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Graphene field effect transistors (GFETs) are promising devices for biochemical sensing. Integrating GFETs onto complex non-planar surfaces could uncap their potential in emerging areas of wearable electronics, such as smart contact lenses and microneedle sensing. However, the fabrication of GFETs on non-planar surfaces is challenging using conventional lithography approaches. Here, we develop a combined spray-coating and photolithography setup for the scalable fabrication of GFETs on non-planar surfaces and demonstrate their application as integrated GFETs on microneedles. We optimize the setup to pattern ∼ 67 μm long GFET channels across the microneedle tips. Graphene is deposited between photo-patterned electrodes by spray-coating a liquid-phase exfoliated graphene ink while monitoring the channel resistance to achieve the required conductivity. The formation of the GFET channels is confirmed by SEM and EDX mapping, and the GFETs are shown to modulate in solution. This demonstrates an approach for manufacturing graphene electronic devices on complex non-planar surfaces like microneedles and opens possibilities for wearable GFET microneedle sensors for real-time monitoring of biomarkers. [ABSTRACT FROM AUTHOR]

Ultraviolet filters (UV-filters) are compounds extensively used in personal care products. These compounds are produced at increasing rates and discharged into marine ecosystems in unknown quantities and with no regulation, making them emerging contaminants. Among those, the UV-filter 4-Methylbenzylidene camphor (4-MBC) is used in a variety of personal care products such as sunscreens, soaps, or lipsticks. This high consumption has resulted in its presence in various environmental matrices at in concentrations ranging from ng to μg L -1 . Very little is known, however, about the possible adverse effects in exposed non-target organisms. Our study presents novel data on the bioconcentration, toxicokinetics, and molecular effects of 4-MBC in a marine bivalve species of commercial interest, Ruditapes philippinarum (Manila clam). Organisms were exposed at two different concentrations (1.34 and 10.79 μg L -1 ) of 4-MBC for 7 days, followed by a 3-day depuration period (clean sea waters). Bioconcentration factors (BCF) were 3562 and 2229 L kg -1 for the low and high exposure concentrations, respectively, making this pollutant bioaccumulative according to REACH criteria. Up to six 4-MBC biotransformation products (BTPs)were identified, 2 of them for the first time. Transcriptomic analysis revealed between 658 and 1310 differently expressed genes (DEGs) after 4-MBC exposure. Functional and enrichment analysis of the DEGs showed the activation of the detoxification pathway to metabolize and excrete the bioconcentrated 4-MBC, which also involved energy depletion and caused an impact on the metabolism of carbohydrates and lipids and in the oxidative phosphorylation pathways. Oxidative stress and immune response were also evidenced through the activation of cathepsins and the complement system. Such elucidation of the mode of action of a ubiquitous pollutant such as 4-MBC at the molecular level is valuable both from an environmental point of view and for the sustainable production of Manila clam, one of the most cultivated mollusk species worldwide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Despite breakthroughs in immune checkpoint inhibitors (ICI), the majority of tumors, including those poorly infiltrated by CD8+ T cells or heavily infiltrated by immunosuppressive immune effector cells, are unlikely to result in clinically meaningful tumor responses. Radiation therapy (RT) has been combined with ICI to potentially overcome this resistance and improve response rates but reported clinical trial results have thus far been disappointing. Novel approaches are required to overcome this resistance and reprogram the immunosuppressive tumor microenvironment (TME) and address this major unmet clinical need. Using diverse preclinical tumor models of prostate and bladder cancer, including an autochthonous prostate tumor (Pten -/- /trp53 -/- ) that respond poorly to radiation therapy (RT) and anti-PD-L1 combinations, the key drivers of this resistance within the TME were profiled and used to develop rationalized combination therapies that simultaneously enhance activation of anti-cancer T cell responses and reprogram the immunosuppressive TME. The addition of anti-CD40mAb to RT resulted in an increase in IFN-y signaling, activation of Th-1 pathways with an increased infiltration of CD8+ T-cells and regulatory T-cells with associated activation of the CTLA-4 signaling pathway in the TME. Anti-CTLA-4mAb in combination with RT further reprogrammed the immunosuppressive TME, resulting in durable, long-term tumor control. Our data provide novel insights into the underlying mechanisms of the immunosuppressive TME that result in resistance to RT and anti-PD-1 inhibitors and inform therapeutic approaches to reprogramming the immune contexture in the TME to potentially improve tumor responses and clinical outcomes., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

