Impact of Peptide Length and Solution Conditions on Tetrameric Coiled Coil Formation.

Unlike naturally derived peptides, computationally designed sequences offer programmed self-assembly and charge display. Herein, new tetrameric, coiled coil-forming peptides were computationally designed ranging from 8 to 29 amino acids in length. Experimental investigations revealed that only the sequences having three or more heptads (i.e., 21 or more amino acids) exhibited coiled coil behavior. The shortest stable coiled coil sequence had a melting temperature (Tm) of approximately 58 ± 1 °C, making it ideal for thermoreversible assembly over moderate temperatures. Effects of pH and monovalent salt were examined, revealing structural stability over a pH range of 4 to 11 and an enhancement in Tm with the addition of salt. The incorporation of the coiled coil as a hydrogel cross-linker results in a thermally and mechanically reversible hydrogel. A subsequent demonstration of the hydrogel printed through a syringe illustrated one of many potential uses from 3D printing to injectable hydrogel drug delivery.

The LTAR-integrated grazing land common experiment at the Texas Gulf.

Extreme weather and climate events have become more frequent and directly affect the ecological structure and function of integrated grazing lands. While the Great Plains have experienced a long history of regular disturbances from drought and floods, grazing, and fires, the increased frequency and magnitude of these disturbances can reduce ecological resilience, largely depending on management practices. Alternative strategies designed to adaptively manage grazing land resources based on the ecology of the system should increase the resistance and resilience to disturbances when compared to prevailing practices. Determining the ecologic and economic value of alternative strategies will require long-term evaluations across large spatial scales. The Long-Term Agroecosystem Research Network has been established to evaluate the differences between alternative and prevailing practices among 18 strategically located sites and across decadal time scales throughout the continental United States. A key integrated grazing land site within this network is the Texas Gulf located at the Riesel Watersheds in the Blackland Prairie of Central Texas. At this study site, the differences between alternative and prevailing grazing management strategies are now being evaluated. The alternative strategy was designed using a combination of knowledge of the site and species ecology with modern-day tools and technologies. Alternatively, the prevailing practice implements a conventional year-round continuous grazing system with heavy reliance on hay and supplemental protein during winter. Results will provide grazing land managers with economically viable adaptive management choices for increasing ecological resilience following extreme and frequent disturbance events.

Genetically Fused Resilin-like Polypeptide-Coiled Coil Bundlemer Conjugates Exhibit Tunable Multistimuli-Responsiveness and Undergo Nanofibrillar Assembly.

Peptide-based materials are diverse candidates for self-assembly into modularly designed and stimuli-responsive nanostructures with precisely tunable compositions. Here, we genetically fused computationally designed coiled coil-forming peptides to the N- and C-termini of compositionally distinct multistimuli-responsive resilin-like polypeptides (RLPs) of various lengths. The successful expression of these hybrid polypeptides in bacterial hosts was confirmed through techniques such as gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism spectroscopy and ultraviolet-visible turbidimetry demonstrated that despite the fusion of disparate structural and responsive units, the coiled coils remained stable in the hybrid polypeptides, and the sequence-encoded differences in thermoresponsive phase separation of the RLPs were preserved. Cryogenic transmission electron microscopy and coarse-grained modeling showed that after thermal annealing in solution, the hybrid polypeptides adopted a closed loop conformation and assembled into nanofibrils capable of further hierarchically organizing into cluster structures and ribbon-like structures mediated by the self-association tendency of the RLPs.

Dynamic Nanocrystal Superlattices with Thermally Triggerable Lubricating Ligands.

