Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus-assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base-pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem of M. mazei pyrrolysyl tRNA (tRNAPyl ), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell-lines for ncAA mutagenesis.

AbstractHeterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus‐assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base‐pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem of M. mazei pyrrolysyl tRNA (tRNAPyl), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell‐lines for ncAA mutagenesis.

The development of antimicrobial peptides (AMPs) as potential therapeutics requires resolving the foundational principles behind their structure-activity relationships. The role of histidine residues within AMPs remains a mystery despite the fact that several potent peptides containing this amino acid are being considered for further clinical development. Gaduscidin-1 (Gad-1) is a potent AMP from Atlantic cod fish that has a total of five His residues. Herein, the role of His residues and metal-potentiated activity of Gad-1 was studied. The five His residues contribute to the broad-spectrum activity of Gad-1. We demonstrated that Gad-1 can coordinate two Cu2+ ions, one at the N-terminus and one at the C-terminus, where the C-terminal binding site is a novel Cu2+ binding motif. High affinity Cu2+ binding at both sites was observed using mass spectrometry and isothermal titration calorimetry. Electron paramagnetic resonance was used to determine the coordination environment of the Cu2+ ions. Cu2+ binding was shown to be responsible for an increase in antimicrobial activity and a new mode of action. Along with the traditional AMP mode of action of pore formation, Gad-1 in the presence of Cu2+ (per)oxidizes lipids. Importantly, His3, His11, His17, and His21 were found to be important to lipid (per)oxidation. This insight will help further understand the inclusion and role of His residues in AMPs, the role of the novel C-terminal binding site, and can contribute to the field of designing potent AMPs that bind metal ions to potentiate activity.

Do stock prices of publicly listed companies respond to changes in transaction costs? Using the SEC’s pilot program that increased the tick size for approximately 1,200 randomly chosen stocks, we find a stock price decrease between 1.75% and 3.2% for small spread stocks affected by the larger tick size relative to a control group. We find that the increase in the present value of transaction costs accounts for a small percentage of the price decrease. We study channels of price variation due to changes in expected returns: information risk, investor horizon, and liquidity risk. The evidence suggests that trading frictions affect the cost of capital.

The small molecule gibberellin JRA-003 was identified as an inhibitor of the NF-kB (nuclear kappa-light-chain-enhancer of activated B cells) pathway. Here we find that JRA-003 binds to and significantly inhibits the nuclear translocation of pathway-activating kinases IKKα (IκB kinase alpha) and IKKβ (IκB kinase beta). Analogs of JRA-003 were synthesized and NF-κB-inhibiting gibberellins were found to be cytotoxic in cancer-derived cell lines (HS 578T, HCC 1599, RC-K8, Sud-HL4, CA 46, and NCIH 4466). Not only was JRA-003 identified as the most potent synthetic gibberellin against cancer-derived cell lines, it displayed no cytotoxicity in cells derived from noncancerous sources (HEK 293T, HS 578BST, HS 888Lu, HS 895Sk, HUVEC). This selectivity suggests a promising approach for the development of new therapeutics.

Dynamic changes in protein structure can be monitored by using a fluorescent probe and a dark quencher. This approach is contingent upon the ability to precisely introduce a fluorophore/quencher pair into two specific sites of a protein of interest. Despite recent advances, there is continued demand for new and convenient approaches to site-selectively label proteins with such optical probes. We have recently developed a chemoselectively rapid azo-coupling reaction (CRACR) for site-specific protein labeling; it relies on rapid coupling between a genetically encoded 5-hydroxytryptophan residue and various aromatic diazonium ions. Herein, it is reported that the product of this conjugation reaction, a highly chromophoric biarylazo group, is a potent fluorescence quencher. The absorption properties of this azo product can be tuned by systematically altering the structure of the aryldiazonium species. A particular "quenchergenic" aryldiazonium has been identified that, upon conjugation, efficiently quenches the fluorescence of green fluorescent protein, which is a widely used genetically encoded fluorescent probe that can be terminally attached to target proteins. This fluorophore/quencher pair was used to evaluate the protein-labeling kinetics of CRACR, as well as to monitor the proteolysis of a fusion protein.

