Recombinetics (United States)

Stereotactic systems have traditionally used Cartesian coordinate combined with linear algebraic mathematical models to navigate the brain. Previously, the development of a novel stereotactic system allowed for improved patient comfort, reduced size, and carried through a simplified interface for surgeons. The system was designed with a work envelope and trajectory range optimized for deep brain stimulation applications only. However, it could be applied in multiple realms of neurosurgery by spanning the entire brain. To this end, a system of translational and rotational adapters was developed to allow total brain navigation capabilities. Adapters were designed to fit onto a Skull Anchor Key of a stereotactic frame system to allow for rotation and translation of the work envelope. Mathematical formulas for the rotations and translations associated with each adapter were developed. Mechanical and image-guided accuracies were examined using a ground truth imaging phantom. The system's clinical workflow and its ability to reliably and accurately be used in a surgical scenario were investigated using a cadaver head and computed tomography guidance. Eight adapters designed and 3D-printed allowed the work envelope to be expanded to the entire head. The mechanical error was 1.75 ± 0.09mm (n = 20 targets), and the cadaver surgical targeting error was 1.18 ± 0.28mm (n = 10 implantations). The novel application of conventional and geometric algebra in conjunction with hardware modifications significantly expands the work envelope of the stereotactic system to the entire cranial cavity. This approach greatly extends the clinical applications by the system.

Spermatogonial stem cell (SSC) transplantation into the testis of a germ cell (GC)-depleted surrogate allows transmission of donor genotype via donor-derived sperm produced by the recipient. Transplantation of gene-edited SSCs provides an approach to propagate gene-edited large animal models. DAZL is a conserved RNA-binding protein important for GC development, and DAZL knockout (KO) causes defects in GC commitment and differentiation. We characterized DAZL-KO pigs as SSC transplantation recipients. While there were GCs in 1-week-old (wko) KO, complete GC depletion was observed by 10 wko. Donor GCs were transplanted into 18 DAZL-KO recipients at 10-13 wko. At sexual maturity, semen and testes were evaluated for transplantation efficiency and spermatogenesis. Approximately 22% of recipient seminiferous tubules contained GCs, including elongated spermatids and proliferating spermatogonia. The ejaculate of 89% of recipients contained sperm, exclusively from donor origin. However, sperm concentration was lower than the wild-type range. Testicular protein expression and serum hormonal levels were comparable between DAZL-KO and wild-type. Intratesticular testosterone and Leydig cell volume were increased, and Leydig cell number decreased in transplanted DAZL-KO testis compared to wild-type. In summary, DAZL-KO pigs support donor-derived spermatogenesis following SSC transplantation, but low spermatogenic efficiency currently limits their use for the production of offspring.

Abstract Glioblastoma (GBM) is an aggressive primary brain tumor with no effective cure. Standard of care treatment offers dismal prognoses for patients with a median survival time of only 15.6 months, and no new drugs have been approved in 15 years. Contributing factors include tumor heterogeneity, high mutation rates, infiltrative growth, a complex tumor-immune microenvironment, and the blood-brain barrier, all of which are obstacles clinicians and researchers must overcome to improve patient outcomes. Research to identify novel therapeutic strategies are ongoing, but treatments that show promise in preclinical studies often do not pass Phase 2 and 3 clinical trials. These costly failures are attributable to a lack of predictive preclinical models that translate to human application, low patient availability for numerous and robust clinical trials, and an “all-comer” approach to clinical trial design where patients are enrolled based on disease presence and not molecular features of the tumor. To advance treatments for GBM, Recombinetics has pioneered somatic gene-editing technologies to the same genetic changes seen in patient tumors in GBM-initiating cells in an immune-competent swine model. These models rapidly and reproducibly develop GBM tumors that resemble human GBMs genetically, clinically and histologically. In addition to the physiological, immunological, and metabolic parallels between swine and humans, the molecular data collected from our swine mesenchymal and classical subtypes of GBM validate accurate disease modeling and we are pursuing additional GBM subtypes. Using RNA-sequencing, we are characterizing molecular profiles of these tumors, as well as identifying and validating drug targets. Studies are ongoing to characterize the tumor immune microenvironment of our models for improved immune-oncology therapeutic development. These large animal models of GBM will usher in a new era of pre-clinical testing that predicts the efficacy of a variety of therapeutic approaches, using precision medicine to bring novel, safe, and effective therapies to patients.

