Publications by authors named "Oscar Molina"

31 Publications

A novel and efficient tandem CD19- and CD22-directed CAR for B cell ALL.

Mol Ther 2021 Sep 1. Epub 2021 Sep 1.

Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Carrer Casanova 143, 4° floor, Barcelona 08036, Spain; RICORS-TERAV, ISCIII, Madrid, Spain; CIBER-ONC, ISCIII, Barcelona, Spain; Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona 08036, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. Electronic address:

CD19-directed chimeric antigen receptor (CAR) T cells have yielded impressive response rates in refractory/relapse B cell acute lymphoblastic leukemia (B-ALL); however, most patients ultimately relapse due to poor CAR T cell persistence or resistance of either CD19 or CD19 B-ALL clones. CD22 is a pan-B marker whose expression is maintained in both CD19 and CD19 relapses. CD22-CAR T cells have been clinically used in B-ALL patients, although relapse also occurs. T cells engineered with a tandem CAR (Tan-CAR) containing in a single construct both CD19 and CD22 scFvs may be advantageous in achieving higher remission rates and/or preventing antigen loss. We have generated and functionally validated using cutting-edge assays a 4-1BB-based CD22/CD19 Tan-CAR using in-house-developed novel CD19 and CD22 scFvs. Tan-CAR-expressing T cells showed similar in vitro expansion to CD19-CAR T cells with no increase in tonic signaling. CRISPR-Cas9-edited B-ALL cells confirmed the bispecificity of the Tan-CAR. Tan-CAR was as efficient as CD19-CAR in vitro and in vivo using B-ALL cell lines, patient samples, and patient-derived xenografts (PDXs). Strikingly, the robust antileukemic activity of the Tan-CAR was slightly more effective in controlling the disease in long-term follow-up PDX models. This Tan-CAR construct warrants a clinical appraisal to test whether simultaneous targeting of CD19 and CD22 enhances leukemia eradication and reduces/delays relapse rates and antigen loss.
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http://dx.doi.org/10.1016/j.ymthe.2021.08.033DOI Listing
September 2021

Engraftment characterization of risk-stratified AML patients in NSGS mice.

Blood Adv 2021 Sep 1. Epub 2021 Sep 1.

Josep Carreras Leukemia Research Institute, Barcelona, Spain.

Acute myeloid leukemia (AML) is the commonest acute leukemia in adults. Disease heterogeneity is well-documented and patient stratification determines treatment decisions. Patient-derived xenografts (PDXs) of risk-stratified AMLs are crucial for studying AML biology and testing novel therapeutics. Despite recent advances in PDX modeling of AML, reproducible engraftment of human AML is mainly limited to high-risk (HR) cases, with inconsistent or very protracted engraftment observed for favorable-risk (FR) and intermediate-risk (IR) patients. We have characterized the engraftment robustness/kinetics in NSGS mice of 28 AML patients grouped according to molecular/cytogenetic classification, and have assessed whether the orthotopic co-administration of patient-matched bone marrow mesenchymal stromal cells (BM-MSCs) improves AML engraftment. PDX event-free survival correlated well with the predictable prognosis of risk-stratified AML patients. The majority (85%-94%) of the mice were engrafted in BM independently of the risk group, although HR-AML patients showed engraftment levels significantly superior to those of FR- and IR-AML patients. Importantly, the engraftment levels observed in NSGS mice by week 6 remained stable overtime. Serial transplantation and long-term culture-initiating cell (LTC-IC) assays revealed long-term engraftment limited to HR-AML patients, fitter leukemia-initiating cells (LICs) in HR- than in FR- or IR-AML samples, and the presence of AML-LICs in the CD34- leukemic fraction, regardless the risk group. Finally, orthotopic co-administration of patient-matched BM-MSCs with AML cells resulted dispensable for BM engraftment levels but favored peripheralization of engrafted AML cells. This comprehensive characterization of human AML engraftment in NSGS mice offers a valuable platform for in vivo testing of targeted therapies in risk-stratified AML patient samples.
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http://dx.doi.org/10.1182/bloodadvances.2020003958DOI Listing
September 2021

Simulium maiaherzogae sp. nov., a new species of black fly (Diptera, Simuliidae) from rock fields of southeastern Brazil.

Acta Trop 2021 Sep 1;221:106030. Epub 2021 Jul 1.

Departamento de Zoologia (DZ), Universidade de Brasília (UnB) / Instituto de Ciências Biológicas (ICB), Brazil.

