Cognate Target Research Articles

Rapid, Sensitive, and Specific Microbial Whole-Cell Biosensor for the Detection of Histamine: A Potential Food Toxin.

Histamine is a biogenic amine; its level indicates food quality, as elevated levels cause food poisoning. Therefore, monitoring food at each step during processing until it reaches the consumer is crucial, but current techniques are complicated and time-consuming. Here, we designed a Pseudomonas putida whole-cell biosensor using a histamine-responsive genetic element expressing a fluorescent protein in the presence of the cognate target. We improved the performance of the proposed biosensor by optimizing the chassis, genetic regulatory element, and reporter gene. A sensitive and rapid biosensor variant was obtained with a limit of detection (LOD) of 0.39 ppm, manifesting a linear response (R2 = 0.98) from 0.28 to 18 ppm in 90 min. The biosensor showed minimal cross-reactivity with other biogenic amines and amino acids prevalent in food, making it highly specific. The biosensor effectively quantified histamine in spiked fish, prawn, and wine samples with a satisfactory recovery. Additionally, a colorimetric sensor variant PAlacZ was developed enabling histamine quantification in seafood via a smartphone application, with an LODgray of 0.23 ppm, exhibiting a linear response from 0 to 2.24 ppm. Overall, this study reports an efficient, specific, and highly sensitive biosensor with strong potential for the on-site detection of histamine, ensuring food safety.

Developmental and housekeeping transcriptional programs display distinct modes of enhancer-enhancer cooperativity in Drosophila

Genomic enhancers are key transcriptional regulators which, upon the binding of sequence-specific transcription factors, activate their cognate target promoters. Although enhancers have been extensively studied in isolation, a substantial number of genes have more than one simultaneously active enhancer, and it remains unclear how these cooperate to regulate transcription. Using Drosophila melanogaster S2 cells as a model, we assay the activities of more than a thousand individual enhancers and about a million enhancer pairs toward housekeeping and developmental core promoters with STARR-seq. We report that housekeeping and developmental enhancers show distinct modes of enhancer-enhancer cooperativity: while housekeeping enhancers are additive such that their combined activity mirrors the sum of their individual activities, developmental enhancers are super-additive and combine multiplicatively. Super-additivity between developmental enhancers is promiscuous and neither depends on the enhancers’ endogenous genomic contexts nor on specific transcription factor motif signatures. However, it can be further boosted by Twist and Trl motifs and saturates for the highest levels of enhancer activity. These results have important implications for our understanding of gene regulation in complex multi-enhancer developmental loci and genomically clustered housekeeping genes, providing a rationale to interpret the transcriptional impact of non-coding mutations at different loci.

The fitness cost of spurious phosphorylation.

The fidelity of signal transduction requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. How such off-target interactions impact fitness is not generally known. Here, we use Saccharomyces cerevisiae to inducibly express tyrosine kinases. Because yeast lacks bona fide tyrosine kinases, the resulting tyrosine phosphorylation is biologically spurious. We engineered 44 yeast strains each expressing a tyrosine kinase, and quantitatively analysed their phosphoproteomes. This analysis resulted in ~30,000 phosphosites mapping to ~3500 proteins. The number of spurious pY sites generated correlates strongly with decreased growth, and we predict over 1000 pY events to be deleterious. However, we also find that many of the spurious pY sites have a negligible effect on fitness, possibly because of their low stoichiometry. This result is consistent with our evolutionary analyses demonstrating a lack of phosphotyrosine counter-selection in species with tyrosine kinases. Our results suggest that, alongside the risk for toxicity, the cell can tolerate a large degree of non-functional crosstalk as interaction networks evolve.

ANTIPASTI: Interpretable prediction of antibody binding affinity exploiting normal modes and deep learning

The high binding affinity of antibodies toward their cognate targets is key to eliciting effective immune responses, as well as to the use of antibodies as research and therapeutic tools. Here, we propose ANTIPASTI, a convolutional neural network model that achieves state-of-the-art performance in the prediction of antibody binding affinity using as input a representation of antibody-antigen structures in terms of normal mode correlation maps derived from elastic network models. This representation captures not only structural features but energetic patterns of local and global residue fluctuations. The learnt representations are interpretable: they reveal similarities of binding patterns among antibodies targeting the same antigen type, and can be used to quantify the importance of antibody regions contributing to binding affinity. Our results show the importance of the antigen imprint in the normal mode landscape, and the dominance of cooperative effects and long-range correlations between antibody regions to determine binding affinity.

The Crystal Structure of the Michaelis-Menten Complex of C1 Esterase Inhibitor and C1s Reveals Novel Insights into Complement Regulation.