We sequenced the sporophyte transcriptome of Killarney fern (Vandenboschia speciosa (Willd.) G. Kunkel). In addition to being a rare endangered Macaronesian-European endemism, this species has a huge genome (10.52 Gb) as well as particular biological features and extreme ecological requirements. These characteristics, together with the systematic position of ferns among vascular plants, make it of high interest for evolutionary, conservation and functional genomics studies. The transcriptome was constructed de novo and contained 36,430 transcripts, of which 17,706 had valid BLAST hits. A total of 19,539 transcripts showed at least one of the 7362 GO terms assigned to the transcriptome, whereas 6547 transcripts showed at least one of the 1359 KEGG assigned terms. A prospective analysis of functional annotation results provided relevant insights on genes involved in important functions such as growth and development as well as physiological adaptations. In this context, a catalogue of genes involved in the genetic control of plant development, during the vegetative to reproductive transition, in stress response as well as genes coding for transcription factors is given. Altogether, this study provides a first step towards understanding the gene expression of a significant fern species and the in silico functional and comparative analyses reported here provide important data and insights for further comparative evolutionary studies in ferns and land plants in general.

Guaiane-type sesquiterpene lactones are naturally occurring compounds which have attracted attention due to their array of biological activities. In this study, chlorinated guaianolides 1–8, isolated from plants of the genus Centaurea, were evaluated against the human leukemia cell lines HL-60, U-937, a specific U-937 cell line that overexpresses the anti-apoptotic Bcl-2 protein and the human melanoma cell line SK-MEL-1. This established the relevant structure-growth inhibition relationships. Chlorohyssopifolins A (1), C (3) and D (4) and linichlorin A (6) were the most potent compounds in terms of inducing growth inhibition in the four cell lines. IC50 values were below 10 μM in all cases. Chlorohyssopifolins A (1) and D (4) and linichlorin A (6) were potent apoptotic inducers in human U-937 leukemia cells, as determined by fluorescent microscopy and flow cytometry, and their mechanism of action was associated with cytochrome c release, caspase activation and poly(ADP-ribose)polymerase cleavage. Overall this study shows that guaianolides induce cytotoxicity against human tumor cells and provides important insights into the cell death pathways that are involved.

Recent pandemic of coronavirus disease caused by a novel coronavirus SARS-CoV-2 in humans is the third outbreak by this family of viruses leading to an acute respiratory infection, which has been a major cause of morbidity and mortality worldwide.The virus belongs to the genus, Betacoronavirus, which has been recently reported to have significant similarity (>89%) to a severe acute respiratory syndrome (SARS)-related member of the Sarbecoviruses. Current researches are not sufficient to understand the etiological and immunopathobiological parameters related to COVID-19 so as to have a therapeutic solution to the problem. A structured search of bibliographic databases for peer-reviewed research literature has been carried out using focused review questions and inclusion/exclusion criteria. Further Standard tools were implied in order to appraise the quality of retrieved papers. The characteristic outcomes of screened research and review articles along with analysis of the interventions and findings of included studies using a conceptual framework have been described employing a deductive qualitative content analysis methodology. This review systematically summarizes the immune-pathobiological characteristics, diagnosis, potential therapeutic options for the treatment and prevention of COVID-19 based on the current published literature and evidence. The current review has covered 125 peerreviewed articles, the majority of which are from high-income technically developed countries providing the most recent updates about the current understanding of the COVID-19 bringing all the significant findings and related researches together at a single platform. In addition, possible therapeutic interventions, treatment strategies and vaccine development initiatives to manage COVID-19 have been proposed. It is anticipated that this review would certainly assist the public in general and scientific community in particular to recognize and effectively deal with COVID-19, providing a reference guide for futuristic studies.

