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Trichothiodystrophy (TTD) is a rare hereditary disease whose prominent feature is brittle hair. Additional clinical signs are physical and neurodevelopmental abnormalities and in about half of the cases hypersensitivity to UV radiation. The photosensitive form of TTD (PS-TTD) is most commonly caused by mutations in the ERCC2/XPD gene encoding a subunit of the transcription/DNA repair complex TFIIH. Here we report novel ERCC2/XPD mutations affecting proper protein folding, which generate thermo-labile forms of XPD associated with thermo-sensitive phenotypes characterized by reversible aggravation of TTD clinical signs during episodes of fever. In patient cells, the newly identified XPD variants result in thermo-instability of the whole TFIIH complex and consequent temperature-dependent defects in DNA repair and transcription. Improving the protein folding process by exposing patient cells to low temperature or to the chemical chaperone glycerol allowed rescue of TFIIH thermo-instability and a concomitant recovery of the complex activities. Besides providing a rationale for the peculiar thermo-sensitive clinical features of these new cases, the present findings demonstrate how variations in the cellular concentration of mutated TFIIH impact the cellular functions of the complex and underlie how both quantitative and qualitative TFIIH alterations contribute to TTD clinical features.

Bi-allelic inactivation of XPD protein, a nucleotide excision repair (NER) signaling pathway component encoded by ERCC2 gene, has been associated with several defective DNA repair phenotypes, including xeroderma pigmentosum, photosensitive trichothiodystrophy, and cerebro-oculo-facio-skeletal syndrome. We report a pediatric patient harboring two compound heterozygous variants in ERCC2 gene, c.361-1G > A and c.2125A > C (p.Thr709Pro), affected by severe postnatal growth deficiency, microcephaly, facial dysmorphisms and pilocytic astrocytoma of the brainstem. Some of these features point to a DNA repair syndrome, and altogether delineate a phenotype differentiating from disorders known to be associated with ERCC2 mutations. The DNA repair efficiency following UV irradiation in the proband's skin fibroblasts was defective indicating that the new set of ERCC2 alleles impacts on NER efficiency. Sequencing analysis on tumor DNA did not reveal any somatic deleterious point variant in cancer-related genes, while SNP-array analysis disclosed a 2 Mb microduplication involving the 7q34 region, spanning from KIAA1549 to BRAF, and resulting in the KIAA1549:BRAF fusion protein, a marker of pilocytic astrocytoma. In conclusion, this report expands the clinical and mutational spectrum of ERCC2-related disorders.

Guided tissue regeneration procedures to treat periodontitis lesions making use of polytetrafluoroethylene (PTFE) membranes exhibit large variability in their surgical outcomes, due to bacterial infection following implantation. This work reports on a facile method to obtain antimicrobial coatings for such PTFE membranes, by exploiting a mussel-inspired approach and in-situ formation of silver nanoparticles (AgNPs). PTFE films were initially coated with self-polymerized 3,4-dihydroxy-DL-phenylalanine (DOPA) (PTFE-DOPA), then incubated with AgNO3 solution. In the presence of catechol moieties, Ag+ ions reduced into Ag0, forming AgNPs of around 68 nm in the polyDOPA coating on PTFE membranes (PTFE-DOPA-Ag). The x-ray photoelectron spectroscopy, atomic force microscopy and scanning electron microscopy analyses indicated that the AgNPs were distributed quite homogeneously in the polymeric membrane. The antimicrobial ability of PTFE-DOPA-Ag membranes against Staphylococcus aureus and Escherichia coli was assessed. In vitro cell assay using NIH 3T3 fibroblasts showed that, although cells were adhered to PTFE-DOPA-Ag membranes, their viability and proliferation were limited demonstrating again the antibacterial activities of PTFE-DOPA-Ag membranes. This work provides proof-of-concept study of a new versatile approach for AgNPs coating, which may be easily applied to many other types of polymeric or metallic implants through exploiting the adhesive behavior of mussel-inspired coatings.

BackgroundCockayne syndrome (CS) is a rare, autosomal recessive multisystem disorder characterised by prenatal or postnatal growth failure, progressive neurological dysfunction, ocular and skeletal abnormalities and premature ageing. About half of the patients with symptoms diagnostic for CS show cutaneous photosensitivity and an abnormal cellular response to UV light due to mutations in either the ERCC8/CSA or ERCC6/CSB gene. Studies performed thus far have failed to delineate clear genotype-phenotype relationships. We have carried out a four-centre clinical, molecular and cellular analysis of 124 patients with CS.Methods and resultsWe assigned 39 patients to the ERCC8/CSA and 85 to the ERCC6/CSB genes. Most of the genetic variants were truncations. The missense variants were distributed non-randomly with concentrations in relatively short regions of the respective proteins. Our analyses revealed several hotspots and founder mutations in ERCC6/CSB. Although no unequivocal genotype-phenotype relationships could be made, patients were more likely to have severe clinical features if the mutation was downstream of the PiggyBac insertion in intron 5 of ERCC6/CSB than if it was upstream. Also a higher proportion of severely affected patients was found with mutations in ERCC6/CSB than in ERCC8/CSA.ConclusionBy identifying >70 novel homozygous or compound heterozygous genetic variants in 124 patients with CS with different disease severity and ethnic backgrounds, we considerably broaden the CSA and CSB mutation spectrum responsible for CS. Besides providing information relevant for diagnosis of and genetic counselling for this devastating disorder, this study improves the definition of the puzzling genotype-phenotype relationships in patients with CS.

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.

