Expanding the Genetic Code for Synthesis of Proteins with Native Biological Modifications and Novel Chemical, Biophysical Probes

Engineering of protein with new modifications essentially expands the protein functions which provide powerful tools for investigating significant biological questions. New modifications also offer scientists the ability to handle protein activity at will. Therefore, development of facile and robust protein modification protocol is highly desirable in almost every aspect of biological research. In combination with chemistry and biology principles, the genetic code expansion technology has emerged as an effective strategy for manipulating proteins and interrogating biological systems. In this regard, the overall objective of the presented research is to expand the chemical biology toolbox based on noncanonical amino acid incorporation and bioorthogonal chemistry. During my graduate study, I was focused on methodology development to genetically incorporate defined, homogeneous native protein posttranslational modifications and new chemical/biophysical probes into proteins for biological investigations, particularly for epigenetic applications. I have established the protocol to incorporate various native lysine acylations into proteins including crotonylation, propionylation and butyrylation. In addition, I also developed a chemical biology approach to study epigenetics, coined “chemical antibody”. This approach allows high throughput fluorescent detection of posttranslational modifications without the use of antibodies. I genetically encoded native lysine posttranslational modification surrogates, such as acryloyllysine, into proteins. Coupled with the bioorthogonal chemistry development, the site-specific detection of the posttranslational modifications was accomplished, essentially offering an alternative route for understanding epigenetic enzyme network. Following the same line of approach, in order to study protein long chain fatty acid acylation, I incorporated long chain unstrained alkene into proteins as chemical reports that are able to be selectively labeled via tetrazine-based bioorthogonal reaction. This development also generates a new direction of research in studying the long chain fatty acid metabolic acylome using chemical biology approach. Lastly, the site-specific incorporation of fluorine-containing noncanonical amino acid probes into proteins was also developed. Fluorine is referred to as “magic bullet” in medicinal organic chemistry and biochemistry. Introducing fluorine into protein allows generating an unrivalled unique probe in a protein without any structural perturbation. The site-specific incorporation of perfluorinated probes is an invaluable tool for the study of noncovalent protein interactions as well as the application on protein designs. Overall, the collective work presented has extensively improved our ability of handling site-specific protein modifications with native biological PTMs and handy chemical/ biophysical probes for diverse medical and biological applications

A Versatile Approach for Siteâ Specific Lysine Acylation in Proteins

Using amber suppression in coordination with a mutant pyrrolysylâ tRNA synthetaseâ tRNAPyl pair, azidonorleucine is genetically encoded in E. coli. Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a siteâ specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4suâ H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified postâ translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.Azidonorleucine, an azideâ containing amino acid, is genetically encoded and incorporated into model proteins. This incorporation followed by traceless Staudinger ligation potentiates the synthesis of proteins with a myriad of siteâ specific lysine acylations.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137311/1/anie201611415-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137311/2/anie201611415_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137311/3/anie201611415.pd

Gut immune system and the implications of oral-administered immunoprophylaxis in finfish aquaculture

The gastrointestinal immune system plays an important role in immune homeostasis regulation. It regulates the symbiotic host-microbiome interactions by training and developing the host’s innate and adaptive immunity. This interaction plays a vital role in host defence mechanisms and at the same time, balancing the endogenous perturbations of the host immune homeostasis. The fish gastrointestinal immune system is armed with intricate diffused gut-associated lymphoid tissues (GALTs) that establish tolerance toward the enormous commensal gut microbiome while preserving immune responses against the intrusion of enteric pathogens. A comprehensive understanding of the intestinal immune system is a prerequisite for developing an oral vaccine and immunostimulants in aquaculture, particularly in cultured fish species. In this review, we outline the remarkable features of gut immunity and the essential components of gut-associated lymphoid tissue. The mechanistic principles underlying the antigen absorption and uptake through the intestinal epithelial, and the subsequent immune activation through a series of molecular events are reviewed. The emphasis is on the significance of gut immunity in oral administration of immunoprophylactics, and the different potential adjuvants that circumvent intestinal immune tolerance. Comprehension of the intestinal immune system is pivotal for developing effective fish vaccines that can be delivered orally, which is less labour-intensive and could improve fish health and facilitate disease management in the aquaculture industry

Long-term follow-up of patients with surgical intractable acromegaly after linear accelerator radiosurgery

