Cockayne's Syndrome A and B Proteins Regulate Transcription Arrest after Genotoxic Stress by Promoting ATF3 Degradation.

Cockayne syndrome (CS) is caused by mutations in CSA and CSB. The CSA and CSB proteins have been linked to both promoting transcription-coupled repair and restoring transcription following DNA damage. We show that UV stress arrests transcription of approximately 70% of genes in CSA- or CSB-deficient cells due to the constitutive presence of ATF3 at CRE/ATF sites. We found that CSB, CSA/DDB1/CUL4A, and MDM2 were essential for ATF3 ubiquitination and degradation by the proteasome. ATF3 removal was concomitant with the recruitment of RNA polymerase II and the restart of transcription. Preventing ATF3 ubiquitination by mutating target lysines prevented recovery of transcription and increased cell death following UV treatment. Our data suggest that the coordinate action of CSA and CSB, as part of the ubiquitin/proteasome machinery, regulates the recruitment timing of DNA-binding factors and provide explanations about the mechanism of transcription arrest following genotoxic stress.

The Cockayne syndrome group A and B proteins are part of a ubiquitin-proteasome degradation complex regulating cell division.

Cytokinesis is monitored by a molecular machinery that promotes the degradation of the intercellular bridge, a transient protein structure connecting the two daughter cells. Here, we found that CSA and CSB, primarily defined as DNA repair factors, are located at the midbody, a transient structure in the middle of the intercellular bridge, where they recruit CUL4 and MDM2 ubiquitin ligases and the proteasome. As a part of this molecular machinery, CSA and CSB contribute to the ubiquitination and the degradation of proteins such as PRC1, the Protein Regulator of Cytokinesis, to ensure the correct separation of the two daughter cells. Defects in CSA or CSB result in perturbation of the abscission leading to the formation of long intercellular bridges and multinucleated cells, which might explain part of the Cockayne syndrome phenotypes. Our results enlighten the role played by CSA and CSB as part of a ubiquitin/proteasome degradation process involved in transcription, DNA repair, and cell division.

The addition of simvastatin administration to cold storage solution of explanted whole liver grafts for facing ischemia/reperfusion injury in an area with a low rate of deceased donation: a monocentric randomized controlled double-blinded phase 2 study.

BACKGROUND: Liver transplantation is the best treatment for end-stage liver disease. The interruption of the blood supply to the donor liver during cold storage damages the liver, affecting how well the liver will function after transplant. The drug Simvastatin may help to protect donor livers against this damage and improve outcomes for transplant recipients. The aim of this study is to evaluate the benefits of treating the donor liver with Simvastatin compared with the standard transplant procedure. PATIENT AND METHODS: We propose a prospective, double-blinded, randomized phase 2 study of 2 parallel groups of eligible adult patients. We will compare 3-month, 6-month, and 12-month graft survival after LT, in order to identify a significant relation between the two homogenous groups of LT patients. The two groups only differ by the Simvastatin or placebo administration regimen while following the same procedure, with identical surgical instruments, and medical and nursing skilled staff. To reach these goals, we determined that we needed to recruit 106 patients. This sample size achieves 90% power to detect a difference of 14.6% between the two groups survival using a one-sided binomial test. DISCUSSION: This trial is designed to confirm the effectiveness of Simvastatin to protect healthy and steatotic livers undergoing cold storage and warm reperfusion before transplantation and to evaluate if the addition of Simvastatin translates into improved graft outcomes. TRIAL REGISTRATION: ISRCTN27083228 .

Congenital pulmonary airway malformations: from prenatal diagnosis to postnatal outcome.