Inert polytetrafluoroethylene (PTFE) membranes for periodontal regeneration suffer from weak osteoconductive properties. In this work, a strategy for hydroxyapatite (HAp) coating on PTFE films through an adhesive layer of self-polymerized 3,4-dihydroxy-DL-phenylalanine (polyDOPA) was developed to improve surface properties. Physico-chemical and morphological analysis demonstrated the deposition of polyDOPA and HAp, with an increase in surface roughness and wettability. A discontinuous coating was present after 14 days in PBS and MC3T3-E1 cells proliferation and adhesion were improved. Results confirmed the potential application of polyDOPA/HAp-coated films for periodontal disease treatments.

Background Trichothiodystrophy (TTD) is a group of rare autosomal recessive disorders that variably affect a wide range of organs derived from the neuroectoderm. The key diagnostic feature is sparse, brittle, sulfur deficient hair that has a ‘tiger-tail’ banding pattern under polarising light microscopy. Patients and methods We describe two male cousins affected by TTD associated with microcephaly, profound intellectual disability, sparse brittle hair, aged appearance, short stature, facial dysmorphism, seizures, an immunoglobulin deficiency, multiple endocrine abnormalities, cerebellar hypoplasia and partial absence of the corpus callosum, in the absence of cellular photosensitivity and ichthyosis. Obligate female carriers showed 100% skewed X-chromosome inactivation. Linkage analysis and Sanger sequencing of 737 X-chromosome exons and whole exome sequencing was used to find the responsible gene and mutation. Results Linkage analysis localised the disease allele to a 7.75 Mb interval from Xq23–q25. We identified a nonsense mutation in the highly conserved RNF113A gene (c.901 C>T, p.Q301*). The mutation segregated with the disease in the family and was not observed in over 100 000 control X chromosomes. The mutation markedly reduced RNF113A protein expression in extracts from lymphoblastoid cell lines derived from the affected individuals. Conclusions The association of RNF113A mutation with non-photosensitive TTD identifies a new locus for these disorders on the X chromosome. The extended phenotype within this family includes panhypopituitarism, cutis marmorata and congenital short oesophagus.

In mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2Wt and DDB2PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity.

Binary blends of poly(L-lactic) acid (PLLA: 201790 Da) and poly(lactide-co-glycolide) (PLGA) (LA:GA = 50:50 mol:mol; 32030 Da) with various compositions were prepared. Physicochemical properties of PLLA/PLGA blends were analyzed. Blends showed a biphasic morphology, with distinct glass transition temperatures, which only slightly approximated compared to pure polymers. Analysis of tensile mechanical properties through the Kerner-Uemura-Takayanagi model showed compatibility for PLLA/PLGA 75/25 blend. Rapid degradation of PLGA phase (2–8 weeks) in PLLA/PLGA 75/25 blend led to porous samples, which appear promising for drug delivery and tissue engineering. A limited inflammatory reaction resulted from subcutaneous implantation of PLLA/PLGA 75/25 in Balb-c mice.

Adrenocortical carcinoma (ACC) is a rare and aggressive tumor characterized by poor prognosis and resistance to conventional chemotherapy. Many chemotherapy agents act determining apoptosis, therefore, studying the responsiveness of ACC to apoptosis inducing molecules, can help to identify possible conditions to promote cancer cell death. Tumor progression is strictly related to the interaction between cancer cells and stroma; yet, extracellular matrix remodeling regulates tumor cell proliferation and apoptosis. At this purpose, we have studied staurosporine-induced apoptosis of ACC cell line H295R adherent to different extracellular matrix molecules. H295R cells grown on plastic showed a low responsiveness to staurosporine, with an apoptotic rate of 24 %, as compared to breast cancer MCF7 cells, with an apoptotic rate of 60 %. The adhesion of H295R cells to type V collagen induced a significant increase of apoptosis up to 52 %; this effect was inhibited by anti-integrin alpha2 antibody. At the same time, the adhesion of H295R cells on polylysine, matrigel, lamimin, fibronectin, and type I-III collagens didn't modify staurosporine-induced apoptosis. Staurosporine-treated H295R cells showed an increase of PARP cleavage and of annexin-V expression, when adherent to type V collagen. Yet, staurosporine induced Akt and Erk activation on H295R cells: the adhesion on type V collagen didn't modify Akt activation, while determined a dramatic inhibition of Erk activation. The described data demonstrate that the adhesion to type V collagen specifically increases the responsiveness of ACC cells to staurosporine-induced apoptosis and that this is probably obtained through the inhibition of Erk activation.

The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.

Mutations in the XPD subunit of the transcription/DNA repair factor (TFIIH) give rise to trichothiodystrophy (TTD), a rare hereditary multisystem disorder with skin abnormalities. Here, we show that TTD primary dermal fibroblasts contain low amounts of collagen type VI alpha1 subunit (COL6A1), a fundamental component of soft connective tissues. We demonstrate that COL6A1 expression is downregulated by the sterol regulatory element-binding protein-1 (SREBP-1) whose removal from the promoter is a key step in COL6A1 transcription upregulation in response to cell confluence. We provide evidence for TFIIH being involved in transcription derepression, thus highlighting a new function of TFIIH in gene expression regulation. The lack of COL6A1 upregulation in TTD is caused by the inability of the mutated TFIIH complexes to remove SREBP-1 from COL6A1 promoter and to sustain the subsequent high rate of COL6A1 transcription. This defect might account for the pathologic features that TTD shares with hereditary disorders because of mutations in COL6A genes.

Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in the process of tumor invasion and metastasis that are found throughout tissues and also in the plasma. The aim of this study was to investigate whether the evaluation of plasma concentrations of MMPs 2, 3 and 9 may have clinical significance in breast cancer. Therefore, sera obtained from 80 patients with breast neoplasia (50 carcinomas and 30 fibroadenomas) were collected before and 96 h after surgery and the concentrations of MMPs 2, 3 and 9 were quantified using an enzyme-linked immunosorbent assay (ELISA). The mean expression level of MMP 2 was significantly higher in carcinoma compared with that in fibroadenoma patients, while there was no significant difference for MMPs 3 and 9. In addition, the group of carcinoma patients was analyzed in order to compare the mean values for each MMP obtained before and after surgery. However, the differences between pre- and post-surgery values for all three MMPs were not statistically significant. Furthermore, the plasma levels of each MMP were correlated with certain clinicopathological parameters of the tumors and we observed a significant and direct correlation between the concentrations of MMPs 2 and 9 and tumor histological grade. These data suggest that the quantification of plasma MMP 2 and MMP 9 levels may provide additional clinical information of the tumor and it is, therefore, a possible prognostic index for breast cancer.

Nucleotide excision repair (NER) is an important DNA repair mechanism through which cells remove bulky DNA lesions. Following DNA damage, the histone acetyltransferase (HAT) p300 (also referred to as lysine acetyltransferase or KAT) is known to associate with proliferating cell nuclear antigen (PCNA), a master regulator of DNA replication and repair processes. This interaction, which results in HAT inhibition, may be dissociated by the cell cycle inhibitor p21(CDKN1A), thereby restoring p300 activity; however, the role of this protein interplay is still unclear. Here, we report that silencing p300 or its homolog CREB-binding protein (CBP) by RNA interference (RNAi) significantly reduces DNA repair synthesis in human fibroblasts. In addition, we determined whether p300 and CBP may associate with and acetylate specific NER factors such as XPG, the 3'-endonuclease that is involved in the incision/excision step and is known to interact with PCNA. Our results show that p300 and CBP interact with XPG, which has been found to be acetylated in vivo. XPG is acetylated by p300 in vitro, and this reaction is inhibited by PCNA. Knocking down both p300/CBP by RNAi or by chemical inhibition with curcumin greatly reduced XPG acetylation, and a concomitant accumulation of the protein at DNA damage sites was observed. The ability of p21 to bind PCNA was found to regulate the interaction between p300 and XPG, and an abnormal accumulation of XPG at DNA damage sites was also found in p21(-/-) fibroblasts. These results indicate an additional function of p300/CBP in NER through the acetylation of XPG protein in a PCNA-p21 dependent manner.

UV-sensitive syndrome (UV SS) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UV SS, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UV SS cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UV SS, one of whom is described for the first time here, formed a separate UV SS-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UV SS-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NERg-deficient disorders., Nature Genetics, 44(5), pp.586-592; 2012

UV-sensitive syndrome (UV S S) is a recently-identified autosomal recessive disorder characterized by mild cutaneous symptoms and defective transcription-coupled repair (TC-NER), the subpathway of nucleotide excision repair (NER) that rapidly removes damage that can block progression of the transcription machinery in actively-transcribed regions of DNA. Cockayne syndrome (CS) is another genetic disorder with sun sensitivity and defective TC-NER, caused by mutations in the CSA or CSB genes. The clinical hallmarks of CS include neurological/developmental abnormalities and premature aging. UV S S is genetically heterogeneous, in that it appears in individuals with mutations in CSB or in a still-unidentified gene. We report the identification of a UV S S patient (UV S S1VI) with a novel mutation in the CSA gene (p.trp361cys) that confers hypersensitivity to UV light, but not to inducers of oxidative damage that are notably cytotoxic in cells from CS patients. The defect in UV S S1VI cells is corrected by expression of the WT CSA gene. Expression of the p.trp361cys-mutated CSA cDNA increases the resistance of cells from a CS-A patient to oxidative stress, but does not correct their UV hypersensitivity. These findings imply that some mutations in the CSA gene may interfere with the TC-NER-dependent removal of UV-induced damage without affecting its role in the oxidative stress response. The differential sensitivity toward oxidative stress might explain the difference between the range and severity of symptoms in CS and the mild manifestations in UV s S patients that are limited to skin photosensitivity without precocious aging or neurodegeneration.

Xeroderma pigmentosum (XP) is a rare DNA repair disorder characterized by increased susceptibility to UV radiation (UVR)-induced skin pigmentation, skin cancers, ocular surface disease, and, in some patients, sunburn and neurological degeneration. Genetically, it is assigned to eight complementation groups (XP-A to -G and variant). For the last 5 y, the UK national multidisciplinary XP service has provided follow-up for 89 XP patients, representing most of the XP patients in the United Kingdom. Causative mutations, DNA repair levels, and more than 60 clinical variables relating to dermatology, ophthalmology, and neurology have been measured, using scoring systems to categorize disease severity. This deep phenotyping has revealed unanticipated heterogeneity of clinical features, between and within complementation groups. Skin cancer is most common in XP-C, XP-E, and XP-V patients, previously considered to be the milder groups based on cellular analyses. These patients have normal sunburn reactions and are therefore diagnosed later and are less likely to adhere to UVR protection. XP-C patients are specifically hypersensitive to ocular damage, and XP-F and XP-G patients appear to be much less susceptible to skin cancer than other XP groups. Within XP groups, different mutations confer susceptibility or resistance to neurological damage. Our findings on this large cohort of XP patients under long-term follow-up reveal that XP is more heterogeneous than has previously been appreciated. Our data now enable provision of personalized prognostic information and management advice for each XP patient, as well as providing new insights into the functions of the XP proteins.

Poly(DL-lactide-co-e-caprolactone) (PLCL) and poly(DL-lactide-co-glycolide) (PLGA) blends of various compositions were prepared. Fractured sections of PLCL/PLGA blends did not evidence phase separation and blend glass transition temperatures suggested some degree of blend compatibility. The elastic modulus showed a negative deviation from the additive law of mixture. Superior biocompatibility in terms of fibroblast NIH 3T3 cell adhesion and proliferation, better mechanical properties, and a more homogeneous phase were obtained with PLCL/PLGA 25/75 blend. Rapid degradation of PLCL phase (4–8 weeks) in PLCL/PLGA 25/75 blend led to a porous structure, which makes it a potential candidate for drug delivery systems.

