Isolation of chromatin from dysfunctional telomeres reveals an important role for Ring1b in NHEJ-mediated chromosome fusions

When telomeres become critically short, DNA damage response factors are recruited at chromosome ends, initiating a cellular response to DNA damage. We performed proteomic isolation of chromatin fragments (PICh) in order to define changes in chromatin composition that occur upon onset of acute telomere dysfunction triggered by depletion of the telomere-associated factor TRF2. This unbiased purification of telomere-associated proteins in functional or dysfunctional conditions revealed the dynamic changes in chromatin composition that take place at telomeres upon DNA damage induction. On the basis of our results, we describe a critical role for the polycomb group protein Ring1b in nonhomologous end-joining (NHEJ)-mediated end-to-end chromosome fusions. We show that cells with reduced levels of Ring1b have a reduced ability to repair uncapped telomeric chromatin. Our data represent an unbiased isolation of chromatin undergoing DNA damage and are a valuable resource to map the changes in chromatin composition in response to DNA damage activation

Mitotic Evolution of Plasmodium falciparum Shows a Stable Core Genome but Recombination in Antigen Families

Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases) revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone). In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1) on chromosome 1. We observed 18 large-scale (greater than 1 kb on average) deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10−6 structural variants per base pair per generation). Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0–9.7×10−9 mutations per base pair per generation), we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum evolves to evade control efforts within both the individual hosts and large populations

Bioinformatics in Italy: BITS2011, the Eighth Annual Meeting of the Italian Society of Bioinformatics

The BITS2011 meeting, held in Pisa on June 20-22, 2011, brought together more than 120 Italian researchers working in the field of Bioinformatics, as well as students in Bioinformatics, Computational Biology, Biology, Computer Sciences, and Engineering, representing a landscape of Italian bioinformatics research

Searching novel therapeutic targets for scleroderma: P2X7-receptor is UP-regulated and promotes a fibrogenic phenotype in systemic sclerosis fibroblasts

Objectives: Systemic sclerosis (SSc) is a connective tissue disorder presenting fibrosis of the skin and internal organs, for which no effective treatments are currently available. Increasing evidence indicates that the P2X7 receptor (P2X7R), a nucleotide-gated ionotropic channel primarily involved in the inflammatory response, may also have a key role in the development of tissue fibrosis in different body districts. This study was aimed at investigating P2X7R expression and function in promoting a fibrogenic phenotype in dermal fibroblasts from SSc patients, also analyzing putative underlying mechanistic pathways. Methods: Fibroblasts were isolated by skin biopsy from 9 SSc patients and 8 healthy controls. P2X7R expression, and function (cytosolic free Ca2+ fluxes, α-smooth muscle actin [α-SMA] expression, cell migration, and collagen release) were studied. Moreover, the role of cytokine (interleukin-1β, interleukin-6) and connective tissue growth factor (CTGF) production, and extracellular signal-regulated kinases (ERK) activation in mediating P2X7R-dependent pro-fibrotic effects in SSc fibroblasts was evaluated. Results: P2X7R expression and Ca2+ permeability induced by the selective P2X7R agonist 2'-3'-O-(4-benzoylbenzoyl)ATP (BzATP) weremarkedly higher in SSc than control fibroblasts. Moreover, increased aSMA expression, cell migration, CTGF, and collagen release were observed in lipopolysaccharides-primed SSc fibroblasts after BzATP stimulation. While P2X7-induced cytokine changes did not affect collagen production, it was completely abrogated by inhibition of the ERK pathway. Conclusion: In SSc fibroblasts, P2X7R is overexpressed and its stimulation induces Ca2+-signaling activation and a fibrogenic phenotype characterized by increased migration and collagen production. These data point to the P2X7R as a potential, novel therapeutic target for controlling exaggerated collagen deposition and tissue fibrosis in patients with SSc

Exercise capacity in children with isolated congenital complete atrioventricular block: does pacing make a difference?