Congenital pulmonary airway malformations (CPAMs) include cystic and non-cystic lung lesions. These represent about 30-40% of developmental lung bud anomaly lesions mainly diagnosed during pregnancy or in newborn infants; or sometimes they remain undetected until adult life. The malformation usually presents as a sporadic, non-hereditary lung abnormality, with no predilection for the right or left lung, sex or race. CPAMs vary in their histological features, epidemiological and clinical presentation, severity and prognosis, supporting the embryologic hypothesis of arrested lung growth during branching morphogenesis. The existence of "hybrid" forms underline the possible common pathogenic mechanism involved in the development of different lesion types; a genetic role has also been proposed in abnormal lung development. Influence of the natural history on pre and postnatal management is relevant. Surgical resection is the standard of therapy for symptomatic CPAMs, while the management of asymptomatic cases remains controversial. The potential risk of infection and malignancy in CPAMs justifies complete surgical resection in the first year of life; while long term follow-up is required in children who do not undergo surgery. A multidisciplinary team including gynecologists, neonatologists, radiologists, pediatricians and pediatric surgeons is recommended in pre, postnatal management and in the postsurgical follow-up of all children with CPAMs.

Mesenchymal stromal cells for cutaneous wound healing in a rabbit model: pre-clinical study applicable in the pediatric surgical setting.

OBJECTIVE: Mesenchymal stromal cells (MSCs) expanded in vitro have been proposed as a potential therapy for congenital or acquired skin defects in pediatrics. The aim of this pre-clinical study was to investigate the effects of intradermal injections of MSC in experimental cutaneous wound repair comparing allogeneic and autologous adipose stem cells (ASCs) and autologous bone marrow-mesenchymal stromal cells (BM-MSCs). METHODS: Mesenchymal stromal cells were in vitro expanded from adipose and BM tissues of young female New Zealand rabbits. MSCs were characterized for plastic adhesion, surface markers, proliferation and differentiation capacity. When an adequate number of cells (ASCs 10 × 10(6) and BM-MSCs 3 × 10(6), because of their low rate of proliferation) was reached, two skin wounds were surgically induced in each animal. The first was topically treated with cell infusions, the second was used as a control. The intradermal inoculation included autologous or allogeneic ASCs or autologous BM-MSCs. For histological examination, animals were sacrificed and wounds were harvested after 11 and 21 days of treatment. RESULTS: Rabbit ASCs were isolated and expanded in vitro with relative abundance, cells expressed typical surface markers (CD49e, CD90 and CD29). Topically, ASC inoculation provided more rapid wound healing than BM-MSCs and controls. Improved re-epithelization, reduced inflammatory infiltration and increased collagen deposition were observed in biopsies from wounds treated with ASCs, with the best result in the autologous setting. ASCs also improved restoration of skin architecture during wound healing. CONCLUSION: The use of ASCs may offer a promising solution to treat extended wounds. Pre-clinical studies are however necessary to validate the best skin regeneration technique, which could be used in pediatric surgical translational research.

CS proteins and ubiquitination: orchestrating DNA repair with transcription and cell division.

To face genotoxic stress, eukaryotic cells evolved extremely refined mechanisms. Defects in counteracting the threat imposed by DNA damage underlie the rare disease Cockayne syndrome (CS), which arises from mutations in the CSA and CSB genes. Although initially defined as DNA repair proteins, recent work shows that CSA and CSB act instead as master regulators of the integrated response to genomic stress by coordinating DNA repair with transcription and cell division. CSA and CSB exert this function through the ubiquitination of target proteins, which are effectors/regulators of these processes. This review describes how the ubiquitination of target substrates is a common denominator by which CSA and CSB participate in different aspects of cellular life and how their mutation gives rise to the complex disease CS.

Cockayne syndrome group A protein localizes at centrosomes during mitosis and regulates Cyclin B1 ubiquitination.

