Programmable RNA-Guided Large DNA Transgenesis by CRISPR/Cas9 and Site-Specific Integrase Bxb1.

The inability to insert large DNA constructs into the genome efficiently and precisely is a key challenge in genomic engineering. Random transgenesis, which is widely used, lacks precision, and comes with a slew of drawbacks. Lentiviral and adeno-associated viral methods are plagued by, respectively, DNA toxicity and a payload capacity of less than 5 kb. Homology-directed repair (HDR) techniques based on CRISPR-Cas9 can be effective, but only in the 1-5 kb range. In addition, long homology arms-DNA sequences that permit construct insertion-of lengths ranging from 0.5 to 5 kb are required by currently known HDR-based techniques. A potential new method that uses Cas9-guided transposases to insert DNA structures up to 10 kb in length works well in bacteria, but only in bacteria. Surmounting these roadblocks, a new toolkit has recently been developed that combines RNA-guided Cas9 and the site-specific integrase Bxb1 to integrate DNA constructs ranging in length from 5 to 43 kb into mouse zygotes with germline transmission and into human cells. This ground-breaking toolkit will give researchers a valuable resource for developing novel, urgently needed mouse and human induced pluripotent stem cell (hiPSC) models of cancer and other genetic diseases, as well as therapeutic gene integration and biopharmaceutical applications, such as the development of stable cell lines to produce therapeutic protein products

Myocardial Mechanics in Hypertensive Disorders of Pregnancy: a Systematic Review and Meta-Analysis

Global longitudinal strain (GLS) is becoming routinely used to direct the medical management of various cardiac diseases, but its application in pregnancy is unclear. Our objective was to perform a meta-analysis and pool multiple study data to consolidate the evidence base for the role of GLS in the assessment of women with hypertensive disorders of pregnancy (HDP). Electronic database searches were performed in PubMed/Medline and EMBASE for research articles reporting GLS in pregnancies complicated by HDP and normotensive pregnancies that have been published up to September 2021. The meta-analysis included 17 studies with a pooled sample size of 1723 participants, which included 951 women with HDP, of which 680 were preeclamptic, and 772 controls. The primary random-effects pooled analysis demonstrated a statistically significant weighted mean difference in GLS between the HDP and control group (mean difference: 3.08% [CI, 2.33–3.82], P <0.001). When analyzed including only preeclamptic studies, there was also a statistically significant mean difference (mean difference: 2.98% [95% CI, 1.97–3.99], P <0.001). This meta-analysis demonstrates that HDP is associated with greater cardiac maladaptation, evidenced by a significantly reduced GLS compared with normal pregnancy. Echocardiography should be considered as a screening tool in women with HDP to enable early cardiovascular risk prevention through national initiatives

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase.

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene landing pad composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies \u3e 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds

K-Rational D-Brane Crystals

In this paper the problem of constructing spacetime from string theory is addressed in the context of D-brane physics. It is suggested that the knowledge of discrete configurations of D-branes is sufficient to reconstruct the motivic building blocks of certain Calabi-Yau varieties. The collections of D-branes involved have algebraic base points, leading to the notion of K-arithmetic D-crystals for algebraic number fields K. This idea can be tested for D0-branes in the framework of toroidal compactifications via the conjectures of Birch and Swinnerton-Dyer. For the special class of D0-crystals of Heegner type these conjectures can be interpreted as formulae that relate the canonical Neron-Tate height of the base points of the D-crystals to special values of the motivic L-function at the central point. In simple cases the knowledge of the D-crystals of Heegner type suffices to uniquely determine the geometry.Comment: 36 page

ADAM17 is essential for ectodomain shedding of the EGF-receptor ligand amphiregulin.

The epidermal growth factor (EGF)-receptor ligand amphiregulin (AREG) is a potent growth factor implicated in proliferative skin diseases and in primary and metastatic epithelial cancers. AREG, synthesized as a propeptide, requires conversion to an active peptide by metalloproteases by a process known as ectodomain shedding. Although (ADAM17) a disintegrin and metalloprotease 17 is a key sheddase of AREG, ADAM8-, ADAM15-, and batimastat (broad metalloprotease inhibitor)-sensitive metalloproteases have also been implicated in AREG shedding. In the present study, using a curly bare (Rhbdf2cub ) mouse model that shows loss-of-hair, enlarged sebaceous gland, and rapid cutaneous wound-healing phenotypes mediated by enhanced Areg mRNA and protein levels, we sought to identify the principal ectodomain sheddase of AREG. To this end, we generated Rhbdf2cub mice lacking ADAM17 specifically in the skin and examined the above phenotypes of Rhbdf2cub mice. We find that ADAM17 deficiency in the skin of Rhbdf2cub mice restores a full hair coat, prevents sebaceous gland enlargement, and impairs the rapid wound-healing phenotype observed in Rhbdf2cub mice. Furthermore, in vitro, stimulated shedding of AREG is abolished in Rhbdf2cub mouse embryonic keratinocytes lacking ADAM17. Thus, our data support previous findings demonstrating that ADAM17 is the major ectodomain sheddase of AREG. FEBS Open Bio 2018; 8(4):702-710

Spontaneous Posterior Segment Vascular Disease Phenotype of a Mouse Model, rnv3, Is Dependent on the Crb1rd8 Allele.