The stainless steel (SS) stents have been used in clinics since 1994. However, typical drawbacks are restenosis and thrombus formation due to limited endothelialisation and hemocompatibility. Surface modification is a smart strategy to enhance antithrombogenicity by promoting endothelialisation. In this work, the layer-by-layer (LbL) technique was applied for coating SS model substrates, after surface priming by functionalisation with 3-aminopropyl triethoxysilane (APTES). A LbL coating made of 14 layers of poly(styrene sulfonate)/poly(diallyldimethylammonium chloride) and heparin as last layer was deposited. FTIR-ATR analysis and contact angle measurements showed that LbL was an effective method to prepare nanostructured coatings. XPS analysis and colorimetric assay employing 1,9-dimethylmethylene blue dye to detect -COOH groups confirmed the successful polyelectrolyte deposition on the coated samples. Preliminary in vitro cell tests, using whole blood and human platelets, were performed to evaluate how surface modification affects platelet activation. Results showed that SS and SS-APTES surfaces induced platelet activation, as indicated by platelet spreading and filopodia formation. After surface modification by LbL coating, the platelets assumed a round shape and no fibrin nets were detected. Data demonstrated that LbL coating is a promising technique to fabricate antithrombogenic surface.

Trichothiodystrophy (TTD) is a rare autosomal recessive disorder whose defining feature is brittle hair. Associated clinical symptoms include physical and mental retardation of different severity, ichthyosis, premature aging, and, in half of the patients, photosensitivity. Recently, C7orf11 (TTDN1) was identified as the first disease gene for the nonphotosensitive form of TTD, being mutated in two unrelated cases and in an Amish kindred. We have evaluated the involvement of TTDN1 in 44 unrelated nonphotosensitive TTD cases of different geographic origin and with different disease severity. Mutations were found in six patients, five of whom are homozygous and one of whom is a compound heterozygote. All five identified mutations are deletions that have not been described before. Three are deletions of a few bases, resulting in frameshifts and premature termination codons. The other two include the whole TTDN1 gene, suggesting that TTDN1 is not essential for cell proliferation and viability. The severity of the clinical features does not correlate with the type of mutation, indicating that other factors besides TTDN1 mutations influence the severity of the disorder. Since only a small proportion of the analyzed cases were mutated in TTDN1, the nonphotosensitive form of TTD is genetically heterogeneous. Mutations in TTDN1 do not affect the response to ultraviolet (UV) light or the steady state level of the repair/transcription factor IIH (TFIIH), which is central to the onset of the photosensitive form of TTD. Hum Mutat 0, 1-5, 2006. (c) 2006 Wiley-Liss, Inc.

Background Xeroderma pigmentosum (XP) is an autosomal recessive disorder of, in most cases, defective nucleotide excision repair (NER) of ultraviolet radiation (UV)- and chemical-induced DNA damage. The condition is characterized by an increased sensitivity of the skin to UV radiation, with early development of pigmentary changes and premalignant lesions in sun-exposed areas of the skin, signs of photoageing and a greatly increased incidence from a young age of skin tumours including melanoma. Approximately 20% of patients with XP show neurological abnormalities of varying severity due to primary neuronal degeneration. Genetic analysis by somatic cell hybridization has led to the identification in the NER-defective form of XP of seven complementation groups, designated XP-A to XP-G. These complementation groups correspond to different proteins involved in the NER process. XP-A classically includes some of the most severely affected patients. Objectives We describe a 61-year-old Punjabi woman with XP. Remarkably she had only mild cutaneous abnormalities, minimal neurological features and unusual longevity, and developed a malignant spindle cell melanoma. There are few previous reports of spindle cell melanoma associated with XP. To gain insight into the aetiology of these unusual features, we sought to analyse the DNA repair properties of the patient and identify the complementation group and the causative mutation in the defective gene. Methods Unscheduled DNA synthesis and the inhibition of RNA synthesis were measured. The complementation group was assigned by fusing the cells of our patient with XP cells of known complementation groups and determining the ability to carry out unscheduled DNA repair. Molecular analysis of the cDNA was carried out by polymerase chain reaction and DNA sequencing. Results Levels of DNA repair were extremely low and complementation analysis assigned the defect to the XP-A group. Sequencing of the XPA gene revealed a novel homozygous mutation of A?G at the eighth nucleotide of intron 4 causing aberrant splicing and a nonfunctional truncated XP-A protein. However, a small amount of normally spliced mRNA was detected at

The cyclin-dependent kinase inhibitor p21CDKN1A plays a fundamental role in the DNA-damage response by inducing cell-cycle arrest, and by inhibiting DNA replication through association with the proliferating cell nuclear antigen (PCNA). However, the role of such an interaction in DNA repair is poorly understood and controversial. Here, we provide evidence that a pool of p21 protein is rapidly recruited to UV-induced DNA-damage sites, where it colocalises with PCNA and PCNA-interacting proteins involved in nucleotide excision repair (NER), such as DNA polymerase δ, XPG and CAF-1. In vivo imaging and confocal fluorescence microscopy analysis of cells coexpressing p21 and PCNA fused to green or red fluorescent protein (p21-GFP, RFP-PCNA), showed a rapid relocation of both proteins at microirradiated nuclear spots, although dynamic measurements suggested that p21-GFP was recruited with slower kinetics. An exogenously expressed p21 mutant protein unable to bind PCNA neither colocalised, nor coimmunoprecipitated with PCNA after UV irradiation. In NER-deficient XP-A fibroblasts, p21 relocation was greatly delayed, concomitantly with that of PCNA. These results indicate that early recruitment of p21 protein to DNA-damage sites is a NER-related process dependent on interaction with PCNA, thus suggesting a direct involvement of p21 in DNA repair.

