Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions

Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure and function. Therefore, the production of 15N- (and 13C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site-specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic DELTAserB mutation, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) DELTAserB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimization and adaption of a high-density culture protocol, we were able to obtain >=10 mg/L homogenously labeled, phosphorylated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with 15N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We propose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses. GCE4All Biomedical Technology Development and Dissemination Center was supported by National Institute of General Medical Science, OSU NMR Facility funded in part by the National Institutes of Health, the Medical Research Foundation at OHSU and the Collins Medical Trust, National Science Foundation EAGER, and by the M. J. Murdock Charitable Trust.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Pathology Education Powered by Virtual and Digital Transformation: Now and the Future

[...]standing on the crest of yet another wave of change, driven by artificial intelligence (AI) and machine learning,2 pathology educators may soon be challenged to convey the best ways to apply these tools to the problems of diagnostic pathology for the coming generation of learners and the present corps of practitioners.3 Hence, this collaborative effort aims to describe the genetic code governing the transmission of pathology knowledge to subsequent generations of medical professionals.4 We aim to expose not just the code but also the supporting array of catalysts, enhancers, and other cofactors now in place to ensure we have a robust and potent supply of pathologists. APPLYING DP IN UNDERGRADUATE MEDICAL, DENTAL, VETERINARY, AND ALLIED HEALTH EDUCATION Beginning in 1985, this technology has been progressively more widely implemented in undergraduate medical, dental, veterinary, and allied health (nursing, pharmacy, medical technology, etc) education platforms in the United States and internationally.5,11-26 As noted above, virtual microscopy laboratories, available on personal devices or in school-based computer labs, have replaced fixed laboratories housing gross specimens, boxes of glass slides, and student microscopes. WSI with links to supplementary resources, such as gross and radiologic images and additional study material, provide enrichment for the teaching and learning experience in the new virtual environment. [...]significant exposure to microanatomy and the laboratory methods of pathology underpinning so much of diagnosis, therapy, and management is foundational.

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression.

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein-protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions.

Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure and function. Therefore, the production of 15N- (and 13C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site-specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic ΔserB mutation, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) ΔserB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimization and adaption of a high-density culture protocol, we were able to obtain ≥10 mg/L homogenously labeled, phosphorylated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with 15N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We propose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses.

Disrupting Six/Eya Signalling as New Therapy for Lung Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common type of interstitial lung disease, with a median survival of 2-4 years from the time of diagnosis [1]. It is estimated that the prevalence of IPF in the US is approximately 10-60 cases per 100,000 people, with limited pharmacological therapies available [2, 3]. IPF is a chronic, progressive disease characterized by alveolar injury, increased extracellular matrix (ECM) deposition and resultant alveolar destruction. Macroscopically, this leads to poor lung compliance, impaired trans-alveolocapillary membrane gas exchange and ultimately, end-stage respiratory failure, necessitating lung transplantation [2, 4, 5]. Several non-genetic risk factors, such as male sex, older age, and smoking, increase the risk of developing IPF [4, 6]. More recently, several genetic risk factors for IPF have also been discovered, including a single-nucleotide polymorphism (rs35705950) in the promoter region of MUC5B [7-9], which codes for an essential protein for airway clearance and innate immune response, along with genes associated with telomere maintenance, such as telomerase RNA component (TERC) and telomerase reverse transcriptase (TERT) [1, 10].

Similarity in Sequences of COVID-19 Genetic Codes over Galois Field

The Coronavirus disease 2019 (COVID-19) outbreak has caused the eco- nomic and health problems for all countries. The origin based on genetic codes of its spreading is a signi cant key for identi cation and solution of the outbreak. The purpose of this research is to study the relation- ships based on similarity measurement over Galois eld amongst genetic codes of COVID-19. A Galois eld is an structure for converting genetic codes to binary codes derived from polynomials and then simi- larity is measured by examing the binary codes. The application is the investigation of the relationships amongst the sequences of genetic codes of COVID-19 particles contaminated from waste water in Brazil, Spain, Italy and the sequences of COVID-19 genetic codes in Thailand and China over Galois eld. The nding shows that the similarity of COVID-19 ge- netic code sequences between China and Brazil is the maximum similarity, 99.9746%. In addition, the relationships amongst the sequences' genetic COVID-19 codes from Wuhan markets, SARS and bats are also investi- gated over a Galois eld. The nding found that the similarity of COVID-19 genetic codes sequences between Bat coronavirus RaTG13-MN996532.1 and Wuhan market- LR757995.1 is the maximum similarity, 55.8548%. In conclusion, the sequence of COVID-19 genetic codes in Brazil is possi- bly signi cant and related to the sequence in China, and vice versa. The sequence of COVID-19 genetic codes at Wuhan market- LR757995.1 is pos- sibly transmitted from Bat coronavirus RaTG13 genetic code to humans in China. © 2022, ECTI Association. All rights reserved.