A new species of black fly from rock fields of southeaster Brazil, Simulium maiaherzogae sp. nov., is described based on morphological features of larval, pupal and adult specimens. Morphological features of this new species were compared with members of Simulium (Trichodagmia) Enderlein 1934, showing a high similarity with S. scutistriatum Lutz 1909 and S. itajara Nascimento, et al. 2020. However, features of the male and female scutum and genitalia, pupal tubercles disposition, pupal gills, larval cephalic appendages, larval gill histoblast, numbers of posterior proleg hooks and rectal papillae lobules, allow to differentiate S. maiaherzogae sp. nov. Moreover, a more detailed analysis using Scanning Electron Microscopy (SEM) showed significant differences and numerical variations in meristic data between the new species and S. scutistriatum. The known distribution of this new species is restricted to the Rio do Salto sub-basin, headland of the Middle Paraíba do Sul River, near the Ibitipoca State Park, a region recognised for its biodiversity richness, but with rare simuliids records.
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http://dx.doi.org/10.1016/j.actatropica.2021.106030DOI Listing
September 2021

Form and function of labral fan-ray microtrichia and posterior proleg barbs in Neotropical black flies larvae (Diptera: Simuliidae).

Acta Trop 2021 Sep 26;221:106024. Epub 2021 Jun 26.

Departamento de Entomologia,Museu Nacional, Universidade Federal do Rio de Janeiro. Quinta da Boa Vista s/n, 20940-040, São Cristóvão, Rio de Janeiro, RJ, Brazil.

The microtrichia of the labral fan primary rays, considered as the finest structure of the larval filter mechanism in Simuliidae, and the posterior proleg hooks, a structure used for substrate attachment, were describe using Scanning Electron Microscopy for the first time in 14 Neotropical species of black flies. Four patterns of primary rays microtrichia and two types of hooks barbs disposition were found. The present study provides new morphological information for Neotropical black fly species, compares it with previous information for worldwide species. Also, shows that the presence of posterior proleg hooks barbs is common among black fly species, that these barbs are not related with the larval habitat or with the primary ray microtrichia pattern, and that flow conditions may influence the last-instar larvae microtrichial patterns, at least in most of the Neotropical species studied here.
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http://dx.doi.org/10.1016/j.actatropica.2021.106024DOI Listing
September 2021

Effects of Bacillus thuringiensis var. israelensis on the Black Fly Communities (Diptera, Simuliidae) in Tropical Streams.

Neotrop Entomol 2021 Apr 10;50(2):269-281. Epub 2021 Feb 10.

Lab de Entomologia, Depto de Zoologia, Univ Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.

Bacillus thuringiensis var. israelensis (Bti) Berliner, 1911 is widely used in the biological control of black fly and mosquito populations. The objective of this study was to evaluate the effects of Bti on the black fly communities in streams in the Atlantic Forest domain. The study was carried out in eight streams of Serra do Mar, in the municipality of Ubatuba, São Paulo. Some parts of the streams in this locality have received applications of Bti fortnightly for more than 25 years by the sanitary agency of the region. In each stream, two sections were sampled, with and without application of Bti (June 2015 and 2016). Black flies were collected and identified to the species level in the laboratory with 1382 larvae being identified, distributed in six species. Of this total, 73% of the individuals were found in sections where Bti was not applied. There was a difference in the abundance of simulids between the sections with and without Bti application, being more abundant in the latter. We measured the head capsule of individuals of the most abundant species, Simulium pertinax Kollar 1832, using a stereoscopic microscope with millimeter lens. These measurements demonstrated that there was a difference between the sections regarding the age structure of Simuliidae populations. In the sections without Bti application, there was a higher proportion of larvae in the last instar, while in the Bti-treated sections, smaller instars were predominant, possibly due to constant recolonization processes.
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http://dx.doi.org/10.1007/s13744-020-00842-2DOI Listing
April 2021

Histology and RNA Sequencing Provide Insights Into Fusarium Head Blight Resistance in AAC Tenacious.

Front Plant Sci 2020 13;11:570418. Epub 2021 Jan 13.

Morden Research and Development Centre, Agriculture and Agri-Food Canada, Morden, MB, Canada.

Fusarium head blight (FHB) is a serious fungal disease affecting wheat and other cereals worldwide. This fungus causes severe yield and quality losses from a reduction in grain quality and contamination of grain with mycotoxins. Intensive breeding efforts led to the release of AAC Tenacious, which was the first spring wheat cultivar registered in Canada with a resistant (R) rating to FHB. To elucidate the physiological mechanisms of resistance, we performed histological and transcriptomic analyses of AAC Tenacious and a susceptible control Roblin after inoculation with (). The spikelet and rachis of infected wheat spikes were hand sectioned and monitored by confocal and fluorescent microscopy. Visible hyphae were observed within the inoculated spikelets for AAC Tenacious; however, the infection was largely restricted to the point of inoculation (POI), whereas the adjacent florets in Roblin were heavily infected. Significant cell wall thickening within the rachis node below the POI was evident in AAC Tenacious compared to Roblin in response to inoculation. Rachis node and rachilla tissues from the POI and the rachis node below the POI were collected at 5 days post inoculation for RNAseq. Significant changes in gene expression were detected in both cultivars in response to infection. The rachis node below the POI in AAC Tenacious had fewer differentially expressed genes (DEGs) when compared to the uninoculated control, likely due to its increased disease resistance. Analysis of DEGs in Roblin and AAC Tenacious revealed the activation of genes and pathways in response to infection, including those putatively involved in cell wall modification and defense response.
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http://dx.doi.org/10.3389/fpls.2020.570418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838103PMC
January 2021

Immune-focused multi-omics analysis of prostate cancer: leukocyte Ig-Like receptors are associated with disease progression.

