APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks

Aprataxin and polynucleotide kinase (PNK) are DNA end processing factors that are recruited into the DNA single- and double-strand break repair machinery through phosphorylation-specific interactions with XRCC1 and XRCC4, respectively. These interactions are mediated through a divergent class of forkhead-associated (FHA) domain that binds to peptide sequences in XRCC1 and XRCC4 that are phosphorylated by casein kinase 2 (CK2). Here, we identify the product of the uncharacterized open reading frame C2orf13 as a novel member of this FHA domain family of proteins and we denote this protein APLF (aprataxin- and PNK-like factor). We show that APLF interacts with XRCC1 in vivo and in vitro in a manner that is stimulated by CK2. Yeast two-hybrid analyses suggest that APLF also interacts with the double-strand break repair proteins XRCC4 and XRCC5 (Ku86). We also show that endogenous and yellow fluorescent protein-tagged APLF accumulates at sites of H(2)O(2) or UVA laser-induced chromosomal DNA damage and that this is achieved through at least two mechanisms: one that requires the FHA domain-mediated interaction with XRCC1 and a second that is independent of XRCC1 but requires a novel type of zinc finger motif located at the C terminus of APLF. Finally, we demonstrate that APLF is phosphorylated in a DNA damage- and ATM-dependent manner and that the depletion of APLF from noncycling human SH-SY5Y neuroblastoma cells reduces rates of chromosomal DNA strand break repair following ionizing radiation. These data identify APLF as a novel component of the cellular response to DNA strand breaks in human cells

APLF (C2orf13) is a novel component of poly(ADP-ribose) signaling in mammalian cells

APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly(ADP-ribose), a polymeric posttranslational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly(ADP-ribosyl)ation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly(ADP-ribosyl)ation in vitro, and this activity is dependent on its capacity to bind the polymer. In addition, transient overexpression in human A549 cells of full-length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly(ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly(ADP-ribose) and is a novel component of poly(ADP-ribose) signaling in mammalian cells

Father-child interactions at 3 months and 24 months: contributions to children's cognitive development at 24 months

The quality of father-child interactions has become a focus of increasing research in the field of child development. We examined the potential contribution of father-child interactions at both 3 months and 24 months to children's cognitive development at 24 months. Observational measures of father-child interactions at 3 and 24 months were used to assess the quality of fathers' parenting (n = 192). At 24 months, the Mental Developmental Index (MDI) of the Bayley Scales of Infant Development, Second Edition (N. Bayley, ) measured cognitive functioning. The association between interactions and cognitive development was examined using multiple linear regression analyses, adjusting for paternal age, education and depression, infant age, and maternal sensitivity. Children whose fathers displayed more withdrawn and depressive behaviors in father-infant interactions at 3 months scored lower on the MDI at 24 months. At 24 months, children whose fathers were more engaged and sensitive as well as those whose fathers were less controlling in their interactions scored higher on the MDI. These findings were independent of the effects of maternal sensitivity. Results indicate that father-child interactions, even from a very young age (i.e., 3 months) may influence children's cognitive development. They highlight the potential significance of interventions to promote positive parenting by fathers and policies that encourage fathers to spend more time with their young children.This research was supported by Wellcome Trust Clinical Research Fellowship No. 078434 to P.G.R

Implementing chlamydia screening: what do women think? A systematic review of the literature

BACKGROUND: Chlamydia trachomatis is a common sexually transmitted infection that can have serious consequences. It is universally agreed that screening for chlamydia infection should be offered to sexually active young women. We undertook a literature review to document the views, attitudes and opinions of women about being screened, tested and diagnosed with Chlamydia trachomatis. METHODS: Online databases (MEDLINE, Meditext, PsycINFO, Web of Science) and reference lists searched up to August 2005. Search terms: chlamydia, attitude, attitude to health, interview, qualitative, women. Eligibility criteria: about chlamydia, included women, involved interviews/surveys/focus groups, looked at women's views/opinions/attitudes, published in English. Thematic analysis identified the main and recurrent themes emerging from the literature. We compared our thematic analysis with the Theory of Planned Behaviour to provide a model that could assist in planning chlamydia screening programs. RESULTS: From 561 identified articles, 25 fulfilled inclusion criteria and were reviewed. 22: USA, UK; 3: Holland, Sweden, Australia. Major themes identified: need for knowledge and information, choice and support; concerns about confidentiality, cost, fear, anxiety and stigma. Women are more likely to find chlamydia screening/testing acceptable if they think chlamydia is a serious, common condition which can cause infertility and if they understand that chlamydia infection can be asymptomatic. Women want a range of options for chlamydia testing including urine tests, self-administered swabs, pelvic exams and clinician-collected swabs, home-testing and community-based testing. Tests should be free, easy and quick. Women want support for dealing with the implications of a chlamydia diagnosis, they feel chlamydia diagnoses need to be normalised and destigmatised and they want assistance with partner notification. Women need to know that their confidentiality will be maintained. CONCLUSION: Our review found that women from various countries and ethnic backgrounds share similar views regarding chlamydia screening, testing and diagnosis. The acknowledged importance of women's views in planning an effective chlamydia screening program is expanded in this review which details the nature and complexity of such views and considers their likely impact

