The optimal mesenchymal stem cell (MSC) culture conditions that would allow clinically viable tissue-engineered devices are still yet to be determined. Most MSCs are found in the bone marrow, an area ...that also contains numerous osteoblasts and adipocytes. Paracrine signalling may be leveraged to modulate MSC differentiation in the preparation of a tissue-engineered device. Thus, the objectives of this study were to determine the effects of adipocyte-conditioned medium (CM) on MSC differentiation to osteoblasts and to determine the effects of osteoblast CM on MSC differentiation to adipocytes. Two groups of murine MSCs were given either an osteogenic differentiation medium or an adipogenic differentiation medium. CM was taken from one group and administered to the opposite group in concentrations of 25% or 50%. Metabolic activity, total protein and alkaline phosphatase (ALP) assays were conducted on the osteogenic group at predefined time points throughout the 21 day study, while metabolic activity, triglyceride and oil red O assays were conducted on the adipogenic group at predefined time points. Adipocyte CM administered at a concentration of 50% increased the ALP production of MSCs undergoing osteogenic differentiation. Additionally, osteoblast CM increased the triglyceride production of MSCs undergoing adipogenic differentiation and enlarged the lipid vesicles that were produced by the cells. The effects of the osteoblast CM were seen at both concentrations, but were greatest at the 50% CM level.
Chemical sites should have well trained and organized emergency response plans to manage an incident within the plant or during transport. The implementation of an incident command system utilizing ...either internal resources or external response through mutual aid agreements is generally sufficient to address the direct impact of an event on the site. When the site resources become overwhelmed in addressing resulting issues such as press releases, medical advice/support, employees and family support, Agency notifications, etc, Corporate should be ready and able to respond.
This paper, taken from an in-depth CCPS workshop led by the author, describes an outline for corporate assistance in the event of a major incident at a site or during transportation.
Objective: To study the effect of in vitro culture on the quality of human testicular sperm and the efficiency of intracytoplasmic sperm injection with in vitro cultured testicular sperm.
Design: ...Clinical study.
Setting: A private IVF center.
Patient(s): Twenty consecutively seen IVF patients undergoing testicular biopsies for ICSI.
Intervention(s): The testicular specimens were cultured in vitro for 24 hours and the isolated spermatozoa were microinjected.
Main Outcome Measure(s): Preincubation and postculture sperm motility, and fertilization, implantation, and pregnancy rates after intracytoplasmic sperm injection.
Result(s): Motility increased from initial nonmotile or twitching sperm to free motile sperm in 18 of 20 cases. The injection of in vitro cultured testicular sperm resulted in a fertilization rate of 58%, an implantation rate of 20%, and a pregnancy rate of 45%.
Conclusion(s): A testicular biopsy procedure can be performed the day before egg retrieval. Despite the low initial sperm quality, a high percentage of the prepared testicular sperm showed increased motility after 24 hours of culture. The injection of in vitro cultured testicular sperm into matured oocytes resulted in fertilization, implantation, and pregnancy rates comparable to those obtained with ejaculated sperm.
''Rethinking Japanese Feminisms'' offers a broad overview of the great diversity of feminist thought and practice in Japan from the early twentieth century to the present. Drawing on methodologies ...and approaches from anthropology, cultural studies, gender and sexuality studies, history, literature, media studies, and sociology, each chapter presents the results of research based on some combination of original archival research, careful textual analysis, ethnographic interviews, and participant observation. Building on more than four decades of scholarship on feminisms in Japanese and English, as well as decades more on women’s history, this book offers a diverse and multivocal approach to scholarship on Japanese feminisms unmatched by existing publications. It will be at home in the hands of students and scholars, as well as activists and others interested in gender, sexuality, and feminist theory and activism in Japan and in Asia more broadly.
Authors' Reply Nabaweesi, Rosemary; Ramakrishnaiah, Raghu H; Aitken, Mary E ...
Journal of the American College of Radiology,
06/2018, Letnik:
15, Številka:
6
Journal Article
Objective: To study the effect of freezing on early stage embryos derived from intracytoplasmic sperm injection (ICSI) or from IVF.
Design: Prospective, controlled clinical study.
Setting: Private ...IVF center.
Patient(s): Sixty-seven consecutive patients undergoing frozen-thawed embryo transfer cycles.
Intervention(s): Early stage embryos were frozen, thawed, and transferred.
Main Outcome Measure(s): Post-thaw survival, implantation and pregnancy rates.
Result(s): We noted an 88% post-thaw survival rate, an 18% implantation rate, and a 52% pregnancy rate in the ICSI group and 81%, 11%, and 25%, respectively, with conventional fertilization.
Conclusion(s): Early stage embryos (either zygote or 2–4 cells) derived from ICSI can be frozen with confidence and higher post-thaw survival and pregnancy rates can be achieved when compared with those from conventional IVF.