The ancient arm of innate immunity known as the complement system is a blood proteolytic cascade involving dozens of membrane-bound and solution-phase components. Although many of these components serve as regulatory molecules to facilitate controlled activation of the cascade, C1 esterase inhibitor (C1-INH) is the sole canonical complement regulator belonging to a superfamily of covalent inhibitors known as serine protease inhibitors (SERPINs). In addition to its namesake role in complement regulation, C1-INH also regulates proteases of the coagulation, fibrinolysis, and contact pathways. Despite this, the structural basis for C1-INH recognition of its target proteases has remained elusive. In this study, we present the crystal structure of the Michaelis-Menten (M-M) complex of the catalytic domain of complement component C1s and the SERPIN domain of C1-INH at a limiting resolution of 3.94 Å. Analysis of the structure revealed that nearly half of the protein/protein interface is formed by residues outside of the C1-INH reactive center loop. The contribution of these residues to the affinity of the M-M complex was validated by site-directed mutagenesis using surface plasmon resonance. Parallel analysis confirmed that C1-INH-interfacing residues on C1s surface loops distal from the active site also drive affinity of the M-M complex. Detailed structural comparisons revealed differences in substrate recognition by C1s compared with C1-INH recognition and highlight the importance of exosite interactions across broader SERPIN/protease systems. Collectively, this study improves our understanding of how C1-INH regulates the classical pathway of complement, and it sheds new light on how SERPINs recognize their cognate protease targets.

Autoantibodies as putative biomarkers and triggers of cell dysfunctions in systemic sclerosis.

Antinuclear autoantibodies represent a serological hallmark of systemic sclerosis (SSc), with anticentromere, antitopoisomerase-I, and anti-RNA polymerase III antibodies routinely assessed for diagnosis, clinical subset classification, and prognosis. In addition, an increasing number of autoantibodies have been demonstrated to play a pathogenic role by mediating different SSc manifestations. This review aims to give an overview on autoantibodies as putative biomarkers in SSc and discuss their possible pathogenic role as triggers of cell dysfunctions. Over the years, different autoantibodies have been proposed as biomarkers aiding in diagnosis, disease subtype classification, disease progression prediction, organ involvement, as well as in understanding treatment response. Increasing literature also indicates functional autoantibodies as direct contributors to SSc pathogenesis by exerting agonistic or antagonistic activities on their specific cognate targets. In SSc, search and validation of novel autoantibodies with higher diagnostic specificity and more accurate predictive values are increasingly needed for early diagnosis and specific follow-up, and to define the best therapeutic option according to different disease subsets. Moreover, since autoantibodies are also emerging as functional pathogenic players, a better unraveling of their possible pathomechanisms becomes essential to identify new targets and develop promising therapeutic agents able to neutralize their effects.

Staphylococcus aureus Proteases: Orchestrators of Skin Inflammation.

Skin homeostasis relies on a delicate balance between host proteases and protease inhibitors along with those secreted from microbial communities, as disruption to this harmony contributes to the pathogenesis of inflammatory skin disorders, including atopic dermatitis and Netherton's syndrome. In addition to being a prominent cause of skin and soft tissue infections, the gram-positive bacterium Staphylococcus aureus is a key player in inflammatory skin conditions due to its array of 10 secreted proteases. Herein we review how S. aureus proteases augment the development of inflammation in skin disorders. These mechanisms include degradation of skin barrier integrity, immune dysregulation and pruritis, and impairment of host defenses. Delineating the diverse roles of S. aureus proteases has the potential to reveal novel therapeutic strategies, such as inhibitors of proteases or their cognate target, as well as neutralizing vaccines to alleviate the burden of inflammatory skin disorders in patients.

Non-Canonical Targets of MicroRNAs: Role in Transcriptional Regulation, Disease Pathogenesis and Potential for Therapeutic Targets.

MicroRNAs are a class of regulatory, non-coding small ribonucleic acid (RNA) molecules found in eukaryotes. Dysregulated expression of microRNAs can lead to downregulation or upregulation of their target gene. In general, microRNAs bind with the Argonaute protein and its interacting partners to form a silencing complex. This silencing complex binds with fully or partial complementary sequences in the 3'-UTR of their cognate target mRNAs and leads to degradation of the transcripts or translational inhibition, respectively. However, recent developments point towards the ability of these microRNAs to bind to the promoters, enhancers or coding sequences, leading to upregulation of their target genes. This review briefly summarizes the various non-canonical binding sites of microRNAs and their regulatory roles in various diseased conditions.