Mutations in CSA and CSB proteins cause Cockayne syndrome, a rare genetic neurodevelopment disorder. Alongside their demonstrated roles in DNA repair and transcription, these two proteins have recently been discovered to regulate cytokinesis, the final stage of the cell division. This last finding allowed, for the first time, to highlight an extranuclear localization of CS proteins, beyond the one already known at mitochondria. In this study, we demonstrated an additional role for CSA protein being recruited at centrosomes in a strictly determined step of mitosis, which ranges from pro-metaphase until metaphase exit. Centrosomal CSA exerts its function in specifically targeting the pool of centrosomal Cyclin B1 for ubiquitination and proteasomal degradation. Interestingly, a lack of CSA recruitment at centrosomes does not affect Cyclin B1 centrosomal localization but, instead, it causes its lasting centrosomal permanence, thus inducing Caspase 3 activation and apoptosis. The discovery of this unveiled before CSA recruitment at centrosomes opens a new and promising scenario for the understanding of some of the complex and different clinical aspects of Cockayne Syndrome.

Promoters of ASCL1- and NEUROD1-dependent genes are specific targets of lurbinectedin in SCLC cells.

Small-Cell Lung Cancer (SCLC) is an aggressive neuroendocrine malignancy with a poor prognosis. Here, we focus on the neuroendocrine SCLC subtypes, SCLC-A and SCLC-N, whose transcription addiction was driven by ASCL1 and NEUROD1 transcription factors which target E-box motifs to activate up to 40% of total genes, the promoters of which are maintained in a steadily open chromatin environment according to ATAC and H3K27Ac signatures. This leverage is used by the marine agent lurbinectedin, which preferentially targets the CpG islands located downstream of the transcription start site, thus arresting elongating RNAPII and promoting its degradation. This abrogates the expression of ASCL1 and NEUROD1 and of their dependent genes, such as BCL2, INSM1, MYC, and AURKA, which are responsible for relevant SCLC tumorigenic properties such as inhibition of apoptosis and cell survival, as well as for a part of its neuroendocrine features. In summary, we show how the transcription addiction of these cells becomes their Achilles's heel, and how this is effectively exploited by lurbinectedin as a novel SCLC therapeutic endeavor.

Defective transcription of ATF3 responsive genes, a marker for Cockayne Syndrome.

Cockayne syndrome (CS) is a rare genetic disorder caused by mutations (dysfunction) in CSA and CSB. CS patients exhibit mild photosensitivity and severe neurological problems. Currently, CS diagnosis is based on the inefficiency of CS cells to recover RNA synthesis upon genotoxic (UV) stress. Indeed, upon genotoxic stress, ATF3, an immediate early gene is activated to repress up to 5000 genes encompassing its responsive element for a short period of time. On the contrary in CS cells, CSA and CSB dysfunction impairs the degradation of the chromatin-bound ATF3, leading to a permanent transcriptional arrest as observed by immunofluorescence and ChIP followed by RT-PCR. We analysed ChIP-seq of Pol II and ATF3 promoter occupation analysis and RNA sequencing-based gene expression profiling in CS cells, as well as performed immunofluorescence study of ATF3 protein stability and quantitative RT-PCR screening in 64 patient cell lines. We show that the analysis of few amount (as for example CDK5RAP2, NIPBL and NRG1) of ATF3 dependent genes, could serve as prominent molecular markers to discriminate between CS and non-CS patient's cells. Such assay can significantly simplify the timing and the complexity of the CS diagnostic procedure in comparison to the currently available methods.

Spleen size evaluation in children: Time to define splenomegaly for pediatric surgeons and pediatricians.

BACKGROUND: We determined the range of normal spleen dimensions evaluated by ultrasonography (US) in children according to sex and age and the relationship between splenic measurements, auxological data and body proportions, in order to define splenomegaly parameters in support of the surgical mini-invasive approaches in pediatrics. METHODS: We prospectively examined 317 caucasian children of both sexes. The patients were divided into three groups: 0-3 years; 4-10 years; 11-18 years. Sex, weight, height/length, body mass index (BMI), waist circumference and xipho-pubic distance were determined for each child. US spleen evaluation included longitudinal/antero-posterior/transverse diameters, transverse area and volume. Spleen volume/abdominal volume, longitudinal spleen diameter/longitudinal left kidney diameter and longitudinal spleen diameter/xipho-pubic distance ratios were also calculated. RESULTS: For caucasian subjects, in different age groups spleen volume, transverse area and diameter increased while the spleen/abdominal volume ratio decreased significantly (p<0.001). A significant (p<0.001) decrease in longitudinal spleen diameter/xipho-pubic distance ratio was noted between the 0-3 years group and both 4-10 and 11-18 years group. Age and auxological data, except BMI, showed a high correlation with spleen dimension (r≥0.8). CONCLUSIONS: The current concept of splenomegaly is not applicable in pediatric surgery. A dedicated classification of splenomegaly is needed for children and would improve the safety and feasibility of treatment.

