Project description:To discriminate between closely related members of a protein family that differ at a limited number of spatially distant positions is a challenge for drug discovery. We describe a combined computational design and experimental selection approach for generating binders targeting functional sites with large, shape complementary interfaces to read out subtle sequence differences for subtype-specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset.
Project description:In most modern bony vertebrates, a considerable portion of the chondrocranium remains cartilaginous only during a relatively small window of embryonic development, making it difficult to study this complex structure. Yet, the transient nature of some chondrocranial elements is precisely why it is so intriguing. Since the chondrocranium has never been lost in any vertebrate, its function is critical to craniofacial development, disease, and evolution. Experimental evidence for the various roles of the chondrocranium is limited, and though snapshots of chondrocranial development in various species at isolated time points are valuable and informative, these cannot provide the data needed to determine the functions of the chondrocranium, or its relationship to the dermatocranium in evolution, in development, or in disease. Observations of the spatiotemporal associations of chondrocranial cartilage, cartilage bone, and dermal bone over early developmental time are available for many vertebrate species and these observations represent the data from which we can build hypotheses. The testing of those hypotheses requires precise control of specific variables like developmental time and molecular signaling that can only be accomplished in a laboratory setting. Here, we employ recent advances in contrast-enhanced micro computed tomography to provide novel 3D reconstructions of the embryonic chondrocranium in relation to forming dermal and cartilage bones in laboratory mice across three embryonic days (E13.5, E14.5, and E15.5). Our observations provide support for the established hypothesis that the vertebrate dermal (exo-) skeleton and endoskeleton evolved as distinct structures and remain distinct. Additionally, we identify spatiotemporal patterning in the development of the lateral wall, roof, and braincase floor of the chondrocranium and the initial mineralization and growth of the bones associated with these cartilages that provides support for the hypothesis that the chondrocranium serves as a scaffold for developing dermatocranial bones. The experimental protocols described and data presented provide tools for further experimental work on chondrocranial development.
Project description:Discovering the interaction mechanism and location of RNA binding proteins (RBPs) on RNA is critical for understanding gene expression regulation. Here, we apply selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) on in vivo transcripts to identify transcriptome-wide footprints (fSHAPE) on RNA when compared to protein-absent transcripts. Structural analyses indicate that fSHAPE precisely detects nucleotides whose base moieties hydrogen bond with protein and that fSHAPE patterns can predict binding sites of RBPs of interest. To illustrate, we demonstrate that fSHAPE correctly identifies known and novel iron response protein RNA elements. Furthermore, we enable selective interrogation of RNA-protein complexes by integrating SHAPE and fSHAPE with crosslinking and immunoprecipitation (eCLIP) of desired RBPs. To demonstrate, histone stem loop elements and their nucleotides that hydrogen bond with stem-loop binding protein were identified by SHAPE-eCLIP and fSHAPE-eCLIP. Together, these technologies greatly expand on strategies for understanding specific cellular RNA interactions in RNA-protein complexes.
Project description:Zn metal holds grand promise as the anodes of aqueous batteries for grid-scale energy storage. However, the rampant zinc dendrite growth and severe surface side reactions significantly impede the commercial implementation. Herein, a universal Zn-metal oxide Ohmic contact interface model is demonstrated for effectively improving Zn plating/stripping reversibility. The high work function difference between Zn and metal oxides enables the building of an interfacial anti-blocking layer for dendrite-free Zn deposition. Moreover, the metal oxide layer can function as a physical barrier to suppress the pernicious side reactions. Consequently, the proof-of-concept CeO2 -modified Zn anode delivers ultrastable durability of over 1300 h at 0.5-5 mA cm-2 and improved Coulombic efficiency, the feasibility of which is also evidenced in MoS2 //Zn full cells. This study enriches the fundamental comprehension of Ohmic contact interfaces on the Zn deposition, which may shed light on the development of other metal battery anodes.
