Fractional Hardy-Sobolev type inequalities for half spaces and John domains

As our main result we prove a variant of the fractional Hardy-Sobolev-Maz'ya inequality for half spaces. This result contains a complete answer to a recent open question by Musina and Nazarov. In the proof we apply a new version of the fractional Hardy-Sobolev inequality that we establish also for more general unbounded John domains than half spaces

Mechanisms of Resistance to Noncovalent Bruton's Tyrosine Kinase Inhibitors

BackgroundCovalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood.MethodsWe performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors.ResultsAmong 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors.ConclusionsResistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.)

Spermatogonial stem cells show an age-dependent and age-independent difference in commitment to self-renewal and differentiation

The lifelong process of spermatogenesis is supported by spermatogonial stem cells (SSCs). Because of their ability to self-renew and produce differentiated cells that transmit genetic information to the next generation, SSCs are an important target cell population to restore male fertility. Pup (6-8 days old) and adult mouse SSCs are believed to possess different characteristics. In order to determine if there is a difference in biological activity of SSCs, in Aim 1, I evaluated the self-renewal and differentiation pattern of pup SSCs and compared it to adult SSCs. Using serial transplantation, I showed that pup SSCs preferentially commit to differentiation during the first month after transplantation compared to adult SSCs. This difference in biological activity of pup and adult SSCs may be due to age-dependent and age-independent effects. To investigate the age-dependent effect, in Aim 2, I used immunomagnetic cell sorting to evaluate the expression pattern of cell-surface molecules on pup and adult SSCs. Transplantation of selected cells showed that pup SSCs preferentially express glial cell line-derived neurotrophic factor (GDNF) family receptor alpha 1, a GDNF receptor, compared to adult SSCs, indicating an age-dependent difference in cell-surface molecule phenotype. In Aim 3, I investigated the age-independent effect of cell cycle activity on SSC commitment to self-renewal and differentiation, based on the fact that pup SSCs are known to cycle more actively than adult SSCs. To investigate the fate decision of cycling SSCs, I used an SSC culture system, which allows for expansion of SSCs in the presence of GDNF and fibroblast growth factor 2 (FGF2). Transplantation of SSC cultures temporally exposed to a retroviral vector carrying the LacZ gene showed a ~100-fold increase in the number of SSCs that divided in the presence of GDNF and FGF2. However, this increase in cell cycle activity stimulated SSCs to produce differentiating cells at the expense of their oLes cellules souches germinales (CSGs) sont responsables de la production à vie de spermatozoïdes et constituent le seul type cellulaire pouvant s'autorenouveler et contribuer au patrimoine génétique de notre progéniture et représentent donc une cible idéale pour restaurer la fertilité masculine. Il est généralement accepté que les CSGs pré-pubères (6 à 8 jours) et adultes possèdent des caractéristiques distinctes. Pour déterminer si leur activité biologique est différente, j'ai évalué leur potentiel d'autorenouvellement et de différenciation par transplantation séquentielle. Contrairement aux CSGs adultes, les CSGs pré-pubères empruntent préférentiellement la voie de différenciation le premier mois post-transplantation. Cette différence entre les CSGs adultes et pré-pubères pourrait découler de facteurs reliés à l'âge. Afin d'éxaminer l'influence de l'âge sur leur activité biologique, j'ai utilisé une approche d'immunosélection cellulaire par tri magnétique pour évaluer l'expression de protéines membranaires des CSGs adultes et pré-pubères. La transplantation des cellules sélectionnées a montré que GFR alpha 1, un récepteur pour GDNF (glial cell-derived neurotrophic factor), est préférentiellement exprimé sur les CSGs pré-pubères, démontrant un effet de l'âge sur le phénotype membranaire des CSGs. Puisque les CSGs pré-pubères se divisent plus activement que les CSGs adultes, j'ai avons ensuite déterminé l'effet de l'activité du cycle cellulaire sur le potentiel d'autorenouvellement et de différenciation des CSGs. Pour évaluer le rôle du cycle cellulaire sur la détermination de la destinée des CSGs, j'ai utilisé un système de culture qui permet l'expansion infinie des CSGs en présence des facteurs de croissance GDNF et FGF2 (fibroblast growth factor 2). Les CSGs ainsi cultivées ont été transduites par un vecteur rétroviral contenant le gène rapporteur LacZ et soumises à une transplan

