Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Human immunoglobulin repertoire analysis guides design of vaccine priming immunogens targeting HIV V2-apex broadly neutralizing antibody precursors.

Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.

Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.

HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies.

Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.

Network pharmacology analysis and molecular docking using the Chinese herbal agent WYHX formula (according to Yan Dexin): Management of coronary artery disease.

BACKGROUND: The therapeutic impact of the Wenyang Huoxue (WYXH) formula on coronary atherosclerotic heart disease (CHD) is well established, yet the precise mechanisms are currently not fully understood. This study provides preliminary insights into the potential mechanisms underlying the therapeutic effects of the formula on CHD by utilizing network pharmacology and molecular docking technology. MATERIALS AND METHODS: The primary active constituents and their corresponding action targets for the formula were retrieved from the TCMSP database. Utilizing Cytoscape 3.9.1 software, a network linking the components of the formula to their respective targets was constructed. Information was collected from Genecards, OMIM, TTD, and DrugBank databases to identify targets related to CHD. The common targets shared by the formula and CHD were then imported into the STRING database to create a protein-protein interaction (PPI) network. Following this, enrichment analyses were performed on the shared targets using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, molecular docking was conducted on the primary active compounds and the core targets. RESULTS: The network encompassing the components and targets of the formula comprises a total of 311 nodes and 895 edges. Compounds exhibiting higher degree centrality consist of quercetin, ß-sitosterol, and kaempferol. In the PPI network, proteins with elevated degree centrality are protein kinase B (AKT1), epidermal growth factor receptor (EGFR), and mitogen-activated protein kinase 3 (MAPK3). The results of GO and KEGG enrichment analyses reveal that the biological processes associated with the efficacy of the formula in treating CHD primarily involve positive regulation of gene expression, hypoxia response, and lipopolysaccharide response, among others. The signaling pathways primarily involved include phosphatidylinositol 3-kinase and protein kinase B (PI3K-AKT), MAPK3, tumor necrosis factor (TNF), and so on. Molecular docking results demonstrate a strong affinity between quercetin, ß-sitosterol, and kaempferol with AKT1, EGFR, and MAPK3. CONCLUSION: We showed for the first time that AKT1, EGFR, and MAPK3 are potential targets influenced by the WYHX formula in CHD treatment. The therapeutic effects could possibly involve signaling pathways such as the PI3K-AKT, MAPK, TNF, and AGE-RAGE pathways.

Effect of Prescription Tongxin on Endothelial Progenitor Cells in Peripheral Blood.

CONTEXT: Coronary heart disease (CHD) refers to a disease where coronary atherosclerosis induces stenosis or obstruction of the blood vessels. Endothelial progenitor cells (EPCs) function to protect and repair the vascular endothelium, and their functional activity state reflects the ability of the body to repair vascular damage. In the peripheral blood of patients with CHD, the density of EPCs decreases, and the function of EPCs is low. OBJECTIVE: This study aimed to investigate the effects of a China Food and Drug Administration (CFDA)-approved prescription medicine, Tongxin, on the density and function of endothelial progenitor cells (EPCs) in peripheral blood. DESIGN: In this study, a randomized, single blind, parallel controlled clinical trial was used. The single blind subjects were subjects. SETTING: The study took place in the Cardiology and Emergency Departments at Shanghai Municipal Hospital of Traditional Chinese Medicine in Shanghai, China. PARTICIPANTS: Participants were 48 patients with coronary heart disease at the hospital. INTERVENTION: Participants were randomly divided into 2 groups (n = 24 each): a control group and an intervention group. Both groups received routine drug treatments, such as platelet inhibitors, nitrates, ß-receptor blockers, statins, angiotensin-converting-enzyme (ACE) inhibitors, angiotensin II receptor antagonists (ARBs), and calcium blockers. The control group was treated with the Shexiang Baoxin Pill, while the intervention group was treated with prescription Tongxin. The course of treatment was 3 months for both groups. OUTCOME MEASURES: Changes in the density and function of EPCs in the peripheral blood of the 2 groups were measured at baseline and postintervention, and the clinical efficacy of the 2 treatments was statistically analyzed. RESULTS: The density of EPCs was significantly higher in both groups after 3 months of treatment, compared to the densities at baseline (P < .05). The change in density between baseline and postintervention was significantly greater for the intervention group than for the control group (P < .05). For the control group, the proliferative vitality [optical density (OD)] value of the EPCs was significantly higher than that at baseline from the fourth day of treatment (P < .05). In the intervention group, the OD value was significantly higher than that at baseline from the first day of treatment (P < .05). Furthermore, the intervention group's cells began to enter the logarithmic growth phase of increase from the fifth day of treatment, and the group's increase as significantly higher than the control group's from the fifth to the seventh dayof treatment (P < .05 for all 3 days). Moreover, the total effective rate was higher in the intervention group than in the control group (P < .05). CONCLUSIONS: Prescription Tongxin can stimulate the release of EPCs from the bone marrow to the peripheral blood of patients with CHD, can significantly increase the proliferation of EPCs in the peripheral blood, and can improve the clinical symptoms of patients. Its curative effect was greater than that of the control treatment.

