Dynamic organization of transcription-coupled DNA repair

The aim of the work described in this thesis is to gain more insight in the role of the Cockayne Syndrome A (GSA) and B (CSB) proteins in the process of transcriptioncoupled DNA repair and transcription. Using biochemical analysis and live cell studies we investigated the molecular behavior of both proteins. First, we analyzed the chromatin remodeling activity of CSB. Secondly, using green fluorescent protein (GFP) technology (Chapter 4) and photobleaching we studied (i) the dynamic behavior of CSB in TCR and transcription in living cells, (ii) the kinetics of GSA in TCR and its molecular connection with the CSB protein and (iii) the cellular localization of CSB with respect to other nuclear processes like transcription and mRNA processing

Lecanora sorediomarginata, a new epiphytic lichen species discovered along the Portuguese coast

Lecanora sorediomarginata Rodrigues, Terron & Elix sp. nov., described as new to science from Portugal, is characterized morphologically by a crustose whitish-grey to greenish thallus developing soralia from small, marginal warts and chemically by the presence of 3,5-dichloro-2'-O-methylnorstenosporic acid [major], 3,5-dichloro-2'-O-methylanziaic acid [minor], 3,5-dichloro-2'-O-methylnordivaricatic acid [minor], 5-chloro-2'-O-methylanziaic acid [trace], atranorin [minor], chloroatranorin [minor], and usnic acid [trace]. It is chemically similar to L. lividocinerea, to which it shows phylogenetic affinities based on ITS rDNA sequence analysis, and to L. sulphurella. Lecanora sorediomarginata is epiphytic on Pious pinaster and P. pinea, in pine forests on sand dunes along the Portuguese coast

CMV infection of liver transplant recipients: comparison of antigenemia and molecular biology assays

BACKGROUND: CMV is a major clinical problem in transplant recipients. Thus, it is important to use sensitive and specific diagnostic techniques to rapidly and accurately detect CMV infection and identify patients at risk of developing CMV disease. In the present study, CMV infection after liver transplantation was monitored retrospectively by two molecular biology assays - a quantitative PCR assay and a qualitative NASBA assay. The results were compared with those obtained by prospective pp65 antigenemia determinations. MATERIALS AND METHODS: 87 consecutive samples from 10 liver transplanted patients were tested for CMV by pp65 antigenemia, and CMV monitor and NASBA pp67 mRNA assay. RESULTS: CMV infection was detected in all patients by antigenemia and CMV monitor, whereas NASBA assay identified only 8/10 patients with viremia. Furthermore, CMV infection was never detected earlier by molecular biology assays than by antigenemia. Only 5/10 patients with CMV infection developed CMV disease. Using a cut off value of 8 cells/50,000, antigenemia was found to be the assay that better identified patients at risk of developing CMV disease. However, the kinetics of the onset of infection detected by NASBA and CMV monitor seemed to have better identified patients at risk of developing CMV disease. Furthermore, before onset of disease, CMV pp67 mRNA was found to have similar or better negative and positive predictive values for the development of CMV disease. CONCLUSIONS: The present data, suggests that the concomitant use of antigenemia and pp67 mRNA assay gives the best identification of patients at risk of developing CMV disease

Incidence of HIV and hepatitis C virus among people who inject drugs, and associations with age and sex or gender: a global systematic review and meta-analysis

Background: Measuring the incidence of HIV and hepatitis C virus (HCV) infection among people who inject drugs (PWID) is key to track progress towards elimination. We aimed to summarise global data on HIV and primary HCV incidence among PWID and associations with age and sex or gender. Methods: In this systematic review and meta-analysis, we updated an existing database of HIV and HCV incidence studies among PWID by searching MEDLINE, Embase, and PsycINFO, capturing studies published between Jan 1, 2000, and Dec 12, 2022, with no language or study design restrictions. We contacted authors of identified studies for unpublished or updated data. We included studies that estimated incidence by longitudinally re-testing people at risk of infection or by using assays for recent infection. We pooled incidence and relative risk (RR; young [generally defined as ≤25 years] vs older PWID; women vs men) estimates using random-effects meta-analysis and assessed risk of bias with a modified Newcastle–Ottawa scale. This study is registered with PROSPERO, CRD42020220884. Findings: Our updated search identified 9493 publications, of which 211 were eligible for full-text review. An additional 377 full-text records from our existing database and five records identified through cross-referencing were assessed. Including 28 unpublished records, 125 records met the inclusion criteria. We identified 64 estimates of HIV incidence (30 from high-income countries [HICs] and 34 from low-income or middle-income countries [LMICs]) and 66 estimates of HCV incidence (52 from HICs and 14 from LMICs). 41 (64%) of 64 HIV and 42 (64%) of 66 HCV estimates were from single cities rather than being multi-city or nationwide. Estimates were measured over 1987–2021 for HIV and 1992–2021 for HCV. Pooled HIV incidence was 1·7 per 100 person-years (95% CI 1·3–2·3; I2=98·4%) and pooled HCV incidence was 12·1 per 100 person-years (10·0–14·6; I2=97·2%). Young PWID had a greater risk of HIV (RR 1·5, 95% CI 1·2–1·8; I2=66·9%) and HCV (1·5, 1·3–1·8; I2=70·6%) acquisition than older PWID. Women had a greater risk of HIV (RR 1·4, 95% CI 1·1–1·6; I2=55·3%) and HCV (1·2, 1·1–1·3; I2=43·3%) acquisition than men. For both HIV and HCV, the median risk-of-bias score was 6 (IQR 6–7), indicating moderate risk. Interpretation: Although sparse, available HIV and HCV incidence estimates offer insights into global levels of HIV and HCV transmission among PWID. Intensified efforts are needed to keep track of the HIV and HCV epidemics among PWID and to expand access to age-appropriate and gender-appropriate prevention services that serve young PWID and women who inject drugs. Funding: Canadian Institutes of Health Research, Fonds de recherche du Québec–Santé, Canadian Network on Hepatitis C, UK National Institute for Health and Care Research, and WHO