In the past two decades, the design and manufacture of nanostructured materials has been of tremendous interest to the scientific community for their application in the biomedical field. Among the available techniques, layer-by-layer (LBL) assembly has attracted considerable attention as a convenient method to fabricate functional coatings. Nowadays, more than 1000 scientific papers are published every year, tens of patents have been deposited and some commercial products based on LBL technology have become commercially available. LBL presents several advantages, such as (1): a precise control of the coating properties; (2) environmentally friendly, mild conditions and low-cost manufacturing; (3) versatility for coating all available surfaces; (4) obtainment of homogeneous film with controlled thickness; and (5) incorporation and controlled release of biomolecules/drugs. This paper critically reviews the scientific challenge of the last 10 years--functionalizing biomaterials by LBL to obtain appropriate properties for biomedical applications, in particular in tissue engineering (TE). The analysis of the state-of-the-art highlights the current techniques and the innovative materials for scaffold and medical device preparation that are opening the way for the preparation of LBL-functionalized substrates capable of modifying their surface properties for modulating cell interaction to improve substitution, repair or enhancement of tissue function.

Trichothiodystrophy (TTD) is a rare hereditary multisystem disorder associated with defects in nucleotide excision repair (NER) as a consequence of mutations in XPD, XPB or TTDA, three genes that are all related to TFIIH, the multiprotein complex involved in NER and transcription. Here we show that all the mutations found in TTD cases, irrespective of whether they are homozygotes, hemizygotes or compound heterozygotes, cause a substantial and specific reduction (by up to 70%) in the cellular concentration of TFIIH. Intriguingly, the degree of reduction in the level of TFIIH does not correlate with the severity of the pathological phenotype, suggesting that the severity of the clinical features in TTD cannot be related solely to the effects of mutations on the stability of TFIIH. We have also measured TFIIH levels in cells in which different mutations in the XPD gene are associated with clinical symptoms not of TTD but of the highly cancer-prone disorder xeroderma pigmentosum (XP). We have found mild reductions (up to 40%) in TFIIH content in some but not all of these cell strains. We conclude that the severity of the clinical features in TTD patients and the clinical outcome of differentially mutated XPD proteins is likely to depend both on the effects that each mutation has on the stability of TFIIH and on the transcriptional activity of the residual TFIIH complexes.

The apoptotic response and the level of expression of p53 and of three genes transcriptionally activated by p53 (Mdm2, p21 and bax) were investigated in UV-sensitive cells from patients with xeroderma pigmentosum (XP) or Cockayne syndrome (CS). These disorders are due to different genetic defects affecting transcription-coupled repair (TCR) and/or global genome repair (GGR), the nucleotide excision repair subpathways which remove UV-induced lesions from the transcribed strand of active genes or from the rest of the genome, respectively. After 20 J/m2 UV light, normal and GGR-defective XP-C fibroblasts showed rapid increase in p53, late induction of Mdm2 and no evidence of apoptosis even 96 h after irradiation. In contrast, in XP-A (defective in GGR and TCR), CS-A and CS-B (defective only in TCR) fibroblasts, the p53 increase was not followed by Mdm2 induction and the persistence of high levels of p53, due to the lack of its degradation by Mdm2, was associated with the appearance of apoptosis. Besides indicating that the persistence of DNA damage in the transcribed strand of active genes leads to apoptosis, these findings provide the first evidence that the lack of activation of Mdm2 plays a key role in the cascade of events leading to apoptosis. Oncogene (2000).

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. Defects in NER result in three different human disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Two cases with the combined features of XP and CS have been assigned to the XP–D complementation group. Despite their extreme UV sensitivity, these cells appeared to incise their DNA as efficiently as normal cells in response to UV damage. These incisions were, however, uncoupled from the rest of the repair process. Using cell-free extracts, we were unable to detect any incision activity in the neighbourhood of the damage. When irradiated plasmids were introduced into unirradiated XP–D/CS cells, the ectopically introduced damage triggered the induction of breaks in the undamaged genomic DNA. XP–D/CS cells thus have a unique response to sensing UV damage, which results in the introduction of breaks into the DNA at sites distant from the damage. We propose that it is these spurious breaks that are responsible for the extreme UV sensitivity of these cells.

Molecular analysis of p53 and patched (PTCH), two candidate tumor suppressor genes for non-melanocytic skin cancer, was performed in skin tumors from six patients affected by the cancer-prone disease xeroderma pigmentosum (XP). UV-specific p53 mutations were detected at a frequency of 38–50% in all the tumor types analysed, including melanomas. Additional analysis of PTCH mutations in the subset of eight basal call carcinomas (BCC) revealed a very high mutation frequency of this gene (90%) which exceeded that detected in the p53 gene in the same tumors (38%). PTCH mutations were predominantly UV-specific C>T transitions. This mutation pattern is different from that reported in BCC from normal donors where PTCH mutation frequency is 27% and mutations are frequently deletions and insertions. These findings suggest that PTCH mutations represent an earlier event in BCC development than p53 alterations and that the inability of XP patients to repair UV-induced PTCH mutations might significantly contribute to the early and frequent appearance of BCC observed in these patients.