Item does not contain fulltextThe management of patients with isolated congenital complete atrioventricular block (CCAVB) has changed during the last decades. The current policy is to pace the majority of patients based on a variety of criteria, among which is limited exercise capacity. Data regarding exercise capacity in this population stems from previous publications reporting small case series of unpaced patients. Therefore, we have investigated the exercise capacity of a group of contemporary children with CCAVB. Sixteen children (mean age 11.5 +/- 4; seven boys, nine girls) with CCAVB were tested. In 13 patients, a median number of three pacemakers were implanted, whereas in three patients no pacemaker was given. All patients had an echocardiogram and completed a cardiopulmonary cycle exercise test. Exercise parameters were determined and compared with reference values obtained from healthy Dutch peers. The peak oxygen uptake/body mass was reduced to 34.4 +/- 9.5 ml kg(-1) min(-1) (79 +/- 24% of predicted) and the ventilatory threshold was reduced to 52 +/- 17% of peak oxygen uptake (78 +/- 21% of predicted), whereas the peak work load/body mass was 2.8 +/- 0.6 W/kg (91 +/- 24% of predicted), which was similar to controls. Importantly, 25% of the paced patients showed upper rate restriction by the pacemaker. In conclusion, children with CCAVB show a reduced peak oxygen uptake and ventilatory threshold, whereas they show normal peak work rates. This indicates that they generate more energy during exercise from anaerobic energy sources. Paced children with CCAVB do not perform better than unpaced children.1 april 201

Regional differences in the quality of maternal and neonatal care during the COVID-19 pandemic in Portugal: Results from the IMAgiNE EURO study

Objective: To compare women's perspectives on the quality of maternal and newborn care (QMNC) around the time of childbirth across Nomenclature of Territorial Units for Statistics 2 (NUTS-II) regions in Portugal during the COVID-19 pandemic. Methods: Women participating in the cross-sectional IMAgiNE EURO study who gave birth in Portugal from March 1, 2020, to October 28, 2021, completed a structured questionnaire with 40 key WHO standards-based quality measures. Four domains of QMNC were assessed: (1) provision of care; (2) experience of care; (3) availability of human and physical resources; and (4) reorganizational changes due to the COVID-19 pandemic. Frequencies for each quality measure within each QMNC domain were computed overall and by region. Results: Out of 1845 participants, one-third (33.7%) had a cesarean. Examples of high-quality care included: low frequencies of lack of early breastfeeding and rooming-in (8.0% and 7.7%, respectively) and informal payment (0.7%); adequate staff professionalism (94.6%); adequate room comfort and equipment (95.2%). However, substandard practices with large heterogeneity across regions were also reported. Among women who experienced labor, the percentage of instrumental vaginal births ranged from 22.3% in the Algarve to 33.5% in Center; among these, fundal pressure ranged from 34.8% in Lisbon to 66.7% in Center. Episiotomy was performed in 39.3% of noninstrumental vaginal births with variations between 31.8% in the North to 59.8% in Center. One in four women reported inadequate breastfeeding support (26.1%, ranging from 19.4% in Algarve to 31.5% in Lisbon). One in five reported no exclusive breastfeeding at discharge (22.1%; 19.5% in Lisbon to 28.2% in Algarve). Conclusion: Urgent actions are needed to harmonize QMNC and reduce inequities across regions in Portugal. © 2022 The Authors. International Journal of Gynecology & Obstetrics published by John Wiley & Sons Ltd on behalf of International Federation of Gynecology and Obstetrics.Funding text 1: This work was supported by the Ministry of Health, Rome - Italy, in collaboration with the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste - Italy. This study was supported by Portuguese fundings through FCT - Fundação para a Ciência e a Tecnologia, IP, in the scope of the projects EPIUnit - UIDB/04750/2020, ITR - LA/P/0064/2020, and HEILab - UIDB/05380/2020, and by the European Social Fund (ESF) and FCT (SFRH/BPD/117597/2016; RC postdoctoral fellowship). We are grateful to the women who dedicated their time to complete the survey, to Associação Portuguesa pelos Direitos da Mulher na Gravidez e Parto (APDMGP) for support with survey dissemination and to nurse Louise Semião for assistance provided in back-translation of the questionnaires. Special thanks to the IMAgiNE EURO study group for their contribution to the development of this project and support for this manuscript.; Funding text 2: This work was supported by the Ministry of Health, Rome ‐ Italy, in collaboration with the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste ‐ Italy. This study was supported by Portuguese fundings through FCT ‐ Fundação para a Ciência e a Tecnologia, IP, in the scope of the projects EPIUnit ‐ UIDB/04750/2020, ITR ‐ LA/P/0064/2020, and HEILab ‐ UIDB/05380/2020, and by the European Social Fund (ESF) and FCT (SFRH/BPD/117597/2016; RC postdoctoral fellowship). We are grateful to the women who dedicated their time to complete the survey, to Associação Portuguesa pelos Direitos da Mulher na Gravidez e Parto (APDMGP) for support with survey dissemination and to nurse Louise Semião for assistance provided in back‐translation of the questionnaires. Special thanks to the IMAgiNE EURO study group for their contribution to the development of this project and support for this manuscript. ; Funding text 3: IMAgiNE EURO project was supported by the Ministry of Health, Rome ‐ Italy, in collaboration with the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste ‐ Italy. This study was supported by Fundação para a Ciência e a Tecnologia; European Social Fund (ESF) Funding informatio