Oncoimmunology 2020 12 1;9(1):1851950. Epub 2020 Dec 1.

Computational Biology Laboratory, CHU de Québec - Université Laval Research Center, Québec City, QC, Canada.

Prostate cancer (PCa) immunotherapy has shown limited efficacy so far, even in advanced-stage cancers. The success rate of PCa immunotherapy might be improved by approaches more adapted to the immunobiology of the disease. The objective of this study was to perform a multi-omics analysis to identify immune genes associated with PCa progression to better characterize PCa immunobiology and propose new immunotherapeutic targets. mRNA, miRNA, methylation, copy number aberration, and single nucleotide variant datasets from The Cancer Genome Atlas PRAD cohort were analyzed after filtering for genes associated with immunity. Sparse partial least squares-discriminant analyses were performed to identify features associated with biochemical recurrence (BCR) in each type of omics data. Selected features predicted BCR with a balanced error rate (BER) of 0.20 to 0.51 in single-omics and of 0.05 in multi-omics analyses. Amongst features associated with BCR were genes from the Immunoglobulin Ig-like Receptor (LILR) family which are immune checkpoints with immunotherapeutic potential. Using Multivariate INTegrative (MINT) analysis, the association of five genes with BCR was quantified in a combination of three RNA-seq datasets and confirmed with Kaplan-Meier analysis in both these and in an independent RNA-seq dataset. Finally, immunohistochemistry showed that a high number of LILRB1 positive cells within the tumors predicted long-term adverse outcomes. Thus, tumors characterized by abnormal expression of genes have an elevated risk of recurring after definitive local therapy. The immunotherapeutic potential of these regulators to stimulate the immune response against PCa should be evaluated in pre-clinical models.
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http://dx.doi.org/10.1080/2162402X.2020.1851950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714461PMC
December 2020

Aneuploidy in Cancer: Lessons from Acute Lymphoblastic Leukemia.

Trends Cancer 2021 01 17;7(1):37-47. Epub 2020 Sep 17.

Josep Carreras Leukaemia Research Institute, School of Medicine, University of Barcelona, Barcelona, Spain; Centro de investigación Biomédica en Red de Cáncer (CIBER-ONC), Instituto de Salud Carlos III (ISCIII), Barcelona, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. Electronic address:

Aneuploidy, the gain or loss of chromosomes in a cell, is a hallmark of cancer. Although our understanding of the contribution of aneuploidy to cancer initiation and progression is incomplete, significant progress has been made in uncovering the cellular consequences of aneuploidy and how aneuploid cancer cells self-adapt to promote tumorigenesis. Aneuploidy is physiologically associated with significant cellular stress but, paradoxically, it favors tumor progression. Although more common in solid tumors, different forms of aneuploidy represent the initiating oncogenic lesion in patients with B cell acute lymphoblastic leukemia (B-ALL), making B-ALL an excellent model for studying the role of aneuploidy in tumorigenesis. We review the molecular mechanisms underlying aneuploidy and discuss its contributions to B-ALL initiation and progression.
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http://dx.doi.org/10.1016/j.trecan.2020.08.008DOI Listing
January 2021

Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope.

J Immunother Cancer 2020 08;8(2)

Josep Carreras Leukemia Research Institute, Barcelona, Spain

Background: There are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19 either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22CD19 B-ALL relapses and CD19 preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied.

Methods: Here, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays.

Results: Conformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy.

Conclusions: We report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22 and CD22 ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22-CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL.
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http://dx.doi.org/10.1136/jitc-2020-000896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422657PMC
August 2020

Impaired condensin complex and Aurora B kinase underlie mitotic and chromosomal defects in hyperdiploid B-cell ALL.

Blood 2020 07;136(3):313-327

Josep Carreras Leukemia Research Institute, Barcelona, Spain.

B-cell acute lymphoblastic leukemia (ALL; B-ALL) is the most common pediatric cancer, and high hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD being an initiating oncogenic event affiliated with childhood B-ALL, the mitotic and chromosomal defects associated with HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated with chromosome-alignment defects at the metaphase plate leading to robust chromosome-segregation defects and nonmodal karyotypes. Mechanistically, biochemical, functional, and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness, and mislocalization of the chromosome passenger complex proteins Aurora B kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and an impaired spindle assembly checkpoint (SAC), thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated with a defective condensin complex, AURKB, and SAC.
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http://dx.doi.org/10.1182/blood.2019002538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413752PMC
July 2020

SUMOylation in development and neurodegeneration.

Development 2020 03 18;147(6). Epub 2020 Mar 18.

Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095-1569, USA

In essentially all eukaryotes, proteins can be modified by the attachment of small ubiquitin-related modifier (SUMO) proteins to lysine side chains to produce branched proteins. This process of 'SUMOylation' plays essential roles in plant and animal development by altering protein function in spatially and temporally controlled ways. In this Primer, we explain the process of SUMOylation and summarize how SUMOylation regulates a number of signal transduction pathways. Next, we discuss multiple roles of SUMOylation in the epigenetic control of transcription. In addition, we evaluate the role of SUMOylation in the etiology of neurodegenerative disorders, focusing on Parkinson's disease and cerebral ischemia. Finally, we discuss the possibility that SUMOylation may stimulate survival and neurogenesis of neuronal stem cells.
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http://dx.doi.org/10.1242/dev.175703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7097199PMC
March 2020

NG2 antigen is a therapeutic target for MLL-rearranged B-cell acute lymphoblastic leukemia.

Leukemia 2019 07 11;33(7):1557-1569. Epub 2019 Jan 11.

Department of Biomedicine, School of Medicine, Josep Carreras Leukemia Research Institute, University of Barcelona, Barcelona, Spain.

B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer, with cure rates of ∼80%. MLL-rearranged (MLLr) B-ALL (MLLr-B-ALL) has, however, an unfavorable prognosis with common therapy refractoriness and early relapse, and therefore new therapeutic targets are needed for relapsed/refractory MLLr-B-ALL. MLLr leukemias are characterized by the specific expression of chondroitin sulfate proteoglycan-4, also known as neuron-glial antigen-2 (NG2). NG2 was recently shown involved in leukemia invasiveness and central nervous system infiltration in MLLr-B-ALL, and correlated with lower event-free survival (EFS). We here hypothesized that blocking NG2 may synergize with established induction therapy for B-ALL based on vincristine, glucocorticoids, and L-asparaginase (VxL). Using robust patient-derived xenograft (PDX) models, we found that NG2 is crucial for MLLr-B-ALL engraftment upon intravenous (i.v.) transplantation. In vivo blockade of NG2 using either chondroitinase-ABC or an anti-NG2-specific monoclonal antibody (MoAb) resulted in a significant mobilization of MLLr-B-ALL blasts from bone marrow (BM) to peripheral blood (PB) as demonstrated by cytometric and 3D confocal imaging analysis. When combined with either NG2 antagonist, VxL treatment achieved higher rates of complete remission, and consequently higher EFS and delayed time to relapse. Mechanistically, anti-NG2 MoAb induces neither antibody-dependent cell-mediated not complement-dependent cytotoxicity. NG2 blockade rather overrides BM stroma-mediated chemoprotection through PB mobilization of MLLr-B-ALL blasts, thus becoming more accessible to chemotherapy. We provide a proof of concept for NG2 as a therapeutic target for MLLr-B-ALL.
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http://dx.doi.org/10.1038/s41375-018-0353-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755967PMC
July 2019

Human Artificial Chromosome with Regulated Centromere: A Tool for Genome and Cancer Studies.

ACS Synth Biol 2018 09 16;7(9):1974-1989. Epub 2018 Aug 16.

Developmental Therapeutics Branch , National Cancer Institute, NIH , Bethesda , Maryland 20892 , United States.

Since their description in the late 1990s, Human Artificial Chromosomes (HACs) bearing functional kinetochores have been considered as promising systems for gene delivery and expression. More recently a HAC assembled from a synthetic alphoid DNA array has been exploited in studies of centromeric chromatin and in assessing the impact of different epigenetic modifications on kinetochore structure and function in human cells. This HAC was termed the alphoid-HAC, as the synthetic monomers each contained a tetO sequence in place of the CENP-B box that can be targeted specifically with tetR-fusion proteins. Studies in which the kinetochore chromatin of the alphoid-HAC was specifically modified, revealed that heterochromatin is incompatible with centromere function and that centromeric transcription is important for centromere assembly and maintenance. In addition, the alphoid-HAC was modified to carry large gene inserts that are expressed in target cells under conditions that recapitulate the physiological regulation of endogenous loci. Importantly, the phenotypes arising from stable gene expression can be reversed when cells are "cured" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. Alphoid-HAC-based technology has also been used to develop new drug screening and assessment strategies to manipulate the CIN phenotype in cancer cells. In summary, the alphoid-HAC is proving to be a versatile tool for studying human chromosome transactions and structure as well as for genome and cancer studies.
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http://dx.doi.org/10.1021/acssynbio.8b00230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6154217PMC
September 2018

A new species of Umanella Gauld (Hymenoptera: Ichneumonidae: Pimplinae) from Colombia.

Zootaxa 2018 Jan 31;4377(2):296-300. Epub 2018 Jan 31.

University of Munich (LMU) Biozentrum Martinsried, Großhadernerstr. 2, 82152 Planegg-Martinsried, Germany SNSB-Zoologische Staatssammlung München, Munchhausenstr. 21, 81247 Munich, Germany;.

