Functional Protein Domains Research Articles

The lysine 2-hydroxyisobutyrylome of Helicobacter pylori: Indicating potential roles of lysine 2-hydroxyisobutyrylation in the bacterial metabolism

Helicobacter pylori (H. pylori) is a pathogen which colonizes the stomach, causing ulcers, chronic gastritis and other related diseases. Protein post-translational modifications (PTMs) in bacteria mainly include glycosylation, ubiquitination, nitrosylation, methylation, phosphorylation and acetylation, all of which have divergent functions in the physiology and pathology of the bacterium. Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered type of PTM in recent years in some kinds of organisms, and this PTM is involved in the regulation of a variety of metabolic process, such as bacterial glucose metabolism, lipid metabolism and protein synthesis. This study performed the first qualitative lysine 2-hydroxyisobutyrylome in H. pylori, and a total of 4419 Khib sites in 812 proteins were identified. The results show that Khib sites are mainly located in the key functional regions or active domains of proteins involved in nickel-trafficking, energy production, virulence factors, anti-oxidation, metal resistance, and ribosome biosynthesis in H. pylori. The study presented here provides new hints in the metabolism and pathology of H. pylori and the proteins with Khib modification may be potentially promising targets for the further development of antibiotics, especially considering the high occurrence of treatment failure of H. pylori failure due to development of antibiotics-resistance.

In‐depth evaluation of protein domains improves classification of SORL1 variant pathogenicity in Alzheimer’s Disease patients

AbstractBackgroundRare pathogenic SORL1 gene variants have been identified as strong risk‐increasing factors in Alzheimer’s disease (AD). Recent analysis indicated that SORL1 loss‐of‐function (LoF) variants associated with a ∼40‐fold increased risk for early‐onset AD, while missense variants that were prioritized using in silico variant pathogenicity‐prediction algorithms associated only with a 2‐fold risk increase. However, current risk‐prediction strategies do not take SORL1‐specific features into account. We aimed to design a variant classification strategy that adds in‐depth knowledge of SORLA structural folding and function to the variant risk prediction.MethodWe identified coding SORL1 variants in a discovery/replication sample of whole exome sequencing data from the ADES/ADSP studies comprising 18,959 cases and 21,893 controls after quality control. We selected only the extremely rare variants (MAF<0.05%) and further divided these into LoF(n = 77) and missense variants(n = 539). With an in‐depth evaluation of protein functional domains (informed by pathogenic variants in homologous genes) combined with their predicted pathogenicity (REVEL algorithm) we prioritized missense variants into ‘high priority’, ‘moderate priority’ and ‘low priority’. We compared the age‐dependent penetrance of missense variants for each priority‐category and LoF variants, in context of APOE genotype.ResultCarriers of prioritized missense variants had a similar age‐dependent penetrance as LoF variants, with median age at AD‐onset of ∼65 years which is significantly lower compared to non‐carriers (∼72 years). In addition, carriers of prioritized SORL1 missense variants have an expedited age at onset by ∼7 years independent of their APOE genotype. If we look at a 80% penetrance level, SORL1 carriers with homozygous ε4 background had AD by age 61 (95%CI 60‐NA, n = 6) compared to age 73 (95%CI 72‐73, n = 1091) for SORL1 WT carriers. Carriers with a heterozygous background had AD by age 71 (95%CI 68‐76, n = 62) compared to 78 (95%CI 78‐78, n = 4816) for WT. For a non‐ε4 background the age at onset for carriers was 83 years (95%CI 78‐90, n = 54) compared to 85 years (95%CI 85‐85, n = 5773) for WT. The moderate and low priority group showed no significant difference compared to WT.ConclusionOur prioritization model can successfully identify likely pathogenic missense SORL1 variants, allowing for better identification of patients with possible SORL1‐associated‐AD.