Background/PurposeRadiotherapy is a crucial treatment for acromegalic patients with growth hormone (GH)-secreting pituitary tumors. However, its effect takes time. We retrospectively reviewed the long-term outcome of linear accelerator stereotactic radiosurgery (LINAC SRS) for patients with acromegaly from the perspective of biochemical remission and associated factors.MethodsTwenty-two patients presenting with residual or recurrent (GH)-secreting functional pituitary tumor between 1994 and 2004 who received LINAC SRS were enrolled and followed up for at least 3 years. Residual or recurrent tumor was defined as persistent elevated GH or insulin-like growth factor-1 (IGF-1) level and image-confirmed tumor after previous surgical treatment. Biochemical remission was defined as fasting GH less than 2.5 ng/mL with normal sex-and-age adjusted IGF-1.ResultsThe mean follow-up period was 94.7 months (range 36–161 months). Overall mean biochemical remission time was 53 months (median 30 months). Biochemical control was achieved in 15 patients (68.2%) over the follow up period. One patient experienced recurrence after SRS and underwent another operation. Initial GH at diagnosis and pre-SRS GH correlated with biochemical control (p = 0.005 and p < 0.0001, respectively). Further evaluation demonstrated that biochemical control stabilized after 7.5 years. Overall post-SRS hormone deficit persisted in five patients (22.7%).ConclusionIn comparison to other radiosurgery modalities, LINAC radiosurgery also provides a satisfactory outcome. SRS has maximum effect over the first 2 years and stabilizes after 7.5 years. Moreover, SRS elicits long-term biochemical effects and requires longer follow-up for better biochemical remission

Effects of lignosulfonates on callus proliferation and shoot induction of recalcitrant indica rice

In vitro culture of recalcitrant indica rice cultivar through intervening callus is difficult due to long regeneration period. Therefore, this study was undertaken to evaluate the growth promoting effects of lignosulfonate (LS) on callus proliferation and shoot induction of Malaysian recalcitrant indica rice cv. MR219. LS is a by-product of wood industry, commonly used as a plant growth enhancer. Seed derived calli were proliferated on Murashige and Skoog (MS) medium supplemented with different ion-chelated LS (calcium LS: CaLS and sodium LS: NaLS) at 50, 100, 150, and 200 mg/L. MS supplemented with 100 mg/L CaLS significantly increased the callus proliferation rate and adventitious root formation. In shoot induction study, both LSs did not enhance the shoot induction efficiency as compared to the control. However, the formation of albino shoot increased in MS fortified with 100 mg/L CaLS. Further chlorophyll and molecular analyses showed that, albino shoots induced from 100 mg/L CaLS had severe reduction in total chlorophyll content and expression of both chlorophyll-associated genes, chlorophyll a/b-binding protein 1 (OsCAB1R) and young seedling albino (OsYSA). Taken together, LS improves callus proliferation rate and modulate different physiological responses during plant growth of recalcitrant indica rice

Effects of lignosulfonates on callus proliferation and shoot induction of recalcitrant Indica rice

In vitro culture of recalcitrant indica rice cultivar through intervening callus is difficult due to long regeneration period. Therefore, this study was undertaken to evaluate the growth promoting effects of lignosulfonate (LS) on callus proliferation and shoot induction of Malaysian recalcitrant indica rice cv. MR219. LS is a by-product of wood industry, commonly used as a plant growth enhancer. Seed derived calli were proliferated on Murashige and Skoog (MS) medium supplemented with different ion-chelated LS (calcium LS: CaLS and sodium LS: NaLS) at 50, 100, 150, and 200 mg/L. MS supplemented with 100 mg/L CaLS significantly increased the callus proliferation rate and adventitious root formation. In shoot induction study, both LSs did not enhance the shoot induction efficiency as compared to the control. However, the formation of albino shoot increased in MS fortified with 100 mg/L CaLS. Further chlorophyll and molecular analyses showed that, albino shoots induced from 100 mg/L CaLS had severe reduction in total chlorophyll content and expression of both chlorophyll-associated genes, chlorophyll a/b-binding protein 1 (OsCAB1R) and young seedling albino (OsYSA). Taken together, LS improves callus proliferation rate and modulate different physiological responses during plant growth of recalcitrant indica rice