The size-dependent and collective physical properties of nanocrystals (NCs) and their self-assembled superlattices (SLs) enable the study of mesoscale phenomena and the design of metamaterials for a broad range of applications. However, the limited mobility of NC building blocks in dried NCSLs often hampers the potential for employing postdeposition methods to produce high-quality NCSLs. In this study, we present tailored promesogenic ligands that exhibit a lubricating property akin to thermotropic liquid crystals. The lubricating ability of ligands is thermally triggerable, allowing the dry solid NC aggregates deposited on the substrates with poor ordering to be transformed into NCSLs with high crystallinity and preferred orientations. The interplay between the dynamic behavior of NCSLs and the molecular structure of the ligands is elucidated through a comprehensive analysis of their lubricating efficacy using both experimental and simulation approaches. Coarse-grained molecular dynamic modeling suggests that a shielding layer from mesogens prevents the interdigitation of ligand tails, facilitating the sliding between outer shells and consequently enhancing the mobility of NC building blocks. The dynamic organization of NCSLs can also be triggered with high spatial resolution by laser illumination. The principles, kinetics, and utility of lubricating ligands could be generalized to unlock stimuli-responsive metamaterials from NCSLs and contribute to the fabrication of NCSLs.

Preoperative and pre-chemotherapy CA-125 levels in high-risk early-stage ovarian cancer - An NRG/GOG study.

OBJECTIVES: To determine clinical significance of preoperative and pre-chemotherapy CA-125 in high-risk early-stage epithelial ovarian cancer patients. METHODS: All patients with stage IA/IB and grade 3, stage IC, clear cell, or completed resected stage II cancer were enrolled in a phase III trial and treated with chemotherapy. Kaplan-Meier method and Cox proportional hazards model were used for statistical analyses. RESULTS: 427 patients with high-risk early-stage ovarian cancer were enrolled. Of 213 patients with preoperative CA-125 data, 79% had elevated CA-125. Median preoperative CA-125 level was 103 U/mL. Patients with ≤10, 11-15, and > 15 cm tumors had median preoperative CA-125 levels of 62, 131 and 158 U/mL, respectively (p = 0.002). For the 350 patients with data for pre-chemotherapy CA-125 level, 69% had elevated pre-chemotherapy CA-125 above 35 U/mL with median value of 65 U/mL. However, age, race, stage, cell type and grade of disease were not correlated with CA-125 levels before and after surgery. On multivariate analysis, elevated pre-chemotherapy CA-125 independently predicted worse recurrence-free survival (HR = 2.13, 95% CI: 1.23-3.69; p = 0.007) and overall survival (HR = 1.99, 95% CI: 1.10-3.59; p = 0.022) after adjusting for age, stage, cell type and grade of disease. Compared to those with normal CA-125, patients with elevated pre-chemotherapy CA-125 had lower recurrence-free survival (RFS, 87% vs. 75%; p = 0.007) and overall survival (OS, 88% vs. 82%; p = 0.02). However, preoperative CA-125 was not prognostic of RFS (p = 0.699) or OS (p = 0.701). CONCLUSIONS: Preoperative CA-125 was elevated in nearly 80% of high-risk early-stage ovarian cancer patients. Pre-chemotherapy CA-125 was associated with recurrence-free and overall survival; however, preoperative CA-125 was not prognostic.

Thermoresponsive Coiled-Coil Peptide-Polymer Grafts.

Alkyl halide side groups are selectively incorporated into monodispersed, computationally designed coiled-coil-forming peptide nanoparticles. Poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) is polymerized from the coiled-coil periphery using photoinitiated atom transfer radical polymerization (photoATRP) to synthesize well-defined, thermoresponsive star copolymer architectures. This facile synthetic route is readily extended to other monomers for a range of new complex star-polymer macromolecules.

Developmental environment has lasting effects on amphibian post-metamorphic behavior and thermal physiology.

Environmental challenges early in development can result in complex phenotypic trade-offs and long-term effects on individual physiology, performance and behavior, with implications for disease and predation risk. We examined the effects of simulated pond drying and elevated water temperatures on development, growth, thermal physiology and behavior in a North American amphibian, Rana sphenocephala. Tadpoles were raised in outdoor mesocosms under warming and drying regimes based on projected climatic conditions in 2070. We predicted that amphibians experiencing the rapid pond drying and elevated pond temperatures associated with climate change would accelerate development, be smaller at metamorphosis and demonstrate long-term differences in physiology and exploratory behavior post-metamorphosis. Although both drying and warming accelerated development and reduced survival to metamorphosis, only drying resulted in smaller animals at metamorphosis. Around 1 month post-metamorphosis, animals from the control treatment jumped relatively farther at high temperatures in jumping trials. In addition, across all treatments, frogs with shorter larval periods had lower critical thermal minima and maxima. We also found that developing under warming and drying resulted in a less exploratory behavioral phenotype, and that drying resulted in higher selected temperatures in a thermal gradient. Furthermore, behavior predicted thermal preference, with less exploratory animals selecting higher temperatures. Our results underscore the multi-faceted effects of early developmental environments on behavioral and physiological phenotypes later in life. Thermal preference can influence disease risk through behavioral thermoregulation, and exploratory behavior may increase risk of predation or pathogen encounter. Thus, climatic stressors during development may mediate amphibian exposure and susceptibility to predators and pathogens into later life stages.

