Tunable synthesis of Prussian Blue in exponentially growing polyelectrolyte multilayer films.

Polyelectrolyte multilayer (PEM) films have become very popular for surface functionalization and the design of functional architectures such as hollow polyelectrolyte capsules. It is known that properties such as permeability to small ionic solutes are strongly dependent on the buildup regime of the PEM films. This permeability can be modified by tuning the ionization degree of the polycations or polyanions, provided the film is made from weak polyelectrolytes. In most previous investigations, this was achieved by playing on the solution pH either during the film buildup or by a postbuildup pH modification. Herein we investigate the functionalization of poly(allylamine hydrochloride)/poly(glutamic acid) (PAH/PGA) multilayers by ferrocyanide and Prussian Blue (PB). We demonstrate that dynamic exchange processes between the film and polyelectrolyte solutions containing one of the component polyelectrolyte allow one to modify its Donnan potential and, as a consequence, the amount of ferrocyanide anions able to be retained in the PAH/PGA film. This ability of the film to be a tunable reservoir of ferrocyanide anions is then used to produce a composite film containing PB particles obtained by a single precipitation reaction with a solution containing Fe(3+) cations in contact with the film. The presence of PB in the PEM films then provides magnetic as well as electrochemical properties to the whole architecture.journal article2009 Dec 15importe

Effect of Combination L-Citrulline and Metformin Treatment on Motor Function in Patients With Duchenne Muscular Dystrophy: A Randomized Clinical Trial

Importance:Nitric oxide precursors, such as the amino acid l-arginine and the biguanide antidiabetic drug metformin, have been associated with metabolism and muscle function in patients with Duchenne muscular dystrophy (DMD). The treatment of DMD remains an unmet medical need.Objective:To evaluate the benefits and harms of a combination of l-citrulline and metformin treatment among patients with DMD.Design, setting, and participants:A single-center randomized double-blind placebo-controlled parallel-group clinical trial was conducted between December 12, 2013, and March 30, 2016, at the University Children's Hospital Basel in Switzerland. A total of 47 ambulant male patients aged 6.5 to 10 years with genetically confirmed DMD were recruited locally and from the patient registries of Switzerland, Germany, Austria, and France. Data were analyzed from April 6, 2016, to September 5, 2019.Interventions:Patients in the treatment group received 2500 mg of l-citrulline and 250 mg of metformin (combination therapy) 3 times a day for 26 weeks compared with patients in the control group, who received placebo.Main outcomes and measures:The primary end point was the change in transfer and standing posture, as assessed by the first dimension of the Motor Function Measure, version 32, from baseline to week 26. Secondary end points included assessments of timed function, quantitative muscle force, biomarkers for muscle necrosis, and adverse events. The 2 prespecified subgroups comprised patients who were able to walk 350 m or more in 6 minutes (stable subgroup) and patients who were not able to walk 350 m in 6 minutes (unstable subgroup) at baseline.Results:Among 49 ambulant male children with DMD who were screened for eligibility, 47 patients with a mean (SD) age of 8.2 (1.1) years were randomized to a treatment group receiving combination therapy (n = 23) or a control group receiving placebo (n = 24), and 45 patients completed the study. No significant differences between groups were found in the results of timed function and muscle force tests for overall, proximal and axial, and distal motor function. Among patients receiving combination therapy, the Motor Function Measure first dimension subscore decrease was 5.5% greater than that of patients receiving placebo (95% CI, -1.0% to 12.1%; P = .09). The administration of combination therapy had significantly favorable effects on the first dimension subscore decrease among the 29 patients in the stable subgroup (6.7%; 95% CI, 0.9%-12.6%; P = .03) but not among the 15 patients in the unstable subgroup (3.9%; 95% CI, -13.2% to 20.9%; P = .63). Overall, the treatment was well tolerated with only mild adverse effects.Conclusions and relevance:Treatment with combination therapy was not associated with an overall reduction in motor function decline among ambulant patients with DMD; however, a reduction in motor function decline was observed among the stable subgroup of patients treated with combination therapy. The statistically nonsignificant difference of distal motor function in favor of combination therapy and the reduced degeneration of muscle tissue appear to support the treatment concept, but the study may have lacked sufficient statistical power. Further research exploring this treatment option with a greater number of patients is warranted

Coreceptor affinity for MHC defines peptide specificity requirements for TCR interaction with coagonist peptide–MHC

Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide–MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4–MHCII interaction is generally weaker than CD8–MHCI.National Institutes of Health (U.S.). Pioneer Awar

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

An Abundant Evolutionarily Conserved CSB-PiggyBac Fusion Protein Expressed in Cockayne Syndrome

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3′ terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1–5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein

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

Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing

BACKGROUND: Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS). METHODS: Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies). RESULTS: We identified causative mutations in 17 out of the 40 patients (43 %). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes. CONCLUSIONS: Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects

Age-Related Neuronal Degeneration: Complementary Roles of Nucleotide Excision Repair and Transcription-Coupled Repair in Preventing Neuropathology

Neuronal degeneration is a hallmark of many DNA repair syndromes. Yet, how DNA damage causes neuronal degeneration and whether defects in different repair systems affect the brain differently is largely unknown. Here, we performed a systematic detailed analysis of neurodegenerative changes in mouse models deficient in nucleotide excision repair (NER) and transcription-coupled repair (TCR), two partially overlapping DNA repair systems that remove helix-distorting and transcription-blocking lesions, respectively, and that are associated with the UV-sensitive syndromes xeroderma pigmentosum (XP) and Cockayne syndrome (CS). TCR–deficient Csa−/− and Csb−/− CS mice showed activated microglia cells surrounding oligodendrocytes in regions with myelinated axons throughout the nervous system. This white matter microglia activation was not observed in NER–deficient Xpa−/− and Xpc−/− XP mice, but also occurred in XpdXPCS mice carrying a point mutation (G602D) in the Xpd gene that is associated with a combined XPCS disorder and causes a partial NER and TCR defect. The white matter abnormalities in TCR–deficient mice are compatible with focal dysmyelination in CS patients. Both TCR–deficient and NER–deficient mice showed no evidence for neuronal degeneration apart from p53 activation in sporadic (Csa−/−, Csb−/−) or highly sporadic (Xpa−/−, Xpc−/−) neurons and astrocytes. To examine to what extent overlap occurs between both repair systems, we generated TCR–deficient mice with selective inactivation of NER in postnatal neurons. These mice develop dramatic age-related cumulative neuronal loss indicating DNA damage substrate overlap and synergism between TCR and NER pathways in neurons, and they uncover the occurrence of spontaneous DNA injury that may trigger neuronal degeneration. We propose that, while Csa−/− and Csb−/− TCR–deficient mice represent powerful animal models to study the mechanisms underlying myelin abnormalities in CS, neuron-specific inactivation of NER in TCR–deficient mice represents a valuable model for the role of NER in neuronal maintenance and survival