Type II Restriction of Bacteriophage DNA With 5hmdU-Derived Base Modifications

To counteract bacterial defense systems, bacteriophages (phages) make extensive base modifications (substitutions) to block endonuclease restriction. Here we evaluated Type II restriction of three thymidine (T or 5-methyldeoxyuridine, 5mdU) modified phage genomes: Pseudomonas phage M6 with 5-(2-aminoethyl)deoxyuridine (5-NedU), Salmonella phage ViI (Vi1) with 5-(2-aminoethoxy)methyldeoxyuridine (5-NeOmdU) and Delftia phage phi W-14 (a.k.a. ΦW-14) with α-putrescinylthymidine (putT). Among &gt;200 commercially available restriction endonucleases (REases) tested, phage M6, ViI, and phi W-14 genomic DNAs (gDNA) show resistance against 48.4, 71.0, and 68.8% of Type II restrictions, respectively. Inspection of the resistant sites indicates the presence of conserved dinucleotide TG or TC (TS, S=C, or G), implicating the specificity of TS sequence as the target that is converted to modified base in the genomes. We also tested a number of DNA methyltransferases (MTases) on these phage DNAs and found some MTases can fully or partially modify the DNA to confer more resistance to cleavage by REases. Phage M6 restriction fragments can be efficiently ligated by T4 DNA ligase. Phi W-14 restriction fragments show apparent reduced rate in E. coli exonuclease III degradation. This work extends previous studies that hypermodified T derived from 5hmdU provides additional resistance to host-encoded restrictions, in parallel to modified cytosines, guanine, and adenine in phage genomes. The results reported here provide a general guidance to use REases to map and clone phage DNA with hypermodified thymidine

Sterile alpha and TIR motif-containing protein 1 is a negative regulator in the anti-bacterial immune responses in nile tilapia (Oreochromis niloticus)

Nile tilapia (Oreochromis niloticus) is one of the most important food fish in the world. However, the farming industry has encountered significant challenges, such as pathogen infections. Toll-like receptors (TLRs) play an essential role in the initiation of the innate immune system against pathogens. Sterile alpha and TIR motif-containing protein 1 (SARM1) is one of the most evolutionarily conserved TLR adaptors, and its orthologs are present in various species from worms to humans. SARM1 plays an important role in negatively regulating TIR domain-containing adaptor proteins inducing IFNβ (TRIF)-dependent TLR signaling in mammals, but its immune function remains poorly understood in fish. In this study, O. niloticus SARM1 (OnSARM1) was cloned and its evolutionary status was verified using bioinformatic analyses. mRNA expression of OnSARM1 was found at a higher level in the trunk kidney and muscle in healthy fish. The examination of its subcellular location showed that the OnSARM1 was detected only in the cytoplasm of THK cells, and colocalized with OnMyD88, OnTRIF and OnTRIF in small speckle-like condensed granules. The transcript levels of OnMyD88, OnTIRAP, OnTRIF, and downstream effectors, including interleukin (IL)-1β, IL-8, IL-12b and type I interferon (IFN)d2.13, were regulated conversely to the expression of OnSARM1 in the head kidney from Aeromonas hydrophila and Streptococcus agalactiae infected fish. Moreover, the treatment of THK cells with lysates from A. hydrophila and S. agalactiae enhanced the activity of the NF-κB promoter, but the effects were inhibited in the OnSARM1 overexpressed THK cells. Overexpression of OnSARM1 alone did not activate the NF-κB-luciferase reporter, but it suppressed OnMyD88- and OnTIRAP-mediated NF-κB promoter activity. Additionally, OnSARM1 inhibited the mRNA expression of proinflammatory cytokines and hepcidin in A. hydrophila lysate stimulated THK cells. Taken together, these findings suggest that OnSARM1 serves as a negative regulator by inhibiting NF-κB activity, thereby influencing the transcript level of proinflammatory cytokines and antimicrobial peptides in the antibacterial responses

Emission Characteristics of Organic Light-Emitting Diodes and Organic Thin-Films with Planar and Corrugated Structures

In this paper, we review the emission characteristics from organic light-emitting diodes (OLEDs) and organic molecular thin films with planar and corrugated structures. In a planar thin film structure, light emission from OLEDs was strongly influenced by the interference effect. With suitable design of microcavity structure and layer thicknesses adjustment, optical characteristics can be engineered to achieve high optical intensity, suitable emission wavelength, and broad viewing angles. To increase the extraction efficiency from OLEDs and organic thin-films, corrugated structure with micro- and nano-scale were applied. Microstructures can effectively redirects the waveguiding light in the substrate outside the device. For nanostructures, it is also possible to couple out the organic and plasmonic modes, not only the substrate mode

Diverse Durham collection phages demonstrate complex BREX defence responses

Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria–phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility