scholarly journals Therapeutic cloning: promises and issues

2020 ◽  
Vol 10 (2) ◽  
Author(s):  
Charlotte Kfoury
Keyword(s):  
De Novo ◽  
Genetic Disorders ◽  
In Vivo Studies ◽  
Therapeutic Cloning ◽  
Ethical Considerations ◽  
Cell Replacement Therapy ◽  
Ethical Implications ◽  
Scientific Principles ◽  
Mitochondrial Heteroplasmy

Advances in biotechnology necessitate both an understanding of scientific principles and ethical implications to be clinically applicable in medicine. In this regard, therapeutic cloning offers significant potential in regenerative medicine by circumventing immunorejection, and in the cure of genetic disorders when used in conjunction with gene therapy. Therapeutic cloning in the context of cell replacement therapy holds a huge potential for de novo organogenesis and the permanent treatment of Parkinson's disease, Duchenne muscular dystrophy, and diabetes mellitus as shown by in vivo studies. Scientific roadblocks impeding advancement in therapeutic cloning are tumorigenicity, epigenetic reprogramming, mitochondrial heteroplasmy, interspecies pathogen transfer, low oocyte availability. Therapeutic cloning is also often tied to ethical considerations concerning the source, destruction and moral status of IVF embryos based on the argument of potential. Legislative and funding issues are also addressed. Future considerations would include a distinction between therapeutic and reproductive cloning in legislative formulations.

scholarly journals Role of SP65 in Assembly of the Dictyostelium discoideum Spore Coat

2007 ◽  
Vol 6 (7) ◽  
pp. 1137-1149 ◽  
Author(s):  
Talibah Metcalf ◽  
Hanke van der Wel ◽  
Ricardo Escalante ◽  
Leandro Sastre ◽  
Christopher M. West
Keyword(s):  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Outer Layer ◽  
Fluorescent Protein ◽  
De Novo ◽  
Middle Layer ◽  
In Vivo Studies ◽  
Permeability Barrier ◽  
Spore Coat

ABSTRACT Like the cyst walls of other protists, the spore coat of Dictyostelium discoideum is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils, resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with green fluorescent protein, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially and that the complex contributes an essential function to outer layer architecture and function.

scholarly journals Insects as a source of phenolic compounds and potential health benefits

2021 ◽  
pp. 1-12
Author(s):  
M.C. Nino ◽  
L. Reddivari ◽  
C. Osorio ◽  
I. Kaplan ◽  
A.M. Liceaga
Keyword(s):  
Phenolic Compounds ◽  
De Novo ◽  
Health Benefits ◽  
Antimicrobial Activities ◽  
In Vivo Studies ◽  
Plant Phenolics ◽  
Potential Health ◽  
Bioactive Potential

The use of insects in traditional medicine and unveiling the chemical structure of the bright pigments in butterfly wings led to the discovery of bioactive phenolic compounds in the insect bodies. These metabolites have been found not only due to the insect absorption and metabolisation of the plant-derived phenolic present in their diet, but also from the ability of insects to synthesise phenolic compounds de novo through the sclerotisation process. Plant phenolics are secondary metabolites involved in the protection of tissues against UV radiation, herbivores, and pathogens, as well as pigmentation of fruits and flowers. These bioactive compounds exhibit antioxidant, anti-inflammatory, anticancer, and antimicrobial activities, demonstrated through in vitro and in vivo studies. This bioactive potential is thought to occur due to their chemical characteristics that allow them to stabilise reactive oxygen species (ROS), chelate prooxidant metal ions, interact with key enzymes and signal cascades involved in biological pathways. Bioactivity of plant phenolics and both in vitro, in vivo studies, suggest that the dietary compounds absorbed by the insect maintain their chemical and bioactive properties. Further characterisation of the phenolic composition in edible insects and evaluation of their bioactive capacity as well as their bioavailability, could result in discovering additional health benefits of entomophagy apart from macro-nutritional (e.g. protein) content.

scholarly journals A Novel FBXO45-Gef-H1 Axis Controls Oncogenic Signaling in B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 711-711
Author(s):  
Anagh Anant Sahasrabuddhe ◽  
Xiaofei Chen ◽  
Kaiyu Ma ◽  
Rui Wu ◽  
Richa Kapoor ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
De Novo ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
In Vivo Studies

Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

The F11 receptor (F11R/JAM-A) in atherothrombosis: Overexpression of F11R in atherosclerotic plaques

2007 ◽  
Vol 97 (02) ◽  
pp. 272-281 ◽  
Author(s):  
Anna Babinska ◽  
Bani Azari ◽  
Moro Salifu ◽  
Ruijie Liu ◽  
Xian-Cheng Jiang ◽  
...  
Keyword(s):  
De Novo ◽  
Human Platelets ◽  
In Vivo Studies ◽  
Atherosclerotic Plaques ◽  
Adhesion Protein ◽  
Heart Attacks ◽  
Therapeutic Drugs ◽  
Cardiovascular Patients ◽  
Enhanced Expression