A new species of Umanella Gauld 1991, U. tricoloripes Herrera-Florez, sp. n., is described from two males collected in Colombia. Umanella tricoloripes sp. nov. differs substantially from the other species of the genus in color pattern.
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http://dx.doi.org/10.11646/zootaxa.4377.2.10DOI Listing
January 2018

Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies.

ACS Synth Biol 2018 04 29;7(4):1116-1130. Epub 2018 Mar 29.

Wellcome Trust Centre for Cell Biology, University of Edinburgh , Edinburgh EH9 3QR, United Kingdom.

It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native chromosomes. To address this problem, in our previous studies we generated a human artificial chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoid-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoid-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins.
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http://dx.doi.org/10.1021/acssynbio.8b00018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951608PMC
April 2018

HP1α targets the chromosomal passenger complex for activation at heterochromatin before mitotic entry.

EMBO J 2018 03 21;37(6). Epub 2018 Feb 21.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK

The chromosomal passenger complex (CPC) is directed to centromeres during mitosis via binding to H3T3ph and Sgo1. Whether and how heterochromatin protein 1α (HP1α) influences CPC localisation and function during mitotic entry is less clear. Here, we alter HP1α dynamics by fusing it to a CENP-B DNA-binding domain. Tethered HP1 strongly recruits the CPC, destabilising kinetochore-microtubule interactions and activating the spindle assembly checkpoint. During mitotic exit, the tethered HP1 traps active CPC at centromeres. These HP1-CPC clusters remain catalytically active throughout the subsequent cell cycle. We also detect interactions between endogenous HP1 and the CPC during G HP1α and HP1γ cooperate to recruit the CPC to active foci in a CDK1-independent process. Live cell tracking with Fab fragments reveals that H3S10ph appears well before H3T3 is phosphorylated by Haspin kinase. Our results suggest that HP1 may concentrate and activate the CPC at centromeric heterochromatin in G before Aurora B-mediated phosphorylation of H3S10 releases HP1 from chromatin and allows pathways dependent on H3T3ph and Sgo1 to redirect the CPC to mitotic centromeres.
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http://dx.doi.org/10.15252/embj.201797677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852645PMC
March 2018

Using human artificial chromosomes to study centromere assembly and function.

Chromosoma 2017 Oct 7;126(5):559-575. Epub 2017 Jul 7.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3QR, UK.

Centromeres are the site of assembly of the kinetochore, which directs chromosome segregation during cell division. Active centromeres are characterized by the presence of nucleosomes containing CENP-A and a specific chromatin environment that resembles that of active genes. Recent work using human artificial chromosomes (HAC) sheds light on the fine balance of different histone post-translational modifications and transcription that exists at centromeres for kinetochore assembly and maintenance. Here, we review the use of HAC technology to understand centromere assembly and function. We put particular emphasis on studies using the alphoid HAC, whose centromere can be specifically modified for epigenetic engineering studies.
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http://dx.doi.org/10.1007/s00412-017-0633-xDOI Listing
October 2017

Molecular basis for Cdk1-regulated timing of Mis18 complex assembly and CENP-A deposition.

EMBO Rep 2017 06 4;18(6):894-905. Epub 2017 Apr 4.

Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, UK

The centromere, a chromosomal locus that acts as a microtubule attachment site, is epigenetically specified by the enrichment of CENP-A nucleosomes. Centromere maintenance during the cell cycle requires HJURP-mediated CENP-A deposition, a process regulated by the Mis18 complex (Mis18α/Mis18β/Mis18BP1). Spatial and temporal regulation of Mis18 complex assembly is crucial for its centromere association and function. Here, we provide the molecular basis for the assembly and regulation of the Mis18 complex. We show that the N-terminal region of Mis18BP1 spanning amino acid residues 20-130 directly interacts with Mis18α/β to form the Mis18 complex. Within Mis18α/β, the Mis18α MeDiY domain can directly interact with Mis18BP1. Mis18α/β forms a hetero-hexamer with 4 Mis18α and 2 Mis18β. However, only two copies of Mis18BP1 interact with Mis18α/β to form a hetero-octameric assembly, highlighting the role of Mis18 oligomerization in limiting the number of Mis18BP1 within the Mis18 complex. Furthermore, we demonstrate the involvement of consensus Cdk1 phosphorylation sites on Mis18 complex assembly and thus provide a rationale for cell cycle-regulated timing of Mis18 assembly and CENP-A deposition.
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http://dx.doi.org/10.15252/embr.201643564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5452045PMC
June 2017

PREditOR: a synthetic biology approach to removing heterochromatin from cells.

Chromosome Res 2016 12 6;24(4):495-509. Epub 2016 Dec 6.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3BF, UK.