Asphaltene Adsorption from Toluene onto Silica through Thin Water Layers.

Asphaltenes in crude oil play a pivotal role in reservoir oil production because they control rock-surface wettability. Upon crude oil invasion into a brine-filled reservoir trap, rock adherence of sticky asphaltene agglomerates formed at the crude oil/brine interface can change the initially water-wet porous medium into mixed-oil wetting. If thick, stable water films coat the rock surfaces, however, asphaltenic-oil adhesion is thought to be prevented. We investigate whether water films influence the uptake of asphaltenes in crude oil onto silica surfaces. Water films of known thickness are formed at a silica surface in a quartz crystal microbalance with dissipation and contacted by toluene-solubilized asphaltene. We confirm that thick water films prevent asphaltene molecular contact with the silica surface blocking asphaltene adhesion. The thicker the water film, the smaller is the amount of asphaltene deposited. Film thickness necessary for complete blockage onto silica is greater than about 500 nm, well beyond the range of molecular-chain contact. Water films of thickness less than 500 nm, sandwiched between toluene and solid silica, apparently rupture into thick water pockets and interposed molecularly thin water layers that permit asphaltene adherence.

RosettaAntibodyDesign (RAbD): A general framework for computational antibody design.

A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228-256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody-antigen complexes, using two design strategies-optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the native amino acid types for residues that contact the antigen in the native structures was 72% in simulations performed in the presence of the antigen and 48% in simulations performed without the antigen, for an ARR of 1.5. For the non-contacting residues, the ARR was 1.08. This shows that the sequence profiles are able to maintain the amino acid types of these conserved, buried sites, while recovery of the exposed, contacting residues requires the presence of the antigen-antibody interface. We tested RAbD experimentally on both a lambda and kappa antibody-antigen complex, successfully improving their affinities 10 to 50 fold by replacing individual CDRs of the native antibody with new CDR lengths and clusters.

An Interfacial Solar Heating Assisted Liquid Sorbent Atmospheric Water Generator.

Harvesting water from air is a promising strategy for fresh-water production, and it is particularly desirable for areas that lack direct access to clean water. While high-concentration liquid sorbent is well-known for high sorption, it has not been widely used for atmospheric water collection, being primarily limited by the difficulty in desorption. Interfacial solar heating based on a salt-resistant GO-based aerogel is now shown to enable a high-concentration liquid sorbent (CaCl2 50 wt % solution) based atmospheric water generator. Fresh water (2.89 kg m-2 day-1 ) can be produced at about 70 % relative humidity, with only solar energy input and energy efficiency of desorption as high as 66.9 %. This low-cost and effective approach provides an attractive pathway to extract water from air, to relieve the thirst of arid, land-locked, and other areas where fresh water is scarce.

De Novo Design of Tetranuclear Transition Metal Clusters Stabilized by Hydrogen-Bonded Networks in Helical Bundles.

De novo design provides an attractive approach to test the mechanism by which metalloproteins define the geometry and reactivity of their metal ion cofactors. While there has been considerable progress in designing proteins that bind transition metal ions including iron-sulfur clusters, the design of tetranuclear clusters with oxygen-rich environments has not been accomplished. Here, we describe the design of tetranuclear clusters, consisting of four Zn2+ and four carboxylate oxygens situated at the vertices of a distorted cube-like structure. The tetra-Zn2+ clusters are bound at a buried site within a four-helix bundle, with each helix donating a single carboxylate (Glu or Asp) and imidazole (His) ligand, as well as second- and third-shell ligands. Overall, the designed site consists of four Zn2+ and 16 polar side chains in a fully connected hydrogen-bonded network. The designed proteins have apolar cores at the top and bottom of the bundle, which drive the assembly of the liganding residues near the center of the bundle. The steric bulk of the apolar residues surrounding the binding site was varied to determine how subtle changes in helix-helix packing affect the binding site. The crystal structures of two of four proteins synthesized were in good agreement with the overall design; both formed a distorted cuboidal site stabilized by flanking second- and third-shell interactions that stabilize the primary ligands. A third structure bound a single Zn2+ in an unanticipated geometry, and the fourth bound multiple Zn2+ at multiple sites at partial occupancy. The metal-binding and conformational properties of the helical bundles in solution, probed by circular dichroism spectroscopy, analytical ultracentrifugation, and NMR, were consistent with the crystal structures.