We demonstrate a novel and versatile sensing platform, based on electrolyte-gated graphene field-effect transistors, for easy, low-cost and scalable production of chemical sensor test strips. The Lab-on-PCB platform is enabled by low-boiling, low-surface-tension sprayable graphene ink deposited on a substrate manufactured using a commercial printed circuit board process. We demonstrate the versatility of the platform by sensing pH and Na + concentrations in an aqueous solution, achieving a sensitivity of 143 ± 4 μA per pH and 131 ± 5 μA per log 10 Na + , respectively, in line with state-of-the-art graphene chemical sensing performance.

A side effect of antibiotic usage is the emergence and dissemination of antibiotic resistance genes (ARGs) within microbial communities. The spread of ARGs among pathogens has emerged as a public health concern. While the distribution of ARGs is documented on a global level, their routes of transmission have not been clarified yet; for example, it is not clear whether and to what extent the emergence of ARGs originates in farms, following the selective pressure exerted by antibiotic usage in animal husbandry, and if they can spread into the environment. Here we address this cutting edge issue by combining data regarding antimicrobial usage and quantitative data from selected ARGs ( bla TEM B, bla CTXM , erm B, van A, qnr S, tet A, sul 2, and mcr -1) encoding for resistance to penicillins, macrolides-lincosamides-streptogramins, glycopeptides, quinolones, tetracyclines, sulfonamides, and colistin at the farm level. Results suggest that dairy farms could be considered a hotspot of ARGs, comprising those classified as the highest risk for human health and that a correlation existed between the usage of penicillins and bla TEM abundances, meaning that, although the antibiotic administration is not exclusive, it remains a certain cause of the ARGs' selection and spread in farms. Furthermore, this study identified the role of calves as the main source of ARGs spread in dairy farms, claiming the need for targeted actions in this productive category to decrease the load of ARGs along the production chain., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Salerno, Cornaggia, Sabatino, Di Cesare, Furlan, Barco, Orsini, Cordioli, Mantovani, Bano and Losasso.)

A breathable tattoo electrode for bio-potential recording based on a Parylene C nanofilm is presented in this study. The proposed approach allows for the fabrication of micro-perforated epidermal submicrometer-thick electrodes that conjugate the unobtrusiveness of Parylene C nanofilms and the very important feature of breathability. The electrodes were fully validated for electrocardiography (ECG) measurements showing performance comparable to that of conventional disposable gelled Ag/AgCl electrodes, with no visible negative effect on the skin even many hours after their application. This result introduces interesting perspectives in the field of epidermal electronics, particularly in applications where critical on-body measurements are involved., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Spanu, Mascia, Baldazzi, Fenech-Salerno, Torrisi, Viola, Bonfiglio, Cosseddu and Pani.)

Printed electronics have been attracting significant interest for their potential to enable flexible and wearable electronic applications. Together with printable semiconductors, solution-processed dielectric inks are key in enabling low-power and high-performance printed electronics. In the quest for suitable dielectrics inks, two-dimensional materials such as hexagonal boron nitride (h-BN) have emerged in the form of printable dielectrics. In this work, we report barium titanate (BaTiO 3 ) nanoparticles as an effective additive for inkjet-printable h-BN inks. The resulting inkjet printed BaTiO 3 /h-BN thin films reach a dielectric constant ( ε r ) of ∼16 by adding 10% of BaTiO 3 nanoparticles (in their volume fraction to the exfoliated h-BN flakes) in water-based inks. This result enabled all-inkjet printed flexible capacitors with C  ∼ 10.39 nF cm -2 , paving the way to future low power, printed and flexible electronics., (Creative Commons Attribution license.)