Project description:The modern age of metagenomics has delivered unprecedented volumes of data describing the genetic and metabolic diversity of bacterial communities, but it has failed to provide information about coincident cellular morphologies. Much like metabolic and biosynthetic capabilities, morphology comprises a critical component of bacterial fitness, molded by natural selection into the many elaborate shapes observed across the bacterial domain. In this essay, we discuss the diversity of bacterial morphology and its implications for understanding both the mechanistic and the adaptive basis of morphogenesis. We consider how best to leverage genomic data and recent experimental developments in order to advance our understanding of bacterial shape and its functional importance.
Project description:An optoelectronic oscillator (OEO) is a microwave photonic system with a positive feedback loop used to create microwave oscillation with ultra-low phase noise thanks to the employment of a high-quality-factor energy storage element, such as a fiber delay line. For many applications, a frequency-tunable microwave signal or waveform, such as a linearly chirped microwave waveform (LCMW), is also needed. Due to the long characteristic time constant required for building up stable oscillation at an oscillation mode, it is impossible to generate an LCMW with a large chirp rate using a conventional frequency-tunable OEO. In this study, we propose and demonstrate a new scheme to generate a large chirp-rate LCMW based on Fourier domain mode locking technique to break the limitation of mode building time in an OEO. An LCMW with a high chirp rate of 0.34 GHz/μs and a large time-bandwidth product of 166,650 is demonstrated.
Project description:One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.
Project description:Current clinical note-taking approaches cannot capture the entirety of information available from patient encounters and detract from patient-clinician interactions. By surveying healthcare providers' current note-taking practices and attitudes toward new clinical technologies, we developed a patient-centered paradigm for clinical note-taking that makes use of hybrid tablet/keyboard devices and artificial intelligence (AI) technologies. PhenoPad is an intelligent clinical note-taking interface that captures free-form notes and standard phenotypic information via a variety of modalities, including speech and natural language processing techniques, handwriting recognition, and more. The output is unobtrusively presented on mobile devices to clinicians for real-time validation and can be automatically transformed into digital formats that would be compatible with integration into electronic health record systems. Semi-structured interviews and trials in clinical settings rendered positive feedback from both clinicians and patients, demonstrating that AI-enabled clinical note-taking under our design improves ease and breadth of information captured during clinical visits without compromising patient-clinician interactions. We open source a proof-of-concept implementation that can lay the foundation for broader clinical use cases.
Project description:The performance of molecular docking can be improved by comparing the shape similarity of the flexibly sampled poses against the target proteins' inverted binding cavities. The effectiveness of these pseudo-ligands or negative image-based models in docking rescoring is boosted further by performing enrichment-driven optimization. Here, we introduce a novel shape-focused pharmacophore modeling algorithm O-LAP that generates a new class of cavity-filling models by clumping together overlapping atomic content via pairwise distance graph clustering. Top-ranked poses of flexibly docked active ligands were used as the modeling input and multiple alternative clustering settings were benchmark-tested thoroughly with five demanding drug targets using random training/test divisions. In docking rescoring, the O-LAP modeling typically improved massively on the default docking enrichment; furthermore, the results indicate that the clustered models work well in rigid docking. The C+ +/Qt5-based algorithm O-LAP is released under the GNU General Public License v3.0 via GitHub ( https://github.com/jvlehtonen/overlap-toolkit ). SCIENTIFIC CONTRIBUTION: This study introduces O-LAP, a C++/Qt5-based graph clustering software for generating new type of shape-focused pharmacophore models. In the O-LAP modeling, the target protein cavity is filled with flexibly docked active ligands, the overlapping ligand atoms are clustered, and the shape/electrostatic potential of the resulting model is compared against the flexibly sampled molecular docking poses. The O-LAP modeling is shown to ensure high enrichment in both docking rescoring and rigid docking based on comprehensive benchmark-testing.
Project description:We show by molecular dynamics that amphiphilic Au nanoparticles (NP) with a diameter of 4 nm generate curvature in phosphatidylcholine lipid membranes. NPs generate negative curvature when they adsorb on the membrane surface but, as they get spontaneously and progressively embedded into the membrane core, the curvature turns positive. As membrane embedding is kinetically slow, both configurations can be observed by Cryo-EM. NP-induced curvature explains the peculiar structure of liposome-liposome interfaces in presence of NPs.