456 research outputs found
Creating Porcine Biomedical Models Through Recombineering
Recent advances in genomics provide genetic information from humans and other
mammals (mouse, rat, dog and primates) traditionally used as models as well
as new candidates (pigs and cattle). In addition, linked enabling technologies,
such as transgenesis and animal cloning, provide innovative ways to design and
perform experiments to dissect complex biological systems. Exploitation of genomic
information overcomes the traditional need to choose naturally occurring models.
Thus, investigators can utilize emerging genomic knowledge and tools to create
relevant animal models. This approach is referred to as reverse genetics. In contrast
to āforward geneticsā, in which gene(s) responsible for a particular phenotype
are identified by positional cloning (phenotype to genotype), the āreverse geneticsā
approach determines the function of a gene and predicts the phenotype of a
cell, tissue, or organism (genotype to phenotype). The convergence of classical
and reverse genetics, along with genomics, provides a working definition of a
āgenetic modelā organism (3). The recent construction of phenotypic maps defining
quantitative trait loci (QTL) in various domesticated species provides insights into
how allelic variations contribute to phenotypic diversity. Targeted chromosomal
regions are characterized by the construction of bacterial artificial chromosome
(BAC) contigs to isolate and characterize genes contributing towards phenotypic
variation. Recombineering provides a powerful methodology to harvest genetic
information responsible for phenotype. Linking recombineering with gene-targeted
homologous recombination, coupled with nuclear transfer (NT) technology can
provide āclonesā of genetically modified animals
Autoreactive T cell profiles are altered following allogeneic islet transplantation with alemtuzumab induction and reāemerging phenotype is associated with graft function
Islet transplantation is an effective therapy for lifeāthreatening hypoglycemia, but graft function gradually declines over time in many recipients. We characterized isletāspecific T cells in recipients within an islet transplant program favoring alemtuzumab (ATZ) lymphodepleting induction and examined associations with graft function. Fiftyāeight recipients were studied: 23 pretransplant and 40 posttransplant (including 5 with pretransplant phenotyping). The proportion with isletāspecific T cell responses was not significantly different over time (preāTx: 59%; 1ā6 m posttransplant: 38%; 7ā12 m: 44%; 13ā24 m: 47%; and >24 m: 45%). However, phenotype shifted significantly, with IFNāĪ³ādominated response in the pretransplant group replaced by ILā10ādominated response in the 1ā6 m posttransplant group, reverting to predominantly IFNāĪ³āoriented response in the >24 m group. Clustering analysis of posttransplant responses revealed two main agglomerations, characterized by IFNāĪ³ and ILā10 phenotypes, respectively. ILā10āoriented posttransplant response was associated with relatively low graft function. Recipients within the ILā10+ cluster had a significant decline in Cāpeptide levels in the period preceding the ILā10 response, but stable graft function following the response. In contrast, an IFNāĪ³ response was associated with subsequently decreased Cāpeptide. Islet transplantation favoring ATZ induction is associated with an initial altered isletāspecific T cell phenotype but reversion toward pretransplant profiles over time. Posttransplant autoreactive T cell phenotype may be a predictor of subsequent graft function
Tethering Telomeric Double- and Single-stranded DNA-binding Proteins Inhibits Telomere Elongation
Mammalian telomeres are composed of G-rich repetitive double-stranded (ds) DNA with a 3' single-stranded (ss) overhang and associated proteins that together maintain chromosome end stability. Complete replication of telomeric DNA requires de novo elongation of the ssDNA by the enzyme telomerase, with telomeric proteins playing a key role in regulating telomerase-mediated telomere replication. In regards to the protein component of mammalian telomeres, TRF1 and TRF2 bind to the dsDNA of telomeres, whereas POT1 binds to the ssDNA portion. These three proteins are linked through either direct interactions or by the proteins TIN2 and TPP1. To determine the biological consequence of connecting telomeric dsDNA to ssDNA through a multiprotein assembly, we compared the effect of expressing TRF1 and POT1 in trans versus in cis in the form of a fusion of these two proteins, on telomere length in telomerase-positive cells. When expressed in trans these two proteins induced extensive telomere elongation. Fusing TRF1 to POT1 abrogated this effect, inducing mild telomere shortening, and generated looped DNA structures, as assessed by electron microscopy, consistent with the protein forming a complex with dsDNA and ssDNA. We speculate that such a protein bridge between dsDNA and ssDNA may inhibit telomerase access, promoting telomere shortening
The pleasures and perils of inheritance
Facing death, reflecting on oneās legacies (material and ethical, personal and political) and the legal and interpersonal attempts to resolve or prevent inheritance conflicts, all bring to the fore constructions of memory and identity, intergenerational relations, and the complexities of doing and undoing family and kinship. Consequently, drawing attention to inheritance, keeping sight of it, and bringing it into play is a useful piece of the puzzle of ageing across a range of disciplines and this article provides an overview of some of the key themes in this emerging field
The impact of time to death in donors after circulatory death on recipient outcome in simultaneous pancreas-kidney transplantation