Effect of Particle Size and Impact Velocity on Collision Behaviors Between Nano-Scale TiN Particles: MD Simulation.

Inter-particle bonding formation which determines qualities of nano-scale ceramic coatings is influenced by particle collision behaviors during high velocity collision processes. In this study, collision behaviors between nano-scale TiN particles with different diameters were illuminated by using Molecular Dynamics simulation through controlling impact velocities. Results show that nano-scale TiN particles exhibit three states depending on particle sizes and impact velocities, i.e., bonding, bonding with localized fracturing, and rebounding. These TiN particles states are summarized into a parameter selection map providing an overview of the conditions in terms of particle sizes and velocities. Microstructure results show that localized atoms displacement and partial fracture around the impact region are main reasons for bonding formation of nano-scale ceramic particles, which shows differences from conventional particles refining and amorphization. A relationship between the adhesion energy and the rebound energy is established to understand bonding formation mechanism for nano-scale TiN particle collision. Results show that the energy relationship is depended on the particle sizes and impact velocities, and nano-scale ceramic particles can be bonded together as the adhesion energy being higher than the rebound energy.

Precise Perforation and Scalable Production of Si Particles from Low-Grade Sources for High-Performance Lithium Ion Battery Anodes.

Alloy anodes, particularly silicon, have been intensively pursued as one of the most promising anode materials for the next generation lithium-ion battery primarily because of high specific capacity (>4000 mAh/g) and elemental abundance. In the past decade, various nanostructures with porosity or void space designs have been demonstrated to be effective to accommodate large volume expansion (∼300%) and to provide stable solid electrolyte interphase (SEI) during electrochemical cycling. However, how to produce these building blocks with precise morphology control at large scale and low cost remains a challenge. In addition, most of nanostructured silicon suffers from poor Coulombic efficiency due to a large surface area and Li ion trapping at the surface coating. Here we demonstrate a unique nanoperforation process, combining modified ball milling, annealing, and acid treating, to produce porous Si with precise and continuous porosity control (from 17% to 70%), directly from low cost metallurgical silicon source (99% purity, ∼ $1/kg). The produced porous Si coated with graphene by simple ball milling can deliver a reversible specific capacity of 1250 mAh/g over 1000 cycles at the rate of 1C, with Coulombic efficiency of first cycle over 89.5%. The porous networks also provide efficient ion and electron pathways and therefore enable excellent rate performance of 880 mAh/g at the rate of 5C. Being able to produce particles with precise porosity control through scalable processes from low-grade materials, it is expected that this nanoperforation may play a role in the next generation lithium ion battery anodes, as well as many other potential applications such as optoelectronics and thermoelectrics.

Agrobacterium tumefaciens recognizes its host environment using ChvE to bind diverse plant sugars as virulence signals.

Agrobacterium tumefaciens is a broad host range plant pathogen that combinatorially recognizes diverse host molecules including phenolics, low pH, and aldose monosaccharides to activate its pathogenic pathways. Chromosomal virulence gene E (chvE) encodes a periplasmic-binding protein that binds several neutral sugars and sugar acids, and subsequently interacts with the VirA/VirG regulatory system to stimulate virulence (vir) gene expression. Here, a combination of genetics, X-ray crystallography, and isothermal calorimetry reveals how ChvE binds the different monosaccharides and also shows that binding of sugar acids is pH dependent. Moreover, the potency of a sugar for vir gene expression is modulated by a transport system that also relies on ChvE. These two circuits tune the overall system to respond to sugar concentrations encountered in vivo. Finally, using chvE mutants with restricted sugar specificities, we show that there is host variation in regard to the types of sugars that are limiting for vir induction.

Simultaneous Purification and Perforation of Low-Grade Si Sources for Lithium-Ion Battery Anode.

Silicon is regarded as one of the most promising candidates for lithium-ion battery anodes because of its abundance and high theoretical capacity. Various silicon nanostructures have been heavily investigated to improve electrochemical performance by addressing issues related to structure fracture and unstable solid-electrolyte interphase (SEI). However, to further enable widespread applications, scalable and cost-effective processes need to be developed to produce these nanostructures at large quantity with finely controlled structures and morphologies. In this study, we develop a scalable and low cost process to produce porous silicon directly from low grade silicon through ball-milling and modified metal-assisted chemical etching. The morphology of porous silicon can be drastically changed from porous-network to nanowire-array by adjusting the component in reaction solutions. Meanwhile, this perforation process can also effectively remove the impurities and, therefore, increase Si purity (up to 99.4%) significantly from low-grade and low-cost ferrosilicon (purity of 83.4%) sources. The electrochemical examinations indicate that these porous silicon structures with carbon treatment can deliver a stable capacity of 1287 mAh g(-1) over 100 cycles at a current density of 2 A g(-1). This type of purified porous silicon with finely controlled morphology, produced by a scalable and cost-effective fabrication process, can also serve as promising candidates for many other energy applications, such as thermoelectrics and solar energy conversion devices.

mRNA-LNP prime boost evolves precursors toward VRC01-like broadly neutralizing antibodies in preclinical humanized mouse models.

Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.

Heterologous prime-boost vaccination drives early maturation of HIV broadly neutralizing antibody precursors in humanized mice.

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

Association between post-stroke cognitive impairment and gut microbiota: A PRISMA-compliant systematic review and meta-analysis.

BACKGROUND: Accumulating evidence has indicated a possible connection between post-stroke cognitive impairment (PSCI) and gut microbiota imbalance. To further investigate this association, the present work was designed to systematically assess the dissimilarity of gut microbiota between PSCI and healthy individuals or stroke patients. METHODS: A meta-analysis and systematic review was conducted by searching various databases including PubMed, Web of Science, Embase, VIP, CNKI, and Wangfang for relevant studies. The pooled outcomes were used to estimate the combined dissimilarity of gut microbiota composition between PSCI and healthy individuals or patients with stroke. RESULTS: Nine eligible studies were included in this meta-analysis. The results showed that there were no significant changes in observed richness indexes (Chao1 and ACE) and Shannon index. Notably, a significant decrease in Simpson index was observed in PSCI patients in comparison to the healthy individuals (-0.31, 95% CI: -0.62 to -0.01, P = 0.04). Moreover, the microbiota composition at the phylum level (increased abundance of Proteobacteria), family level (increased abundance of Bacteroidaceae, Lachnospiraceae, and Veillonellaceae; decreased abundance of Enterobacteriaceae), and genus level (increased abundance of Bacteroides, Clostridium XIVa, and Parabacteroides; decreased abundance of Prevotella and Ruminococcus) was found to be significantly different between PSCI and controls. CONCLUSION: This meta-analysis suggests a significant shift of observed species and microbiota composition in PSCI compared to healthy individuals or patients with stroke.

Microstructure and Corrosion Behavior of Zinc/Hydroxyapatite Multi-Layer Coating Prepared by Combining Cold Spraying and High-Velocity Suspension Flame Spraying.

The study aims to enhance the corrosion resistance and bioactivity of Mg alloy substrates through the development of a zinc/hydroxyapatite multi-layer (Zn/HA-ML) coating. The Zn/HA-ML coating was prepared by depositing a cold-sprayed (CS) Zn underlayer and a high-velocity suspension flame sprayed (HVSFS) Zn/HA multi-layer and was compared with the CS Zn coating and the Zn/HA dual-layer (Zn/HA-DL) coating. Phase, microstructure, and bonding strength were examined, respectively, by X-ray diffraction, scanning electron microscopy, and tensile bonding testing. Corrosion behavior and bioactivity were investigated using potentiodynamic polarization, electrochemical impedance spectroscopy, and immersion testing. Results show that the HVSFS Zn/HA composite layers were mainly composed of Zn, HA, and ZnO and were well bonded to the substrate. The HVSFS HA upper layer on the CS Zn underlayer in the Zn/HA-DL coating exhibited microcracks due to their mismatched thermal expansion coefficient (CTE). The Zn/HA-ML coating exhibited good bonding within different layers and showed a higher bonding strength of 27.3 ± 2.3 MPa than the Zn/HA-DL coating of 20.4 ± 2.7 MPa. The CS Zn coating, Zn/HA-DL coating, and Zn/HA-ML coating decreased the corrosion current density of the Mg alloy substrate by around two-fourfold from 3.12 ± 0.75 mA/cm2 to 1.41 ± 0.82mA/cm2, 1.06 ± 0.31 mA/cm2, and 0.88 ± 0.27 mA/cm2, respectively. The Zn/HA-ML coating showed a sixfold decrease in the corrosion current density and more improvements in the corrosion resistance by twofold after an immersion time of 14 days, which was mainly attributed to newly formed apatite and corrosion by-products of Zn particles. The Zn/HA-ML coating effectively combined the advantages of the corrosion resistance of CS Zn underlayer and the bioactivity of HVSFS Zn/HA multi-layers, which proposed a low-temperature strategy for improving corrosion resistance and bioactivity for implant metals.

Development of α-helical calpain probes by mimicking a natural protein-protein interaction.

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs will be useful for future investigation of calpains, while the cross-linking technique will enable exploration of other protein-protein interactions.