First narrow-band search for continuous gravitational waves from known pulsars in advanced detector data

Spinning neutron stars asymmetric with respect to their rotation axis are potential sources of continuous gravitational waves for ground-based interferometric detectors. In the case of known pulsars a fully coherent search, based on matched filtering, which uses the position and rotational parameters obtained from electromagnetic observations, can be carried out. Matched filtering maximizes the signalto- noise (SNR) ratio, but a large sensitivity loss is expected in case of even a very small mismatch between the assumed and the true signal parameters. For this reason, narrow-band analysis methods have been developed, allowing a fully coherent search for gravitational waves from known pulsars over a fraction of a hertz and several spin-down values. In this paper we describe a narrow-band search of 11 pulsars using data from Advanced LIGO’s first observing run. Although we have found several initial outliers, further studies show no significant evidence for the presence of a gravitational wave signal. Finally, we have placed upper limits on the signal strain amplitude lower than the spin-down limit for 5 of the 11 targets over the bands searched; in the case of J1813-1749 the spin-down limit has been beaten for the first time. For an additional 3 targets, the median upper limit across the search bands is below the spin-down limit. This is the most sensitive narrow-band search for continuous gravitational waves carried out so far

Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms

UVSSA and USP7, a new couple in transcription-coupled DNA repair

Transcription-coupled nucleotide excision repair (TC-NER) specifically removes transcription-blocking lesions from our genome. Defects in this pathway are associated with two human disorders: Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS). Despite a similar cellular defect in the UV DNA damage response, patients with these syndromes exhibit strikingly distinct symptoms; CS patients display severe developmental, neurological, and premature aging features, whereas the phenotype of UVSS patients is mostly restricted to UV hypersensitivity. The exact molecular mechanism behind these clinical differences is still unknown; however, they might be explained by additional functions of CS proteins beyond TC-NER. A short overview of the current hypotheses addressing possible molecular mechanisms and the proteins involved are presented in this review. In addition, we will focus on two new players involved in TC-NER which were recently identified: UV-stimulated scaffold protein A (UVSSA) and ubiquitin-specific protease 7 (USP7). UVSSA has been found to be the causative gene for UVSS and, together with USP7, is implicated in regulating TC-NER activity. We will discuss the function of UVSSA and USP7 and how the discovery of these proteins contributes to a better understanding of the molecular mechanisms underlying the clinical differences between UVSS and the more severe CS

Environmental and genetic influences on early attachment

Attachment theory predicts and subsequent empirical research has amply demonstrated that individual variations in patterns of early attachment behaviour are primarily influenced by differences in sensitive responsiveness of caregivers. However, meta-analyses have shown that parenting behaviour accounts for about one third of the variance in attachment security or disorganisation. The exclusively environmental explanation has been challenged by results demonstrating some, albeit inconclusive, evidence of the effect of infant temperament. In this paper, after reviewing briefly the well-demonstrated familial and wider environmental influences, the evidence is reviewed for genetic and gene-environment interaction effects on developing early attachment relationships. Studies investigating the interaction of genes of monoamine neurotransmission with parenting environment in the course of early relationship development suggest that children's differential susceptibility to the rearing environment depends partly on genetic differences. In addition to the overview of environmental and genetic contributions to infant attachment, and especially to disorganised attachment relevant to mental health issues, the few existing studies of gene-attachment interaction effects on development of childhood behavioural problems are also reviewed. A short account of the most important methodological problems to be overcome in molecular genetic studies of psychological and psychiatric phenotypes is also given. Finally, animal research focusing on brain-structural aspects related to early care and the new, conceptually important direction of studying environmental programming of early development through epigenetic modification of gene functioning is examined in brief

Quantitative nucleolar proteomics reveals nuclear re-organization during stress- induced senescence in mouse fibroblast

<p>Abstract</p> <p>Background</p> <p>Nucleolus is the most prominent mammalian organelle within the nucleus which is also the site for ribosomal biogenesis. There have been many reports indicating the involvement of nucleolus in the process of aging. Several proteins related to aging have been shown to localize in the nucleolus, which suggests the role of this organelle in senescence.</p> <p>Results</p> <p>In this study, we used quantitative mass spectrometry to map the flux of proteins into and out of the nucleolus during the induction of senescence in cultured mammalian cells. Changes in the abundance of 344 nucleolar proteins in sodium butyrate-induced senescence in NIH3T3 cells were studied by SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry. Biochemically, we have validated the proteomic results and confirmed that B23 (nucleophosmin) protein was down-regulated, while poly (ADP-ribose) polymerase (PARP) and nuclear DNA helicase II (NDH II/DHX9/RHA) were up-regulated in the nucleolus upon treatment with sodium butyrate. Accumulation of chromatin in the nucleolus was also observed, by both proteomics and microscopy, in sodium butyrate-treated cells. Similar observations were found in other models of senescence, namely, in mitoxantrone- (MTX) treated cells and primary fibroblasts from the Lamin A knockout mice.</p> <p>Conclusion</p> <p>Our data indicate an extensive nuclear organization during senescence and suggest that the redistribution of B23 protein and chromatin can be used as an important marker for senescence.</p