Corticotropin-releasing hormone neurons in the central nucleus of amygdala are required for chronic stress-induced hypertension.

AIMS: Chronic stress is a well-known risk factor for the development of hypertension. However, the underlying mechanisms remain unclear. Corticotropin-releasing hormone (CRH) neurons in the central nucleus of the amygdala (CeA) are involved in the autonomic responses to chronic stress. Here, we determined the role of CeA-CRH neurons in chronic stress-induced hypertension. METHODS AND RESULTS: Borderline hypertensive rats (BHRs) and Wistar-Kyoto (WKY) rats were subjected to chronic unpredictable stress (CUS). Firing activity and M-currents of CeA-CRH neurons were assessed, and a CRH-Cre-directed chemogenetic approach was used to suppress CeA-CRH neurons. CUS induced a sustained elevation of arterial blood pressure (ABP) and heart rate (HR) in BHRs, while in WKY rats, CUS-induced increases in ABP and HR quickly returned to baseline levels after CUS ended. CeA-CRH neurons displayed significantly higher firing activities in CUS-treated BHRs than unstressed BHRs. Selectively suppressing CeA-CRH neurons by chemogenetic approach attenuated CUS-induced hypertension and decreased elevated sympathetic outflow in CUS-treated BHRs. Also, CUS significantly decreased protein and mRNA levels of Kv7.2 and Kv7.3 channels in the CeA of BHRs. M-currents in CeA-CRH neurons were significantly decreased in CUS-treated BHRs compared with unstressed BHRs. Blocking Kv7 channel with its blocker XE-991 increased the excitability of CeA-CRH neurons in unstressed BHRs but not in CUS-treated BHRs. Microinjection of XE-991 into the CeA increased sympathetic outflow and ABP in unstressed BHRs but not in CUS-treated BHRs. CONCLUSIONS: CeA-CRH neurons are required for chronic stress-induced sustained hypertension. The hyperactivity of CeA-CRH neurons may be due to impaired Kv7 channel activity, which represents a new mechanism involved in chronic stress-induced hypertension.

Genetic Fusion of Thermoresponsive Polypeptides with UCST-type Behavior Mediates 1D Assembly of Coiled-Coil Bundlemers.

Thermoresponsive resilin-like polypeptides (RLPs) of various lengths were genetically fused to two different computationally designed coiled coil-forming peptides with distinct thermal stability, to develop new strategies to assemble coiled coil peptides via temperature-triggered phase separation of the RLP units. Their successful production in bacterial expression hosts was verified via gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism (CD) spectroscopy, ultraviolet-visible (UV/Vis) turbidimetry, and dynamic light scattering (DLS) measurements confirmed the stability of the coiled coils and showed that the thermosensitive phase behavior of the RLPs was preserved in the genetically fused hybrid polypeptides. Cryogenic-transmission electron microscopy and coarse-grained modeling revealed that functionalizing the coiled coils with thermoresponsive RLPs leads to their thermally triggered noncovalent assembly into nanofibrillar assemblies.

The clinical and prognostic significance of pre-chemotherapy serum CA-125 in high-risk early stage ovarian cancer: An NRG/GOG ancillary study.