Abstract
Large scale sequencing efforts are currently being applied to numerous cancers and although significant numbers of mutations are being identified, functional validation to identify driver ...mutations is lagging. To accelerate progress in the identification of genetic drivers in hematological malignances, we are taking an integrated approach of deep sequencing coupled with functional screens using molecularly targeted drugs and siRNA screening of primary patient leukemia samples. The functional screens allow prioritization of mutations for validation. Using this approach, we identified a CSF3R mutation in a patient with chronic neutrophilic leukemia (CNL). Patients with CNL and the related disorder, atypical chronic myeloid leukemia (aCML), have neoplastic expansion of granulocytic cells with the diagnosis including exclusion of genetic drivers known to occur in other types of World Health Organization-defined myeloproliferative neoplasms (MPN) or myelodysplastic syndrome/MPN overlap disorders. After identifying a CSF3R mutation in our index case, we examined 21 additional cases of CNL and aCML and found that 59% of patients with CNL/aCML harbor CSF3R mutations. Two distinct regions of mutations within the CSF3R gene were identified. Expression of these classes of mutations in a growth factor-dependent myeloid cell line, BaF3, transformed these cells to growth factor independence. Interestingly, these two mutational classes produce preferential downstream kinase signaling (via TNK2/SRC or JAK kinases) and differential sensitivity to kinase inhibitors. Specifically, mutants signaling through TNK2/SRC are sensitive to SRC inhibitors such as dasatinib while mutants signaling thought JAK kinases were sensitive to JAK inhibitors such as ruxolitinib. One patient with CNL carrying a CSF3R mutation that signals through JAK kinases showed a dramatic and durable clinical improvement after treatment with the JAK1/2 kinase inhibitor, ruxolitinib. Mutations in the CSF3R define a large subset of patients with CNL/aCML and testing for these mutations will complement histopathologic diagnosis of CNL and aCML. Patients with CSF3R mutations may benefit from clinical administration of TNK2/SRC inhibitors (such as dasatinib) or JAK inhibitors (such as ruxolitinib). Our studies demonstrate that combining functional screens with genome sequencing can significantly accelerate target validation to define driving mutations that can be matched with therapies.
Citation Format: Julia Maxson, Jason Gotlib, Daniel Pollyea, Angela Fleischman, Christopher Eide, Daniel Bottomly, Beth Wilmot, Shannon McWeeney, Cristina Tognon, J. Blake Pond, Robert Collins, Michael Deininger, Bill Chang, Marc Loriaux, Brian Druker, Jeffrey Tyner. Rapid identification of targetable CSF3R mutations that define neutrophilic leukemia by combining functional and genomic screens. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2282. doi:10.1158/1538-7445.AM2013-2282
▪
We have recently identified mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) in ∼60% of chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) patients ...(Maxson et al, NEJM 2013). These mutations fall into two categories: membrane proximal point mutations (the most common of which is T618I) and truncation mutations. Drug and siRNA screening of primary patient samples revealed that the two classes of CSF3R mutations exhibit differential sensitivity to inhibition of SRC or JAK kinases. CSF3R truncation mutations conferred sensitivity to SRC family kinase inhibition, while CSF3R membrane proximal mutations (T618I) conferred sensitivity to JAK kinase inhibition. A patient with the T618I membrane proximal mutation responded to treatment with the FDA approved JAK inhibitor, ruxolitinib. CSF3R truncation mutations have also been observed in a subset of severe congenital neutropenia patients who are at high risk for development of acute myeloid leukemia. Prior studies in this context have shown that truncation mutations result in loss of endocytic and degradation motifs, leading to increased expression of the receptor. The differences in signaling and drug sensitivity of these mutation classes suggest that membrane proximal mutations may activate CSF3R signaling through a distinct, as-yet unknown mechanism. Furthermore, a subset of CNL patients harbor both membrane proximal and truncation mutations on the same allele, though the consequences of these compound mutations are not yet known.
CSF3R expression level and banding pattern were assessed by immunoblot of lysates from 293T17 cells transfected with wild type, membrane proximal mutant, or truncation mutant CSF3R. O-linked glycosylation was removed from the receptor by treatment with O-glycosidase and neuraminidase. Ligand independence of the CSF3R mutants was analyzed in murine interleukin-3 (IL3)-dependent Ba/F3 cells. CSF3R dimerization was assessed by co-transfecting CSF3R-Flag and CSF3R-V5 tagged constructs and then immunoprecipitating CSF3R-Flag and detecting co-immunoprecipitation of the CSF3R-V5 by immunoblot. Transforming potential of the CSF3R compound mutations relative to the corresponding point or truncation mutations was assessed by analyzing IL3-independent growth of Ba/F3 cells or mouse bone marrow colony formation.
To better understand the functional and biochemical differences between membrane proximal and truncation mutant CSF3R, we examined transformation potential, requirement for ligand, and expression patterns in Ba/F3 and 293T17 cells. We found membrane proximal mutations to exhibit rapid transformation potential and ligand independence, while truncation mutations exhibited delayed transformation and ligand hypersensitivity. Unlike the truncation mutations, which induce dramatic overexpression of CSF3R, the T618I mutation did not result in overexpression of the receptor but instead induced a shifted banding pattern, indicative of altered protein modification. We examined the amino acid sequence surrounding the membrane proximal mutations and found residue T618 to be part of a consensus motif for O-glycosylation, wherein wild type CSF3R is O-glycosylated and the T618I mutation abrogates this O-glycosylation event. Furthermore, the T618I mutation exhibited increased receptor dimerization compared to wild type CSF3R, which likely explains its ligand independence. Finally, we found that CSF3R compound mutations have increased transforming potential in Ba/F3 and murine bone marrow colony assays compared with either class of single mutation, further underscoring the different mechanisms of action of the membrane proximal and truncation mutations.
CSF3R represents a promising therapeutic target for patients with CNL. We show that T618I, the most common CSF3R mutation in CNL, is part of an O-linked glycosylation site. Mutation of this residue leads to loss of O-linked glycosylation and represents a novel mechanism of homodimeric cytokine receptor activation. CSF3R compound mutations are more rapidly transforming relative to the membrane proximal or truncation mutations alone, warranting their further investigation for patient prognosis and therapy.
Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Gotlib:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel Support Other. Fleischman:Incyte: Speakers Bureau. Collins:Genoptix: Membership on an entity's Board of Directors or advisory committees. Oh:Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Deininger:Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding. Druker:Bristol-Myers Squibb: PI or co-investigator on BMS clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Novartis: PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Incyte: PI or co-investigator on clinical trials., PI or co-investigator on clinical trials. Other. Tyner:Incyte Corporation: Research Funding.
PDF