Catalytic action and bactericidal behavior of samarium/carbon spheres-doped manganese oxide nanostructures and their molecular docking analysis

The co-precipitation technique was adapted to synthesize different concentrations (2 % and 4 %) of samarium (Sm) doped constant equates of carbon spheres (Cs) and manganese oxide (MnO2). The principal objective of this investigation is to demonstrate confirmation that Sm/Cs-doped MnO2 nanostructures (NSs) owned antibacterial and catalytic attributes. Reduction in surface area manifested to agglomeration, NSs faces become inaccessible to initiate a reaction, can be overcome upon doping of Sm. Sm has the potential to increase the activity of metal oxide ascribed to its electron trapping effect. The structural morphologies, optical properties, functional groups, elemental composition, and d-spacing were determined by applying various characterizations. With the incorporation of CS and Sm, UV–vis spectra shifted towards lower wavelength and band gap energy (Eg) was reduced. MnO2 possessed orthorhombic structure, according to the XRD pattern and TEM exhibited long Burr-like morphology of undoped MnO2. SAED image illustrated that MnO2 is polycrystalline. 4 % Sm/CS doped MnO2 revealed the highest degradation (91 %) in a neutral environment. Furthermore, 4 % Sm/Cs doped-MnO2 revealed an inhibitory zone of 2.85 mm against Escherichia coli (E. coli). Additionally, an analysis of molecular docking revealed a binding interface with NRs and the functional domains of certain cellular proteins. Results indicated that Cs-doped MnO2 and Cs/Sm-doped MnO2 NRs are the most potent DNA gyrase and FabB enzyme inhibitors.

The Distribution of the Germ Plasm Gene <i>germes</i> among Anurans

The germes gene is a marker of germ plasm and primordial germ cells (PGC) described in the African clawed frog Xenopus laevis. It is known that overexpression of its mutant form negatively affects the formation and migration of PGC. However, until now it was not known how widely this gene is represented in animals of different phylogenetic groups. In this work, we performed bioinformatic analysis of genomic and transcriptome sequences of animals with germ plasm. It turned out that germes homologs are present only in representatives of the genera Xenopus and Hymenochirus of the family Pipidae (order Anura). The obtained results were confirmed by RT-PCR analysis of the expression of germes orthologs in the ovaries of six representatives of different Anura families. Phylogenetic analysis of cloned sequences of germes homologs suggests the appearance of this gene in the ancestors of Pipidae and its secondary loss in the genus Pseudohymenochirus. It is also identified that the amino acid sequences of the functional domains of the Germes protein are rather conservative.

Multiscale simulations reveal TDP-43 molecular-level interactions driving condensation

The RNA-binding protein TDP-43 is associated with mRNA processing and transport from the nucleus to the cytoplasm. TDP-43 localizes in the nucleus as well as accumulating in cytoplasmic condensates such as stress granules. Aggregation and formation of amyloid-like fibrils of cytoplasmic TDP-43 are hallmarks of numerous neurodegenerative diseases, most strikingly present in >90% of amyotrophic lateral sclerosis (ALS) patients. If excessive accumulation of cytoplasmic TDP-43 causes, or is caused by, neurodegeneration is presently not known. In this work, we use molecular dynamics simulations at multiple resolutions to explore TDP-43 self- and cross-interaction dynamics. A full-length molecular model of TDP-43, all 414 amino acids, was constructed from select structures of the protein functional domains (N-terminal domain, and two RNA recognition motifs, RRM1 and RRM2) and modeling of disordered connecting loops and the low complexity glycine-rich C-terminus domain. All-atom CHARMM36m simulations of single TDP-43 proteins served as guides to construct a coarse-grained Martini 3 model of TDP-43. The Martini model and a coarser implicit solvent C⍺ model, optimized for disordered proteins, were subsequently used to probe TDP-43 interactions; self-interactions from single-chain full-length TDP-43 simulations, cross-interactions from simulations with two proteins and simulations with assemblies of dozens to hundreds of proteins. Our findings illustrate the utility of different modeling scales for accessing TDP-43 molecular-level interactions and suggest that TDP-43 has numerous interaction preferences or patterns, exhibiting an overall strong, but dynamic, association and driving the formation of biomolecular condensates.

Chromatin gatekeeper and modifier CHD proteins in development, and in autism and other neurological disorders.