Current tissue engineering (TE) strategies are aimed at the design of biomimetic scaffolds whose structural and mechanical properties are provided by biocompatible and bioresorbable synthetic polymers while the cell response is mediated by the presence of biomimetic natural polymers (proteins and polysaccharides) or short peptide sequences derived from intact extracellular matrix (ECM) proteins. The chapter reviews current approaches for the preparation of bioartificial materials with biomimetic properties for TE applications. Bulk functionalization techniques are based on blending between natural and synthetic polymers or on the synthesis of bioartificial copolymers for the preparation of biomimetic hydrogels. Surface functionalization techniques preserve the biomaterial bulk characteristics and only modify the surface properties at the micro- or nanoscale. Several surface modification techniques are available involving physical adsorption, affinity bonding, and covalent grafting. Besides the traditional approaches using natural polymers or peptides, a new trend is evolving based on scaffold functionalization with decellularized ECM.

A 9-month-old girl presented with psychomotor regression and acquired microcephaly beginning at age 6 months. Clinical evaluation showed axial hypotonia and increased muscle tone in all limbs, brisk tendon reflexes, and absent head control. Peripheral neuropathy was evident on neurophysiologic studies. Brain and spine MRI findings are shown in the figure. Molecular analysis identified homozygous c.162delT mutation in the ERCC8 gene, confirming the diagnosis of Cockayne syndrome (CS).1 Cranial nerve and cauda equina contrast enhancement (CE) might be related to either altered vascular permeability due to perivascular inflammation or to areas of active myelin breakdown. CS should be included in the differential diagnosis of infantile-onset white matter disorders with positive CE.2

Thyroid hormone (TH) plays an important role in the modulation of cardiac function, including contractility and systemic vascular resistance (SVR). 3,5,3'-triiodothyronine (T(3)), the active form of TH, induces the activation of endothelial nitric oxide synthase via PI3K/AKT non-genomic signaling. Hypothyroidism is associated with an increase in SVR and serum low-density lipoproteins (LDL) levels, and accumulation of oxidized LDL (oxLDL) may impair endothelial-dependent vascular relaxation. The aim of this study was to investigate the effects of both native LDL (nLDL) and oxLDL on T(3)-mediated AKT phosphorylation, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP) production in human endothelial cells.Human umbilical vein endothelial cells were exposed to either nLDL or oxLDL for 3 hours and then stimulated with T(3) (10(-7) M) or pretreated with an antioxidant mixture of vitamins E and C for 12 hours before treatment with LDL. An analysis of AKT phosphorylation was performed by Western blot, and NO production was evaluated by using 4,5-diaminofluorescein diacetate. Intracellular production of cGMP was measured by enzymatic immunoassay. LDL oxidation was carried out by incubating LDL with CuSO(4), and α-tocopherol content of LDL was evaluated by high-performance liquid chromatography.OxLDL impaired T(3)-mediated AKT phosphorylation at serine 473 and significantly decreased the production of both NO (oxLDL+T(3) vs. T(3), 9.79±0.5 AU vs. 80.75±2.8 AU, mean±standard deviation, p0.0001) and cGMP. Furthermore, pretreatment with the antioxidant mixture obviated the inhibitory effect of LDL on T(3) action.The results of this study demonstrate that oxLDL may contribute to a blunting of the non-genomic action of T(3) and impair the effect of T(3) on NO and cGMP production in endothelial cells. These data suggest that oxLDL, apart from inducing the atherosclerotic process, may also promote a mechanism of peripheral resistance to T(3,) further amplifying the impact of hypothyroidism on endothelial function by increasing SVR.

Cockayne syndrome (CS) is a genetic disorder characterized by developmental abnormalities and photodermatosis resulting from the lack of transcription-coupled nucleotide excision repair, which is responsible for the removal of photodamage from actively transcribed genes. To date, all identified causative mutations for CS have been in the two known CS-associated genes, ERCC8 (CSA) and ERCC6 (CSB). For the rare combined xeroderma pigmentosum (XP) and CS phenotype, all identified mutations are in three of the XP-associated genes, ERCC3 (XPB), ERCC2 (XPD), and ERCC5 (XPG). In a previous report, we identified several CS cases who did not have mutations in any of these genes. In this paper, we describe three CS individuals deficient in ERCC1 or ERCC4 (XPF). Remarkably, one of these individuals with XP complementation group F (XP-F) had clinical features of three different DNA-repair disorders--CS, XP, and Fanconi anemia (FA). Our results, together with those from Bogliolo et al., who describe XPF alterations resulting in FA alone, indicate a multifunctional role for XPF.

The significant progress made over the last few years on the pathogenesis of Cockayne syndrome (CS) greatly improved our knowledge on several aspects crucial for development and ageing, demonstrating that this disorder, even if rare, represents a valuable tool to clarify key aspects of human health. Primary cells from patients have been instrumental to elucidate the multiple roles of CS proteins and to approach the dissection of the complex interplay between repair and transcription that is central to the CS clinical phenotype. Here we discuss the results of the cellular assays applied for confirmation of the clinical diagnosis as well as the results of genetic and molecular studies in DNA repair defective patients. Furthermore, we provide a general overview of recent in vivo and in vitro studies indicating that both CSA and CSB proteins are involved in distinct aspects of the cellular responses to UV and oxidative stress, transcription and regulation of gene expression, chromatin remodelling, redox balance and cellular bioenergetics. In light of the literature data, we will finally discuss how inactivation of specific functional roles of CS proteins may differentially affect the phenotype, thus explaining the wide range in type and severity of symptoms reported in CS patients.

