Correlation between Phenotype and Genotype in CTNNB1 Syndrome: A Systematic Review of the Literature.

The CTNNB1 Syndrome is a rare neurodevelopmental disorder associated with developmental delay, intellectual disability, and delayed or absent speech. The aim of the present study is to systematically review the available data on the prevalence of clinical manifestations and to evaluate the correlation between phenotype and genotype in published cases of patients with CTNNB1 Syndrome. Studies were identified by systematic searches of four major databases. Information was collected on patients' genetic mutations, prenatal and neonatal problems, head circumference, muscle tone, EEG and MRI results, dysmorphic features, eye abnormalities, early development, language and comprehension, behavioral characteristics, and additional clinical problems. In addition, the mutations were classified into five groups according to the severity of symptoms. The study showed wide genotypic and phenotypic variability in patients with CTNNB1 Syndrome. The most common moderate-severe phenotype manifested in facial dysmorphisms, microcephaly, various motor disabilities, language and cognitive impairments, and behavioral abnormalities (e.g., autistic-like or aggressive behavior). Nonsense and missense mutations occurring in exons 14 and 15 were classified in the normal clinical outcome category/group because they had presented an otherwise normal phenotype, except for eye abnormalities. A milder phenotype was also observed with missense and nonsense mutations in exon 13. The autosomal dominant CTNNB1 Syndrome encompasses a wide spectrum of clinical features, ranging from normal to severe. While mutations cannot be more generally categorized by location, it is generally observed that the C-terminal protein region (exons 13, 14, 15) correlates with a milder phenotype.

The role of the C-terminal D0 domain of flagellin in activation of Toll like receptor 5.

Flagellin is a wide-spread bacterial virulence factor sensed by the membrane-bound Toll-like receptor 5 (TLR5) and by the intracellular NAIP5/NLRC4 inflammasome receptor. TLR5 recognizes a conserved region within the D1 domain of flagellin, crucial for the interaction between subunits in the flagellum and for bacterial motility. While it is known that a deletion of the D0 domain of flagellin, which lines the interior of flagella, also completely abrogates activation of TLR5, its functional role remains unknown. Using a protein fusion strategy, we propose a role for the D0 domain in the stabilization of an active dimeric signaling complex of flagellin-TLR5 at a 2:2 stoichiometric ratio. Alanine-scanning mutagenesis of flagellin revealed a previously unidentified region of flagellin, the C-terminal D0 domain, to play a crucial role in TLR5 activation. Interestingly, we show that TLR5 recognizes the same hydrophobic motif of the D0 domain of flagellin as the intracellular NAIP5/NLRC4 inflammasome receptor. Further, we show that residues within the D0 domain play a previously unrecognized role in the evasion of TLR5 recognition by Helicobacter pylori. These findings demonstrate that TLR5 is able to simultaneously sense several spatially separated sites of flagellin that are essential for its functionality, hindering bacterial evasion of immune recognition. Our findings significantly contribute to the understanding of the mechanism of TLR5 activation, which plays an important role in host defense against several pathogens, but also in several diseases, such as Crohn's disease, cystic fibrosis and rheumatoid arthritis.

The art of designed coiled-coils for the regulation of mammalian cells.

Synthetic biology aims to engineer complex biological systems using modular elements, with coiled-coil (CC) dimer-forming modules are emerging as highly useful building blocks in the regulation of protein assemblies and biological processes. Those small modules facilitate highly specific and orthogonal protein-protein interactions, offering versatility for the regulation of diverse biological functions. Additionally, their design rules enable precise control and tunability over these interactions, which are crucial for specific applications. Recent advancements showcase their potential for use in innovative therapeutic interventions and biomedical applications. In this review, we discuss the potential of CCs, exploring their diverse applications in mammalian cells, such as synthetic biological circuit design, transcriptional and allosteric regulation, cellular assemblies, chimeric antigen receptor (CAR) T cell regulation, and genome editing and their role in advancing the understanding and regulation of cellular processes.

Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing.

The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool, revolutionizing biological and biomedical sciences, where an improvement of efficiency could have a strong impact. Here we present a strategy to enhance gene editing based on the concerted action of Cas9 and an exonuclease. Non-covalent recruitment of exonuclease to Cas9/gRNA complex via genetically encoded coiled-coil based domains, termed CCExo, recruited the exonuclease to the cleavage site and robustly increased gene knock-out due to progressive DNA strand recession at the cleavage site, causing decreased re-ligation of the nonedited DNA. CCExo exhibited increased deletion size and enhanced gene inactivation efficiency in the context of several DNA targets, gRNA selection, Cas variants, tested cell lines and type of delivery. Targeting a sequence-specific oncogenic chromosomal translocation using CCExo in cells of chronic myelogenous leukemia patients and in an animal model led to the reduction or elimination of cancer, establishing it as a highly specific tool for treating CML and potentially other appropriate diseases with genetic etiology.

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and ß-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small ß-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The ß-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the ß-annulus peptide for vaccine design.

Increased gene translation stringency in mammalian cells by nonsense suppression at multiple permissive sites with a single noncanonical amino acid.

The considerable potential of engineered cells compels the development of strategies for the stringent control of gene expression. A promising approach is the introduction of a premature stop codon (PTC) into a selected gene that is expressed only in the presence of noncanonical amino acids through nonsense suppression. Here, different strategies of amber PTC readthrough in mammalian cells were tested. The use of a tRNA synthetase together with a TAG codon-specific tRNA achieved PTC readthrough depending on the addition of a noncanonical amino acid (4-benzoyl-L-phenylalanine; Bpa). While single TAG codon incorporation exhibited detectable expression of the reporter protein even in the absence of Bpa, the use of a double PTC enabled virtually leakage-free functional gene translation. The introduction of an additional 5'-PTC, therefore, represents a generally applicable strategy to increase stringency in gene translation.