It is widely accepted that heterochromatin is necessary to maintain genomic stability. However, direct experimental evidence supporting this is slim. Previous studies using either enzyme inhibitors, gene knockout or knockdown studies all are subject to the caveat that drugs may have off-target effects and enzymes that modify chromatin proteins to support heterochromatin formation may also have numerous other cellular targets as well. Here, we describe PREditOR (protein reading and editing of residues), a synthetic biology approach that allows us to directly remove heterochromatin from cells without either drugs or global interference with gene function. We find that removal of heterochromatin perturbs mitotic progression and causes a dramatic increase in chromosome segregation defects, possibly as a result of interfering with the normal centromeric localization of the chromosomal passenger complex.
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http://dx.doi.org/10.1007/s10577-016-9539-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167778PMC
December 2016

Epigenetic engineering reveals a balance between histone modifications and transcription in kinetochore maintenance.

Nat Commun 2016 11 14;7:13334. Epub 2016 Nov 14.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, EH9 3QR, UK.

Centromeres consist of specialized centrochromatin containing CENP-A nucleosomes intermingled with H3 nucleosomes carrying transcription-associated modifications. We have designed a novel synthetic biology 'in situ epistasis' analysis in which H3 dimethylated on lysine 4 (H3K4me2) demethylase LSD2 plus synthetic modules with competing activities are simultaneously targeted to a synthetic alphoid HAC centromere. This allows us to uncouple transcription from histone modifications at the centromere. Here, we report that H3K4me2 loss decreases centromeric transcription, CENP-A assembly and stability and causes spreading of H3K9me3 across the HAC, ultimately inactivating the centromere. Surprisingly, CENP-28/Eaf6-induced transcription of the alphoid array associated with H4K12 acetylation does not rescue the phenotype, whereas p65-induced transcription associated with H3K9 acetylation does rescue. Thus mitotic transcription plus histone modifications including H3K9ac constitute the 'epigenetic landscape' allowing CENP-A assembly and centrochromatin maintenance. H3K4me2 is required for the transcription and H3K9ac may form a barrier to prevent heterochromatin spreading and kinetochore inactivation at human centromeres.
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http://dx.doi.org/10.1038/ncomms13334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114538PMC
November 2016

3D-CLEM Reveals that a Major Portion of Mitotic Chromosomes Is Not Chromatin.

Mol Cell 2016 11 10;64(4):790-802. Epub 2016 Nov 10.

Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, EH9 3BF Edinburgh, UK. Electronic address:

Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%-47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes.
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http://dx.doi.org/10.1016/j.molcel.2016.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5128728PMC
November 2016

Occurrence of Ditylenchus weischeri and Not D. dipsaci in Field Pea Harvest Samples and Cirsium arvense in the Canadian Prairies.

J Nematol 2014 Dec;46(4):376-84

Plant Pest Diagnostic Center, California Department of Food and Agriculture, 3294 Meadowview Road, Sacramento, CA 95832-1448.

The stem nematode, a parasite of the herbaceous perennial weed, Cirsium arvense (L.) Scop. and identified as Ditylenchus dipsaci (Kühn) Filipjev, was reported in the Canadian prairies in 1979. Recently, D. weischeri Chizhov parasitizing Cirsium arvense was described in Russia, and it has been shown that this species is not an agricultural pest. In this study, we examined Ditylenchus species found in field pea (Pisum sativum L.) grain harvest samples in 2009 and 2010 and from C. arvense shoots in pea fields in the Saskatchewan, Alberta, and Manitoba provinces. Samples from 538 fields (mainly yellow pea) were provided by 151 growers throughout the main pea-growing area of the Canadian prairies. Of the samples collected, 2% were positive for Ditylenchus. The population density of the nematode ranged between 4 and 1,500 nematodes kg(-1) pea harvest sample and related to presence of C. arvense seeds. Positive samples occurred in 2009 but not in 2010 and were from throughout the pea-growing area of the Canadian prairies and not related to cropping history. C. arvense collected from yellow pea fields in Saskatchewan and Manitoba, but not Alberta, were infested with Ditylenchus. Morphological and molecular (ITS-PCR-RFLP) traits indicated that this species belongs to D. weischeri. The results indicated the stem nematode found in yellow pea grain is D. weischeri which resided with C. arvense seeds and debris to pea samples. Unlike D. dipsaci, D. weischeri is not a nematode pest of economic importance; therefore, its presence in the pea harvest samples was not a concern.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4284090PMC
December 2014

Deletions and duplications of the 22q11.2 region in spermatozoa from DiGeorge/velocardiofacial fathers.

Mol Cytogenet 2014 25;7(1):86. Epub 2014 Nov 25.

Unitat de Biologia Cellular (Facultat de Biociències). Universitat Autònoma de Barcelona, 08193-Bellaterra, Cerdanyola del Vallès, Spain.

Background: DiGeorge/velocardiofacial syndrome (DGS/VCFS) is the most common deletion syndrome in humans. Low copy repeats flanking the 22q11.2 region confer a substrate for non-allelic homologous recombination (NAHR) events leading to rearrangements. This study sought to identify DGS/VCFS fathers with increased susceptibility to deletions and duplications at the 22q11.2 region in spermatozoa and to assess the particular contribution of intra-chromatid and/or inter-chromatid NAHR. Semen samples from nine DGS/VCFS fathers were analyzed by triple-color FISH using a probe combination that discriminated between normal, deleted and duplicated genotypes. Microsatellite analysis were performed in the parents and the affected children to determine the parental origin of the deleted chromosome 22.