Abstractβ‐Boryl carbonyl compounds are produced by a Ni‐catalyzed cross‐coupling of vinylboron “ate” complexes and acid chloride or acid anhydride electrophiles. The reactions are efficient, being complete in as little as two minutes, and can be applied to a broad range of substrates.

AbstractThe orthogonal sulfur–fluoride exchange reaction (SuFEx) and copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) are employed to synthesize sequence‐regulated synthetic polymers. The high efficiency and broad tolerance of SuFEx and CuAAC to diverse chemical functionalities enable the one‐pot synthesis of polydispersed sequence‐controlled polymers by step‐growth copolymerization in high yield and sequence complexity. Furthermore, iterative SuFEx and CuAAC coupling reactions on a solid support, without the need of protecting groups, afford monodispersed sequence‐defined oligomers. The use of this orthogonal pair of click reactions provides new opportunities to facilely access sequence‐regulated synthetic polymers with a high degree of structural diversity.

In August 2008, the American Printing House for the Blind (APH) brought together an advisory group to provide guidance and clarity on a range of issues related to cortical visual impairment (CVI) as those issues relate to the development of products. The internationally drawn CVI Advisory Group, with members suggested by APH ex officio trustees and consultants, represents a continuing effort by APH to serve the growing group of students with CVI who are registered for Federal Quota funds. Previous activities conducted by APH on behalf of these students have included improving the Federal Quota registration process by better defining eligibility criteria; hosting a CVI Synergy meeting in Louisville, Kentucky; identifying existing APH products that serve the unique needs of this population; developing and manufacturing new products for students with CVI; creating a CVI web site; and providing information on APH products that are related to students with CVI through workshops of the APH National Instructional Partnership. DEFINITION OF CVI: A WORKING DEFINITION FOR EDUCATIONAL SERVICES CVI is defined as impaired vision that is due to bilateral dysfunction of the optic radiations or visual cortex or both. It can coexist with ocular and ocular motor disorders and can be the result of perinatal brain dysfunction or be caused by trauma. Approximately 30%-40% of children with visual impairments have CVI (see Figure 1, which can be viewed in an area dedicated to CVI on the American Foundation for the Blind's web site at ). One concern of professionals in the field of education of students with visual impairments is to establish a standard definition of CVI. Accordingly, the purpose of this article is to clarify the differences between children who qualify for services from vision educators and those who have visual processing difficulties that are not considered visual impairment. Our perspective is that all children who have CVI should be classified as visually impaired and receive the necessary services, regardless of the severity of the degree of CVI or additional disabilities. A child with CVI is distinguished from a child with learning disabilities or developmental disabilities by the following criteria: (1) an eye examination that cannot fully explain the child's use of vision; (2) a history or presence of neurological problems, even when the child's brain-imaging studies may appear normal (Dutton, 2008); and (3) the presence of the behavioral or visual responses that are collectively associated with CVI. In most North American jurisdictions, low vision is defined as a reduction in visual acuity no better than 20/70 (6/21) but better than 20/200 (6/60) in the better eye with the best correction. Legal blindness is defined as visual acuity no better than 20/200 (6/60) in the better eye with the best correction. Legal blindness is also defined as a central visual field that is no greater than 20 degrees. Using this framework, CVI should be defined, albeit arbitrarily, by a reduction in visual acuity, in the visual fields, or in a child's ability to see compared to other children of the same age. Unfortunately, traditional methods of precisely determining acuity or visual field function in children with CVI are problematic. Because children with CVI frequently have additional disabilities, it is often difficult to measure visual acuity. When it is possible to do so, standard visual acuity testing should be performed. Electrophysiological measures, such as visual evoked potential acuities, may also be used. When warranted, visual acuities should be measured using forced-choice preferential looking acuities or estimating visual function through the identification of sized objects at specific distances. Dutton (2008) and others have proposed a theoretical construct for classifying higher-level visual processing. This framework considers the effects of damage to the dorsal and ventral streams of the brain to explain visual dysfunction. …