Do postoperative outcomes vary between midshaft clavicle fractures of 2, 3, or more than 3 fragments?

OBJECTIVE: This study aims to assess differences in clinical and surgical outcomes associated with the surgical treatment of midshaft clavicle fractures of different complexities based on fragment number. Additionally, the investigation seeks to present the outcomes of a series of patients who underwent surgery at our institution. MATERIALS AND METHODS: A retrospective analysis was conducted on the medical records of patients aged over 18 who underwent midshaft clavicle fracture surgery at our center from November 2009 to May 2021. Patients were categorized based on the number of fracture fragments into groups of two, three, or more than three fragments. Consolidation, implant removal, complications, surgical duration, and functional outcomes (assessed through VAS, ASES, and Constant-Murley scale) were evaluated for each specific group and for the overall cohort. RESULTS: In total, 260 patients were analyzed. There were no significant differences in any of the parameters between the three groups except for surgical time, which was shorter in simple fractures than in those with more than three fragments (68.2 min vs. 75.3 min; p = 0.01). Pseudoarthrosis rate was 2.69%, implant removal rate was 9.61%, and 4.23% of patients presented with complications other than the previous ones. Functional results were excellent, with averages of 97.3 (72.7-100) for the ASES score, 97.5 (75-100) for the Constant score, and 0.6 (0-8) on the VAS. CONCLUSION: According to our results, there were no differences in postoperative results between simple and multifragmentary midshaft clavicle fractures. Patients across all groups reported satisfactory results.

Adherence and impact of an oral nutritional supplement enriched in leucine, EVOO, EPA and DHA, and beta-glucans on the coverage of energy and protein requirements in patients with cancer and malnutrition: Alisenoc study.

OBJECTIVE: The aim of this study was to evaluate the impact of an enhanced ONS (enriched in EPA, DHA, leucine, and beta-glucans) on the dietary intake of cancer patients. METHODS: A randomized, double-blind, parallel, controlled, and multicenter clinical trial was conducted in patients with cancer and malnutrition. The trial compared prescribed dietary advice and two packs per day, for 8 weeks, of a hypercaloric (400 kcal/pack) and hyperproteic ONS (20 g/pack) with fiber and specific ingredients (leucine, EPA and DHA, and beta-glucans) (enhanced-ONS) versus an isocaloric and isoproteic formula (standard-ONS) without specific ingredients. Food intake was assessed with a 3-day dietary survey, and adherence to the supplement with a patient self-completed diary. RESULTS: Thirty-seven patients completed the intervention period. The combined intervention of dietary advice and ONS managed to increase the energy intake of the overall cohort by 792.55 (378.57) kcal/day, protein by 40.72 (19.56) g/day. Increases in energy and nutrient intakes were observed in both groups, both in dietary intake and associated exclusively with the supplement. The group that received the enhanced-ONS ingested a greater volume of product when there was a greater severity of malnutrition; a tumor location in the head, neck, upper digestive area, liver, or pancreas; more advanced stages of the tumor; or the receipt of more than one antineoplastic treatment. CONCLUSION: The use of an enhanced-ONS helps meet the nutritional requirements of cancer patients, especially those who have a more compromised clinical condition, with high adherence, good tolerance, and acceptance.

Retinal shadows produced by myopia control spectacles.

PURPOSE: To analyse ocular coherence tomography (OCT) images of the retinal shadows caused by defocus and diffusion optics spectacles. METHODS: One eye was fitted successively with the Hoya Defocus Incorporated Multiple Segments (DIMS) spectacle lens, two variations of the +3.50 D peripheral add spectacle (DEFOCUS) and the low-contrast dot lens (Diffusion Optics Multiple Segments, DOMS); each at a vertex distance of 12 mm. Simultaneously, a retinal image of the macular region with central fixation was obtained using infrared OCT. The corneal power and intraocular distances were determined using an optical biometer. RESULTS: The retinal images for the DIMS and DOMS lenses showed patterns of obvious retinal shadows in the periphery, while the central 10-11° remained clear. The DEFOCUS lens produced a darkened peripheral area. Dividing the size of the retinal pattern, measured with the calliper of the OCT software, by the actual size on the spectacle lens gave a magnification of -0.57 times. This is consistent with the incoming OCT beam being imaged to a position approximately 31 mm beyond the front of the eye. [Correction added on 26 October 2023 after first online publication: The preceding paragraph was corrected.] CONCLUSION: With device-specific correction, retinal OCT images can help visualise the regions affected by the defocus or lowered contrast induced by myopia control spectacles. This is of potential value for improving myopia therapies.

Identifying the needs of older people living with HIV (≥ 50 years old) from multiple centres over the world: a descriptive analysis.

BACKGROUND: Older People Living with HIV (OPWH) combine both aging and HIV-infection features, resulting in ageism, stigma, social isolation, and low quality of life. This context brings up new challenges for healthcare professionals, who now must aid patients with a significant comorbidity burden and polypharmacy treatments. OPWH opinion on their health management is hardly ever considered as a variable to study, though it would help to understand their needs on dissimilar settings. METHODS: We performed a cross-sectional, comparative study including patients living with HIV aged ≥50 years old from multiple centers worldwide and gave them a survey addressing their perception on overall health issues, psychological problems, social activities, geriatric conditions, and opinions on healthcare. Data was analyzed through Chisquared tests sorting by geographical regions, age groups, or both. RESULTS: We organized 680 participants data by location (Center and South America [CSA], Western Europe [WE], Africa, Eastern Europe and Israel [EEI]) and by age groups (50- 55, 56-65, 66-75, >75). In EEI, HIV serostatus socializing and reaching undetectable viral load were the main problems. CSA participants are the least satisfied regarding their healthcare, and a great part of them are not retired. Africans show the best health perception, have financial problems, and fancy their HIV doctors. WE is the most developed region studied and their participants report the best scores. Moreover, older age groups tend to live alone, have a lower perception of psychological problems, and reduced social life. CONCLUSIONS: Patients' opinions outline region- and age-specific unmet needs. In EEI, socializing HIV and reaching undetectable viral load were the main concerns. CSA low satisfaction outcomes might reflect high expectations or profound inequities in the region. African participants results mirror a system where general health is hard to achieve, but HIV clinics are much more appealing to them. WE is the most satisfied region about their healthcare. In this context, age-specific information, education and counseling programs (i.e. Patient Reported Outcomes, Patient Centered Care, multidisciplinary teams) are needed to promote physical and mental health among older adults living with HIV/AIDS. This is crucial for improving health-related quality of life and patient's satisfaction.

Injerto de cresta ilíaca como cirugía de revisión de Latarjet fallida por inestabilidad glenohumeral en deportistas / Iliac crest graft for previous failed Latarjet surgery with glenohumeral instability in athletes

Introducción: El objetivo de este estudio es reportar los resultados funcionales, el retorno al deporte, la tasa de consolidación y las complicaciones en deportistas jóvenes con una cirugía de Latarjet previa fallida, tratados con injerto autólogo de cresta ilíaca.Materiales y métodos: entre el 2017 y el 2020, se operaron en nuestra institución doce pacientes con inestabilidad glenohumeral recurrente luego de una estabilización previa fallida con cirugía de Latarjet, con injerto autólogo de cresta ilíaca como cirugía de revisión. La evaluación funcional se realizó con el score de Rowe, la escala visual análoga (EVA) y el score de ASOSS. Evaluamos el porcentaje de retorno al deporte, el nivel alcanzado y el tiempo que tardaron los deportistas en volver a competir. La consolidación ósea y la posición adecuada del injerto se analizó en todos los casos con radiografías de hombro frente y perfil y tomografía axial computada con reconstrucción 3D. Se registraron las complicaciones y las revisiones.Resultados: el seguimiento promedio fue de 42.6 meses (rango 24 a 92 meses). El score de Rowe, la EVA y el ASOSS mejoraron significativamente luego de la cirugía (p <0.1). Nueve pacientes retornaron al deporte, ocho de ellos al mismo nivel. El injerto óseo consolidó en todos los pacientes. No hubo recurrencias. No se reportaron complicaciones.Conclusión: el injerto autólogo de cresta ilíaca es una opción válida para el tratamiento de deportistas con inestabilidad glenohumeral recurrente luego de una estabilización previa fallida con cirugía de Latarjet. Nivel de Evidencia: IV

Introduction: The purpose of this study was to report the functional results, return to sport, consolidation rate and complications in young athletes with a previous failed Latarjet surgery, treated with an autologous iliac crest graft.Materials and methods: between 2017 and 2020, twelve patients with recurrent glenohumeral instability were operated on at our institution after previous failed stabilization with Latarjet surgery with autologous iliac crest graft as revision surgery. Functional evaluation was performed with the Rowe score, the VAS, and the ASOSS score. We evaluated the percentage of return to sport, the level reached, and the time it took the athletes to compete again. Bone consolidation and the adequate position of the graft were evaluated in all cases with front and profile X-rays of the shoulder and computed tomography with 3D reconstruction. Complications and revisions were recorded.Results: the average follow-up was 42.6 months (range 24 to 92 months). The Rowe score, visual analog scale, and ASOSS were significantly improved after surgery (p <0.1). Nine patients returned to sport, eight of them at the same level. The bone graft consolidated in all patients. There were no recurrences. No complications were reported.Conclusion: autologous iliac crest grafting is a valid option for the treatment of athletes with recurrent glenohumeral instability after previous failed stabilization with a Latarjet procedure. Level of Evidence: IV

Technologies for the nitrogen oxides reduction from flue gas: A review.

The required energy of the global industry is mostly generated from fossil fuel sources, such as natural gas, gasoline, diesel, oil, and coal. Nitrogen oxides are one of the main air pollutants that are produced from the combustion of fossil fuels in stationary and mobile sources. Development of new technologies to decrease the NOx emission from exhaust gases is essential due to the harmful effect of NOx on the environment and human health. Compared with pre-combustion and combustion methods (with <50% NOx removal efficiency), the post-combustion methods with higher efficiency (above 80%) have attracted more attention in NOx elimination. This review describes the currently used technologies of NOx abatement. Different available post-combustion methods of NOx removal, including selective catalytic reduction (using different types of reducing reagents, including ammonia, hydrogen, hydrocarbons, and carbon monoxide), selective noncatalytic reduction, wet scrubbing, adsorption, electron beam, nonthermal plasma, and electrochemical reduction of NOx, are discussed.

Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains.

Cell motility and migration requires the reorganization of the cortical cytoskeleton at the leading edge of cells and extracellular Ca2+ entry is essential for this reorganization. However the molecular nature of the regulators of this pathway is unknown. This work contributes to understanding the role of STIM1 and ORAI1 in the promotion of membrane ruffling by showing that phospho-STIM1 localizes at the leading edge of cells, and that both phospho-STIM1 and ORAI1 co-localize with cortactin (CTTN), a regulator of the cytoskeleton at membrane ruffling areas. STIM1-KO and ORAI1-KO cell lines were generated by CRISPR/Cas9 genome editing in U2OS cells. In both cases, KO cells presented a notable reduction of store-operated Ca2+ entry (SOCE) that was rescued by expression of STIM1-mCherry and ORAI1-mCherry. These results demonstrated that SOCE regulates membrane ruffling at the leading edge of cells. Moreover, endogenous ORAI1 and overexpressed ORAI1-GFP co-immunoprecipitated with endogenous CTTN. This latter result, in addition to the KO cells' phenotype, the preservation of ORAI1-CTTN co-localization during ruffling, and the inhibition of membrane ruffling by the Ca2+-channel inhibitor SKF96365, further supports a functional link between SOCE and membrane ruffling.

IL-1α is a DNA damage sensor linking genotoxic stress signaling to sterile inflammation and innate immunity.

Environmental signals can be translated into chromatin changes, which alter gene expression. Here we report a novel concept that cells can signal chromatin damage from the nucleus back to the surrounding tissue through the cytokine interleukin-1alpha (IL-1α). Thus, in addition to its role as a danger signal, which occurs when the cytokine is passively released by cell necrosis, IL-1α could directly sense DNA damage and act as signal for genotoxic stress without loss of cell integrity. Here we demonstrate localization of the cytokine to DNA-damage sites and its subsequent secretion. Interestingly, its nucleo-cytosolic shuttling after DNA damage sensing is regulated by histone deacetylases (HDAC) and IL-1α acetylation. To demonstrate the physiological significance of this newly discovered mechanism, we used IL-1α knockout mice and show that IL-1α signaling after UV skin irradiation and DNA damage is important for triggering a sterile inflammatory cascade in vivo that contributes to efficient tissue repair and wound healing.

Phospho-STIM1 is a downstream effector that mediates the signaling triggered by IGF-1 in HEK293 cells.

STIM1 is a Ca(2+) sensor of the endoplasmic reticulum (ER) that triggers the activation of plasma membrane Ca(2+) channels upon depletion of Ca(2+) levels within the ER. During thapsigargin-triggered Ca(2+) store depletion, ERK1/2 phosphorylates STIM1 at Ser575, Ser608, and Ser621. This phosphorylation plays a role in the regulation of STIM1 dissociation from the microtubule plus-end binding protein EB1, an essential step for STIM1 activation by thapsigargin. However, little is known regarding the physiological role of this phosphorylation. Because IGF-1 triggers the activation of the RAF-MEK-ERK and the phosphoinositide pathways, the role of STIM1 phosphorylation in IGF-1 stimulation was studied. There was found to be phosphorylation of ERK1/2 in both the presence and the absence of extracellular Ca(2+), demonstrating that Ca(2+) influx is not essential for ERK1/2 activation. In parallel, IGF-1 triggered STIM1 phosphorylation at the aforementioned sites, an effect that was blocked by PD0325901, a MEK1/2 inhibitor used to block ERK1/2 activation. Also, STIM1-GFP was found in clusters upon IGF-1 stimulation, and STIM1-S575A/S608A/S621A-GFP strongly reduced this multimerization. Interestingly, phospho-STIM1 was mainly found in clusters when cells were treated with IGF-1, and IGF-1 triggered the dissociation of STIM1 from EB1, similarly to what has been observed for thapsigargin, suggesting that STIM1 mediates the IGF-1 signaling pathway. A study of IGF-1-stimulated NFAT translocation was therefore performed, finding that STIM1-S575A/S608A/S621A blocked this translocation, as did the fusion protein STIM1-EB1, confirming that both STIM1 phosphorylation and STIM1-EB1 dissociation are required for IGF-1-triggered Ca(2+)-dependent signaling, and demonstrating that STIM1 phosphorylation plays a role as a downstream effector of the RAF-MEK-ERK pathway and an upstream activator of Ca(2+) entry.

STIM1 phosphorylation triggered by epidermal growth factor mediates cell migration.

STIM1 is a key regulator of store-operated calcium entry (SOCE), and therefore a mediator of Ca²âº entry-dependent cellular events. Phosphorylation of STIM1 at ERK1/2 target sites has been described as enhancing STIM1 activation during intracellular Ca²âº emptying triggered by the inhibition of the sarco(endo)plasmic Ca²âº -ATPase with thapsigargin. However, no physiological function is known for this specific phosphorylation. The present study examined the role of STIM1 phosphorylation in cell signaling triggered by EGF. Using a human endometrial adenocarcinoma cell line (Ishikawa cells) EGF or H-Ras(G12V), an active mutant of H-Ras, was found to trigger STIM1 phosphorylation at residues Ser575, Ser608, and Ser621, and this process was sensitive to PD0325901, an inhibitor of ERK1/2. Both, ERK1/2 activation and STIM1 phosphorylation took place in the absence of extracellular Ca²âº, indicating that both events are upstream steps for Ca²âºentry activation. Also, EGF triggered the dissociation of STIM1 from EB1 (a regulator of microtubule plus-ends) in a manner similar to that reported for the activation of STIM1 by thapsigargin. Migration of the Ishikawa cells was impaired when STIM1 phosphorylation was targeted by Ser-to-Ala substitution mutation of ERK1/2 target sites. This effect was also observed with the Ca²âº channel blocker SKF96365. Phosphomimetic mutation of STIM1 restored the migration to levels similar to that found for STIM1-wild type. Finally, the increased vimentin expression and relocalization of E-cadherin triggered by EGF were largely inhibited by targeting STIM1 phosphorylation, while STIM1-S575E/S608E/S621E normalized the profiles of these two EMT markers.