A Case Report of Acute Diverticulitis in "Pseudodiverticulosis" after Hemorpex System® Procedure.

Introduction. In the last years many mini-invasive approaches were developed in order to reduce postoperative pain and complication after haemorrhoid surgery: one of these alternatives is represented by Hemorpex System, a relatively young technique that combines transanal dearterialization with mucopexy through a dedicated proctoscope. Case Presentation. A 78-year-old male patient was admitted to the Emergency Department for acute urinary retention and elevated temperature. Hemorpex procedure was performed 4 years before. Clinical, endoscopic, and radiological findings demonstrated the presence of multiple diverticula-like structures fulfilled by purulent fluid and a deep alteration of the normal anatomy of the rectum. He was treated following the standard protocol of acute diverticulitis and full recovery from symptoms was achieved. Discussion. Hemorpex System is a young technique, and nowadays-available studies lack long-term follow-up data. Anatomical changes induced by the procedure are consistent and definitive. Our patient luckily demonstrated a prompt response to conservative treatment, but it must be taken into account that, in case of medical treatment failure, surgical approach would be necessary and the actual patient anatomical changes could lead the surgeon to unavoidable threatening maneuvers.

Congenital pulmonary malformations: metabolomic profile of lung phenotype in infants.

BACKGROUND: The main hydrosoluble metabolites in three different human congenital pulmonary malformations are described by nuclear magnetic resonance (NMR) spectroscopy. METHODS: Bronchogenic cyst (BC), congenital lobar emphysema (CLE) and intrapulmonary sequestration (IPS), were analyzed with respect to a control sample. The extracted metabolites were submitted to high-resolution (1)H NMR-spectroscopy. RESULTS: Congenital lung malformations showed free choline, phosphocoline and myoinositol high levels. IPS and CLE were found increased in lactic acid/glucose ratio. Lactic acid and glucose values resulted to be more elevated in control sample. CONCLUSIONS: Congenital lung lesions showed different metabolomic profiles useful for early diagnosis.

Fetal surgery and maternal cortisol response to stress. The myelomeningocele sheep model.

BACKGROUND: Prenatal surgery represents a multifactorial stressor event for mother and fetus. The stress response to fetal surgery was evaluated by measuring maternal plasma and amniotic fluid (AF) cortisol levels in the myelomeningocele (MMC) sheep model. SUBJECTS AND METHODS: Pregnant ewes (n = 8) underwent general anesthesia for MMC-induction (step 1: 75 d gestation), surgical defect repair (step 2: 110 d gestation), and delivery (step 3: 140 d gestation). Maternal blood samples were taken before surgery (surgical stage T1), after laparotomy and uterine exposure (surgical stage T2), at the end of the procedure (surgical stage T3). Fetal stress was evaluated by measuring cortisol levels in AF after hysterotomy at steps 1-3. RESULTS: Maternal cortisol concentrations at T2 and T3 increased compared with T1 (p = 0.019 and p = 0.046). AF cortisol response increased from 1 to 3 surgical steps and during pregnancy. The AF cortisol level was lower than maternal serum levels (induction p < 0.001; repair p < 0.001; caesarean section p < 0.001). CONCLUSIONS: Hysterotomy was the most stressful event in the ewes. Fetuses seemed to be partially protected from the high maternal cortisol levels. The fetal stress response to prenatal surgery increased with gestational age. Pain perception development, fetal maturation, and "pain memory" are probably associated with this increase.