Fertility Preservation in Female Cancer Patients

With improved survival rates among cancer patients, fertility preservation is now being recognized as an issue of great importance. There are currently several methods of fertility preservation available in female cancer patients and the options and techniques via assisted reproduction and cryopreservation are increasing, but some are still experimental and continues to be evaluated. The established means of preserving fertility include embryo cryopreservation, gonadal shielding during radiation therapy, ovarian transposition, conservative gynecologic surgery such as radical trachelectomy, donor embryos/oocytes, gestational surrogacy, and adoption. The experimental methods include oocyte cryopreservation, ovarian cryopreservation and transplantation, in vitro maturation, and ovarian suppression. With advances in methods for the preservation of fertility, providing information about risk of infertility and possible options of fertility preservation to all young patients with cancer, and discussing future fertility with them should be also considered as one of the important parts of consultation at the time of cancer diagnosis

Acid-sensing ion channel 1a drives AMPA receptor plasticity following ischemia and acidosis in hippocampal CA1 neurons

The CA1 region of the hippocampus is particularly vulnerable to ischemic damage. While NMDA receptors play a major role in excitotoxicity, it is thought to be exacerbated in this region by two forms of post-ischemic AMPA receptor (AMPAR) plasticity - namely, anoxic long-term potentiation (a-LTP), and a delayed increase in the prevalence of Ca2+ -permeable GluA2-lacking AMPARs (CP-AMPARs). The acid-sensing ion channel 1a (ASIC1a) which is expressed in CA1 pyramidal neurons, is also known to contribute to post-ischemic neuronal death and to physiologically induced LTP. This raises the question - does ASIC1a activation drive the post-ischemic forms of AMPAR plasticity in CA1 pyramidal neurons? We have tested this by examining organotypic hippocampal slice cultures (OHSCs) exposed to oxygen glucose deprivation (OGD), and dissociated cultures of hippocampal pyramidal neurons (HPN) exposed to low pH (acidosis). We find that both a-LTP and the delayed increase in the prevalence of CP-AMPARs are dependent on ASIC1a activation during ischemia. Indeed, acidosis alone is sufficient to induce the increase in CP-AMPARs. We also find that inhibition of ASIC1a channels circumvents any potential neuroprotective benefit arising from block of CP-AMPARs. By demonstrating that ASIC1a activation contributes to post-ischemic AMPAR plasticity, our results identify a functional interaction between acidotoxicity and excitotoxicity in hippocampal CA1 cells, and provide insight into the role of ASIC1a and CP-AMPARs as potential drug targets for neuroprotection. We thus propose that ASIC1a activation can drive certain forms of CP-AMPAR plasticity, and that inhibiting ASIC1a affords neuroprotection

Similarity of slow stripe fluctations between Sr-doped cuprates and oxygen-doped nickelates

Stripe fluctuations in La2NiO4.17 have been studied by 139La NMR using the field and temperature dependence of the linewidth and relaxation rates. In the formation process of the stripes the NMR line intensity is maximal below 230K, starts to diminish around 140K, disappears around 50K and recovers at 4K. These results are shown to be consistent with, but completely complementary to neutron measurements, and to be generic for oxygen doped nickelates and underdoped cuprates.Comment: 4 pages including 4 figure

Gene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD. Institutode Investigacio ' n Sanitaria de la Fundacio '

Gene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA

TheauthorswouldliketothankMiguelA.MartinforthecarefulmaintenanceofNSGmice,andRebecaSa ́nchezandOmairaAlberquillafortheirtechnicalassistanceinflowcytometry.TheauthorsalsothankFundacio ́n Botı ́n forpromotingtranslationalresearchattheHemato-poieticInnovativeTherapiesDivisionoftheCIEMATPyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD.S

Expression of mitofusin 2(R94Q) in a transgenic mouse leads to Charcot-Marie-Tooth neuropathy type 2A.

Charcot-Marie-Tooth disease type 2A is an autosomal dominant axonal form of peripheral neuropathy caused by mutations in the mitofusin 2 gene. Mitofusin 2 encodes a mitochondrial outer membrane protein that participates in mitochondrial fusion in mammalian cells. How mutations in this protein lead to Charcot-Marie-Tooth disease type 2A pathophysiology remains unclear. We have generated a transgenic mouse expressing either a mutated (R94Q) or wild-type form of human mitofusin 2 in neurons to evaluate whether the R94Q mutation was sufficient for inducing a Charcot-Marie-Tooth disease type 2A phenotype. Only mice expressing mitofusin 2(R94Q) developed locomotor impairments and gait defects thus mimicking the Charcot-Marie-Tooth disease type 2A neuropathy. In these animals, the number of mitochondria per axon was significantly increased in the distal part of the sciatic nerve axons with a diameter smaller than 3.5 microm. Importantly, the analysis of R94Q transgenic animals also revealed an age-related shift in the size of myelinated axons leading to an over-representation of axons smaller than 3.5 microm. Together these data suggest a link between an increased number of mitochondria in axons and a shift in axonal size distribution in mitofusin 2(R94Q) transgenic animals that may contribute to their neurological phenotype

GUIDANCE: a web server for assessing alignment confidence scores

Evaluating the accuracy of multiple sequence alignment (MSA) is critical for virtually every comparative sequence analysis that uses an MSA as input. Here we present the GUIDANCE web-server, a user-friendly, open access tool for the identification of unreliable alignment regions. The web-server accepts as input a set of unaligned sequences. The server aligns the sequences and provides a simple graphic visualization of the confidence score of each column, residue and sequence of an alignment, using a color-coding scheme. The method is generic and the user is allowed to choose the alignment algorithm (ClustalW, MAFFT and PRANK are supported) as well as any type of molecular sequences (nucleotide, protein or codon sequences). The server implements two different algorithms for evaluating confidence scores: (i) the heads-or-tails (HoT) method, which measures alignment uncertainty due to co-optimal solutions; (ii) the GUIDANCE method, which measures the robustness of the alignment to guide-tree uncertainty. The server projects the confidence scores onto the MSA and points to columns and sequences that are unreliably aligned. These can be automatically removed in preparation for downstream analyses. GUIDANCE is freely available for use at http://guidance.tau.ac.il

Specialized dynamical properties of promiscuous residues revealed by simulated conformational ensembles

The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein-protein interaction prediction and design methods. © 2013 American Chemical Society

Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells

Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.info:eu-repo/semantics/publishedVersio

Mid-Infrared Conductivity from Mid-Gap States Associated with Charge Stripes

The optical conductivity of La(2-x)Sr(x)NiO(4) has been interpreted in various ways, but so far the proposed interpretations have neglected the fact that the holes doped into the NiO(2) planes order in diagonal stripes, as established by neutron and X-ray scattering. Here we present a study of optical conductivity in La(2)NiO(4+d) with d=2/15, a material in which the charge stripes order three-dimensionally. We show that the conductivity can be decomposed into two components, a mid-infrared peak that we attribute to transitions from the filled valence band into empty mid-gap states associated with the stripes, and a Drude peak that appears at higher temperatures as carriers are thermally excited into the mid-gap states. The shift of the mid-IR peak to lower energy with increasing temperature is explained in terms of the Franck-Condon effect. The relevance of these results to understanding the optical conductivity in the cuprates is discussed.Comment: final version of paper (minor changes from previous version