The Application of Biomarkers of Spermatogonial Stem Cells for Restoring Male Fertility

Spermatogonial stem cells (SSCs) are defined by their ability to both self-renew and produce differentiated germ cells that will develop into functional spermatozoa. Because of this ability, SSCs can reestablish spermatogenesis after testicular damage caused by cytotoxic agents or after transplantation into an infertile recipient. Therefore, SSCs are an important target cell for restoring male fertility, particularly for cancer patients who have to undergo sterilizing cancer therapies. In the mouse, the identification of SSC markers allows for the isolation of a highly enriched population of stem cells. This enriched stem cell population can be expanded in culture for an indefinite period of time, cryopreserved, and transplanted into infertile recipients to restore fertility. Thus, the identification of markers and the establishment of a long-term culture system for human SSCs will be crucial for realizing the potential of these cells in a clinical setting. In this article, we focus on the markers that have been identified for mouse SSCs and discuss how human SSC markers may be used in the restoration of fertility

Bivalent Chromatin Marks Developmental Regulatory Genes in the Mouse Embryonic Germline In Vivo

Developmental regulatory genes have both activating (H3K4me3) and repressive (H3K27me3) histone modifications in embryonic stem cells (ESCs). This bivalent configuration is thought to maintain lineage commitment programs in a poised state. However, establishing physiological relevance has been complicated by the high number of cells required for chromatin immunoprecipitation (ChIP). We developed a low-cell-number chromatin immunoprecipitation (low-cell ChIP) protocol to investigate the chromatin of mouse primordial germ cells (PGCs). Genome-wide analysis of embryonic day 11.5 (E11.5) PGCs revealed H3K4me3/H3K27me3 bivalent domains highly enriched at developmental regulatory genes in a manner remarkably similar to ESCs. Developmental regulators remain bivalent and transcriptionally silent through the initiation of sexual differentiation at E13.5. We also identified >2,500 “orphan” bivalent domains that are distal to known genes and expressed in a tissue-specific manner but silent in PGCs. Our results demonstrate the existence of bivalent domains in the germline and raise the possibility that the somatic program is continuously maintained as bivalent, potentially imparting transgenerational epigenetic inheritance

Vitamin C Induces Specific Demethylation of H3K9me2 in Mouse Embryonic Stem Cells via Kdm3a/b

Histone methylation patterns regulate gene expression and are highly dynamic during development. The erasure of histone methylation is carried out by histone demethylase enzymes. We had previously shown that vitamin C enhances the activity of Tet enzymes in embryonic stem (ES) cells, leading to DNA demethylation and activation of germline genes. We report here that vitamin C induces a remarkably specific demethylation of histone H3 lysine 9 dimethylation (H3K9me2) in ES cells. Vitamin C treatment reduces global levels of H3K9me2, but not other histone methylation marks analyzed, as measured by western blot, immunofluorescence and mass spectrometry. Vitamin C leads to widespread loss of H3K9me2 at large chromosomal domains as well as gene promoters and repeat elements. Vitamin C-induced loss of H3K9me2 occurs rapidly within 24 hours and is reversible. Importantly, we found that the histone demethylases Kdm3a and Kdm3b are required for vitamin C-induced demethylation of H3K9me2. Moreover, we show that vitamin C-induced Kdm3a/b-mediated H3K9me2 demethylation and Tet-mediated DNA demethylation are independent processes. Lastly, we document Kdm3a/b are partially required for the up-regulation of germline genes by vitamin C. These results reveal a specific role for vitamin C in histone demethylation in ES cells, and document that DNA methylation and H3K9me2 cooperate to silence germline genes in pluripotent cells

Vitamin C induces specific demethylation of H3K9me2 in mouse embryonic stem cells via Kdm3a/b