Chromatin, a protein-DNA complex, is a dynamic structure that stores genetic information within the nucleus and responds to molecular/cellular changes in its structure, providing conditional access to the genetic machinery. ATP-dependent chromatin modifiers regulate access of transcription factors and RNA polymerases to DNA by either "opening" or "closing" the structure of chromatin, and its aberrant regulation leads to a variety of neurodevelopmental disorders. The chromodomain helicase DNA-binding (CHD) proteins are ATP-dependent chromatin modifiers involved in the organization of chromatin structure, act as gatekeepers of genomic access, and deposit histone variants required for gene regulation. In this review, we first discuss the structural and functional domains of the CHD proteins, and their binding sites, and phosphorylation, acetylation, and methylation sites. The conservation of important amino acids in SWItch/sucrose non-fermenting (SWI/SNF) domains, and their protein and mRNA tissue expression profiles are discussed. Next, we convey the important binding partners of CHD proteins, their protein complexes and activities, and their involvements in epigenetic regulation. We also show the ChIP-seq binding dynamics for CHD1, CHD2, CHD4, and CHD7 proteins at promoter regions of histone genes, as well as several genes that are critical for neurodevelopment. The role of CHD proteins in development is also discussed. Finally, this review provides information about CHD protein mutations reported in autism and neurodevelopmental disorders, and their pathogenicity. Overall, this review provides information on the progress of research into CHD proteins, their structural and functional domains, epigenetics, and their role in stem cell, development, and neurological disorders.

Sequence Characterization of Extra-Chromosomal Circular DNA Content in Multiple Blackgrass (Alopecurus myosuroides) Populations.

Alopecurus myosuroides (blackgrass) is a problematic weed of Western European winter wheat, and its success is largely due to widespread multiple-herbicide resistance. Previous analysis of F2 seed families derived from two distinct blackgrass populations exhibiting equivalent non-target site resistance (NTSR) phenotypes shows resistance is polygenic and evolves from standing genetic variation. Using a CIDER-seq pipeline, we show that herbicide-resistant (HR) and herbicide-sensitive (HS) F3 plants from these F2 seed families as well as the parent populations they were derived from carry extra-chromosomal circular DNA (eccDNA). We identify the similarities and differences in the coding structures within and between resistant and sensitive populations. Although the numbers and size of detected eccDNAs varied between the populations, comparisons between the HR and HS blackgrass populations identified shared and unique coding content, predicted genes, and functional protein domains. These include genes related to herbicide detoxification such as Cytochrome P450s, ATP-binding cassette transporters, and glutathione transferases including AmGSTF1. eccDNA content was mapped to the A. myosuroides reference genome, revealing genomic regions at the distal end of chromosome 5 and the near center of chromosomes 1 and 7 as regions with a high number of mapped eccDNA gene density. Mapping to 15 known herbicide-resistant QTL regions showed that the eccDNA coding sequences matched twelve, with four QTL matching HS coding sequences; only one region contained HR coding sequences. These findings establish that, like other pernicious weeds, blackgrass has eccDNAs that contain homologs of chromosomal genes, and these may contribute genetic heterogeneity and evolutionary innovation to rapidly adapt to abiotic stresses, including herbicide treatment.

CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination.

DNA repair in mammalian cells involves the coordinated action of a range of complex cellular repair machinery. Our understanding of these DNA repair processes has advanced to the extent that they can be leveraged to improve the efficacy and precision of Cas9-assisted genome editing tools. Here, we review how the fusion of CRISPR-Cas9 to functional domains of proteins that directly or indirectly impact the DNA repair process can enhance genome editing. Such studies have allowed the development of diverse technologies that promote efficient gene knock-in for safer genome engineering practices.

Divergence time estimates for the hypoxia-inducible factor-1 alpha (HIF1α) reveal an ancient emergence of animals in low-oxygen environments.

Unveiling the tempo and mode of animal evolution is necessary to understand the links between environmental changes and biological innovation. Although the earliest unambiguous metazoan fossils date to the late Ediacaran period, molecular clock estimates agree that the last common ancestor (LCA) of all extant animals emerged ~850 Ma, in the Tonian period, before the oldest evidence for widespread ocean oxygenation at ~635-560 Ma in the Ediacaran period. Metazoans are aerobic organisms, that is, they are dependent on oxygen to survive. In low-oxygen conditions, most animals have an evolutionarily conserved pathway for maintaining oxygen homeostasis that triggers physiological changes in gene expression via the hypoxia-inducible factor (HIFa). However, here we confirm the absence of the characteristic HIFa protein domain responsible for the oxygen sensing of HIFa in sponges and ctenophores, indicating the LCA of metazoans lacked the functional protein domain as well, and so could have maintained their transcription levels unaltered under the very low-oxygen concentrations of their environments. Using Bayesian relaxed molecular clock dating, we inferred that the ancestral gene lineage responsible for HIFa arose in the Mesoproterozoic Era, ~1273 Ma (Credibility Interval 957-1621 Ma), consistent with the idea that important genetic machinery associated with animals evolved much earlier than the LCA of animals. Our data suggest at least two duplication events in the evolutionary history of HIFa, which generated three vertebrate paralogs, products of the two successive whole-genome duplications that occurred in the vertebrate LCA. Overall, our results support the hypothesis of a pre-Tonian emergence of metazoans under low-oxygen conditions, and an increase in oxygen response elements during animal evolution.

