Genome expansion by a CRISPR trimmer-integrase.

CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity1. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient2-5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation6,7, but many CRISPR-Cas systems lack Cas48. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited9,10 exonucleases for faithful acquisition of new CRISPR immune sequences.

A Functional Mini-Integrase in a Two-Protein-type V-C CRISPR System.

CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.

PEGylation of anti-MerTK Antibody Modulates Ocular Biodistribution.

Here, we explore whether PEGylation of antibodies can modulate their biodistribution to the eye, an organ once thought to be immune privileged but has recently been shown to be accessible to IV-administered large molecules, such as antibodies. We chose to PEGylate an anti-MerTK antibody, a target with known potential for ocular toxicity, to minimize biodistribution to retinal pigment epithelial cells (RPEs) in the eye by increasing the hydrodynamic volume of the antibody. We used site-specific conjugation to an engineered cysteine on anti-MerTK antibody to chemically attach 40-kDa branched or linear PEG polymers. Despite reduced binding to MerTK on cells, site-specifically PEGylated anti-MerTK retained similar potency in inhibiting MerTK-mediated macrophage efferocytosis of apoptotic cells. Importantly, we found that PEGylation of anti-MerTK significantly reduced MerTK receptor occupancy in RPE cells in both naïve mice and MC-38 tumor-bearing mice, with the branched PEG exhibiting a greater effect than linear PEG. Furthermore, similar to unconjugated anti-MerTK, PEGylated anti-MerTK antibody triggered type I IFN response and exhibited antitumor effect in syngeneic mouse tumor studies. Our results demonstrate the potential of PEGylation to control ocular biodistribution of antibodies.

A Motivational Interviewing Intervention for Adolescents in Accelerated High School Curricula: Applicability and Acceptability in a Second Sample.

The paper describes the applicability and acceptability of a selective intervention-Motivation, Assessment, and Planning (MAP)-for high school students that was developed based on the principles of motivational interviewing (MI) and tailored to the unique needs and strengths of students taking accelerated coursework, specifically Advanced Placement (AP) and International Baccalaureate (IB) classes. In addition to detailing the intervention in terms of MI spirit, processes, and relational and technical skills, we report applicability and acceptability data from a second iteration of MAP implementation in eight AP/IB programs in a Southeastern state during spring 2018. We analyzed quantitative and qualitative acceptability data from 121 high school freshmen (97 from AP and 24 from IB courses), as well as the seven MAP coaches who were trained using the Motivational Interview Training and Assessment System (Frey et al. 2017). To gain perspectives from the intended end users of the refined MAP, 12 school counselors and school psychologists who were not trained in MAP evaluated the intervention and provided qualitative and quantitative data on applicability and acceptability. All three stakeholder groups (students, coaches, and school mental health staff) rated and described the intervention as highly acceptable and appropriate for addressing the social-emotional needs of adolescents in AP/IB classes.

Molecular imaging of biological systems with a clickable dye in the broad 800- to 1,700-nm near-infrared window.

Fluorescence imaging multiplicity of biological systems is an area of intense focus, currently limited to fluorescence channels in the visible and first near-infrared (NIR-I; ∼700-900 nm) spectral regions. The development of conjugatable fluorophores with longer wavelength emission is highly desired to afford more targeting channels, reduce background autofluorescence, and achieve deeper tissue imaging depths. We have developed NIR-II (1,000-1,700 nm) molecular imaging agents with a bright NIR-II fluorophore through high-efficiency click chemistry to specific molecular antibodies. Relying on buoyant density differences during density gradient ultracentrifugation separations, highly pure NIR-II fluorophore-antibody conjugates emitting ∼1,100 nm were obtained for use as molecular-specific NIR-II probes. This facilitated 3D staining of ∼170-µm histological brain tissues sections on a home-built confocal microscope, demonstrating multicolor molecular imaging across both the NIR-I and NIR-II windows (800-1,700 nm).

Lactic Acid and an Acidic Tumor Microenvironment suppress Anticancer Immunity.

Immune evasion and altered metabolism, where glucose utilization is diverted to increased lactic acid production, are two fundamental hallmarks of cancer. Although lactic acid has long been considered a waste product of this alteration, it is now well accepted that increased lactic acid production and the resultant acidification of the tumor microenvironment (TME) promotes multiple critical oncogenic processes including angiogenesis, tissue invasion/metastasis, and drug resistance. We and others have hypothesized that excess lactic acid in the TME is responsible for suppressing anticancer immunity. Recent studies support this hypothesis and provide mechanistic evidence explaining how lactic acid and the acidic TME impede immune cell functions. In this review, we consider lactic acid's role as a critical immunoregulatory molecule involved in suppressing immune effector cell proliferation and inducing immune cell de-differentiation. This results in the inhibition of antitumor immune responses and the activation of potent, negative regulators of innate and adaptive immune cells. We also consider the role of an acidic TME in suppressing anticancer immunity. Finally, we provide insights to help translate this new knowledge into impactful anticancer immune therapies.