Nucleotide excision repair (NER) is the most flexible of all known DNA-repair mechanisms, and XPG is a 3′-endonuclease that participates in NER. Mutations in this gene (ERCC5) may result in the human syndrome xeroderma pigmentosum (XP) and, in some cases, in the complex phenotype of Cockayne syndrome (CS). Two Brazilian XP siblings, who were mildly affected, were investigated and classified into the XP-G group. The cells from these patients were highly ultraviolet (UV) sensitive but not sensitive to photosensitized methylene blue, an agent that causes oxidative stress. This phenotype is in contrast to XP-G/CS cells, which are highly sensitive to this oxidative agent. Sequencing revealed a compound heterozygous genotype with two novel missense mutations: c.83C>A (p.Ala28Asp) and c.2904G>C (p.Trp968Cys). The first mutation maps to the catalytic site of the XPG protein, whereas the second may compromise binding to DNA. Functional assays indicated that the mutated alleles were unable to perform the complete repair of UV-irradiated plasmids; however, full correction was observed for oxidatively damaged plasmids. Therefore, the XP phenotype of these patients is caused by novel missense mutations that specifically affect DNA repair for UV- but not oxidative-stress-induced DNA damage, and implications for XP versus XP/CS phenotype are discussed.

Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair, mental and growth retardation, peculiar face, ichthyosis, and in 20% of the reported cases photosensitivity. Cellular photosensitivity due to the same genetic defect present in xeroderma pigmentosum group D (XP-D) has been described in several patients. Nine patients with clinical symptoms diagnostic for TTD have been identified in Italy to date. We report the results of DNA repair investigations performed in cultured fibroblasts from these patients and 8 TTD parents. Survival, DNA repair synthesis and RNA synthesis following UV irradiation were all normal in the 8 TTD heterozygous cell strains. Among the 9 TTD-affected individuals, normal cellular UV sensitivity was observed in the 2 patients without signs of clinical photosensitivity. In contrast, the other 7 TTD cell strains showed a notable reduction in UV-induced DNA repair synthesis (UDS) levels, ranging between 40% and 5–15% of normal values. Complementation analysis indicated that in the repair-deficient TTD cell strains the genetic defect is the same as that present in XP-D cells. The biochemical heterogeneity of the XP-D defect in TTD patients characterized by different degrees of defective UDS results in different patterns of response to the killing effect of UV light in non-proliferating cells.

The inhibitor of cyclin-dependent kinases p21CDKN1A plays a fundamental role in several pathways involved in the DNA damage response, like checkpoint-mediated cell cycle arrest, transcription, apoptosis, and DNA repair. Although p21 protein level is regulated by proteasomal degradation, the relationship of this process with DNA repair pathways is not yet clear. In addition, the role of protein/protein interaction in regulating turnover of p21 protein, is controversial. Here, we show that in human fibroblasts treated with agents inducing lesions repaired through nucleotide excision repair (NER), or base excision repair (BER), p21 degradation was triggered more by the extent, than by the type of DNA damage, or consequent DNA repair pathway. In fact, lowering the amount of DNA damage resulted in an increased stability of p21 protein. Overexpression of p21 was found to obscure degradation, both for p21wt and a p21 mutant unable to bind PCNA (p21PCNA-). However, when expressed to lower levels, turnover of p21 protein after DNA damage was greatly influenced by interaction with PCNA, since p21PCNA- was more efficiently degraded than wild-type protein. Interestingly, a p21 mutant protein unable to localize in the nucleus because of mutations in the NLS region, was not degraded after DNA damage, thus indicating that nuclear localization is necessary for p21 turnover. Removal of p21 was not required for NER activity, since inhibition of p21 degradation by caffeine did not affect the UV-induced recruitment of repair proteins, such as PCNA and DNA polymerase delta, nor significantly influence DNA repair synthesis, as determined by autoradiography. These results indicate that degradation of p21 is not dependent on a particular DNA repair pathway, and is not required for efficient DNA repair.

Trichothiodystrophy (TTD) is a rare, autosomal recessive neurodevelopmental disorder most commonly caused by mutations in ERCC2 (XPD), a gene that encodes a subunit of the transcription/repair factor IIH (TFIIH). Here, we describe two TTD cases in which detailed biochemical and molecular investigations offered a clue to explain their moderately affected phenotype. Patient TTD22PV showed new mutated XPD alleles: one contains a nonsense mutation (c.1984C>T) encoding a nonfunctional truncated product (p.Gln662X) whereas the second carries a genomic deletion (c.2191-18_c.2213del) that affects the splicing of intron 22 and generates multiple out-of-frame transcripts from codon 731. XPD mRNA from the second allele corresponds to 20% of the total. The predicted proteins, which are longer than normal, affect the cellular repair activity but only partially interfere with TFIIH stability, suggesting that the observed changes in the C-ter region of XPD cause minor structural changes that do not drastically compromise the transcriptional activity of TFIIH. Patient TTD24PV was compound heterozygous for a typical TTD allele (c.2164C>T, p.Arg722Trp) and for a new XPD allele with a mutation that partially affects intron 10 splicing, resulting in both mutated and normal XPD transcripts (that together represent 15% of the total XPD mRNA). Compared to the previously described TTD compound heterozygotes for the Arg722Trp change, Patient TTD24PV's cells show similar level of TFIIH but increased repair activity, suggesting that even low amounts of normal XPD subunits are able to partially rescue the functionality of TFIIH complexes.

Laboratory diagnosis for DNA repair diseases has been performed in western Europe from the early seventies for xeroderma pigmentosum (XP) and from the mid-eighties for Cockayne syndrome (CS) and trichothiodystrophy (TTD). The combined data from the DNA repair diagnostic centres in France, (West) Germany, Italy, the Netherlands and the United Kingdom have been investigated for three groups of diseases: XP (including XP-variant), CS (including XP/CS complex) and TTD. Incidences in western Europe were for the first time established at 2.3 per million livebirths for XP, 2.7 per million for CS and 1.2 per million for TTD. As immigrant populations were disproportionately represented in the patients' groups, incidences were also established for the autochthonic western European population at: 0.9 per million for XP, 1.8 per million for CS and 1.1 per million for TTD. Perhaps contrary to general conceptions, compared to XP the incidence of CS appears to be somewhat higher and the incidence of TTD to be quite similar in the native West-European population. (c) 2008 Elsevier B.V. All rights reserved.