Antibiotic resistance is a serious concern for public health. Farm environments are relevant reservoirs of antibiotic resistant bacteria and antibiotic resistance genes (ARGs), thus strategies to limit the spread of ARGs from farms to the environment are needed. In this study a broiler farm, where antibiotics have never been used for any purpose, was selected to evaluate if this measure is effective in reducing the ARGs load in farm environment (FE) and in meat processing environment (MPE). Faecal samples from FE and MPE were processed for DNA extraction. Detection and quantification of the 16S rRNA gene and selected ARGs (bla TEM , qnrS, sul2, and tetA) were carried out by PCR and digital droplet PCR (ddPCR), respectively. Generally, the relative abundance of the quantified ARGs in FE was similar or higher than that measured in intensive farms. Furthermore, apart for tetA, no differences in relative abundances of the other ARGs between FE and MPE were determined. These results suggest that the choice to not use antibiotics in broiler farming is not so effective to limit the ARGs spread in MPE and that further sources of ARGs should be considered including the preceding production phase with particular reference to the breeding stage., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Tetrodotoxins (TTXs) are a group of potent neurotoxins named after the Tetraodontidae fish family (pufferfish). TTXs have been reported in several animal taxa, both terrestrial and marine. The ingestion of TTX-contaminated flesh can cause serious neurotoxic symptomatology and can eventually lead to death. Traditionally, TTXs have been associated with Asian countries, in particular with pufferfish consumption. However, they have also been reported in bivalve mollusks farmed in the Pacific area and, recently, in European seas. In Europe, different countries have reported TTXs, especially those bordering the Mediterranean Sea. As a consequence, in 2017 the European Food Safety Authority (EFSA) released an opinion with reference to TTX present in marine gastropods and bivalves, proposing a safety limit of 44 µg/kg TTXs in shellfish meat, below which no adverse effects should be observed in humans. Nevertheless, this limit has been exceeded on many occasions in European shellfish and, while for bivalves there have been no registered human intoxications, that is not the case for marine gastropods. However, TTXs have not yet been included in the list of marine biotoxins officially monitored in live bivalve mollusks within the European Union (EU). Thus, the aims of this manuscript are to discuss the increasing occurrence of TTXs in live bivalve mollusks from European sea waters, to acknowledge the still ongoing knowledge gaps that should be covered and to stimulate constructive debate on the eventuality of adopting a shared regulatory context, at least in the EU, for monitoring and managing this potential threat to food safety., (© 2021 The Authors. Comprehensive Reviews in Food Science and Food Safety published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

The growing cancer burden necessitates the development of cost-effective solutions that provide rapid, precise and personalised information to improve patient outcome. The aim of this study was to develop a novel, Lab-on-Chip compatible method for the detection and quantification of DNA methylation for MGMT, a well-established molecular biomarker for glioblastoma, with direct clinical translation as a predictive target. A Lab-on-Chip compatible isothermal amplification method (LAMP) was used to test its efficacy for detection of sequence-specific methylated regions of MGMT, with the method's specificity and sensitivity to have been compared against gold-standards (MethyLight, JumpStart). Our LAMP primer combinations were shown to be specific to the MGMT methylated region, while sensitivity assays determined that the amplification methods were capable of running at clinically relevant DNA concentrations of 0.2 - 20 ng/µL. For the first time, the ability to detect the presence of DNA methylation on bisulfite converted DNA was demonstrated on a Lab-on-Chip setup, laying the foundation for future applications of this platform to other epigenetic biomarkers in a point-of-care setting.