Fabrication of a 3D-printed interim obturator prosthesis: A contemporary approach.

This report describes the combination of analog and digital techniques for the fabrication of a 2-piece hollow bulb maxillary obturator. The procedure described provides an accurate representation of the surgical defect while avoiding the discomfort associated with analog impressions. The manipulation of a routine postoperative computed tomography (CT) scanner in conjunction with a 3D printer allowed for the fabrication of a 3D-printed anatomic cast from which the 2-piece hollow bulb obturator was fabricated. The clinical and laboratory steps involved are described in this article.

Search for 5'-leader regulatory RNA structures based on gene annotation aided by the RiboGap database.

The discovery of noncoding RNAs (ncRNAs) and their importance for gene regulation led us to develop bioinformatics tools to pursue the discovery of novel ncRNAs. Finding ncRNAs de novo is challenging, first due to the difficulty of retrieving large numbers of sequences for given gene activities, and second due to exponential demands on calculation needed for comparative genomics on a large scale. Recently, several tools for the prediction of conserved RNA secondary structure were developed, but many of them are not designed to uncover new ncRNAs, or are too slow for conducting analyses on a large scale. Here we present various approaches using the database RiboGap as a primary tool for finding known ncRNAs and for uncovering simple sequence motifs with regulatory roles. This database also can be used to easily extract intergenic sequences of eubacteria and archaea to find conserved RNA structures upstream of given genes. We also show how to extend analysis further to choose the best candidate ncRNAs for experimental validation.

A custom positioning aid for an auricular prosthesis: A clinical report.

This report describes the treatment of a 68-year-old man with basosquamous cell carcinoma of the left auricular area. His oncologic treatment resulted in the loss of his left ear and much of the left temporal bone. The loss of a significant portion of the temporal bone precluded the use of extraoral implants. Owing to the lack of anatomic landmarks after his surgical treatment, positioning his auricular prosthesis proved challenging. The fabrication of his prosthesis and a custom positioning aid are described.

Radiological evolution of peri-odontoid pannus in a patient with Chiari I malformation: a case-based review.

INTRODUCTION: The Chiari I malformation (CIM) is commonly encountered by neurosurgeons and can have different etiologies and clinical presentations. CASE REPORT: We report a CIM patient who presented with symptoms of ventral brain stem compression and was found to have a large peri-odontoid pannus. Posterior fossa decompression was performed with a planned second-stage odontoidectomy. However, at the 6-month follow-up, postoperative images demonstrated a mostly resolved pannus and improvement of the brain stem compression symptoms, and the patient progressed uneventfully without the need for odontoidectomy. CONCLUSIONS: This case illustrates the resolution of a significant and symptomatic peri-odontoid pannus in a patient with CIM without craniocervical fusion or odontoidectomy. Such a case indicates that not all peri-odontoid pannus formations in CIM patients are due to hypermobility at the craniocervical junction.

Subrenal capsule grafting technology in human cancer modeling and translational cancer research.

Patient-derived xenograft (PDX) cancer models with high fidelity are in great demand. While the majority of PDXs are grafted under the skin of immunodeficient mice, the Living Tumor Laboratory (LTL), using unique subrenal capsule grafting techniques, has successfully established more than 200 transplantable PDX models of various low to high grade human cancers. The LTL PDX models retain key biological properties of the original malignancies, including histopathological and molecular characteristics, tumor heterogeneity, metastatic ability, and response to treatment. The PDXs are stored frozen at early transplant generations in a resurrectable form, which eliminates continuous passaging in mice, thus ensuring maintenance of the high biologic and molecular fidelity and reproducibility of the models. The PDX models have been demonstrated to be powerful tools for (i) studies of cancer progression, metastasis and drug resistance, (ii) evidenced-based precision cancer therapy, (iii) preclinical drug efficacy testing and discovery of new anti-cancer drug candidates. To better provide resources for the research community, an LTL website (www.livingtumorlab.com) has been designed as a publicly accessible database which allows researchers to identify PDX models suitable for translational/preclinical cancer research. In summary, subrenal capsule grafting technology maximizes both tumor engraftment rate and retention of human cancer heterogeneity. Moreover, the method makes possible the recovery of PDXs from frozen stocks for further applications, thus providing a powerful platform for translational cancer research.