Eukaryotic cells respond to a variety of DNA insults by triggering a common signal transduction cascade, known as checkpoint response, which temporarily halts cell-cycle progression. Although the main players involved in the cascade have been identified, there is still uncertainty about the nature of the structures that activate these surveillance mechanisms. To understand the role of nucleotide excision repair (NER) in checkpoint activation, we analyzed the UV-induced phosphorylation of the key checkpoint proteins Chk1 and p53, in primary fibroblasts from patients with xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy (TTD), or UV light-sensitive syndrome. These disorders are due to defects in transcription-coupled NER (TC-NER) and/or global genome NER (GG-NER), the NER subpathways repairing the transcribed strand of active genes or the rest of the genome, respectively. We show here that in G 0 /G 1 and G 2 /M phases of the cell cycle, triggering of the DNA damage cascade requires recognition and processing of the lesions by the GG-NER. Loss of TC-NER does not affect checkpoint activation. Mutations in XPD , XPB , and in TTDA , encoding subunits of the TFIIH complex, involved in both transcription and NER, impair checkpoint triggering. The only exception is represented by mutations in XPD , resulting in combined features of XP and CS (XP/CS) that lead to activation of the checkpoint cascade after UV radiation. Inhibition of RNA polymerase II transcription significantly reduces the phosphorylation of key checkpoint factors in XP/CS fibroblasts on exposure to UV damage.

International audience; How subunits of the transcription/repair factor TFIIH cooperate to allow for the removal of DNA lesions or for the transcription of genes is crucial to understand the functioning of this complex. Here, we reveal that p8/TTD-A, the tenth subunit of TFIIH, has a critical role in DNA repair where it triggers DNA opening by stimulating XPB ATPase activity together with the damage recognition factor XPC-hHR23B. Fluorescent antibody labeling shows that such opening is needed for the recruitment of XPA to the site of the damage. By contrast, p8 is dispensable for RNA synthesis and doesn't interfere with the transcriptional function of CAK, although both interact with the XPD subunit. Interestingly, p8 overexpression in TTD-XPD cells counteracts the detrimental effect of XPD mutations by restoring the cellular TFIIH concentration. These findings resolve the primary functions of p8 and unveil how TFIIH components specifically direct the complex toward repair or transcription.

Xeroderma pigmentosum (XP) is a skin cancer-prone autosomal recessive disease characterized by inability to repair UV-induced DNA damage. The major form of XP is defective in nucleotide excision repair (NER) and comprises seven complementation groups (A-G). The genes defective in all groups have been identified unambiguously with the exception of group E. The cells of some XP-E patients are deficient in a protein complex (consisting of two subunits: p127/DDBI and p48/DDB2) which binds to UV-damaged DNA (UV-DDB) and is specifically involved in the removal of photoproducts from the non-transcribed regions of the genome. However, other XP-E patients have been reported not to lack UV-damaged DNA binding activity (DDB(+)). Here we describe several genetically unrelated XP-E patients, not previously analyzed in depth, each carrying two mutated alleles for DDB2, causing either a single amino acid change or a protein truncation or internal deletion. These defects result in a severe decrease of detectable p48 protein, abolish interaction with the p127 subunit, and produce a deficiency in UV-DDB binding activity (DDB(-)). The role of p48 in the repair defect of these patients was demonstrated in vivo and in vitro. Investigation of four DDB(+) cell strains from patients previously assigned to XP-E, allowed us to reclassify all of them into other groups and to show that they do not share the molecular and biochemical features typical for XP-E. Besides confirming that the true XP-E phenotype is DDB(-), resulting from defects in a single gene, DDB2, our results identify the functional domains of the corresponding p48 protein.

The MMS19 gene of the yeast Saccharomyces cerevisiae encodes a polypeptide of unknown function which is required for both nucleotide excision repair (NER) and RNA polymerase II (RNAP II) transcription. Here we report the molecular cloning of human and mouse orthologs of the yeast MMS19 gene. Both human and Drosophila MMS19 cDNAs correct thermosensitive growth and sensitivity to killing by UV radiation in a yeast mutant deleted for the MMS19 gene, indicating functional conservation between the yeast and mammalian gene products. Alignment of the translated sequences of MMS19 from multiple eukaryotes, including mouse and human, revealed the presence of several conserved regions, including a HEAT repeat domain near the C-terminus. The presence of HEAT repeats, coupled with functional complementation of yeast mutant phenotypes by the orthologous protein from higher eukaryotes, suggests a role of Mms19 protein in the assembly of a multiprotein complex(es) required for NER and RNAP II transcription. Both the mouse and human genes are ubiquitously expressed as multiple transcripts, some of which appear to derive from alternative splicing. The ratio of different transcripts varies in several different tissue types.

International audience; Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are two hereditary disorders in which photosensitivity is associated with distinct clinical and cellular phenotypes and results from genetically different defects. We have identified the primary molecular alteration in two patients in whom clinical manifestations strongly reminiscent of a severe form of XP were unexpectedly associated with the CS cellular phenotype and with a defect in the CSB gene. Sequencing of the CSB -coding region in both cDNA and genomic DNA showed that these patients had identical alterations to those in a patient with the clinical features of the classical form of CS. These data, together with fluorescence in situ hybridization analysis, demonstrated that the two siblings with XP as well as the CS patient were homozygous for the same CSB mutated allele, containing a silent C2830T change and a nonsense mutation C2282T converting Arg735 to a stop codon. The finding that the same inactivating mutation underlies different pathological phenotypes indicates that there is no simple correlation between the molecular defect and the clinical features. Therefore, alterations in the CSB gene give rise to the same repair defect at the cellular level but other genetic and/or environmental factors determine the pathological phenotype.