The Functionality of Membrane-Inserting Proteins and Peptides: Curvature Sensing, Generation, and Pore Formation.

Proteins and peptides with hydrophobic and amphiphilic segments are responsible for many biological functions. The sensing and generation of membrane curvature are the functions of several protein domains or motifs. While some specific membrane proteins play an essential role in controlling the curvature of distinct intracellular membranes, others participate in various cellular processes such as clathrin-mediated endocytosis, where several proteins sort themselves at the neck of the membrane bud. A few membrane-inserting proteins form nanopores that permeate selective ions and water to cross the membrane. In addition, many natural and synthetic small peptides and protein toxins disrupt the membrane by inducing nonspecific pores in the membrane. The pore formation causes cell death through the uncontrolled exchange between interior and exterior cellular contents. In this article, we discuss the insertion depth and orientation of protein/peptide helices, and their role as a sensor and inducer of membrane curvature as well as a pore former in the membrane. We anticipate that this extensive review will assist biophysicists to gain insight into curvature sensing, generation, and pore formation by membrane insertion.

Molecular cloning and functional analysis of flavanone 3-hydroxylase (f3h）gene in Passiflora edulis Sims

Flavanone 3-hydroxylase (F3H) plays a crucial role in the biosynthesis of flavonoids. In the present study, one F3H gene (P_edulia040010337.g) from Passiflora edulis Sims, which has a coding sequence (CDS) of 1161 bp, encoding a protein consisting of 386 amino acid residues was cloned. The PeF3H protein contains a non-heme dioxygenase (DIOX-N superfamily) domain and a typical F3H protein functional domain (2OG-FeII-Oxy dioxygenase). Phylogenetic analysis revealed that the PeF3H protein shared high similarity with F3H proteins in Turnera subulata, Populus alba, and Populus tomentosa, with 88% identities of amino acid sequences. The PeF3H protein lacks a transmembrane structure, indicating it is likely to be expressed in the mitochondria. Additionally, 3D structure, protein and protein interaction, and KEGG pathway of PeF3H were anticipated based on homologous proteins. qRT-PCR analysis showed that PeF3H was highly expressed in leaves, followed by stems and roots. These studies have provided insights into the molecular mechanisms underlying flavonoid biosynthesis and predicted potential targets for genetic engineering to improve the nutritional and medicinal properties of passion fruit. Bangladesh J. Bot. 52(2): 613-623, 2023 (June) Special

Molecular cloning and functional analysis of flavanone 3-hydroxylase (f3h）gene in Passiflora edulis Sims

Flavanone 3-hydroxylase (F3H) plays a crucial role in the biosynthesis of flavonoids. In the present study, one F3H gene (P_edulia040010337.g) from Passiflora edulis Sims, which has a coding sequence (CDS) of 1161 bp, encoding a protein consisting of 386 amino acid residues was cloned. The PeF3H protein contains a non-heme dioxygenase (DIOX-N superfamily) domain and a typical F3H protein functional domain (2OG-FeII-Oxy dioxygenase). Phylogenetic analysis revealed that the PeF3H protein shared high similarity with F3H proteins in Turnera subulata, Populus alba, and Populus tomentosa, with 88% identities of amino acid sequences. The PeF3H protein lacks a transmembrane structure, indicating it is likely to be expressed in the mitochondria. Additionally, 3D structure, protein and protein interaction, and KEGG pathway of PeF3H were anticipated based on homologous proteins. qRT-PCR analysis showed that PeF3H was highly expressed in leaves, followed by stems and roots. These studies have provided insights into the molecular mechanisms underlying flavonoid biosynthesis and predicted potential targets for genetic engineering to improve the nutritional and medicinal properties of passion fruit. Bangladesh J. Bot. 52(2): 613-623, 2023 (June) Special

Diverse Clinical Phenotypes of CASK-Related Disorders and Multiple Functional Domains of CASK Protein.

