An efficient mRNA display protocol yields potent bicyclic peptide inhibitors for FGFR3c: outperforming linear and monocyclic formats in affinity and stability.

Macrocyclization has positioned itself as a powerful method for engineering potent peptide drug candidates. Introducing one or multiple cyclizations is a common strategy to improve properties such as affinity, bioavailability and proteolytic stability. Consequently, methodologies to create large libraries of polycyclic peptides by phage or mRNA display have emerged, allowing the rapid identification of binders to virtually any target. Yet, within those libraries, the performance of linear vs. mono- or bicyclic peptides has rarely been studied. Indeed, a key parameter to perform such a comparison is to use a display protocol and cyclization chemistry that enables the formation of all 3 formats in equal quality and diversity. Here, we developed a simple, efficient and fast mRNA display protocol which meets these criteria and can be used to generate highly diverse libraries of thioether cyclized polycyclic peptides. As a proof of concept, we selected peptides against fibroblast growth factor receptor 3c (FGFR3c) and compared the different formats regarding affinity, specificity, and human plasma stability. The peptides with the best KD's and stability were identified among bicyclic peptide hits, further strengthening the body of evidence pointing at the superiority of this class of molecules and providing functional and selective inhibitors of FGFR3c.

Identification and engineering of potent cyclic peptides with selective or promiscuous binding through biochemical profiling and bioinformatic data analysis.

As our understanding of biological systems grows, so does the need to selectively target individual or multiple members of specific protein families in order to probe their function. Many targets of current biological and pharmaceutical interest are part of a large family of closely related proteins and achieving ligand selectivity often remains either an elusive or time-consuming endeavour. Cyclic peptides (CPs) occupy a key niche in ligand space, able to achieve high affinity and selectivity while retaining synthetic accessibility. De novo cyclic peptide ligands can be rapidly generated against a given target using mRNA display. In this study we harness mRNA display technology and the wealth of next generation sequencing (NGS) data generated to explore both experimental approaches and bioinformatic, statistical data analysis of peptide enrichment in cross-screen selections to rapidly generate high affinity CPs with differing intra-family protein selectivity profiles against fibroblast growth factor receptor (FGF-R) family proteins. Using these methods, CPs with distinct selectivity profiles can be generated which can serve as valuable tool compounds to decipher biological questions.

An integrated platform approach enables discovery of potent, selective and ligand-competitive cyclic peptides targeting the GIP receptor.

In any drug discovery effort, the identification of hits for further optimisation is of crucial importance. For peptide therapeutics, display technologies such as mRNA display have emerged as powerful methodologies to identify these desired de novo hit ligands against targets of interest. The diverse peptide libraries are genetically encoded in these technologies, allowing for next-generation sequencing to be used to efficiently identify the binding ligands. Despite the vast datasets that can be generated, current downstream methodologies, however, are limited by low throughput validation processes, including hit prioritisation, peptide synthesis, biochemical and biophysical assays. In this work we report a highly efficient strategy that combines bioinformatic analysis with state-of-the-art high throughput peptide synthesis to identify nanomolar cyclic peptide (CP) ligands of the human glucose-dependent insulinotropic peptide receptor (hGIP-R). Furthermore, our workflow is able to discriminate between functional and remote binding non-functional ligands. Efficient structure-activity relationship analysis (SAR) combined with advanced in silico structural studies allow deduction of a thorough and holistic binding model which informs further chemical optimisation, including efficient half-life extension. We report the identification and design of the first de novo, GIP-competitive, incretin receptor family-selective CPs, which exhibit an in vivo half-life up to 10.7 h in rats. The workflow should be generally applicable to any selection target, improving and accelerating hit identification, validation, characterisation, and prioritisation for therapeutic development.

Seizure Characteristics, Outcome, and Risk of Epilepsy in Pediatric Anti-N-Methyl-d-Aspartate Receptor Encephalitis.