SummaryF11R is the gene name for an adhesion protein, called the F11-receptor, aka JAM-A, which under normal physiological conditions is expressed constitutively on the surface of platelets and localized within tight junctions of endothelial cells (EC). Previous studies of the interactions between human platelets and EC suggested that F11R/JAM-A plays a crucial role in inflammatory thrombosis and atherosclerosis. The study reported here obtained in-vivo confirmation of this conclusion by investigating F11R/JAM-A protein and mRNA in patients with aortic and peripheral vascular disease and in an animal model of atherosclerosis. Molecular and immunofluorescence determinations revealed very high levels of F11R/JAM-A mRNA and F11R/JAM-A protein in atherosclerotic plaques of cardiovascular patients. Similar results were obtained with 12-week-old atherosclerosis-prone apoE-/- mice, an age in which atherosclerotic plaques are well established. Enhanced expression of the F11R/JAM-A message in cultured EC from human aortic and venous vessels was observed following exposure of the cells to cytokines. Determinations of platelet adhesion to cultured EC inflamed by combined cytokine treatment in the presence of F11R/JAM-A – antagonists provided data indicating that de novo expression of F11R/JAM-A on the luminal surface of inflamed EC has an important role in the conversion of EC to a thrombogenic surface. Further studies of these interactions under flow conditions and under in-vivo settings could provide a final proof of a causal role for F11R/JAM-A in the initiation of thrombosis. Based on our invitro and in-vivo studies to date, we propose that therapeutic drugs which antagonize the function of F11R/JAM-A should be tested as novel means for the prevention and treatment of atherosclerosis, heart attacks and stroke.

scholarly journals Histologic Comparison of the Dura Mater among Species

2020 ◽  
Vol 70 (2) ◽  
pp. 170-175 ◽  
Author(s):  
Ahmet Kinaci ◽  
Wilhelmina Bergmann ◽  
Ronald LAW Bleys ◽  
Albert van der Zwan ◽  
Tristan PC van Doormaal
Keyword(s):  
Dura Mater ◽  
Animal Species ◽  
Porcine Model ◽  
Gross Anatomy ◽  
In Vivo Studies ◽  
Ethical Considerations ◽  
Border Cell ◽  
Hematoxylin And Eosin ◽  
Basic Characteristics

The biocompatibility, biodegradation, feasibility, and efficacy of medical devices like dural sealants and substitutes are often evaluated in various animal models. However, none of these studies explain the rationale for choosing a particular species, and a systematic interspecies comparison of the dura is not available. We hypothesized that histologic characteristics of the dura would differ among species. We systematically investigated basic characteristics of the dura, including thickness, composition, and fibroblast orientation of the dura mater, in 34 samples representing 10 animal species and compared these features with human dura by using hematoxylin and eosin staining and light microscopy. Dura showed many similarities between species in terms of composition. In all species, dura consisted of at least one fibrovascular layer, which contained collagen, fibroblasts, and blood vessels, and a dural border cell layer beneath the fibrovascular layer. Differences between species included the number of fibrovascular layers, fibroblast orientation, and dural thickness. Human dura was the thickest (564 μm) followed by equine (313 μm), bovine (311 μm), and porcine (304 μm) dura. Given the results of this study and factors such as gross anatomy, feasibility, housing, and ethical considerations, we recommend the use of a porcine model for dural research, especially for in vivo studies.

scholarly journals Variants in PRKAR1B cause a neurodevelopmental disorder with autism spectrum disorder, apraxia, and insensitivity to pain

2020 ◽  
Author(s):  
Felix Marbach ◽  
Georgi Stoyanov ◽  
Florian Erger ◽  
Jill A. Rosenfeld ◽  
Erin Torti ◽  
...  
Keyword(s):  
Autism Spectrum Disorder ◽  
De Novo ◽  
Neurodevelopmental Disorder ◽  
Autism Spectrum ◽  
Spectrum Disorder ◽  
In Vivo Studies ◽  
Global Developmental Delay ◽  
Missense Variants ◽  
Significant Enrichment

Purpose: We characterize the phenotypes of six unrelated individuals with intellectual disability and autism spectrum disorder, who carry heterozygous missense-variants of the PRKAR1B gene. Methods: Variants of PRKAR1B were identified by single-exome or trio-exome analysis. We contacted the families and physicians of the six individuals in order to collect clinical and phenotypic information. Results: PRKAR1B encodes the R1β subunit of the cyclic AMP-dependent protein kinase A (PKA), and is predominantly expressed in the central nervous system. Recent studies of patient cohorts with neurodevelopmental disorders found significant enrichment of de novo missense variants in PRKAR1B, and in vivo studies of the murine ortholog demonstrated altered hippocampal function and reduced neurogenic inflammation and long-term nociceptive pain in R1β-deficient mice. In our study, de novo origin of the PRKAR1B-variants could be confirmed in five out of six individuals, and four carried the same heterozygous de novo variant c.1003C>T (p. Arg335Trp; NM_001164760). Global developmental delay, autism spectrum disorder, and apraxia/dyspraxia has been reported in all six, and reduced pain sensitivity was found in three individuals carrying the c.1003C>T variant. Conclusion: Our study provides strong evidence for a novel, PRKAR1B-related neurodevelopmental disorder.