OBJECTIVES: To determine the clinical and prognostic significance of CA-125 trends prior to, during, and after chemotherapy in high-risk early-stage epithelial ovarian cancer patients. METHODS: All patients were enrolled in a phase III randomized trial (GOG 157) following upfront surgery for grade 3 stage IA/IB, stage IC, or stage II disease, and had been treated with either three or six cycles of carboplatin/paclitaxel. Kaplan-Meier method and Cox proportional hazards model were used to evaluate recurrence-free survival (RFS) and overall survival (OS). RESULTS: Of 350 patients, the median pre-chemotherapy CA-125 was 65 (IQR: 31-129). 71% of Whites had an elevated CA-125 compared to 47% of non-Whites (p = 0.006). Following the first cycle of chemotherapy, 74% of those with elevated CA-125 had normalization. Those who had normalization of CA-125 after 1 cycle had significantly better 5-year RFS (81% vs. 65%, p = 0.003) and OS (87% vs. 75%, p = 0.009) compared to those who did not normalize (defined as ≤35 U/mL). The pattern of CA-125 change following chemotherapy cycle 1, from normal to normal vs. elevated to normal vs. elevated to elevated had corresponding RFS of 87% vs. 80% vs. 68% (p = 0.013), and OS of 92% vs. 88% vs. 77% (p = 0.009). However, the percent decline (p = 0.993) and absolute nadir normal value of CA-125 (0-10 vs. 11-35 U/mL) were not predictive of outcome (p = 0.4). CONCLUSIONS: Normal baseline CA125 and normalization of this biomarker after the first cycle of chemotherapy were associated with better survival in high-risk early-stage epithelial ovarian cancer patients.

Mutations of the DNA repair gene PNKP in a patient with microcephaly, seizures, and developmental delay (MCSZ) presenting with a high-grade brain tumor.

Polynucleotide Kinase-Phosphatase (PNKP) is a bifunctional enzyme that possesses both DNA 3'-phosphatase and DNA 5'-kinase activities, which are required for processing termini of single- and double-strand breaks generated by reactive oxygen species (ROS), ionizing radiation and topoisomerase I poisons. Even though PNKP is central to DNA repair, there have been no reports linking PNKP mutations in a Microcephaly, Seizures, and Developmental Delay (MSCZ) patient to cancer. Here, we characterized the biochemical significance of 2 germ-line point mutations in the PNKP gene of a 3-year old male with MSCZ who presented with a high-grade brain tumor (glioblastoma multiforme) within the cerebellum. Functional and biochemical studies demonstrated these PNKP mutations significantly diminished DNA kinase/phosphatase activities, altered its cellular distribution, caused defective repair of DNA single/double stranded breaks, and were associated with a higher propensity for oncogenic transformation. Our findings indicate that specific PNKP mutations may contribute to tumor initiation within susceptible cells in the CNS by limiting DNA damage repair and increasing rates of spontaneous mutations resulting in pediatric glioma associated driver mutations such as ATRX and TP53.

Computational Design of Homotetrameric Peptide Bundle Variants Spanning a Wide Range of Charge States.

With the ability to design their sequences and structures, peptides can be engineered to realize a wide variety of functionalities and structures. Herein, computational design was used to identify a set of 17 peptides having a wide range of putative charge states but the same tetrameric coiled-coil bundle structure. Calculations were performed to identify suitable locations for ionizable residues (D, E, K, and R) at the bundle's exterior sites, while interior hydrophobic interactions were retained. The designed bundle structures spanned putative charge states of -32 to +32 in units of electron charge. The peptides were experimentally investigated using spectroscopic and scattering techniques. Thermal stabilities of the bundles were investigated using circular dichroism. Molecular dynamics simulations assessed structural fluctuations within the bundles. The cylindrical peptide bundles, 4 nm long by 2 nm in diameter, were covalently linked to form rigid, micron-scale polymers and characterized using transmission electron microscopy. The designed suite of sequences provides a set of readily realized nanometer-scale structures of tunable charge that can also be polymerized to yield rigid-rod polyelectrolytes.

Computational Design of Single-Peptide Nanocages with Nanoparticle Templating.