BACKGROUND: We identified seizure characteristics, long-term outcome, and predictors of persistent seizures in children with anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis. METHOD: Data were analyzed from patients with anti-NMDAR encephalitis who presented with seizures at our center between August 2012 and June 2018. RESULTS: Sixty-two of 86 patients with anti-NMDAR encephalitis experienced seizures. Seizures occurred within two weeks of disease onset in 58 of 62 (93.6%) patients; 36 of 62 (58.1%) had seizures as the initial symptom. Males were more likely to exhibit seizures as the initial symptom (P = 0.039). More than a quarter of patients (17 of 62, 27.4%) manifested two or more seizure types. Focal seizures were the most common (46 of 62, 74.2%). Status epilepticus occurred in 27 of 62 (43.5%) patients, and nonconvulsive status epilepticus, in two of 62 (3.2%) patients. No patient developed refractory status epilepticus. No systemic tumors were found. Electroencephalographic abnormalities included background slowing (77.4%), absence of a posterior dominant rhythm (62.9%), interictal epileptic discharges (50.0%), and extreme delta brush (6.5%). In the acute phase, 45 patients (45 of 62, 72.6%) received antiepileptic drugs. Persistent seizures occurred in only five of 62 (8%) patients. On univariate analysis, status epilepticus and combination antiepileptic drug treatment were associated with persistent seizures, but neither independently predicted persistent seizures. CONCLUSIONS: Multiple seizure types may develop at any stage of anti-N-methyl-d-aspartate receptor encephalitis. Refractory status epilepticus, systemic tumors, and extreme delta brush in electroencephalography are rare in pediatric patients. Anti-NMDAR encephalitis-associated seizures appear to have good prognosis, without the need for long-term antiepileptic drug treatment.

Enhanced microphase separation of thin films of low molecular weight block copolymer by the addition of an ionic liquid.

We report on the use of a selective, non-volatile ionic liquid (IL) to enhance the self-assembly via solvent annealing of a low molecular weight block copolymer (BCP) of styrene and 2-vinylpyridine (2VP) suitable for generating sub-10 nm features. Diblock and triblock copolymers of different molecular weights of styrene and 2VP are individually blended with the IL and then solvent annealed in acetone, a non-preferential solvent for the BCPs. Differential scanning calorimetry indicates that the IL selectively resides in the 2VP block of the BCP, resulting in a decrease of the block's Tg and an increase of the effective Flory-Huggins parameter (χeff) of the BCP. The influence of the IL on the non-preferential window of a random copolymer brush used to treat the substrate for self-assembly of the BCPs is also analyzed. Well-defined lamellar patterns form when the optimal weight ratio of IL (∼1%) is added to the BCPs. A detailed analysis of the orientational correlation length and pitch size of the BCPs quantitatively shows that the addition of the IL enhanced the microphase separation of the low molecular weight version of the BCP. Subsequent treatment of the self-assembled BCP with sequential infiltration synthesis yields sub-10 nm AlOx lines.

Aminosilanization nanoadhesive layer for nanoelectric circuits with porous ultralow dielectric film.

An ultrathin layer is investigated for its potential application of replacing conventional diffusion barriers and promoting interface adhesion for nanoelectric circuits with porous ultralow dielectrics. The porous ultralow dielectric (k ≈ 2.5) substrate is silanized by 3-aminopropyltrimethoxysilane (APTMS) to form the nanoadhesive layer by performing oxygen plasma modification and tailoring the silanization conditions appropriately. The high primary amine content is obtained in favor of strong interaction between amino groups and copper. And the results of leakage current measurements of metal-oxide-semiconductor capacitor structure demonstrate that the aminosilanization nanoadhesive layer can block copper diffusion effectively and guarantee the performance of devices. Furthermore, the results of four-point bending tests indicate that the nanoadhesive layer with monolayer structure can provide the satisfactory interface toughness up to 6.7 ± 0.5 J/m(2) for Cu/ultralow-k interface. Additionally, an annealing-enhanced interface toughness effect occurs because of the formation of Cu-N bonding and siloxane bridges below 500 °C. However, the interface is weakened on account of the oxidization of amines and copper as well as the breaking of Cu-N bonding above 500 °C. It is also found that APTMS nanoadhesive layer with multilayer structure provides relatively low interface toughness compared with monolayer structure, which is mainly correlated to the breaking of interlayer hydrogen bonding.