KLF2 enhancer variant rs4808485 increases lupus risk by modulating inflammasome machinery and cellular homoeostasis

ObjectiveA recent genome-wide association study linked KLF2 as a novel Asian-specific locus for systemic lupus erythematosus (SLE) susceptibility. However, the underlying causal functional variant(s), cognate target gene(s) and genetic mechanisms...

PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are "open" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin "pioneering factor" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.

Bilingual parafoveal processing: Children and adults preprocess orthographic information of the upcoming word during sentence reading in their first and second language.

Readers of different ages and across different languages routinely process information of upcoming words in a sentence, before their eyes move to fixate them directly (parafoveal processing). However, there is inconsistent evidence of similar parafoveal processing in a reader's second language (L2). In this eye movement study, the gaze-contingent boundary paradigm (Rayner, 1975a) was used to test whether parafoveal processing of orthographic information is an integral part of both beginning and proficient L2 reading. The eye movements of beginning L2-learners (n = 53, aged 11-14 years) and highly proficient L2-users (n = 56, aged 19-65 years) were recorded while they read sentences in their first language (L1) German and L2 English. Sentences each contained a cognate target word (e.g., English: tunnel, German: Tunnel). The parafoveal preview of the targets either (a) preserved the spelling and meaning of the target (identity condition), (b) preserved letter identities but transposed the position of two adjacent letters (transposed-letter [TL] condition, e.g., tunenl/Tunenl), or substituted the identity of two adjacent letters (substituted-letter condition, e.g., tunocl/Tunocl). TL previews elicited longer early first-pass reading times than identity previews in both L1 and L2 reading in children and adults, suggesting that letter position was processed parafoveally. Substituted-letter previews resulted in longer reading times than TL previews in children and adults in L1 and L2, suggesting that letter identity information was processed independently of position information. These results suggest that letter position and identity information are extracted from the parafovea during L1 and L2 reading, facilitating word recognition in children and adults. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Bi-isotype immunoglobulins enhance antibody-mediated neutrophil activity against Plasmodium falciparum parasites.

Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay. We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG-IgA Fc region chimera would enhance the level of ADRB activity. We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG-IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31. We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites.

EXPRESSION ANALYSIS OF COLD RESPONSIVE GENES AND MECHANISMS INVOLVED DURING COLD STRESS IN SUGARCANE CELLULAR ORGANELLES

Sugarcane is a major cash crops cultivated in tropical and subtropical regions around the globe. This crop is mainly considered cold sensitive, although some cultivars showed resistance to cold stress. The crops respond to cold stress in diverse mechanisms and among these mechanisms, genes expression is one of the significant biological pathways. Our goal was to study the involvement of gene expression in cell organelles and cognate targets in the tolerance to low temperatures in sugarcane. The results reported here indicated that 2,324 genes were found upregulated in cultivar GT08-1108 organelles in a total of 5,649 upregulated genes, while 1,252 genes were found downregulated in a total of 3,289 downregulated genes. Furthermore, the cultivar ROC22 gene expression analysis showed that a total of 2,223 genes were found upregulated in 5,558 total upregulated genes, while 1,449 genes were found downregulated in a total of 3,252 downregulated genes. The gene expression analysis in both cultivars showed that during cold stress, the gene expression in the vacuole was found significantly downregulated. Hence, from these findings, we concluded that during cold stress, the most affected organelle is the vacuole. Collectively, our findings suggested that cold stress seriously affected cellular organelles in sugarcane. Hence, more research is needed to identify more cold tolerant cultivars using transcriptomics approaches in sugarcane, which could be significant for better production and quality of sugarcane. Keywords: Sugarcane; Cellular organelles; Gene expression; Sequence Read Archive; Cold stress.