Purpose: To determine the molecular basis of lesion development in a murine model of spontaneous retinal vascularization, rnv3 (retinal vascularization 3, aka JR5558). Methods: Disease progression of rnv3 was examined in longitudinal studies by clinical evaluation, electroretinography (ERG) and light microscopy analyses. The chromosomal position for the recessive rnv3 mutation was determined by DNA pooling and genome-wide linkage analysis. The causative mutation was discovered by comparison of whole exome sequences of rnv3 mutant and wild-type (WT) controls. In order to confirm the causative mutation, transcription activator-like effector nuclease (TALEN)-mediated oligonucleotide directed repair (ODR) was utilized to correct the mutant allele. Phenotypic correction was assessed by fundus imaging and optical coherence tomography of live mice. Results: rnv3 exhibits early-onset, multifocal depigmented retinal lesions observable by fundus examination starting at 18 days of age. The retinal lesions are associated with fluorescein leakage around 25 days of age, with peak leakage at about 4 weeks of age. ERG responses deteriorate as rnv3 mutants age, concomitant with progressive photoreceptor disruption and loss that is observable by histology. Genetic analysis localized rnv3 to mouse chromosome (Chr) 1. By high throughput sequencing of a whole exome capture library of a rnv3/rnv3 mutant and subsequent sequence analysis, a single base deletion (del) in the Crb1 [crumbs family member 1] gene, which was previously reported to cause retinal degeneration 8, was identified. The TALEN-mediated ODR rescued the posterior segment vascularization phenotype; heterozygous Crb1rd8+em1Boc/Crb1rd8 and homozygous Crb1rd8+em1Boc/Crb1rd8+em1Boc mice showed a normal retinal phenotype. Additionally, six novel disruptions of Crb1 that were generated through aberrant non-homologous end joining induced by TALEN exhibited variable levels of vascularization, suggesting allelic effects. Conclusions: The rnv3 model and the models of six novel disruptions of Crb1 are all reliable, novel mouse models for the study of both early and late events associated with posterior segment vascularization and can also be used to test the effects of pharmacological targets for treating human ocular vascular disorders. Further study of these models may provide a greater understanding about how different Crb1 alleles result in aberrant angiogenesis

Computations in non-commutative Iwasawa theory

We study special values of L-functions of elliptic curves over Q twisted by Artin representations that factor through a false Tate curve extension $Q(\mu_p^\infty,\sqrt[p^\infty]{m})/Q$. In this setting, we explain how to compute L-functions and the corresponding Iwasawa-theoretic invariants of non-abelian twists of elliptic curves. Our results provide both theoretical and computational evidence for the main conjecture of non-commutative Iwasawa theory.Comment: 60 pages; with appendix by John Coates and Ramdorai Sujath

Role of MicroRNA in Inflammatory Bowel Disease: Clinical Evidence and the Development of Preclinical Animal Models.

The dysregulation of microRNA (miRNA) is implicated in cancer, inflammation, cardiovascular disorders, drug resistance, and aging. While most researchers study miRNA\u27s role as a biomarker, for example, to distinguish between various sub-forms or stages of a given disease of interest, research is also ongoing to utilize these small nucleic acids as therapeutics. An example of a common pleiotropic disease that could benefit from miRNA-based therapeutics is inflammatory bowel disease (IBD), which is characterized by chronic inflammation of the small and large intestines. Due to complex interactions between multiple factors in the etiology of IBD, development of therapies that effectively maintain remission for this disease is a significant challenge. In this review, we discuss the role of dysregulated miRNA expression in the context of clinical ulcerative colitis (UC) and Crohn\u27s disease (CD)-the two main forms of IBD-and the various preclinical mouse models of IBD utilized to validate the therapeutic potential of targeting these miRNA. Additionally, we highlight advances in the development of genetically engineered animal models that recapitulate clinical miRNA expression and provide powerful preclinical models to assess the diagnostic and therapeutic promise of miRNA in IBD

Inactive rhomboid proteins RHBDF1 and RHBDF2 (iRhoms): a decade of research in murine models.

Rhomboid proteases, first discovered in Drosophila, are intramembrane serine proteases. Members of the rhomboid protein family that are catalytically deficient are known as inactive rhomboids (iRhoms). iRhoms have been implicated in wound healing, cancer, and neurological disorders such as Alzheimer\u27s and Parkinson\u27s diseases, inflammation, and skin diseases. The past decade of mouse research has shed new light on two key protein domains of iRhoms-the cytosolic N-terminal domain and the transmembrane dormant peptidase domain-suggesting new ways to target multiple intracellular signaling pathways. This review focuses on recent advances in uncovering the unique functions of iRhom protein domains in normal growth and development, growth factor signaling, and inflammation, with a perspective on future therapeutic opportunities