Accelerating PERx reaction enables covalent nanobodies for potent neutralization of SARS-CoV-2 and variants.

The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity, yet their noncovalent interactions often lead to drug dissociation and incomplete inhibition. Here, we have developed covalent nanobodies capable of binding with SARS-CoV-2 irreversibly via a proximity-enabled reactive therapeutic (PERx) mechanism. A latent bioreactive amino acid (FFY) was designed and genetically encoded into nanobodies to accelerate the PERx reaction rate. Compared with the noncovalent wild-type nanobody, the FFY-incorporated covalent nanobodies neutralized both wild-type SARS-CoV-2 and its Alpha, Delta, Epsilon, Lambda, and Omicron variants with drastically higher potency. This PERx-enabled covalent-nanobody strategy and the related insights into increased potency can be valuable to developing effective therapeutics for various viral infections.

2019 Novel Coronavirus Disease (Covid-19): Toward a Novel Design for Nasopharyngeal and Oropharyngeal Swabbing Robot

The world still suffers from the global pandemic of corona virus. The COVID 19 is a disease that majorly influence the patient's respiratory system and degrade the performance of the immune system. The Polymerase Chain Reactions (PCRs) is commonly used method to assist the health care service in detection the viral disease. It permits identifying the defined chains of the virus genetic code in sputum samples. The front-line staffs are susceptible to infection by COVID 19 during direct swab test that unfortunately subjects them to a high level of risk. To remedy this problem, this paper introduces a flexible swabbing robot for oropharyngeal and nasopharyngeal. This robot is teleworked-based to perform swab sampling with consisting of a flexible manipulator, an integrated endoscope monitor, and the main device. The additional components incorporate a passive locating arm, an active end effector, and a detachable swab handles capable to sense forces. The achieved results revealed the capability of the proposed system in the detection COVID 19 of PCRs specimens based on little data acquired from the clinicians. © 2022 IEEE.

The Algorithm that Mapped Omicron: With Antigenic Maps, Vaccines Can Evolve with COVID-19 Variants

When Omicron, the now ubiquitous COVID-19 variant, first emerged in South Africa in November, scientists were immediately worried. Genetic sequencing showed that Omicron boasted dozens of mutations in key regions of its genetic code—about four times as many as previous variants. Still, they did not know how much Omicron differed physically, not just genetically, from previous variants. That's crucial information in the fight against SARS-CoV-2, the virus that causes COVID-19. It's the physical changes—alterations in how a virus looks and functions—that enable such a pathogen to cause infections by evading the immune systems of people vaccinated or infected with prior strains.

[The mechanism and techniques of vaccination]. / Le mécanisme et les techniques de la vaccination.

From the discovery of the first vaccines to the development of the latest ones, such as the one against Covid-19, research has improved the service rendered against infectious diseases by diversifying vaccination techniques to evolve responses to aggression by microorganisms.

Protective efficacy of a SARS-CoV-2 DNA vaccine in wild type and immunosuppressed Syrian hamsters

A worldwide effort to counter the COVID-19 pandemic has resulted in hundreds of candidate vaccines moving through various stages of research and development, including several vaccines in phase 1, 2 and 3 clinical trials. A relatively small number of these vaccines have been evaluated in SARS-CoV-2 disease models, and fewer in a severe disease model. Here, a SARS-CoV-2 DNA targeting the spike protein and delivered by jet injection, nCoV-S(JET), elicited neutralizing antibodies in hamsters and was protective in both wild-type and transiently immunosuppressed hamster models. This study highlights the DNA vaccine, nCoV-S(JET), we developed has a great potential to move to next stage of preclinical studies, and it also demonstrates that the transiently immunosuppressed Syrian hamsters, which recapitulate severe and prolonged COVID-19 disease, can be used for preclinical evaluation of the protective efficacy of spike-based COVID-19 vaccines.

Uncover New Reactivity of Genetically Encoded Alkyl Bromide Non-Canonical Amino Acids.