Thymic uptake after high-dose I-131 treatment in patients with differentiated thyroid carcinoma: a brief review of possible causes and management.

PURPOSE: To report five cases of patients diagnosed with differentiated thyroid carcinoma (DTC) with uptake in the thymic area after high-dose treatment with I-131 and to evaluate the potential causes and therapeutic management. METHODS: Five cases of young female patients with a mean age of 36.6 years (24-43) who had been treated with a mean dose of 106 mCi of I-131 (100-150 mCi) showing tracer uptake in the thymic area are reported. An I-131 whole-body scan (131I-WBS) was performed 7 days after therapeutic dose administration to each patient. Anterior and posterior planar images, followed by SPECT/CT of the head, neck and superior mediastinum were acquired in all patients. Thyroglobulin levels were measured with and without hormone replacement therapy in all cases. Samples taken from the superior mediastinum were sent to pathology for analysis, which confirmed the presence of thymic tissue. RESULTS: Two patients underwent elective total thymectomy due to the gross characteristics of the gland, local 131-I uptake, and high thyroglobulin levels. The remaining three patients had already undergone thymectomy as part of neck dissection during initial surgery, and no further invasive interventions were therefore performed. Pathological examination revealed no metastases in these five patients. CONCLUSIONS: Thymus visualization in young patients after administration of therapeutic doses of I-131 seems to be a more common finding than usually thought. Absence of metastasis in the thymus despite high thyroglobulin levels was confirmed in all cases. Based on these results, we suggest that a more expectant and less aggressive therapeutic approach could be used. We also suggest that I-131 therapy for DTC should be considered in classification of the potential causes of true thymic hyperplasia in the subgroup of patients recovering from a stressor.

Bone signaling in middle ear development: a genome-wide differential expression analysis.

Common middle ear diseases may affect bone behavior in the middle ear air cell system. To understand this pathologic pneumatization, the normal development of bone in the middle ear should be investigated. The objective of this study was to analyze gene expression of bone-related signaling factors and gene sets in the developing middle ear. Microarray technology was used to identify bone-related genes and gene sets, which were differentially expressed between the lining tissue of adult (quiescent) bulla and young (resorbing/forming) bulla. Data were analyzed using tools of bioinformatics and expression levels of selected genes were validated using quantitative polymerase chain reaction. The candidate gene products were compared with previously published data on middle ear bone metabolism. No differentially expressed genes were found on the outer surface of bulla. On the inner lining a total of 260 genes were identified of which 22 genes were involved in bone metabolism. Gene set analysis revealed five enriched bone-related gene sets. The identified differentially expressed bone-related mRNAs and gene sets are of potential significance in the normally developing bulla. These factors and gene sets may also play important roles during pathologic pneumatization of the middle ear air cell system in common middle ear diseases. In addition, this study suggests that the control of growth rate and wall thickness from resorptive as well as formative signals all originate from the inner lining cells of the bulla wall.

SET7/9-dependent methylation of ARTD1 at K508 stimulates poly-ADP-ribose formation after oxidative stress.

ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein-protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo. ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo. Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.

Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling.

Our understanding of the mechanisms governing the response to DNA damage in higher eucaryotes crucially depends on our ability to dissect the temporal and spatial organization of the cellular machinery responsible for maintaining genomic integrity. To achieve this goal, we need experimental tools to inflict DNA lesions with high spatial precision at pre-defined locations, and to visualize the ensuing reactions with adequate temporal resolution. Near-infrared femtosecond laser pulses focused through high-aperture objective lenses of advanced scanning microscopes offer the advantage of inducing DNA damage in a 3D-confined volume of subnuclear dimensions. This high spatial resolution results from the highly non-linear nature of the excitation process. Here we review recent progress based on the increasing availability of widely tunable and user-friendly technology of ultrafast lasers in the near infrared. We present a critical evaluation of this approach for DNA microdamage as compared to the currently prevalent use of UV or VIS laser irradiation, the latter in combination with photosensitizers. Current and future applications in the field of DNA repair and DNA-damage dependent chromatin dynamics are outlined. Finally, we discuss the requirement for proper simulation and quantitative modeling. We focus in particular on approaches to measure the effect of DNA damage on the mobility of nuclear proteins and consider the pros and cons of frequently used analysis models for FRAP and photoactivation and their applicability to non-linear photoperturbation experiments.