In recent years, graphene has found its use in numerous industrial applications due to its unique properties. While its impermeable and conductive nature can replace currently used anticorrosive toxic pigments in coating systems, due to its large strength to weight ratio, graphene can be an important component as a next-generation additive for automotive, aerospace and construction applications. The current bottlenecks in using graphene and graphene oxide and other two-dimensional materials are the availability of cost-effective, high-quality materials and their effective incorporation (functionalization and dispersion) into the product matrices. On overcoming these factors, graphene may attract significant demands in terms of volume consumption. Graphene can be produced on industrial scales and through cost-effective top-down routes such as chemical, electrochemical and/or high-pressure mechanical exfoliation. Graphene, depending on end applications, can be chemically tuned and modified via functionalization so that easy incorporation into product matrices is possible. This paper discusses different production methods and their impact on the quality of graphene produced in terms of energy input. Graphene with an average thickness below five layers was produced by both methods with varied defects. However, a higher yield of graphene with a lower number of layers was produced via the high-pressure exfoliation route. This article is part of a discussion meeting issue 'A cracking approach to inventing new tough materials: fracture stranger than friction'.

Background: The delay between the occurrence of symptoms and the call seeking medical assistance is an important component of the pain-to-balloon delay in patients with ST-segment elevation myocardial infarction (STEMI). Factors affecting this "patient delay" have been poorly studied, and campaigns to raise emergency call awareness have barely been evaluated., Aims: To evaluate the factors related to patient delay, and the effects of public awareness campaigns undertaken in our region., Methods: Data from the regional registry of STEMI in Limousin, France, were analysed, and we performed a survey to assess medical history, clinical signs, context, socioeconomic situation and perception and behaviour of the patient. "Late callers" (i.e. third tertile,>154minutes) were compared with "early callers" (i.e. first and second tertiles,≤154minutes) using univariate and multivariable statistical methods. The influence of public awareness campaigns was studied by comparing the patient delays before and after a regional campaign., Results: Among 481 patients, the median patient delay was 87minutes. "Late callers" were older (odds ratio [OR] 1.02 per year, 95% confidence interval [CI] 1.00 to 1.03), more often had symptom onset between 00:00 and 05:59 a.m. (OR 1.86, 95% CI 1.10 to 3.12) and more often sought assistance from a general practitioner (OR 2.58, 95% CI 1.66 to 4.04) or attended the emergency room (OR 4.10, 95% CI 2.04 to 8.32). Sweats and considering the situation to be severe were factors associated with a reduced delay. After awareness campaigns, there was no change in patient delay, but the proportion of patients calling the Emergency Medical Services increased from 55% to 62% (P<0.001)., Conclusions: Patient delay is multifactorial. The impact of previous campaigns is mixed. Psychological and behavioural aspects are determinant, and should be taken into consideration to develop awareness messages that target specific groups., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Abstract: To realize the full gamut of functions that are envisaged for electronic textiles (e-textiles) a range of semiconducting, conducting and electrochemically active materials are needed. This article will discuss how metals, conducting polymers, carbon nanotubes, and two-dimensional (2D) materials, including graphene and MXenes, can be used in concert to create e-textile materials, from fibers and yarns to patterned fabrics. Many of the most promising architectures utilize several classes of materials (e.g., elastic fibers composed of a conducting material and a stretchable polymer, or textile devices constructed with conducting polymers or 2D materials and metal electrodes). While an increasing number of materials and devices display a promising degree of wash and wear resistance, sustainability aspects of e-textiles will require greater attention., Competing Interests: Conflict of interestOn behalf of all authors, the corresponding author states that there is no conflict of interest., (© The Author(s) 2021.)

In this report, we present the whole-genome sequence of a Campylobacter jejuni strain isolated recursively for the last 3 years from an Italian poultry farm., (Copyright © 2020 Peruzzo et al.)