Protein complexes perform a diversity of functions in natural biological systems. While computational protein design has enabled the development of symmetric protein complexes with spherical shapes and hollow interiors, the individual subunits often comprise large proteins. Peptides have also been applied to self-assembly, and it is of interest to explore such short sequences as building blocks of large, designed complexes. Coiled-coil peptides are promising subunits as they have a symmetric structure that can undergo further assembly. Here, an α-helical 29-residue peptide that forms a tetrameric coiled coil was computationally designed to assemble into a spherical cage that is approximately 9 nm in diameter and presents an interior cavity. The assembly comprises 48 copies of the designed peptide sequence. The design strategy allowed breaking the side chain conformational symmetry within the peptide dimer that formed the building block (asymmetric unit) of the cage. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques showed that one of the seven designed peptide candidates assembled into individual nanocages of the size and shape. The stability of assembled nanocages was found to be sensitive to the assembly pathway and final solution conditions (pH and ionic strength). The nanocages templated the growth of size-specific Au nanoparticles. The computational design serves to illustrate the possibility of designing target assemblies with pre-determined specific dimensions using short, modular coiled-coil forming peptide sequences.

Impaired Kv7 channel activity in the central amygdala contributes to elevated sympathetic outflow in hypertension.

AIMS: Elevated sympathetic outflow is associated with primary hypertension. However, the mechanisms involved in heightened sympathetic outflow in hypertension are unclear. The central amygdala (CeA) regulates autonomic components of emotions through projections to the brainstem. The neuronal Kv7 channel is a non-inactivating voltage-dependent K+ channel encoded by KCNQ2/3 genes involved in stabilizing the neuronal membrane potential and regulating neuronal excitability. In this study, we investigated if altered Kv7 channel activity in the CeA contributes to heightened sympathetic outflow in hypertension. METHODS AND RESULTS: The mRNA and protein expression levels of Kv7.2/Kv7.3 in the CeA were significantly reduced in spontaneously hypertensive rats (SHRs) compared with Wistar-Kyoto (WKY) rats. Lowering blood pressure with coeliac ganglionectomy in SHRs did not alter Kv7.2 and Kv7.3 channel expression levels in the CeA. Fluospheres were injected into the rostral ventrolateral medulla (RVLM) to retrogradely label CeA neurons projecting to the RVLM (CeA-RVLM neurons). Kv7 channel currents recorded from CeA-RVLM neurons in brain slices were much smaller in SHRs than in WKY rats. Furthermore, the basal firing activity of CeA-RVLM neurons was significantly greater in SHRs than in WKY rats. Bath application of specific Kv7 channel blocker 10, 10-bis (4-pyridinylmethyl)-9(10H)-anthracnose (XE-991) increased the excitability of CeA-RVLM neurons in WKY rats, but not in SHRs. Microinjection of XE-991 into the CeA increased arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA), while microinjection of Kv7 channel opener QO-58 decreased ABP and RSNA, in anaesthetized WKY rats but not SHRs. CONCLUSIONS: Our findings suggest that diminished Kv7 channel activity in the CeA contributes to elevated sympathetic outflow in primary hypertension. This novel information provides new mechanistic insight into the pathogenesis of neurogenic hypertension.

Colloid-like solution behavior of computationally designed coiled coil bundlemers.

The use of isotropic potential models of simple colloids for describing complex protein-protein interactions is a topic of ongoing debate in the biophysical community. This contention stems from the unavailability of synthetic protein-like model particles that are amenable to systematic experimental characterization. In this article, we test the utility of colloidal theory to capture the solution structure, interactions and dynamics of novel globular protein-mimicking, computationally designed peptide assemblies called bundlemers that are programmable model systems at the intersection of colloids and proteins. Small-angle neutron scattering (SANS) measurements of semi-dilute bundlemer solutions in low and high ionic strength solution indicate that bundlemers interact locally via repulsive interactions that can be described by a screened repulsive potential. We also present neutron spin echo (NSE) spectroscopy results that show high-Q freely-diffusive dynamics of bundlemers. Importantly, formation of clusters due to short-range attractive, inter-bundlemer interactions is observed in SANS even at dilute bundlemer concentrations, which is indicative of the complexity of the bundlemer charged surface. The similarities and differences between bundlemers and simple colloidal as well as complex protein-protein interactions is discussed in detail.

Design of a Superpositively Charged Enzyme: Human Carbonic Anhydrase II Variant with Ferritin Encapsulation and Immobilization.