CASK-related disorders are a form of rare X-linked neurological diseases and most of the patients are females. They are characterized by several symptoms, including microcephaly with pontine and cerebellar hypoplasia (MICPCH), epilepsy, congenital nystagmus, and neurodevelopmental disorders. Whole-genome sequencing has identified various mutations, including nonsense and missense mutations, from patients with CASK-related disorders, revealing correlations between specific mutations and clinical phenotypes. Notably, missense mutations associated with epilepsy and intellectual disability were found throughout the whole region of the CASK protein, while missense mutations related to microcephaly and MICPCH were restricted in certain domains. To investigate the pathophysiology of CASK-related disorders, research groups have employed diverse methods, including the generation of CASK knockout mice and the supplementation of CASK to rescue the phenotypes. These approaches have yielded valuable insights into the identification of functional domains of the CASK protein associated with a specific phenotype. Additionally, recent advancements in the AI-based prediction of protein structure, such as AlphaFold2, and the application of genome-editing techniques to generate CASK mutant mice carrying missense mutations from patients with CASK-related disorders, allow us to understand the pathophysiology of CASK-related disorders in more depth and to develop novel therapeutic methods for the fundamental treatment of CASK-related disorders.

Host Factor Nucleophosmin 1 (NPM1/B23) Exerts Antiviral Effects against Chikungunya Virus by Its Interaction with Viral Nonstructural Protein 3.

Chikungunya virus (CHIKV) hijacks host cell machinery to support its replication. Nucleophosmin 1 (NPM1/B23), a nucleolar phosphoprotein, is one of the host proteins known to restrict CHIKV infection; however, the mechanistic details of the antiviral role of NPM1 are not elucidated. It was seen in our experiments that the level of NPM1 expression affected the expression levels of interferon-stimulated genes (ISGs) that play antiviral roles in CHIKV infection, such as IRF1, IRF7, OAS3, and IFIT1, indicating that one of the antiviral mechanisms could be through modulation of interferon-mediated pathways. Our experiments also identified that for CHIKV restriction, NPM1 must move from the nucleus to the cytoplasm. A deletion of the nuclear export signal (NES), which confines NPM1 within the nucleus, abolishes its anti-CHIKV action. We observed that NPM1 binds CHIKV nonstructural protein 3 (nsP3) strongly via its macrodomain, thereby exerting a direct interaction with viral proteins to limit infection. Based on site-directed mutagenesis and coimmunoprecipitation studies, it was also observed that amino acid residues N24 and Y114 of the CHIKV nsP3 macrodomain, known to be involved in virus virulence, bind ADP-ribosylated NPM1 to inhibit infection. Overall, the results show a key role of NPM1 in CHIKV restriction and indicate it as a promising host target for developing antiviral strategies against CHIKV. IMPORTANCE Chikungunya, a recently reemerged mosquito-borne infection caused by a positive-sense, single-stranded RNA virus, has caused explosive epidemics in tropical regions. Unlike the classical symptoms of acute fever and debilitating arthralgia, incidences of neurological complications and mortality were reported. Currently there are no antivirals or commercial vaccines available against chikungunya. Like all viruses, CHIKV uses host cellular machinery for establishment of infection and successful replication. To counter this, the host cell activates several restriction factors and innate immune response mediators. Understanding these host-virus interactions helps to develop host-targeted antivirals against the disease. Here, we report the antiviral role of the multifunctional host protein NPM1 against CHIKV. The significant inhibitory effect of this protein against CHIKV involves its increased expression and movement from its natural location within the nucleus to the cytoplasm. There, it interacts with functional domains of key viral proteins. Our results support ongoing efforts toward development of host-directed antivirals against CHIKV and other alphaviruses.

Phylobone: a comprehensive database of bone extracellular matrix proteins in human and model organisms

The bone extracellular matrix (ECM) contains minerals deposited on highly crosslinked collagen fibrils and hundreds of non-collagenous proteins. Some of these proteins are key to the regulation of bone formation and regeneration via signaling pathways, and play important regulatory and structural roles. However, the complete list of bone extracellular matrix proteins, their roles, and the extent of individual and cross-species variations have not been fully captured in both humans and model organisms. Here, we introduce the most comprehensive resource of bone extracellular matrix (ECM) proteins that can be used in research fields such as bone regeneration, osteoporosis, and mechanobiology. The Phylobone database (available at https://phylobone.com) includes 255 proteins potentially expressed in the bone extracellular matrix (ECM) of humans and 30 species of vertebrates. A bioinformatics pipeline was used to identify the evolutionary relationships of bone ECM proteins. The analysis facilitated the identification of potential model organisms to study the molecular mechanisms of bone regeneration. A network analysis showed high connectivity of bone ECM proteins. A total of 214 functional protein domains were identified, including collagen and the domains involved in bone formation and resorption. Information from public drug repositories was used to identify potential repurposing of existing drugs. The Phylobone database provides a platform to study bone regeneration and osteoporosis in light of (biological) evolution, and will substantially contribute to the identification of molecular mechanisms and drug targets.