Results: A significant increase in 22q11.2 deletions was observed in the sperm of two out of nine DGS/VCFS fathers (odds ratio 2.03-fold, P < 0.01), and in both cases the deletion in the offspring was transmitted by the father. Patients with significant increases in sperm anomalies presented a disturbed deletion:duplication 1:1 ratio (P < 0.01).

Conclusions: Altogether, results support that intra-chromatid NAHR is the mechanism responsible for the higher rate of sperm deletions, which is directly related to the transmission of the deleted chromosome 22 to offspring. Accordingly, the screening of sperm anomalies in the 22q11.2 region should be taken into account in the genetic counseling of DGS/VCFS families.
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http://dx.doi.org/10.1186/s13039-014-0086-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4247602PMC
December 2014

Histone H4 Lys 20 monomethylation of the CENP-A nucleosome is essential for kinetochore assembly.

Dev Cell 2014 Jun;29(6):740-9

Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka 411-8540, Japan. Electronic address:

In vertebrate cells, centromeres are specified epigenetically through the deposition of the centromere-specific histone CENP-A. Following CENP-A deposition, additional proteins are assembled on centromeric chromatin. However, it remains unknown whether additional epigenetic features of centromeric chromatin are required for kinetochore assembly. Here, we used ChIP-seq analysis to examine centromere-specific histone modifications at chicken centromeres, which lack highly repetitive sequences. We found that H4K20 monomethylation (H4K20me1) is enriched at centromeres. Immunofluorescence and biochemical analyses revealed that H4K20me1 is present at all centromeres in chicken and human cells. Based on immunoprecipitation data, H4K20me1 occurs primarily on the histone H4 that is assembled as part of the CENP-A nucleosome following deposition of CENP-A into centromeres. Targeting the H4K20me1-specific demethylase PHF8 to centromeres reduces the level of H4K20me1 at centromeres and results in kinetochore assembly defects. We conclude that H4K20me1 modification of CENP-A nucleosomes contributes to functional kinetochore assembly.
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http://dx.doi.org/10.1016/j.devcel.2014.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4081567PMC
June 2014

Effects of glucans and eicosapentaenoic acid on differential regulation of phenylpropanoid and mevalonic pathways during potato response to Phytophthora infestans.

Plant Physiol Biochem 2012 Nov 8;60:119-28. Epub 2012 Aug 8.

Department of Plant Science, Agriculture Building, University of Manitoba, Winnipeg, Canada R3T 2N2.

The effects of Phytophthora infestans glucans, eicosapentaenoic acid (EPA) and isolates of this pathogen, on the differential expression of eight genes from the phenylpropanoid and the mevalonate (Ac-MVA) pathways were analyzed in potato by semi-quantitative RT-PCR and qRT-PCR. The application of EPA had an elicitor effect in Russet Burbank (RB) and Defender (DF) in response to inoculation with a US8 isolate of P. infestans, thereby reducing symptoms of late blight. Such effect was associated with the expression of PAL-1 and PAL-2, since the latter occurred only when EPA was followed by inoculation, whereas these genes were down-regulated in individual treatments RB + EPA, RB + US8, DF + EPA, and DF + US8. The glucan fraction did not by itself suppress phenylpropanoid genes, but its combination with the pathogen resulted in a down-regulation of PAL-1, PAL-2 and CHS. The addition of the glucan fraction to the elicitor EPA, had a negative effect (RB + EPA + GL + US8) since plants showed higher disease symptoms than the ones pretreated with water then infected with US8, and in comparison with RB + EPA + US8 and RB + GL + US8. Exclusive up-regulation of 4CL in DF + US11 and of CHS in DF + EPA + GL + US8, DF + EPA + US11, DF + GL + US11 and DF + EPA + GL + US11, where late blight lesions were not detected, could be associated with potato protection against late blight. Along with previous findings in this pathosystem, these data suggest that genetic resistance in potato against P. infestans is not the result of isolated reactions against the pathogen, but rather the combination of many factors in-line with a polygenic/horizontal resistance.
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http://dx.doi.org/10.1016/j.plaphy.2012.07.027DOI Listing
November 2012

High rates of de novo 15q11q13 inversions in human spermatozoa.

Mol Cytogenet 2012 Feb 6;5(1):11. Epub 2012 Feb 6.

Unitat de Biologia Cel·lular (Facultat de Biociències), Universitat Autònoma de Barcelona, 08193-Bellaterra (Cerdanyola del Vallès), SPAIN.