Objective: To assess the performance of transthoracic echocardiographic parameters to predict operative mortality and morbidity in patients undergoing coronary artery bypass grafting, and to assess its incremental prognostic value as compared to the Society of Thoracic Surgeons (STS) score., Materials and Methods: We prospectively collected the clinical and biological data required to calculate the STS score in patients hospitalised for coronary artery bypass grafting. Preoperative transthoracic echocardiography was performed for each patient. The primary endpoint was 30-day mortality or major morbidity (i.e. stroke, renal failure, prolonged ventilation, deep sternal wound infection, reoperation) as defined by the STS. The secondary endpoint was prolonged hospitalisation for over 14 days., Results: A total of 172 patients was included (mean age 66.1±10.2 years, 12.2% were women). The primary endpoint occurred in 33 patients (19.2%), and 28 patients (16.3%) had a prolonged hospital stay. Independent predictive factors for the primary endpoint were an increased left atrial volume (>31 mL/m²; odds ratio (OR) 3.55, 95% confidence interval (CI) 1.38-9.12; P =0.004) and a decreased tricuspid annular plane systolic excursion (<20 mm; OR 3.45, 95% CI 1.47-8.21; P =0.008). The predictive value of the multivariate model increased when the two echocardiographic parameters were added to the STS score (area under the curve 0.598 vs. 0.695, P =0.001; integrated discrimination improvement 7.44%)., Conclusion: In patients undergoing coronary artery bypass grafting, preoperative assessment of left atrial size and tricuspid annular plane systolic excursion should be performed systematically, as it provides additional prognostic information to the STS score.

UV filters are a class of emerging contaminants with an annual estimated production of 10,000 tons worldwide that continuously enter aquatic environments. Among UV filters, 4-methylbenzylidenecamphor (4-MBC) is an organic camphor derivative used in the cosmetic industry for its ability to protect the skin against UV, specifically UV B radiation. Individuals of the Japanese clam, Ruditapes philippinarum, were exposed to 4-MBC at environmentally relevant and slightly higher concentrations (nominal: 0, 1, 10, 100 μg L -1 ) using a semi-static exposure system over a 7-days period followed by a 3-days depuration period (total 10 days) where no 4-MBC was added to the tanks. Assessed mortality reached up to 100 % at the highest exposure concentration and a LC50 value of 7.71 μg·L -1 4-MBC was derived. Environmental risk assessment carried out in a site specific environment, the Cadiz bay in the south of Spain, revealed a potential risk produced by the presence of 4-MBC. Digestive glands tissues were analysed for differential expression of genes encoding proteins involved in the stress response (SOD, MT, GST, EIF1, BCL2, TP53, CAT, 18S, GADPH, GPX, GADD45, THIO9) by RT-qPCR for relative quantification. Results showed that the presence of 4-MBC at environmentally relevant concentrations induced the expression of genes that encode for antioxidant enzymes (GST) and for proteins related to the inhibition of apoptosis (BCL2) and cellular stress (GADD), suggesting a physiological stress response., Competing Interests: Declaration of Competing Interest The authors, Melania Santonocito, Barbara Salerno, Chiara Trombini, Federico Tonini, Marina G. Pintado-Herrera, Gonzálo Martínez-Rodríguez, Julián Blasco, Pablo Antonio Lara-Martín and Miriam Hampel are responsible for the research and declare that they have participated in the concept and design, analysis and interpretation of the data, writing and correction of the manuscript, and that they approve the final text that has been sent to the Special Issue PRIMO 2020 of Aquatic Toxicology and that this document is sent exclusively to Aquatic Toxicology and that it has not been sent to another journal (or submitted for consideration) and that it has not been published in full or in part. This study has no conflict of interest. The persons or entities mentioned in the acknowledgments agree with the final presentation of the article and its appearance in the same., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective:: Postoperative atrial fibrillation is a major complication following coronary artery bypass graft. We hypothesized that, beyond clinical and electrocardiogram (ECG) data, transthoracic echocardiography could improve the prediction of postoperative atrial fibrillation., Methods:: We prospectively studied 169 patients in sinus rhythm who underwent isolated coronary artery bypass graft in our institution. Clinical, biological, ECG and transthoracic echocardiography data were collected within 24 h before surgery. The patients were continuously monitored during the first five days, and then had daily 12-lead ECG afterwards until discharge. Postoperative atrial fibrillation was defined by any episode >10 min., Results:: Postoperative atrial fibrillation was found in 65 patients (38%). Compared with those without, patients with postoperative atrial fibrillation were significantly older ( p=0.008), had more frequently a history of hypertension ( p=0.009), history of atrial fibrillation ( p<0.001) and New York Heart Association class ⩾III ( p=0.004). They also had longer PR interval ( p=0.005), higher preoperative NT-pro brain natriuretic peptide level ( p=0.006), left ventricle end-diastolic volume ( p=0.002), indexed left ventricle mass ( p<0.0001), indexed maximal left atrial volume ( p<0.0001), maximal right atrial area ( p<0.001) and lower left ventricle ejection fraction ( p=0.04). In multivariate analysis, history of atrial fibrillation (odds ratio =6.1, 95% confidence interval: 1.4-26.0, p=0.02) and indexed maximal left atrial volume (odds ratio =1.13, 95% confidence interval: 1.1-1.2, p=0.001) were the only two independent predictive factors of postoperative atrial fibrillation. The addition of echocardiographic parameters improved the predictive value (χ 2 ) of the model, from 34 to 57., Conclusion:: A history of atrial fibrillation and indexed left atrial maximal volume are the best predictors of the occurrence of postoperative atrial fibrillation following coronary artery bypass graft. The identification of high risk population of postoperative atrial fibrillation using these two factors could lead to the development of targeted strategies to limit this frequent complication in these patients.