Quality of facility-based maternal and newborn care around the time of childbirth during the COVID-19 pandemic: online survey investigating maternal perspectives in 12 countries of the WHO European Region

Background Multi-country studies assessing the quality of maternal and newborn care (QMNC) during the COVID-19 pandemic, as defined by WHO Standards, are lacking. Methods Women who gave birth in 12 countries of the WHO European Region from March 1, 2020 - March 15, 2021 answered an online questionnaire, including 40 WHO Standard-based Quality Measures. Findings 21,027 mothers were included in the analysis. Among those who experienced labour (N=18,063), 41·8% (26·1%- 63·5%) experienced difficulties in accessing antenatal care, 62% (12·6%-99·0%) were not allowed a companion of choice, 31·1% (16·5%-56·9%) received inadequate breastfeeding support, 34·4% (5·2%-64·8%) reported that health workers were not always using protective personal equipment, and 31·8% (17·8%-53·1%) rated the health workers’ number as “insufficient”. Episiotomy was performed in 20·1% (6·1%-66·0%) of spontaneous vaginal births and fundal pressure applied in 41·2% (11·5% -100%) of instrumental vaginal births. In addition, 23·9% women felt they were not treated with dignity (12·8%-59·8%), 12·5% (7·0%-23·4%) suffered abuse, and 2·4% (0·1%-26·2%) made informal payments. Most findings were significantly worse among women with prelabour caesarean birth (N=2,964). Multivariate analyses confirmed significant differences among countries, with Croatia, Romania, Serbia showing significant lower QMNC Indexes and Luxemburg showing a significantly higher QMNC Index than the total sample. Younger women and those with operative births also reported significantly lower QMNC Indexes. Interpretation Mothers reports revealed large inequities in QMNC across countries of the WHO European Region. Quality improvement initiatives to reduce these inequities and promote evidence-based, patient-centred respectful care for all mothers and newborns during the COVID-19 pandemic and beyond are urgently needed. Funding The study was financially supported by the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy. Study registration ClinicalTrials.gov Identifier: NCT04847336This research was funded by the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste Italy

A siRNA-Based Screen for Genes Involved in Chromosome End Protection

Telomeres are nucleoprotein complexes which protect the ends of linear chromosomes from detection as DNA damage and provide a sequence buffer against replication-associated shortening. In mammals, telomeres consist of repetitive DNA sequence (TTAGGG) and associated proteins. The telomeric core complex is called shelterin and is comprised of the proteins TRF1, TRF2, POT1, TIN2, TPP1 and RAP1. Excessive telomere shortening or de-protection of telomeres through the loss of shelterin subunits allows the detection of telomeres as DNA damage, which can be visualized as DNA damage protein foci at chromosome ends called TIF (Telomere Dysfunction-Induced Foci). We sought to exploit the TIF phenotype as marker for telomere dysfunction to identify novel genes involved in telomere protection by siRNA-mediated knock-down of a set of 386 candidates. Here we report the establishment, specificity and feasibility of such a screen and the results of the genes tested. Only one of the candidate genes showed a unique TIF phenotype comparable to the suppression of the main shelterin components TRF2 or TRF1 and that gene was identified as a TRF1-like pseudogene. We also identified a weak TIF phenotype for SKIIP (SNW1), a splicing factor and transcriptional co-activator. However, the knock-down of SKIIP also induced a general, not telomere-specific DNA damage response, which complicates conclusions about a telomeric role. In summary, this report is a technical demonstration of the feasibility of a cell-based screen for telomere deprotection with the potential of scaling it to a high-throughput approach

