received high, medium, and low doses, respectively, of Sabin-IPV, while groups D and E received trivalent oral polio vaccine and Salk strain–based IPV, respectively, all on the same schedule. Immune sera were collected after the third dose of primary immunization, and tested in cross-neutralization assays against 19 poliovirus strains of all 3 types. Results. All immune sera from all 5 groups interacted with the 19 poliovirus strains with various titers and in a dose-dependent manner. One type 2 immunodeficiency-associated vaccine-derived poliovirus strain was not recognized by these immune sera. Conclusions. Sabin-IPV vaccine can induce protective antibodies against currently circulating and reference wild poliovirus strains and most vaccine-derived poliovirus strains, with rare exceptions. Clinical Trials Registration. NCT01056705