\ua9 2024 The AuthorsThe time to arrest donors after circulatory death is unpredictable and can vary. This leads to variable periods of warm ischemic damage prior to pancreas transplantation. There is little evidence supporting procurement team stand-down times based on donor time to death (TTD). We examined what impact TTD had on pancreas graft outcomes following donors after circulatory death (DCD) simultaneous pancreas-kidney transplantation. Data were extracted from the UK transplant registry from 2014 to 2022. Predictors of graft loss were evaluated using a Cox proportional hazards model. Adjusted restricted cubic spline models were generated to further delineate the relationship between TTD and outcome. Three-hundred-and-seventy-five DCD simultaneous kidney-pancreas transplant recipients were included. Increasing TTD was not associated with graft survival (adjusted hazard ratio HR 0.98, 95% confidence interval 0.68-1.41, P = .901). Increasing asystolic time worsened graft survival (adjusted hazard ratio 2.51, 95% confidence interval 1.16-5.43, P = .020). Restricted cubic spline modeling revealed a nonlinear relationship between asystolic time and graft survival and no relationship between TTD and graft survival. We found no evidence that TTD impacts pancreas graft survival after DCD simultaneous pancreas-kidney transplantation; however, increasing asystolic time was a significant predictor of graft loss. Procurement teams should attempt to minimize asystolic time to optimize pancreas graft survival rather than focus on the duration of TTD
A Genetic Porcine Model of Cancer
The large size of the pig and its similarity in anatomy, physiology, metabolism, and genetics to humans make it an ideal platform to develop a genetically defined, large animal model of cancer. To this end, we created a transgenic oncopig line encoding Cre recombinase inducible porcine transgenes encoding KRASG12D and TP53R167H, which represent a commonly mutated oncogene and tumor suppressor in human cancers, respectively. Treatment of cells derived from these oncopigs with the adenovirus encoding Cre (AdCre) led to KRASG12D and TP53R167H expression, which rendered the cells transformed in culture and tumorigenic when engrafted into immunocompromised mice. Finally, injection of AdCre directly into these oncopigs led to the rapid and reproducible tumor development of mesenchymal origin. Transgenic animals receiving AdGFP (green fluorescent protein) did not have any tumor mass formation or altered histopathology. This oncopig line could thus serve as a genetically malleable model for potentially a wide spectrum of cancers, while controlling for temporal or spatial genesis, which should prove invaluable to studies previously hampered by the lack of a large animal model of cancer
Extra-Activism: Counter-Mapping and Data Justice
Neither big data, nor data justice are particularly new. Data collection, in the form of land surveys and mapping, was key to successive projects of European imperialist and then capitalist extraction of natural resources. Geo-spatial instruments have been used since the fifteenth century to highlight potential sites of mineral, oil, and gas extraction, and inscribe European economic, cultural and political control across indigenous territories. Although indigenous groups consistently challenged maintained their territorial sovereignty, and resisted corporate and state surveillance practices, they were largely unable to withstand the combined onslaught of surveyors, armed personnel, missionaries and government bureaucrats. This article examines the use of counter-mapping by indigenous nations in Canada, one of the globeās hubs of extractivism, as part of the exercise of indigenous territorial sovereignty. After a brief review of the colonial period, I then compare the use of counter-mapping during two cycles of indigenous mobilization. During the 1970s, counter-mapping projects were part of a larger repertoire of negotiations with the state over land claims, and served to re-inscribe first nationās long-standing history of economic, social and cultural relations in their territories, and contribute to new collective imaginaries and identities. In the current cycle of contests over extractivism and indigenous sovereignty, the use, scope and geographic scale of counter-mapping has shifted; maps are used as part of larger trans-media campaigns of Indigenous sovereignty. During both cycles, counter-mapping as data justice required fusion within larger projects of redistributive, transformative and restorative justice
Telomerase Efficiently Elongates Highly Transcribing Telomeres in Human Cancer Cells
RNA polymerase II transcribes the physical ends of linear eukaryotic chromosomes into a variety of long non-coding RNA molecules including telomeric repeat-containing RNA (TERRA). Since TERRA discovery, advances have been made in the characterization of TERRA biogenesis and regulation; on the contrary its associated functions remain elusive. Most of the biological roles so far proposed for TERRA are indeed based on in vitro experiments carried out using short TERRA-like RNA oligonucleotides. In particular, it has been suggested that TERRA inhibits telomerase activity. We have exploited two alternative cellular systems to test whether TERRA and/or telomere transcription influence telomerase-mediated telomere elongation in human cancer cells. In cells lacking the two DNA methyltransferases DNMT1 and DNMT3b, TERRA transcription and steady-state levels are greatly increased while telomerase is able to elongate telomeres normally. Similarly, telomerase can efficiently elongate transgenic inducible telomeres whose transcription has been experimentally augmented. Our data challenge the current hypothesis that TERRA functions as a general inhibitor of telomerase and suggest that telomere length homeostasis is maintained independently of TERRA and telomere transcription
A universal telomerase RNA core structure includes structured motifs required for binding the telomerase reverse transcriptase protein
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