DrosOMA: the Drosophila Orthologous Matrix browser

Background: Comparative genomic analyses to delineate gene evolutionary histories inform the understanding of organismal biology by characterising gene and gene family origins, trajectories, and dynamics, as well as enabling the tracing of speciation, duplication, and loss events, and facilitating the transfer of gene functional information across species. Genomic data are available for an increasing number of species from the genus Drosophila, however, a dedicated resource exploiting these data to provide the research community with browsable results from genus-wide orthology delineation has been lacking. Methods: Using the OMA Orthologous Matrix orthology inference approach and browser deployment framework, we catalogued orthologues across a selected set of Drosophila species with high-quality annotated genomes. We developed and deployed a dedicated instance of the OMA browser to facilitate intuitive exploration, visualisation, and downloading of the genus-wide orthology delineation results. Results: DrosOMA - the Drosophila Orthologous Matrix browser, accessible from https://drosoma.dcsr.unil.ch/ - presents the results of orthology delineation for 36 drosophilids from across the genus and four outgroup dipterans. It enables querying and browsing of the orthology data through a feature-rich web interface, with gene-view, orthologous group-view, and genome-view pages, including comprehensive gene name and identifier cross-references together with available functional annotations and protein domain architectures, as well as tools to visualise local and global synteny conservation. Conclusions: The DrosOMA browser demonstrates the deployability of the OMA browser framework for building user-friendly orthology databases with dense sampling of a selected taxonomic group. It provides the Drosophila research community with a tailored resource of browsable results from genus-wide orthology delineation.

Characterization, Directed Evolution, and Targeting of DNA Virus-Encoded RNA Capping Enzymes Using Phenotypic Yeast Platforms.

The constant and the sudden emergence of zoonotic human and animal viruses is a significant threat to human health, the world economy, and the world food supply. This has necessitated the development of broad-spectrum therapeutic strategies to combat these emerging pathogens. Mechanisms that are essential for viral replication and propagation have been successfully targeted in the past to develop broad-spectrum therapeutics that can be readily repurposed to combat new zoonotic pathogens. Because of the importance of viral RNA capping enzymes to viral replication and pathogenesis, as well as their presence in both DNA and RNA viruses, these viral proteins have been a long-standing therapeutic target. Here, we use genome sequencing information and yeast-based platforms (YeRC0M) to identify, characterize, and target viral genome-encoded essential RNA capping enzymes from emerging strains of DNA viruses, i.e., Monkeypox virus and African Swine Fever Virus, which are a significant threat to human and domestic animal health. We first identified and biochemically characterized these viral RNA capping enzymes and their necessary protein domains. We observed significant differences in functional protein domains and organization for RNA capping enzymes from emerging DNA viruses in comparison to emerging RNA viruses. We also observed several differences in the biochemical properties of these viral RNA capping enzymes using our phenotypic yeast-based approaches (YeRC0M) as compared to the previous in vitro studies. Further, using directed evolution, we were able to identify inactivation and attenuation mutations in these essential viral RNA capping enzymes; these data could have implications on virus biocontainment as well as live attenuated vaccine development. We also developed methods that would facilitate high-throughput phenotypic screening to identify broad-spectrum inhibitors that selectively target viral RNA capping enzymes over host RNA capping enzymes. As demonstrated here, our approaches to identify, characterize, and target viral genome-encoded essential RNA capping enzymes are highly modular and can be readily adapted for targeting emerging viral pathogens as well as their variants that emerge in the future.

Expression of SRP-9001 dystrophin and stabilization of motor function up to 2 years post-treatment with delandistrogene moxeparvovec gene therapy in individuals with Duchenne muscular dystrophy.