Low-Copy Repeats predispose the 15q11-q13 region to non-allelic homologous recombination. We have already demonstrated that a significant percentage of Prader-Willi syndrome (PWS) fathers have an increased susceptibility to generate 15q11q13 deletions in spermatozoa, suggesting the participation of intrachromatid exchanges. This work has been focused on assessing the incidence of de novo 15q11q13 inversions in spermatozoa of control donors and PWS fathers in order to determine the basal rates of inversions and to confirm the intrachromatid mechanism as the main cause of 15q11q13 anomalies.Semen samples from 10 control donors and 16 PWS fathers were processed and analyzed by triple-color FISH. Three differentially labeled BAC-clones were used: one proximal and two distal of the 15q11-q13 region. Signal associations allowed the discrimination between normal and inverted haplotypes, which were confirmed by laser-scanning confocal microscopy.Two types of inversions were detected which correspond to the segments involved in Class I and II PWS deletions. No significant differences were observed in the mean frequencies of inversions between controls and PWS fathers (3.59% ± 0.46 and 9.51% ± 0.87 vs 3.06% ± 0.33 and 10.07% ± 0.74). Individual comparisons showed significant increases of inversions in four PWS fathers (P < 0.05) previously reported as patients with increases of 15q11q13 deletions.Results suggest that the incidence of heterozygous inversion carriers in the general population could reach significant values. This situation could have important implications, as they have been described as predisposing haplotypes for genomic disorders. As a whole, results confirm the high instability of the 15q11-q13 region, which is prone to different types of de novo reorganizations by intrachromatid NAHR.
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http://dx.doi.org/10.1186/1755-8166-5-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3293048PMC
February 2012

Sperm rates of 7q11.23, 15q11q13 and 22q11.2 deletions and duplications: a FISH approach.

Hum Genet 2011 Jan 8;129(1):35-44. Epub 2010 Oct 8.

Unitat de Biologia Cel·lular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193, Bellaterra (Cerdanyola del Vallès), Spain.

Genomic disorders are human diseases caused by meiotic chromosomal rearrangements of unstable regions flanked by Low Copy Repeats (LCRs). LCRs act as substrates for Non-Allelic Homologous Recombination (NAHR) leading to deletions and duplications. The aim of this study was to assess the basal frequency of deletions and duplications of the 7q11.23, 15q11-q13 and 22q11.2 regions in spermatozoa from control donors to check differences in the susceptibility to generate anomalies and to assess the contribution of intra- and inter-chromatid NAHR events. Semen samples from ten control donors were processed by FISH. A customized combination of probes was used to discriminate among normal, deleted and duplicated sperm genotypes. A minimum of 10,000 sperm were assessed per sample and region. There were no differences in the mean frequency of deletions and duplications (del + dup) among the 7q11.23, 15q11-q13 and 22q11.2 regions (frequency ± SEM, 0.37 ± 0.02; 0.46 ± 0.07 and 0.27 ± 0.07%, respectively) (P = 0.122). Nevertheless, hierarchical cluster analysis reveals interindividual differences suggesting that particular haplotypes could be the main source of variability in NAHR rates. The mean frequency of deletions was not different from the mean frequency of duplications in the 7q11.23 (P = 0.202) and 15q11-q13 (P = 0.609) regions, indicating a predominant inter-chromatid NAHR. By contrast, in the 22q11.2 region the frequency of deletions slightly exceed duplications (P = 0.032), although at the individual level any donor showed differences. Altogether, our results support the inter-chromatid NAHR as the predominant mechanism involved in the generation of sperm deletions and duplications.
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http://dx.doi.org/10.1007/s00439-010-0894-4DOI Listing
January 2011

FISH on sperm: spot-counting to stop counting? Not yet.

Fertil Steril 2009 Oct 17;92(4):1474-1480. Epub 2008 Oct 17.

Unitat de Biologia Cel·lular (Facultat de Biociències), Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), Spain. Electronic address:

Objective: To evaluate the reliability and applicability of the spot-counting system (Cytovision Spot AX workstation) which offers an alternative to the tedious manual analysis of sperm fluorescence in situ hybridization (FISH).

Design: Manual and automatic analyses were performed and compared.

Setting: Universitat Autònoma de Barcelona.

Patient(s): Twenty-four men who asked for information on infertility showing different seminal parameters.

Intervention(s): A semen sample for each patient was collected and prepared for FISH.

Main Outcome Measure(s): A dual-color FISH using specific probes for chromosomes 13 and 21 and a triple-color FISH with centromeric probes for chromosomes 18, X, and Y were used (Vysis). Standard FISH analysis was carried out. Automatic analysis was subsequently performed using a Spot AX system.

Result(s): Overall, we performed 120 comparisons. In 116 out of 120 (96.67%), the percentage of anomalies reported using manual counting fell within the incidence detected using the automatic system. In the remaining comparisons, statistical differences were detected (4 out of 120; 3.33%). Time consumed by the automatic analysis was always higher than the manual one, being influenced by the characteristics of the preparations.

Conclusion(s): The spot-counting system has potential, but before the service is ready to be offered, we still need to overcome some limitations associated with it.
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http://dx.doi.org/10.1016/j.fertnstert.2008.07.1779DOI Listing
October 2009
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