A Multilayered Post-Genome-Wide Association Study Analysis Pipeline Defines Functional Variants and Target Genes for Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE), an autoimmune disease with incompletely understood etiology, has a strong genetic component. Although genome-wide association studies (GWASs) have revealed multiple SLE susceptibility loci and associated single-nucleotide polymorphisms (SNPs), the precise causal variants, target genes, cell types, tissues, and mechanisms of action remain largely unknown. Here, we report a comprehensive post-GWAS analysis using extensive bioinformatics, molecular modeling, and integrative functional genomic and epigenomic analyses to optimize fine-mapping. We compile and cross-reference immune cell-specific expression quantitative trait loci (cis- and trans-expression quantitative trait loci) with promoter capture high-throughput capture chromatin conformation (PCHi-C), allele-specific chromatin accessibility, and massively parallel reporter assay data to define predisposing variants and target genes. We experimentally validate a predicted locus using CRISPR/Cas9 genome editing, quantitative polymerase chain reaction, and Western blot. Anchoring on 452 index SNPs, we selected 9,931 high linkage disequilibrium (r2 > 0.8) SNPs and defined 182 independent non-human leukocyte antigen (HLA) SLE loci. The 3,746 SNPs from 143 loci were identified as regulating 564 unique genes. Target genes are enriched in lupus-related tissues and associated with other autoimmune diseases. Of these, 329 SNPs (106 loci) showed significant allele-specific chromatin accessibility and/or enhancer activity, indicating regulatory potential. Using CRISPR/Cas9, we validated reference SNP identifier 57668933 (rs57668933) as a functional variant regulating multiple targets, including SLE-risk gene ELF1 in B cells. We demonstrate and validate post-GWAS strategies for using multidimensional data to prioritize likely causal variants with cognate gene targets underlying SLE pathogenesis. Our results provide a catalog of significantly SLE-associated SNPs and loci, target genes, and likely biochemical mechanisms to guide experimental characterization.

Abstract 1687: Targeted degradation of MECOM in high-risk acute myeloid leukemia reveals a novel repressive function that is amenable to therapeutic small-molecule rewiring

Abstract Hematopoiesis relies on an intricate balance between stem-cell self-renewal and terminal maturation of blood cells. Disrupting this balance can lead to the development of deadly blood cancers, like acute myeloid leukemia (AML). Increasing evidence suggests that acquiring hematopoietic stem cell (HSC) gene expression programs in AML confers a particularly poor prognosis and increases risk of relapse with conventional therapy. Acquisition of these features in AML is commonly driven by dysregulation of MECOM, a transcription factor and master regulator of HSC self-renewal. To define the role of MECOM in AMLs, we engineered cell-line models of high-risk AML by endogenously tagging MECOM with an FKBP12F36V degron. These models enable targeted degradation of MECOM which we employed in concert with multi-omic readouts to assess changes in nascent transcription and chromatin accessibility to identify direct transcriptional targets of MECOM. We demonstrate that MECOM degradation results in a rapid increase in chromatin accessibility at MECOM-bound sites and increased expression of cognate genes to establish a pro-differentiation gene program. These findings suggest a previously unappreciated mechanism for MECOM in repressing differentiation-promoting cis-regulatory elements (cisREs) and their cognate target genes in AML. We then employed a pooled CRISPR inhibition (CRISPRi) screen to functionally characterize the role that each cisRE plays in promoting high-risk features in AML. Utilizing CRISPRi to repress MECOM-bound cisREs in concert with dTAG-mediated MECOM degradation, we examined if repression of over 500 individual MECOM-bound cisREs is sufficient to maintain AML progenitors in the absence of MECOM. This functional dissection revealed that the individual repression of three cisREs that regulate CEBPA, GFI1B, and RUNX1 is sufficient to maintain AML progenitor cells in the absence of MECOM. This result implicates MECOM as a direct repressor of known regulators of myeloid differentiation. Given these findings, we hypothesized that small molecule-based recruitment of a transcriptional coactivator to MECOM could rewire its repressive function to activate myeloid differentiation and subsequent leukemia cell death. To test this approach, we treated a MECOM-FKBP12F36V AML degron model with the bifunctional small molecule NICE-01 (AP1867-PEG2-JQ1) to recruit an epigenetic activator (BRD4) to MECOM to functionally rewire this transcriptional network. NICE-01 treatment induced significant differentiation of AML progenitor cells relative to JQ1 treatment alone. Our work highlights the utility of targeted protein degradation to mechanistically interrogate the function of a key driver of high-risk AMLs and suggests the potential for small molecule-rewiring of stem cell gene regulatory networks to confer therapeutic benefit. Citation Format: Travis J. Fleming, Michael Gundry, Richard Voit, Mateusz Antoszewski, William J. Gibson, Ananthan Sadagopan, Vijay G. Sankaran. Targeted degradation of MECOM in high-risk acute myeloid leukemia reveals a novel repressive function that is amenable to therapeutic small-molecule rewiring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1687.

High-throughput evaluation of genetic variants with prime editing sensor libraries.

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.