Tracking genomic cancer evolution for precision medicine: The Lung TRACERx Study

The importance of intratumour genetic and functional heterogeneity is increasingly recognised as a driver of cancer progression and survival outcome. Understanding how tumour clonal heterogeneity impacts upon therapeutic outcome, however, is still an area of unmet clinical and scientific need. TRACERx (TRAcking non-small cell lung Cancer Evolution through therapy [Rx]), a prospective study of patients with primary non-small cell lung cancer (NSCLC), aims to define the evolutionary trajectories of lung cancer in both space and time through multiregion and longitudinal tumour sampling and genetic analysis. By following cancers from diagnosis to relapse, tracking the evolutionary trajectories of tumours in relation to therapeutic interventions, and determining the impact of clonal heterogeneity on clinical outcomes, TRACERx may help to identify novel therapeutic targets for NSCLC and may also serve as a model applicable to other cancer types

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology

Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies

The role of a divergent FHA domain in DNA single-strand break repair

XRCC1 plays a major role in the repair of these lesions in mammalian cells by binding and/or activating many components of the single-strand break repair (SSBR) pathway. One such component is polynucleotide kinase (PNK) which possesses a divergent forkhead associated (FHA) domain that binds CK2-phosphorylated XRCC1. Aprataxin has a similar divergent FHA domain that also interacts with XRCC1 and which has been implicated in SSBR. In this thesis, yeast two-hybrid analysis indicated that PNK interacted with the pro-apoptotic protein Hippi in a manner dependent on the PNK FHA domain. In addition, a novel protein containing a similar FHA domain to PNK and aprataxin was identified and denoted APLF (Aprataxin and PNK-Like Factor). APLF was also shown to bind XRCC1 in a manner dependent on its FHA domain. Furthermore, this interaction was greatly stimulated by CK2-phosphorylation of XRCC1. APLF interacted with the double-strand break repair (DSBR) factor XRCC4. APLF was modified following DNA damage, presumably by phosphorylation. Nuclear localisation of YFP-APLF was promoted by the presence of XRCC1. Moreover, YFP-APLF colocalised with RFP-XRCC1 in DNA damage-induced nuclear foci following H₂O₂ treatment. Novel interaction partners of APLF identified by employing a yeast two-hybrid library screen included Ku86/XRCC5 and KEAP1. These data suggest a role for APLF in the cellular response to DNA strand breaks.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Father-child interactions at 3 months and 24 months: contributions to children's cognitive development at 24 months

The quality of father-child interactions has become a focus of increasing research in the field of child development. We examined the potential contribution of father-child interactions at both 3 months and 24 months to children's cognitive development at 24 months. Observational measures of father-child interactions at 3 and 24 months were used to assess the quality of fathers' parenting (n = 192). At 24 months, the Mental Developmental Index (MDI) of the Bayley Scales of Infant Development, Second Edition (N. Bayley, ) measured cognitive functioning. The association between interactions and cognitive development was examined using multiple linear regression analyses, adjusting for paternal age, education and depression, infant age, and maternal sensitivity. Children whose fathers displayed more withdrawn and depressive behaviors in father-infant interactions at 3 months scored lower on the MDI at 24 months. At 24 months, children whose fathers were more engaged and sensitive as well as those whose fathers were less controlling in their interactions scored higher on the MDI. These findings were independent of the effects of maternal sensitivity. Results indicate that father-child interactions, even from a very young age (i.e., 3 months) may influence children's cognitive development. They highlight the potential significance of interventions to promote positive parenting by fathers and policies that encourage fathers to spend more time with their young children.This research was supported by Wellcome Trust Clinical Research Fellowship No. 078434 to P.G.R

Measurement of chromosomal DNA single-strand breaks and replication fork progression rates

Chromosomal single-strand breaks (SSBs) are the most common lesions arising in cells, but are normally rapidly repaired by multiprotein complexes centered around the scaffold protein, XRCC1. Here, we describe protocols to measure chromosomal SSBs in cells and for recovering and identifying novel components of SSBR complexes in vitro and in vivo. We also describe an assay we employ to measure the rate of replication fork progression in mammalian/vertebrate cells in the presence or absence of DNA damage