Background: Histone methylation patterns regulate gene expression and are highly dynamic during development. The erasure of histone methylation is carried out by histone demethylase enzymes. We had previously shown that vitamin C enhances the activity of Tet enzymes in embryonic stem (ES) cells, leading to DNA demethylation and activation of germline genes. Results: We report here that vitamin C induces a remarkably specific demethylation of histone H3 lysine 9 dimethylation (H3K9me2) in naïve ES cells. Vitamin C treatment reduces global levels of H3K9me2, but not other histone methylation marks analyzed, as measured by western blot, immunofluorescence and mass spectrometry. Vitamin C leads to widespread loss of H3K9me2 at large chromosomal domains as well as gene promoters and repeat elements. Vitamin C-induced loss of H3K9me2 occurs rapidly within 24 h and is reversible. Importantly, we found that the histone demethylases Kdm3a and Kdm3b are required for vitamin C-induced demethylation of H3K9me2. Moreover, we show that vitamin C-induced Kdm3a/b-mediated H3K9me2 demethylation and Tet-mediated DNA demethylation are independent processes at specific loci. Lastly, we document Kdm3a/b are partially required for the upregulation of germline genes by vitamin C. Conclusions: These results reveal a specific role for vitamin C in histone demethylation in ES cells and document that DNA methylation and H3K9me2 cooperate to silence germline genes in pluripotent cells.Science, Faculty ofOther UBCNon UBCMicrobiology and Immunology, Department ofReviewedFacult

Maternal vitamin C regulates reprogramming of DNA methylation and germline development

Development is often assumed to be hardwired in the genome, but several lines of evidence indicate that it is susceptible to environmental modulation with potential long-term consequences, including in mammals1,2. The embryonic germline is of particular interest because of the potential for intergenerational epigenetic effects. The mammalian germline undergoes extensive DNA demethylation3–7 that occurs in large part by passive dilution of methylation over successive cell divisions, accompanied by active DNA demethylation by TET enzymes3,8–10. TET activity has been shown to be modulated by nutrients and metabolites, such as vitamin C11–15. Here we show that maternal vitamin C is required for proper DNA demethylation and the development of female fetal germ cells in a mouse model. Maternal vitamin C deficiency does not affect overall embryonic development but leads to reduced numbers of germ cells, delayed meiosis and reduced fecundity in adult offspring. The transcriptome of germ cells from vitamin-C-deficient embryos is remarkably similar to that of embryos carrying a null mutation in Tet1. Vitamin C deficiency leads to an aberrant DNA methylation profile that includes incomplete demethylation of key regulators of meiosis and transposable elements. These findings reveal that deficiency in vitamin C during gestation partially recapitulates loss of TET1, and provide a potential intergenerational mechanism for adjusting fecundity to environmental conditions.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Maternal vitamin C regulates reprogramming of DNA methylation and germline development

Development is often assumed to be hardwired in the genome, but several lines of evidence indicate that it is susceptible to environmental modulation with potential long-term consequences, including in mammals1,2. The embryonic germline is of particular interest because of the potential for intergenerational epigenetic effects. The mammalian germline undergoes extensive DNA demethylation3-7 that occurs in large part by passive dilution of methylation over successive cell divisions, accompanied by active DNA demethylation by TET enzymes3,8-10. TET activity has been shown to be modulated by nutrients and metabolites, such as vitamin C11-15. Here we show that maternal vitamin C is required for proper DNA demethylation and the development of female fetal germ cells in a mouse model. Maternal vitamin C deficiency does not affect overall embryonic development but leads to reduced numbers of germ cells, delayed meiosis and reduced fecundity in adult offspring. The transcriptome of germ cells from vitamin-C-deficient embryos is remarkably similar to that of embryos carrying a null mutation in Tet1. Vitamin C deficiency leads to an aberrant DNA methylation profile that includes incomplete demethylation of key regulators of meiosis and transposable elements. These findings reveal that deficiency in vitamin C during gestation partially recapitulates loss of TET1, and provide a potential intergenerational mechanism for adjusting fecundity to environmental conditions