Supercharged proteins exhibit high solubility and other desirable properties, but no engineered superpositively charged enzymes have previously been made. Superpositively charged variants of proteins such as green fluorescent protein have been efficiently encapsulated within Archaeoglobus fulgidus thermophilic ferritin (AfFtn). Encapsulation by supramolecular ferritin can yield systems with a variety of sequestered cargo. To advance applications in enzymology and green chemistry, we sought a general method for supercharging an enzyme that retains activity and is compatible with AfFtn encapsulation. The zinc metalloenzyme human carbonic anhydrase II (hCAII) is an attractive encapsulation target based on its hydrolytic activity and physiologic conversion of carbon dioxide to bicarbonate. A computationally designed variant of hCAII contains positively charged residues substituted at 19 sites on the protein's surface, resulting in a shift of the putative net charge from -1 to +21. This designed hCAII(+21) exhibits encapsulation within AfFtn without the need for fusion partners or additional reagents. The hCAII(+21) variant retains esterase activity comparable to the wild type and spontaneously templates the assembly of AfFtn 24mers around itself. The AfFtn-hCAII(+21) host-guest complex exhibits both greater activity and thermal stability when compared to hCAII(+21). Upon immobilization on a solid support, AfFtn-hCAII(+21) retains enzymatic activity and exhibits an enhancement of activity at elevated temperatures.

Peptide Design and Self-assembly into Targeted Nanostructure and Functional Materials.

Peptides have been extensively utilized to construct nanomaterials that display targeted structure through hierarchical assembly. The self-assembly of both rationally designed peptides derived from naturally occurring domains in proteins as well as intuitively or computationally designed peptides that form ß-sheets and helical secondary structures have been widely successful in constructing nanoscale morphologies with well-defined 1-d, 2-d, and 3-d architectures. In this review, we discuss these successes of peptide self-assembly, especially in the context of designing hierarchical materials. In particular, we emphasize the differences in the level of peptide design as an indicator of complexity within the targeted self-assembled materials and highlight future avenues for scientific and technological advances in this field.

Rapid endovascular bailout revascularization of the renal arteries with a steerable guiding sheath after endovascular abdominal aortic aneurysm repair.

Advancements in endovascular therapy have made it increasingly available for patients with complex cases but not without complications. Unintentional coverage of the renal arteries is a rare occurrence during endovascular aortic aneurysm repair. Given the potentially devastating repercussions, it is important that surgeons understand the suitability and the risks and benefits of the available revascularization options. We have described two cases of unintentional renal coverage, with subsequent successful bailout via direct manipulation of the stent-graft with a steerable sheath. We also conducted a review of the reported data, discussed the breadth of management options and their technical aspects, and provided several distinct solutions.

Novel variants of engineered water soluble mu opioid receptors with extensive mutations and removal of cysteines.

We have shown that water-soluble variants of the human mu opioid receptor (wsMOR) containing a reduced number of hydrophobic residues at the lipid-facing residues of the transmembrane (TM) helices can be expressed in E. coli. In this study, we tested the consequences of increasing the number of mutations on the surface of the transmembrane domain on the receptor's aqueous solubility and ligand binding properties, along with mutation of 11 cysteine residues regardless of their solvent exposure value and location in the protein. We computationally engineered 10 different variants of MOR, and tested four of them for expression in E. coli. We found that all four variants were successfully expressed and could be purified in high quantities. The variants have alpha helical structural content similar to that of the native MOR, and they also display binding affinities for the MOR antagonist (naltrexone) similar to the wsMOR variants we engineered previously that contained many fewer mutations. Furthermore, for these full-length variants, the helical content remains unchanged over a wide range of pH values (pH 6 ~ 9). This study demonstrates the flexibility and robustness of the water-soluble MOR variants with respect to additional designed mutations in the TM domain and changes in pH, whereupon the protein's structural integrity and its ligand binding affinity are maintained. These variants of the full-length MOR with less hydrophobic surface residues and less cysteines can be obtained in large amounts from expression in E. coli and can serve as novel tools to investigate structure-function relationships of the receptor.