Riboswitches that sense S-adenosylhomocysteine and activate genes involved in coenzyme recycling.

We have identified a highly conserved RNA motif that occurs upstream of genes involved in S-adenosyl-L-methionine (SAM) recycling in many Gram-positive and Gram-negative species of bacteria. The phylogenetic distribution and the conserved structural features of representatives of this motif are indicative of riboswitch function. Riboswitches are widespread metabolite-sensing gene control elements that are typically found in the 5' untranslated regions (UTRs) of bacterial mRNAs. We experimentally verified that examples of this RNA motif specifically recognize S-adenosylhomocysteine (SAH) in protein-free in vitro assays, and confirmed that these RNAs strongly discriminate against SAM and other closely related analogs. A representative SAH motif was found to activate expression of a downstream gene in vivo when the metabolite is bound. These observations confirm that SAH motif RNAs are distinct ligand-binding aptamers for a riboswitch class that selectively binds SAH and controls genes essential for recycling expended SAM coenzymes.

Riboswitches in eubacteria sense the second messenger c-di-AMP.

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered bacterial second messenger implicated in the control of cell wall metabolism, osmotic stress responses and sporulation. However, the mechanisms by which c-di-AMP triggers these physiological responses have remained largely unknown. Notably, a candidate riboswitch class called ydaO associates with numerous genes involved in these same processes. Although a representative ydaO motif RNA recently was reported to weakly bind ATP, we report that numerous members of this noncoding RNA class selectively respond to c-di-AMP with subnanomolar affinity. Our findings resolve the mystery regarding the primary ligand for this extremely common riboswitch class and expose a major portion of the super-regulon of genes that are controlled by the widespread bacterial second messenger c-di-AMP.

Spontaneous stepwise self-assembly of a polyoxometalate-organic hybrid into catalytically active one-dimensional anisotropic structures.

An inorganic-organic hybrid surfactant with a hexavanadate cluster as the polar head group was designed and observed to assemble into micelle structures, which further spontaneously coagulate into a 1D anisotropic structure in aqueous solutions. Such a hierarchical self-assembly process is driven by the cooperation of varied noncovalent interactions, including hydrophobic, electrostatic, and hydrogen-bonding interactions. The hydrophobic interaction drives the quick formation of the micelle structure; electrostatic interactions involving counterions leads to the further coagulation of the micelles into larger assemblies. This process is similar to the crystallization process, but the specific counterions and the directional hydrogen bonding lead to the 1D growth of the final assemblies. Since most of the hexavanadates are exposed to the surface, the 1D assembly with nanoscale thickness is a highly efficient heterogeneous catalyst for the oxidation of organic sulfides with appreciable recyclability.

IL-7 functionally segregates the pro-B cell stage by regulating transcription of recombination mediators across cell cycle.

Ag receptor diversity involves the introduction of DNA double-stranded breaks during lymphocyte development. To ensure fidelity, cleavage is confined to the G(0)-G(1) phase of the cell cycle. One established mechanism of regulation is through periodic degradation of the RAG2 recombinase protein. However, there are additional levels of protection. In this paper, we show that cyclical changes in the IL-7R signaling pathway functionally segregate pro-B cells according to cell cycle status. In consequence, the level of a downstream effector of IL-7 signaling, phospho-STAT5, is inversely correlated with cell cycle expression of Rag, a key gene involved in recombination. Higher levels of phopho-STAT5 in S-G(2) correlate with decreased Rag expression and Rag relocalization to pericentromeric heterochromatin. These cyclical changes in transcription and locus repositioning are ablated upon transformation with v-Abl, which renders STAT5 constitutively active across the cell cycle. We propose that this activity of the IL-7R/STAT5 pathway plays a critical protective role in development, complementing regulation of RAG2 at the protein level, to ensure that recombination does not occur during replication. Our data, suggesting that pro-B cells are not a single homogeneous population, explain inconsistencies in the role of IL-7 signaling in regulating Igh recombination.

A highly specialized flavin mononucleotide riboswitch responds differently to similar ligands and confers roseoflavin resistance to Streptomyces davawensis.