CRISPRa-mediated FOXP3 gene upregulation in mammalian cells.

BACKGROUND: Forkhead box P3+ (FOXP3 +) regulatory T cells (Tregs) are a subset of lymphocytes, critical for the maintenance of immune homeostasis. Loss-of-function mutations of the FOXP3 gene in animal models and humans results in loss of differentiation potential into Treg cells and are responsible for several immune-mediated inflammatory diseases. Strategies of increasing FOXP3 expression represent a potential approach to increase the pool of Tregs within the lymphocyte population and may be employed in therapies of diverse autoimmune conditions. In the present study, a dCas9 CRISPR-based method was systematically employed to achieve upregulation and sustained high expression of endogenous FOXP3 in HEK293 and human Jurkat T cell lines through targeting of the core promotor, three known regulatory regions of the FOXP3 gene (CNS1-3), and two additional regions selected through extensive bioinformatics analysis (Cage1 and Cage2). RESULTS: Using an activator-domain fusion based dCas9 transcription activator, robust upregulation of FOXP3 was achieved, and an optimal combination of single guide RNAs was selected, which exerted an additive effect on FOXP3 gene upregulation. Simultaneous targeting of FOXP3 and EOS, a transcription factor known to act in concert with FOXP3 in initiating a Treg phenotype, resulted in upregulation of FOXP3 downstream genes CD25 and TNFR2. When compared to ectopic expression of FOXP3 via plasmid electroporation, upregulation of endogenous FOXP3 via the Cas9-based method resulted in prolonged expression of FOXP3 in Jurkat cells. CONCLUSIONS: Transfection of both HEK293 and Jurkat cells with dCas9-activators showed that regulatory regions downstream and upstream of FOXP3 promoter can be very potent transcription inducers in comparison to targeting the core promoter. While introduction of genes by conventional methods of gene therapy may involve a risk of insertional mutagenesis due to viral integration into the genome, transient up- or down-regulation of transcription by a CRISPR-dCas9 approach may resolve this safety concern. dCas9-based systems provide great promise in DNA footprint-free phenotype perturbations (perturbation without the risk of DNA damage) to drive development of transcription modulation-based therapies.

Extension and refinement of the recognition motif for Toll-like receptor 5 activation by flagellin.

TLRs sense conserved and essential molecular components of microbes that invade multicellular organisms. The wide range of TLR agonists, differing in size and shape, is recognized either through a single or a pair of binding sites on the ectodomains of TLRs. TLR5 recognizes bacterial flagellin through two distinct binding sites on the ectodomain, the first facilitating primary binding of flagellin and the second guiding receptor dimerization necessary for signaling. The regions of flagellin recognized by TLR5 encompass key functional regions within the D1 domain of flagellin, which is also required for the assembly of functional flagella. In addition to previously identified binding sites at the N-terminal and central segment of the TLR5 ectodomain, we extended the TLR5'-D1 interaction interface on TLR5 and showed a species-specific recognition relevance of this extended region. In addition, we showed that the loop and following ß-hairpin region of flagellin, previously proposed to participate in the TLR5-flagellin dimerization interface, is not accountable for these species-specific differences. We further identified residues that contribute to the interaction between two TLR5 ectodomains in an active signaling complex. Our work demonstrates that flagellin is recognized by TLR5 through a more extensive interaction surface than previously characterized.

Distinctive Recognition of Flagellin by Human and Mouse Toll-Like Receptor 5.

Toll-like receptor 5 (TLR5) is a receptor of the innate immune system that recognizes flagellin from certain bacterial species and triggers an inflammatory response. The Salmonella dublin flagellin in complex with zebrafish TLR5 has been crystallized previously. In the present study, we extrapolate the structure of this complex using structure-guided mutagenesis to determine the recognition modes of human and mouse TLR5 receptors and demonstrate species-specific differences in flagellin recognition. In general, the recognition mode of the mouse receptor can be said to be more robust in comparison to that of the human receptor. All-atom molecular dynamics simulation showed differences between the two receptors within the primary binding region. Using a functional motility assay, we show that although the highly conserved area of the flagellin analyzed in this study encompasses key structural requirements for flagella formation, a direct correlation between immune recognition and structure on the level of amino acid residues is not observed.

A bistable genetic switch based on designable DNA-binding domains.

Bistable switches are fundamental regulatory elements of complex systems, ranging from electronics to living cells. Designed genetic toggle switches have been constructed from pairs of natural transcriptional repressors wired to inhibit one another. The complexity of the engineered regulatory circuits can be increased using orthogonal transcriptional regulators based on designed DNA-binding domains. However, a mutual repressor-based toggle switch comprising DNA-binding domains of transcription-activator-like effectors (TALEs) did not support bistability in mammalian cells. Here, the challenge of engineering a bistable switch based on monomeric DNA-binding domains is solved via the introduction of a positive feedback loop composed of activators based on the same TALE domains as their opposing repressors and competition for the same DNA operator site. This design introduces nonlinearity and results in epigenetic bistability. This principle could be used to employ other monomeric DNA-binding domains such as CRISPR for applications ranging from reprogramming cells to building digital biological memory.