scholarly journals ESCRT-III acts in scissioning new peroxisomes from the ER

2017 ◽  
Author(s):  
Fred D. Mast ◽  
Thurston Herricks ◽  
Kathleen M. Strehler ◽  
Leslie R. Miller ◽  
Ramsey A. Saleem ◽  
...  
Keyword(s):  
De Novo ◽  
Genetic Disorders ◽  
Dynamic Control ◽  
Eukaryotic Cell ◽  
Peroxisome Biogenesis ◽  
Endosomal Sorting ◽  
Obesity And Diabetes ◽  
A Cell ◽  
And Function

AbstractDynamic control of peroxisome proliferation is integral to the peroxisome’s many functions. A breakdown in the ability of cells to form peroxisomes is linked to many human health issues, including defense against infectious agents, cancer, aging, heart disease, obesity and diabetes, and forms the basis of a spectrum of peroxisomal genetic disorders that cause severe neuropathologies. The ER serves as a source for preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and to support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. Here we show that the evolutionarily ancient endosomal sorting complexes required for transport (ESCRT)-III are peroxisome biogenesis factors that function to cleave PPVs budding from the ER into the cytosol. Using comprehensive morphological and genetic assays of peroxisome formation and function we find that absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay we show that ESCRT-III proteins Vps20 and Snf7 are required to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and towards the cytosol, a finding that has important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.

scholarly journals RARE-22. THERAPEUTIC TARGETING OF PURINE METABOLISM IN DIPG

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i45-i45
Author(s):  
Ian Mersich ◽  
Biplab Dasgupta
Keyword(s):  
Cell Lines ◽  
De Novo ◽  
Treatment Strategies ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Purine Metabolism ◽  
In Vivo Studies ◽  
Molecule Inhibitor ◽  
Clinical Benefits

Abstract Diffuse intrinsic pontine glioma is a universally lethal disease primarily impacting pediatric patients. There are currently no targeted therapies increasing overall for patients with these tumors; therefore, our lab set out to elucidate metabolic dependencies in DIPG patient-derived cell lines with the ultimate goal of identifying novel therapeutic targets. Through untargeted metabolomics and gene expression analyses, we have identified the purine metabolism gene ATIC to be important for DIPG tumor cell survival and proliferation. Anti-folate drugs such as methotrexate target de novo purine biosynthesis and are used to treat other pediatric cancers; however, we have identified a small molecule inhibitor of ATIC that may offer clinical benefits over other inhibitors of this pathway. In vitro cell viability experiments have demonstrated DIPG cell lines are much more sensitive to the ATIC inhibitor relative to normal neural stem cells and glial cell lines. Furthermore, we have started in vivo studies on pre-clinical mouse models of DIPG with promising results. Treatment with the ATIC inhibitor has significantly increased overall survival relative to control and vehicle treated mice. The dosage we started at was well tolerated in these mice so we are following up on this in vivo work through dose-escalation studies as well as combination treatment strategies. Mechanistically, the ATIC inhibitor works differently than anti-folate compounds such as methotrexate; therefore we are also elucidating why cancer cells are much more sensitive to this compound.

scholarly journals The CbiB Protein of Salmonella enterica Is an Integral Membrane Protein Involved in the Last Step of the De Novo Corrin Ring Biosynthetic Pathway

2007 ◽  
Vol 189 (21) ◽  
pp. 7697-7708 ◽  
Author(s):  
Carmen L. Zayas ◽  
Kathy Claas ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Membrane Protein ◽  
Salmonella Enterica ◽  
De Novo ◽  
Integral Membrane Protein ◽  
In Vivo Studies ◽  
Single Amino Acid ◽  
Serovar Typhimurium ◽  
Ethanolamine Phosphate ◽  
Coenzyme B

ABSTRACT We report results of studies of the conversion of adenosylcobyric acid (AdoCby) to adenosylcobinamide-phosphate, the last step of the de novo corrin ring biosynthetic branch of the adenosylcobalamin (coenzyme B12) pathway of Salmonella enterica serovar Typhimurium LT2. Previous reports have implicated the CbiB protein in this step of the pathway. Hydropathy analysis predicted that CbiB would be an integral membrane protein. We used a computer-generated topology model of the primary sequence of CbiB to guide the construction of CbiB-LacZ and CbiB-PhoA protein fusions, which were used to explore the general topology of CbiB in the cell membrane. A refined model of CbiB as an integral membrane protein is presented. In vivo analyses of the effect of single-amino-acid changes showed that periplasm- and cytosol-exposed residues are critical for CbiB function. Results of in vivo studies also show that ethanolamine-phosphate (EA-P) is a substrate of CbiB, but l-Thr-P is not, and that CbiB likely activates AdoCby by phosphorylation. The latter observation leads us to suggest that CbiB is a synthetase not a synthase enzyme. Results from mass spectrometry and bioassay experiments indicate that serovar Typhimurium synthesizes norcobalamin (cobalamin lacking the methyl group at C176) when EA-P is the substrate of CbiB.

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