Genetically encoded non-canonical amino acids (ncAAs) with electrophilic moieties are excellent tools to investigate protein-protein interactions (PPIs) both in vitro and in vivo. These ncAAs, including a series of alkyl bromide-based ncAAs, mainly target cysteine residues to form protein-protein cross-links. Although some reactivities towards lysine and tyrosine residues have been reported, a comprehensive understanding of their reactivity towards a broad range of nucleophilic amino acids is lacking. Here we used a recently developed OpenUaa search engine to perform an in-depth analysis of mass spec data generated for Thioredoxin and its direct binding proteins cross-linked with an alkyl bromide-based ncAA, BprY. The analysis showed that, besides cysteine residues, BprY also targeted a broad range of nucleophilic amino acids. We validated this broad reactivity of BprY with Affibody/Z protein complex. We then successfully applied BprY to map a binding interface between SUMO2 and SUMO-interacting motifs (SIMs). BprY was further applied to probe SUMO2 interaction partners. We identified 264 SUMO2 binders, including several validated SUMO2 binders and many new binders. Our data demonstrated that BprY can be effectively used to probe protein-protein interaction interfaces even without cysteine residues, which will greatly expand the power of BprY in studying PPIs.

Deep Gene Networks and Response to Stress

We consider systems of differential equations with polynomial and rational nonlinearities and with a dependence on a discrete parameter. Such systems arise in biological and ecological applications, where the discrete parameter can be interpreted as a genetic code. The genetic code defines system responses to external perturbations. We suppose that these responses are defined by deep networks. We investigate the stability of attractors of our systems under sequences of perturbations (for example, stresses induced by environmental changes), and we introduce a new concept of biosystem stability via gene regulation. We show that if the gene regulation is absent, then biosystems sooner or later collapse under fluctuations. By a genetic regulation, one can provide attractor stability for large times. Therefore, in the framework of our model, we prove the Gromov–Carbone hypothesis that evolution by replication makes biosystems robust against random fluctuations. We apply these results to a model of cancer immune therapy.

Antisense Peptide Technology for Diagnostic Tests and Bioengineering Research.

Antisense peptide technology (APT) is based on a useful heuristic algorithm for rational peptide design. It was deduced from empirical observations that peptides consisting of complementary (sense and antisense) amino acids interact with higher probability and affinity than the randomly selected ones. This phenomenon is closely related to the structure of the standard genetic code table, and at the same time, is unrelated to the direction of its codon sequence translation. The concept of complementary peptide interaction is discussed, and its possible applications to diagnostic tests and bioengineering research are summarized. Problems and difficulties that may arise using APT are discussed, and possible solutions are proposed. The methodology was tested on the example of SARS-CoV-2. It is shown that the CABS-dock server accurately predicts the binding of antisense peptides to the SARS-CoV-2 receptor binding domain without requiring predefinition of the binding site. It is concluded that the benefits of APT outweigh the costs of random peptide screening and could lead to considerable savings in time and resources, especially if combined with other computational and immunochemical methods.

Genome recoding strategies to improve cellular properties: mechanisms and advances.

The genetic code, once believed to be universal and immutable, is now known to contain many variations and is not quite universal. The basis for genome recoding strategy is genetic code variation that can be harnessed to improve cellular properties. Thus, genome recoding is a promising strategy for the enhancement of genome flexibility, allowing for novel functions that are not commonly documented in the organism in its natural environment. Here, the basic concept of genetic code and associated mechanisms for the generation of genetic codon variants, including biased codon usage, codon reassignment, and ambiguous decoding, are extensively discussed. Knowledge of the concept of natural genetic code expansion is also detailed. The generation of recoded organisms and associated mechanisms with basic targeting components, including aminoacyl-tRNA synthetase-tRNA pairs, elongation factor EF-Tu and ribosomes, are highlighted for a comprehensive understanding of this concept. The research associated with the generation of diverse recoded organisms is also discussed. The success of genome recoding in diverse multicellular organisms offers a platform for expanding protein chemistry at the biochemical level with non-canonical amino acids, genetically isolating the synthetic organisms from the natural ones, and fighting viruses, including SARS-CoV2, through the creation of attenuated viruses. In conclusion, genome recoding can offer diverse applications for improving cellular properties in the genome-recoded organisms.

The endless frontier of tRNA synthetases.

This chapter calls out the following contributed articles, and gives a sense of why the tRNA synthetases are an endless frontier for scientific research and the unveiling of a vast world of new biology.