Identification and characterization of novel drought-responsive lncRNAs in stone apple (Aegle marmelos L.) through whole-transcriptome analysis

Stone apple (Aegle marmelos L.) is a subtropical fruit tree of the Rutaceae family, highly valued in traditional medicine across the Indian subcontinent. We conceived this study with the objective of developing a comprehensive transcriptome dataset, identifying SSRs for marker-assisted breeding, and delineating regulators of gene expression, with a specific emphasis on non-coding RNA (ncRNA), particularly related to drought stress. To achieve this, RNA-seq was conducted using RNA pooled from various tissues, including roots, leaves, inflorescence, and developing seeds from stone apple, and the clean reads were assembled into 40,886 unigenes. Subsequently, the unigenes were categorized into gene ontology categories encompassing biological processes, molecular functions, and cellular components. Within the unigenes, we identified a total of 9174 perfect simple sequence repeats (SSRs), 2167 transcription factors (TFs) distributed among 69 families, and 415 transcription regulators (TRs) across 27 families. Additionally, 19 microRNAs (miRNAs) from 12 families, 16,811 potential long noncoding RNAs (lncRNAs), and six functional endogenous target mimics (eTMs) were detected. Analysis of lncRNA-miRNA-mRNA interactions unveiled multiple regulatory nodes, elucidating lncRNA/miRNA-driven gene expression control in stone apple. The increased co-expression of selected drought-related lncRNAs and their cognate target mRNAs supported the aforementioned findings under drought conditions. Overall, this study significantly advances our understanding of stone apple genomics and lays a foundation for future omics-based studies, thereby facilitating the deployment of climate-resilient strategies in the species.

Designing Potential Antisense Oligonucleotides (ASO) against Lung Cancer Associated microRNA and Studying their Interaction

Abstract: MicroRNAs (miRNAs) are an abundant class of small non-protein-coding RNAs that function as gene regulators. They regulate diverse biological processes and bioinformatics data indicates that each miRNA can control hundreds of gene targets, underscoring the potential influence of miRNAs on almost every genetic pathway. miRNA mutations or mis-expression correlate with various human cancers and miRNAs can function as tumor suppressors and oncogenes. miRNAs have been shown to repress the expression of important cancer-related genes and might prove useful in the diagnosis and treatment of cancer. In this context, we identified various miRNAs associated with the prevalent cancer phenotype NSCLC/SCLC and used the respective target miRNA sequences as templates for designing antisense oligonucleotides (ASOs). We investigated the interaction of this cognate oligonucleotides and target miRNAs to hypotheses the inhibitory potential of such oligonucleotides through molecular docking studies.

Intein-based thermoregulated meganucleases for containment of genetic material.

Limiting the spread of synthetic genetic information outside of the intended use is essential for applications where biocontainment is critical. In particular, biocontainment of engineered probiotics and plasmids that are excreted from the mammalian gastrointestinal tract is needed to prevent escape and acquisition of genetic material that could confer a selective advantage to microbial communities. Here, we built a simple and lightweight biocontainment system that post-translationally activates a site-specific DNA endonuclease to degrade DNA at 18°C and not at higher temperatures. We constructed an orthogonal set of temperature-sensitive meganucleases (TSMs) by inserting the yeast VMA1 L212P temperature-sensitive intein into the coding regions of LAGLIDADG homing endonucleases. We showed that the TSMs eliminated plasmids carrying the cognate TSM target site from laboratory strains of Escherichia coli at the permissive 18°C but not at higher restrictive temperatures. Plasmid elimination is dependent on both TSM endonuclease activity and intein splicing. TSMs eliminated plasmids from E. coli Nissle 1917 after passage through the mouse gut when fecal resuspensions were incubated at 18°C but not at 37°C. Collectively, our data demonstrates the potential of thermoregulated meganucleases as a means of restricting engineered plasmids and probiotics to the mammalian gut.

Fine-tuning the antigen sensitivity of CAR T cells: emerging strategies and current challenges.

Chimeric antigen receptor (CAR) T cells are "living drugs" that specifically recognize their target antigen through an antibody-derived binding domain resulting in T cell activation, expansion, and destruction of cognate target cells. The FDA/EMA approval of CAR T cells for the treatment of B cell malignancies established CAR T cell therapy as an emerging pillar of modern immunotherapy. However, nearly every second patient undergoing CAR T cell therapy is suffering from disease relapse within the first two years which is thought to be due to downregulation or loss of the CAR target antigen on cancer cells, along with decreased functional capacities known as T cell exhaustion. Antigen downregulation below CAR activation threshold leaves the T cell silent, rendering CAR T cell therapy ineffective. With the application of CAR T cells for the treatment of a growing number of malignant diseases, particularly solid tumors, there is a need for augmenting CAR sensitivity to target antigen present at low densities on cancer cells. Here, we discuss upcoming strategies and current challenges in designing CARs for recognition of antigen low cancer cells, aiming at augmenting sensitivity and finally therapeutic efficacy while reducing the risk of tumor relapse.