Progressive Telomere Dysfunction Causes Cytokinesis Failure and Leads to the Accumulation of Polyploid Cells

Most cancer cells accumulate genomic abnormalities at a remarkably rapid rate, as they are unable to maintain their chromosome structure and number. Excessively short telomeres, a known source of chromosome instability, are observed in early human-cancer lesions. Besides telomere dysfunction, it has been suggested that a transient phase of polyploidization, in most cases tetraploidization, has a causative role in cancer. Proliferation of tetraploids can gradually generate subtetraploid lineages of unstable cells that might fire the carcinogenic process by promoting further aneuploidy and genomic instability. Given the significance of telomere dysfunction and tetraploidy in the early stages of carcinogenesis, we investigated whether there is a connection between these two important promoters of chromosomal instability. We report that human mammary epithelial cells exhibiting progressive telomere dysfunction, in a pRb deficient and wild-type p53 background, fail to complete the cytoplasmatic cell division due to the persistence of chromatin bridges in the midzone. Flow cytometry together with fluorescence in situ hybridization demonstrated an accumulation of binucleated polyploid cells upon serial passaging cells. Restoration of telomere function through hTERT transduction, which lessens the formation of anaphase bridges by recapping the chromosome ends, rescued the polyploid phenotype. Live-cell imaging revealed that these polyploid cells emerged after abortive cytokinesis due to the persistence of anaphase bridges with large intervening chromatin in the cleavage plane. In agreement with a primary role of anaphase bridge intermediates in the polyploidization process, treatment of HMEC-hTERT cells with bleomycin, which produces chromatin bridges through illegimitate repair, resulted in tetraploid binucleated cells. Taken together, we demonstrate that human epithelial cells exhibiting physiological telomere dysfunction engender tetraploid cells through interference of anaphase bridges with the completion of cytokinesis. These observations shed light on the mechanisms operating during the initial stages of human carcinogenesis, as they provide a link between progressive telomere dysfunction and tetraploidy

53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility

Double-strand breaks activate the ataxia telangiectasia mutated (ATM) kinase, which promotes the accumulation of DNA damage factors in the chromatin surrounding the break. The functional significance of the resulting DNA damage foci is poorly understood. Here we show that 53BP1 (also known as TRP53BP1), a component of DNA damage foci, changes the dynamic behaviour of chromatin to promote DNA repair. We used conditional deletion of the shelterin component TRF2 (also known as TERF2) from mouse cells (TRF2fl/-) to deprotect telomeres, which, like double-strand breaks, activate the ATM kinase, accumulate 53BP1 and are processed by non-homologous end joining (NHEJ). Deletion of TRF2 from 53BP1-deficient cells established that NHEJ of dysfunctional telomeres is strongly dependent on the binding of 53BP1 to damaged chromosome ends. To address the mechanism by which 53BP1 promotes NHEJ, we used time-lapse microscopy to measure telomere dynamics before and after their deprotection. Imaging showed that deprotected telomeres are more mobile and sample larger territories within the nucleus. This change in chromatin dynamics was dependent on 53BP1 and ATM but did not require a functional NHEJ pathway. We propose that the binding of 53BP1 near DNA breaks changes the dynamic behaviour of the local chromatin, thereby facilitating NHEJ repair reactions that involve distant sites, including joining of dysfunctional telomeres and AID (also known as AICDA)-induced breaks in immunoglobulin class-switch recombination