The neuromuscular disorder, spinal muscular atrophy (SMA), results from insufficient levels of the survival motor neuron (SMN) protein. Together with Gemins 2-8 and Unrip, SMN forms the large macromolecular SMN-Gemins complex, which is known to be indispensable for chaperoning the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). It remains unclear whether disruption of this function is responsible for the selective neuromuscular degeneration in SMA. In the present study, we first show that loss of wmd, the Drosophila Unrip orthologue, has a negative impact on the motor system. However, due to lack of a functional relationship between wmd/Unrip and Gemin3, it is likely that Unrip joined the SMN-Gemins complex only recently in evolution. Second, we uncover that disruption of either Tgs1 or pICln, two cardinal players in snRNP biogenesis, results in viability and motor phenotypes that closely resemble those previously uncovered on loss of the constituent members of the SMN-Gemins complex. Interestingly, overexpression of both factors leads to motor dysfunction in Drosophila, a situation analogous to that of Gemin2. Toxicity is conserved in the yeast S. pombe where pICln overexpression induces a surplus of Sm proteins in the cytoplasm, indicating that a block in snRNP biogenesis is partly responsible for this phenotype. Importantly, we show a strong functional relationship and a physical interaction between Gemin3 and either Tgs1 or pICln. We propose that snRNP biogenesis is the pathway connecting the SMN-Gemins complex to a functional neuromuscular system, and its disturbance most likely leads to the motor dysfunction that is typical in SMA., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Context: Roux-en-Y gastric bypass (RYGB) is among the most effective treatments for extreme obesity and obesity-related complications. However, despite its potential efficacy, many patients do not achieve and/or maintain sufficient weight loss., Objective: Our objective was to identify genetic factors underlying the variability in weight loss outcomes after RYGB surgery., Design: We conducted a genome-wide association study using a 2-stage phenotypic extreme study design., Setting: Patients were recruited from a comprehensive weight loss program at an integrated health system., Patients: Eighty-six obese (body mass index >35 kg/m(2)) patients who had the least percent excess body weight loss (%EBWL) and 89 patients who had the most %EBWL at 2 years after surgery were genotyped using Affymetrix version 6.0 single-nucleotide polymorphism (SNP) arrays. A second group from the same cohort consisting of 164 patients in the lower quartile of %EBWL and 169 from the upper quartile were selected for evaluation of candidate regions using custom SNP arrays., Intervention: We performed RYGB surgery., Main Outcome Measures: We assessed %EBWL at 2 years after RYGB and SNPs., Results: We identified 111 SNPs in the first-stage analysis whose frequencies were significantly different between 2 phenotypic extremes of weight loss (allelic χ(2) test P < .0001). Linear regression of %EBWL at 2 years after surgery revealed 17 SNPs that approach P < .05 in the validation stage and cluster in or near several genes with potential biological relevance including PKHD1, HTR1A, NMBR, and IGF1R., Conclusions: This is the first genome-wide association study of weight loss response to RYGB. Variation in weight loss outcomes after RYGB may be influenced by several common genetic variants.