Calcium-binding capacity of centrin2 is required for linear POC5 assembly but not for nucleotide excision repair.

Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC), stabilising it, and its presence slightly increases nucleotide excision repair (NER) activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.

Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1.

STIM1 (stromal interaction molecule 1) is a key regulator of store-operated calcium entry (SOCE). Upon depletion of Ca(2+) concentration within the endoplasmic reticulum (ER), STIM1 relocalizes at ER-plasma membrane junctions, activating store-operated calcium channels (SOCs). Although the molecular details for STIM1-SOC binding is known, the regulation of SOCE remains largely unknown. A detailed list of phosphorylated residues within the STIM1 sequence has been reported. However, the molecular pathways controlling this phosphorylation and its function are still under study. Using phosphospecific antibodies, we demonstrate that ERK1/2 mediates STIM1 phosphorylation at Ser575, Ser608 and Ser621 during Ca(2+) store depletion, and that Ca(2+) entry and store refilling restore phosphorylation to basal levels. This phosphorylation occurs in parallel to the dissociation from end-binding protein 1 (EB1), a regulator of growing microtubule ends. Although Ser to Ala mutation of residues 575, 608 and 621 showed a constitutive binding to EB1 even after Ca(2+) store depletion, Ser to Glu mutation of these residues (to mimic the phosphorylation profile attained after store depletion) triggered full dissociation from EB1. Given that wild-type STIM1 and STIM1(S575E/S608E/S621E) activate SOCE similarly, a model is proposed to explain how ERK1/2-mediated phosphorylation of STIM1 regulates SOCE. This regulation is based on the phosphorylation of STIM1 to trigger dissociation from EB1 during Ca(2+) store depletion, an event that is fully reversed by Ca(2+) entry and store refilling.

Imaging of the DNA damage-induced dynamics of nuclear proteins via nonlinear photoperturbation.

Understanding the cellular response to DNA strand breaks is crucial to decipher the mechanisms maintaining the integrity of our genome. We present a novel method to visualize how the mobility of nuclear proteins changes in response to localized DNA damage. DNA strand breaks are induced via nonlinear excitation with femtosecond laser pulses at λ = 1050 nm in a 3D-confined subnuclear volume. After a time delay of choice, protein mobility within this volume is analysed by two-photon photoactivation of PA-GFP fusion proteins at λ = 775 nm. By changing the position of the photoactivation spot with respect to the zone of lesion the influence of chromatin structure and of the distance from damage are investigated. As first applications we demonstrate a locally confined, time-dependent mobility increase of histone H1.2, and a progressive retardation of the DNA repair factor XRCC1 at damaged sites. This assay can be used to map the response of nuclear proteins to DNA damage in time and space.

Dissection of the xeroderma pigmentosum group C protein function by site-directed mutagenesis.

Xeroderma pigmentosum group C (XPC) protein is a sensor of helix-distorting DNA lesions, the function of which is to trigger the global genome repair (GGR) pathway. Previous studies demonstrated that XPC protein operates by detecting the single-stranded character of non-hydrogen-bonded bases opposing lesion sites. This mode of action is supported by structural analyses of the yeast Rad4 homologue that identified critical side chains making close contacts with a pair of extrahelical nucleotides. Here, alanine substitutions of the respective conserved residues (N754, F756, F797, F799) in human XPC were tested for DNA-binding activity, accumulation in tracks and foci of DNA lesions, nuclear protein mobility, and the induction of downstream GGR reactions. This study discloses a dynamic interplay between XPC protein and DNA, whereby the association with one displaced nucleotide in the undamaged strand mediates the initial encounter with lesion sites. The additional flipping-out of an adjacent nucleotide is necessary to hand over the damaged site to the next GGR player. Surprisingly, this mutagenesis analysis also reveals that the rapid intranuclear trafficking of XPC protein depends on constitutive interactions with native DNA, implying that the search for base damage takes place in living cells by a facilitated diffusion process.