Streptomyces davawensis is the only organism known to synthesize the antibiotic roseoflavin, a riboflavin (vitamin B2) analog. Roseoflavin is converted to roseoflavin mononucleotide (RoFMN) and roseoflavin adenine dinucleotide in the cytoplasm of target cells. (Ribo-)Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for the biosynthesis and transport of riboflavin. Streptomyces davawensis is roseoflavin resistant, and the closely related bacterium Streptomyces coelicolor is roseoflavin sensitive. The two bacteria served as models to investigate roseoflavin resistance of S. davawensis and to analyze the mode of action of roseoflavin in S. coelicolor. Our experiments demonstrate that the ribB FMN riboswitch of S. davawensis (in contrast to the corresponding riboswitch of S. coelicolor) is able to discriminate between the two very similar flavins FMN and RoFMN and shows opposite responses to the latter ligands.

COVID-19 school closures and Chinese children's school readiness: Results from the natural experimental data.

AIMS: To determine the associations between COVID-19 school closures and school readiness skills for Chinese kindergarteners. DESIGN: We utilized the natural experimental condition created by local COVID-19 outbreaks in 2022 (Study 1) to compare school readiness skills of children whose kindergartens were closed for 5 months (Group 1) with children whose kindergartens stayed open (Group 2). We further compared the school readiness skills of one pre-COVID-19 cohort (Cohort 2019) with one COVID-19 cohort (Cohort 2021) from a fifth kindergarten (Study 2). SAMPLES: For Study 1, Group 1 included 445 children and Group 2 included 584 children aged 4-6 years. For Study 2, Cohort 2019 included 156 children and Cohort 2021 included 228 children aged 3-6 years. MEASURES: For both studies, survey data on four school readiness skills were collected from parents. Additionally, Study 1 collected parental locus of control data from parents. RESULTS: Controlling for covariates, Study 1 revealed that Group 1 and Group 2 did not differ in terms of language and emergent literacy or approaches to learning. However, Group 1 scored lower than Group 2 on health and well-being and arts and imagination. Study 2 revealed that Cohort 2021 scored higher than Cohort 2019 on language and emergent literacy but lower on the other three skills. CONCLUSIONS: The associations of COVID-19 school closures with Chinese children's school readiness skills were not uniform, with a positive relation with language and emergent literacy and negative associations with health and well-being, approaches to learning, as well as arts and imagination.

Corrigendum: Therapeutically targeting type I interferon directly to XCR1+ dendritic cells reveals the role of cDC1s in anti-drug antibodies.

[This corrects the article DOI: 10.3389/fimmu.2023.1272055.].

A Bispecific Modeling Framework Enables the Prediction of Efficacy, Toxicity, and Optimal Molecular Design of Bispecific Antibodies Targeting MerTK.

Inhibiting MerTK on macrophages is a promising therapeutic strategy for augmenting anti-tumor immunity. However, blocking MerTK on retinal pigment epithelial cells (RPEs) results in retinal toxicity. Bispecific antibodies (bsAbs) containing an anti-MerTK therapeutic and anti-PD-L1 targeting arm were developed to reduce drug binding to MerTK on RPEs, since PD-L1 is overexpressed on macrophages but not RPEs. In this study, we present a modeling framework using in vitro receptor occupancy (RO) and pharmacokinetics (PK) data to predict efficacy, toxicity, and therapeutic index (TI) of anti-MerTK bsAbs. We first used simulations and in vitro RO data of anti-MerTK monospecific antibody (msAb) to estimate the required MerTK RO for in vivo efficacy and toxicity. Using these estimated RO thresholds, we employed our model to predict the efficacious and toxic doses for anti-MerTK bsAbs with varying affinities for MerTK. Our model predicted the highest TI for the anti-MerTK/PD-L1 bsAb with an attenuated MerTK binding arm, which was consistent with in vivo efficacy and toxicity observations. Subsequently, we used the model, in combination with sensitivity analysis and parameter scans, to suggest an optimal molecular design of anti-MerTK bsAb with the highest predicted TI in humans. Our prediction revealed that this optimized anti-MerTK bsAb should contain a MerTK therapeutic arm with relatively low affinity, along with a high affinity targeting arm that can bind to a low abundance target with slow turnover rate. Overall, these results demonstrated that our modeling framework can guide the rational design of bsAbs.

CRISPR technology: A decade of genome editing is only the beginning.

The advent of clustered regularly interspaced short palindromic repeat (CRISPR) genome editing, coupled with advances in computing and imaging capabilities, has initiated a new era in which genetic diseases and individual disease susceptibilities are both predictable and actionable. Likewise, genes responsible for plant traits can be identified and altered quickly, transforming the pace of agricultural research and plant breeding. In this Review, we discuss the current state of CRISPR-mediated genetic manipulation in human cells, animals, and plants along with relevant successes and challenges and present a roadmap for the future of this technology.