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

Abstract Background : Goats are distributed worldwide and include many breeds with marked phenotypic variation. Both natural and human driven processes have shaped their genomes. Here, 52K genome-wide SNP genotypes were used to investigate signatures of divergent selection between indigenous goats from an arid hot environment in Egypt and breeds developed under temperate environments in Europe. Three selection signature approaches were used, the di derivative of F ST , iHS and Rsb. Results: Out of a total of 36 candidate regions that were detected to be under selection by the three approaches, the analysis detected nine regions with the strongest selection signatures spanning 134 genes of which 68 were the most significantly functionally enriched. In addition to well-known genes affecting dairy traits ( LAP3 , ABCG2 , MEPE , IBSP , MED28 ) and body weight and stature ( LCORL , NCAPG , CCSER1 , DCAF16 ) in several mammalian species, we found evidence for selection in regions spanning genes implicated in the regulation of fatty acid synthesis and metabolic pathways ( PI4K2B and PPARGC1A ), inflammatory gene expression and autoimmune response pathways ( CXCL8 , HERC5 , RGS18 , TROVE2 ) and spermatogenesis ( SPATA18 , LAP3 ). Other genes not reported before in goats included SLIT2 , PACRGL , GRP125 , DHX15 , SOD3 and KCNIP4 which have been associated with thermal nociception in mice. We also detected a paralog of TBC1D12 , namely TBC1D14 , in one of the candidate regions. TBC1D12 has been linked to environmental adaptation in sheep. Gene ontology analysis revealed the 68 candidate genes were highly enriched in two biological processes viz regulation of G protein-coupled receptor signaling pathway (GO:0045744) and cellular response to stress (GO:0033554). Conclusions: Our results provide evidence that goat genomes have evolved, in part, via diverse positive selection directed at breed formation, at past and recent genetic improvement and adaptation to environments. Based on their uniqueness, these breed-group specific signatures of selection can be considered to be footprints of divergence which may be useful in characterizing genome architecture and diversity in domestic animals and for targeted genetic improvement.

The prolactin receptor (PRLR) have been described as a candidate gene with a variant allele containing a nonsense/dominant mutation causative of the slick coat (SC) phenotype (chromosome 20:39136558GC>G; p.L462*) in Senepol and SC Holstein cattle of Puerto Rico (PR). However, inconsistencies between p.L462* genotypes and SC phenotype had been observed in Senepol. Therefore, a total of 661 animals [Holstein (230), Senepol (357), Charolais (4), Senepol x Charolais (13), Jersey (34), and Creole (23)] were genotyped by DNA sequencing of a 645 bp PRLR exon 11 fragment that includes 5 reported polymorphic positions. A non-reported cytosine by thymine single nucleotide polymorphism (SNP) was identified at the chromosome 20:39136612 position; 53 bp downstream of the reported p.L462* cytosine insertion/deletion (InDel), also resulting in a nonsense mutation. This SNP incorporates 16 amino acids to the shortened PRLR. Five genotype combinations (CC/CC, CC/CT, C-/CC, C-/CT and --/CC) resulting from both PRLR exon 11 polymorphisms were observed and allelic heterogeneity resulting in SC phenotype was exclusively present in Senepol and Senepol x Charolais. The PRLR InDel/SNP genotype combination CC/CC, were present in non-slick: Senepol x Charolais (3), Charolais (4), Holstein (127), and Jersey (33) cattle with an overall frequency of 0.25. Heterozygote SC animals, genotypes combination CC/CT [Senepol (4), Senepol x Charolais (2)] and C-/CC [Senepol (49), Senepol x Charolais (7), Holstein (103), Jersey (1), and Creole (23)] had frequencies of 0.01 and 0.28, respectively. Meanwhile, homozygous SC animals, C-/CT [Senepol (45) and Senepol x Charolais (1)] and --/CC [Senepol (259)] presented frequencies of 0.07 and 0.39, individually. The novel pyrimidine transition reported herein in SC Senepol that do not possess the PRLR p.L462* responsible InDel suggest a possible more accurate scenario for slick genotype calling within such tropically adapted breed if selection is to be made in favor of such coat type.

Genomic selection has revolutionized dairy cattle breeding. Since 2000, assays have been developed to genotype large numbers of single-nucleotide polymorphisms (SNPs) at relatively low cost. The first commercial SNP genotyping chip was released with a set of 54,001 SNPs in December 2007. Over 15,000 genotypes were used to determine which SNPs should be used in genomic evaluation of US dairy cattle. Official USDA genomic evaluations were first released in January 2009 for Holsteins and Jerseys, in August 2009 for Brown Swiss, in April 2013 for Ayrshires, and in April 2016 for Guernseys. Producers have accepted genomic evaluations as accurate indications of a bull's eventual daughter-based evaluation. The integration of DNA marker technology and genomics into the traditional evaluation system has doubled the rate of genetic progress for traits of economic importance, decreased generation interval, increased selection accuracy, reduced previous costs of progeny testing, and allowed identification of recessive lethals.