Background: Streptococcus pneumoniae has two paralogous, homotetrameric, single-stranded DNA binding (SSB) proteins, designated SsbA and SsbB. Previous studies demonstrated that SsbA and SsbB have different solution-dependent binding mode preferences with variable DNA binding capacities. The impact of these different binding properties on the assembly of multiple SsbAs and SsbBs onto single-stranded DNA was investigated., Methodology/principal Findings: The complexes that were formed by the SsbA and SsbB proteins on dT(n) oligomers of defined lengths were examined by polyacrylamide gel electrophoresis. Complexes containing either two SsbAs or two SsbBs, or mixed complexes containing one SsbA and one SsbB, could be formed readily, provided the dT(n) oligomer was long enough to satisfy the full binding mode capacities of each of the bound proteins under the particular solution conditions. Complexes containing two SsbAs or two SsbBs could also be formed on shorter dT(n) oligomers via a "shared-strand binding" mechanism in which one or both proteins were bound using only a portion of their potential binding capacity. Mixed complexes were not formed on these shorter oligomers, however, indicating that SsbA and SsbB were incompatible for shared-strand binding. Additional experiments suggested that this shared-strand binding incompatibility may be due in part to differences in the structure of a loop region on the outer surface of the subunits of the SsbA and SsbB proteins., Conclusion/significance: These results indicate that the SsbA and SsbB proteins may co-assemble on longer DNA segments where independent binding is possible, but not on shorter DNA segments where coordinated interactions between adjacent SSBs are required. The apparent compatibility requirement for shared-strand binding could conceivably serve as a self-recognition mechanism that regulates the manner in which SsbA and SsbB interact in S. pneumoniae.

Nonhomologous end-joining (NHEJ) is an important pathway for the repair of DNA double-strand breaks (DSBs) and plays a critical role in maintaining genomic stability in mammalian cells. While Ku70/80 (Ku) functions in NHEJ as part of the DNA-dependent protein kinase (DNA-PK), genetic evidence indicates that the role of Ku in NHEJ goes beyond its participation in DNA-PK. Inositol hexakisphosphate (IP(6)) was previously found to stimulate NHEJ in vitro and Ku was identified as an IP(6)-binding factor. Through mutational analysis, we identified a bipartite IP(6)-binding site in Ku and generated IP(6)-binding mutants that ranged from 1.22% to 58.48% of wild-type binding. Significantly, these Ku IP(6)-binding mutants were impaired for participation in NHEJ in vitro and we observed a positive correlation between IP(6) binding and NHEJ. Ku IP(6)-binding mutants were separation-of-function mutants that bound DNA and activated DNA-PK as well as wild-type Ku. Our observations identify a hitherto undefined IP(6)-binding site in Ku and show that this interaction is important for DSB repair by NHEJ in vitro. Moreover, these data indicate that in addition to binding of exposed DNA termini and activation of DNA-PK, the Ku heterodimer plays a role in mammalian NHEJ that is regulated by binding of IP(6).