Introduction: Delandistrogene moxeparvovec (SRP-9001) is an investigational gene transfer therapy designed for targeted expression of SRP-9001 dystrophin protein, a shortened dystrophin retaining key functional domains of the wild-type protein. Methods: This Phase 2, double-blind, two-part (48weeks per part) crossover study (SRP-9001-102 [Study 102]; NCT03769116) evaluated delandistrogene moxeparvovec in patients, aged ≥4 to <8years with Duchenne muscular dystrophy. Primary endpoints (Part 1) were change from baseline (CFBL) in SRP-9001 dystrophin expression (Week 12), by Western blot, and in North Star Ambulatory Assessment (NSAA) score (Week 48). Safety assessments included treatment-related adverse events (TRAEs). Patients were randomized and stratified by age to placebo (n = 21) or delandistrogene moxeparvovec (n = 20) and crossed over for Part 2. Results: SRP-9001 dystrophin expression was achieved in all patients: mean CFBL to Week 12 was 23.82% and 39.64% normal in Parts 1 and 2, respectively. In Part 1, CFBL to Week 48 in NSAA score (least-squares mean, LSM [standard error]) was +1.7 (0.6) with treatment versus +0.9 (0.6) for placebo; p = 0.37. Disparity in baseline motor function between groups likely confounded these results. In 4- to 5-year-olds with matched baseline motor function, CFBL to Week 48 in NSAA scores was significantly different (+2.5 points; p = 0.0172), but not significantly different in 6-to-7-year-olds with imbalanced baseline motor function (-0.7 points; p = 0.5384). For patients treated with delandistrogene moxeparvovec in Part 2, CFBL to Week 48 in NSAA score was +1.3 (2.7), whereas for those treated in Part 1, NSAA scores were maintained. As all patients in Part 2 were exposed to treatment, results were compared with a propensity-score-weighted external control (EC) cohort. The LSM difference in NSAA score between the Part 2 treated group and EC cohort was statistically significant (+2.0 points; p = 0.0009). The most common TRAEs were vomiting, decreased appetite, and nausea. Most occurred within the first 90days and all resolved. Discussion: Results indicate robust expression of SRP-9001 dystrophin and overall stabilization in NSAA up to 2years post-treatment. Differences in NSAA between groups in Part 1 were not significant for the overall population, likely because cohorts were stratified only by age, and other critical prognostic factors were not well matched at baseline.

The recombinase activating genes: architects of immune diversity during lymphocyte development.

The mature lymphocyte population of a healthy individual has the remarkable ability to recognise an immense variety of antigens. Instead of encoding a unique gene for each potential antigen receptor, evolution has used gene rearrangements, also known as variable, diversity, and joining gene segment (V(D)J) recombination. This process is critical for lymphocyte development and relies on recombination-activating genes-1 (RAG1) and RAG2, here collectively referred to as RAG. RAG serves as powerful genome editing tools for lymphocytes and is strictly regulated to prevent dysregulation. However, in the case of dysregulation, RAG has been implicated in cases of cancer, autoimmunity and severe combined immunodeficiency (SCID). This review examines functional protein domains and motifs of RAG, describes advances in our understanding of the function and (dys)regulation of RAG, discuss new therapeutic options, such as gene therapy, for RAG deficiencies, and explore in vitro and in vivo methods for determining RAG activity and target specificity.

Genomic Characteristics of the New HIV-1 CRF07_BC K28E32 Variant.

Accompanied with the appearance and prevalence of the new K28E32 variant among men who have sex with men, HIV-1 circulating recombinant form 07_BC (CRF07_BC) was becoming the most predominant subtype circulating in China. The K28E32 variant with five specific mutations in reverse transcriptase coding region appears to have significantly higher in vitro HIV-1 replication ability than the wild-type strain. In this study, we characterized the special mutations/substitutions in the K28E32 variant at the genomic level. Ten specific mutations that rarely appeared in other six main HIV-1 subtypes/CRFs (A-D, CRF01_AE, and CRF02_AG) were identified in the coding genes/regions of the K28E32 variant, including S77L and a novel seven-amino acid detection (32DKELYPL38) (p6Δ7) in p6, I135L in integrase, T189S in Vif, H/Y15L/F in Vpr, I264V/A and LV/LI328-329VG in gp41, and H82C and S97P in Rev. The special locations of the novel p6Δ7, and gp41 mutations I264V/A and LV/LI328-329VG in crucial protein functional domains suggest that these mutations might be functionally important to the K28E32 variant. Furthermore, eight specific substitutions were identified in Rev responsive element (RRE) of the K28E32 variant, and were revealed to increase the stability of RRE structure with a lower minimum free energy. Whether these mutations/substitutions contribute to improved transmissibility of the CRF07_BC K28E32 variant needs to be further confirmed.