Nanoembossing induced ferroelectric lithography on PZT films for silver particle patterning.

The concept of growing nanosize particles on polarized ferroelectric domain areas is known as ferroelectric lithography (FL). Here, a further step of technical development was achieved by combining nanoembossing technique with the FL to realize the selective growth of silver on the polarized areas induced by nanoembossing. The induced rearrangements of domain distributions by embossing in the ferroelectric films have been characterized by piezoresponse force microscopy (PFM). The selective photochemical reduction of silver particles on the embossed nanostructures associated with the underlying domain patterns created by the nanoembossing process has been successfully demonstrated. This nanoembossing induced ferroelectric lithography (NIFL) developed in this work is expected to create an alternative route for nanoscale patterning of metals.

Nano-embossing technology on ferroelectric thin film Pb(Zr0.3,Ti0.7)O3 for multi-bit storage application.

In this work, we apply nano-embossing technique to form a stagger structure in ferroelectric lead zirconate titanate [Pb(Zr0.3, Ti0.7)O3 (PZT)] films and investigate the ferroelectric and electrical characterizations of the embossed and un-embossed regions, respectively, of the same films by using piezoresponse force microscopy (PFM) and Radiant Technologies Precision Material Analyzer. Attributed to the different layer thickness of the patterned ferroelectric thin film, two distinctive coercive voltages have been obtained, thereby, allowing for a single ferroelectric memory cell to contain more than one bit of data.

Spatially selective photochemical reduction of silver on nanoembossed ferroelectric PZT nanowires.

It is well known that photochemical reaction in an aqueous solution can be chosen by selectively patterning the domain structures of ferroelectrics. In this work, we investigate the photochemically induced deposition of Ag particles on ferroelectric lead zirconate titanate [Pb(Zr(x),Ti(1 - x))O(3)] nanowires fabricated by nanoembossing technology. The photochemical reduction of Ag particles is found to occur preferentially along the embossed nanowires. By imaging domain configurations of the embossed films using the piezoresponse force microscope, the spatially selective deposition of Ag particles can be associated with the underlying ferroelectric domain structures created by the nanoembossing process. The controllable and selective deposition of metal species onto nanoembossed ferroelectric nanostructures without the need for an external electrical field is promising for providing a new route to nanoferroelectric lithography.

A new chemically amplified resist for high resolution patterning by E-beam lithography.

In this work, we have undertaken evaluation of the lithography property of a recently available chemically amplified resist (CAR) resist, UV1116 supplied by Rohm and Haas Company. Systematic study of the EBL property such as sensitivity, contrast, high resolution limit and dense capability, as well as resistance to plasma dry etching has been carried out. In comparison with the performance of UVIII, we conclude that the UV1116 can be a good alternative with better lithography quality.

Fabrication of 150 nm half-pitch grating templates for nanoimprint lithography.

We present the fabrication of 150 nm half-pitch Si grating templates by reactive ion etch (RIE), which are used in nanoimprint lithography (NIL) for high groove density gratings in SU-8 plastic. The etch properties such as the etch rate, profile and etching selectivity of Si over Cr as etch mask were carefully studied. Under the optimum condition Si gratings with 150 nm in linewidth, 480 nm in height and nearly 90 degree in verticality of the sidewall have been achieved. 150 nm half-pitch gratings on SU-8 were then successfully imprinted using the fabricated templates. The diffraction pattern of zeroth and first order from the SU-8 gratings was observed using a 266 nm laser beam. The developed nanofabrication technique in this work is applicable not only for templates but also for other nanostructures in silicon.