Transforming growth factor beta (TGFβ) signalling is involved in both tumour suppression and tumour progression. The mRNA expression levels of the TGFβ isoforms and receptors in breast tumours may have prognostic value and clinical implications.TGFB1, TGFB2, TGFB3, TGFBR1 and TGFBR2 were analysed in primary breast tumours and adjacent normal breast tissues, and the associations with tumour characteristics and patients’ overall and relapse-free survival were evaluated, using the public gene expression microarray data from The Cancer Genome Atlas (n = 520) and the Gene Expression Omnibus (four datasets) and our quantitative real-time PCR validation data (n = 71).The mRNA levels of TGFB1 and TGFB3 mRNA levels and lower TGFBR2 mRNA levels were observed in primary tumours compared with their paired normal tissues. TGFB1 mRNA expression was seemly lower in triple-negative tumours and in tumours from lymph node-negative patients. TGFB3 mRNA expression was significantly lower in estrogen receptor-negative/progesterone receptor-negative/Basal-like/Grade 3 tumours. High TGFB2, TGFB3 and TGFBR2 mRNA levels in tumours were generally associated with better prognosis for patients, especially those diagnosed with lymph node-negative diseases. High TGFBR1 mRNA levels in tumours were associated with poorer clinical outcomes for patients diagnosed with small (diameter ≤2 cm) tumours.Significantly higher TGFB1 in malignant tumours and a preferential up-regulation of TGFB3 in premalignant tumours. The results may not only provide prognostic value for patients but also assist in classifying tumours according to their potential responses to TGFβ and selecting patients for TGFβ signalling pathway targeted therapies.The results indicate a reduced responsiveness of tumour cells to TGFβ, a preferential up-regulation of  Transforming growth factor beta (TGFβ) signalling is involved in the maintenance of tissue homeostasis and suppression of premalignant tumour cells, however, when the regulations are circumvented, TGFβ signalling can be advantageously exploited by the tumour cells to promote tumour progression and metastasis . Three iExtensive efforts have been made to investigate the role of TGFβ signalling in the development and progression of cancer and to develop drugs targeting the TGFβ signalling pathway for a number of cancer types, including breast cancer , 5–7. ThTGFB1, TGFB2, TGFB3, TGFBR1 and TGFBR2 in primary tumours and tumour-adjacent normal tissues, and to evaluate the associations between the mRNA levels and the clinical, pathological and molecular tumour characteristics and the patients’ overall and relapse-free survival. We also validated the gene expression profiles on an independent set of breast cancer surgical specimens by reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR).Here, we used the public breast cancer gene expression microarray datasets from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) to compare the mRNA expression levels of The TCGA breast cancer level 3 mRNA expression Agilent microarray data  and the patients’ clinical data were downloaded from the Broad Genome Data Analysis Centre Firehose April 2015 version). The TCGA cohort  from 71 females (age range 33–85 years) diagnosed with breast cancer (Table TGFB1), 220406_at (TGFB2), 209747_at (TGFB3), 206943_at (TGFBR1) and 208944_at (TGFBR2) from each GEO dataset and each subset of the Ivshina dataset  were log2-transformed and standardized to mean = 0 and standard deviation (SD) = 1. The Ivshina cohort was renamed as “GEO cohort, All”, which was used to represent unselected population. The log2-transformed and standardized gene expression data of the Schmidt cohort, the Wang cohort and the lymph node-negative subset of the Ivshina cohort were merged to form “GEO cohort, N neg.”; and the log2-transformed and standardized gene expression data of the Symmans cohort and the ER-positive subset of the Ivshina cohort were merged to form “GEO cohort, ER pos.”. The T ≤2 cm, T >2 cm, lymph node-positive and ER-negative subsets of the Ivshina cohort were named as “GEO cohort, T ≤2 cm” “GEO cohort, T >2 cm”, “GEO cohort, N pos.” and “GEO cohort, ER neg.”, respectively. The Ivshina dataset contained recurrence-free survival data , while the other three datasets contained distant relapse-free survival data. Thus the merged survival data was referred as relapse-free survival data but with bias to more distant relapse-free survival data.The gene expression data of the four GEO datasets were all based on Affymetrix U133A and U133A&B array sets. The data of the probe sets 203084_at , according to the manufacturer’s instructions. RNA concentration, integrity and purity were analysed using NanoDrop 1000 Spectrophotometer (Thermo Scientific) and agarose gel electrophoresis. Quality-checked 500 ng RNA per specimen was reverse transcribed using high capacity RNA-to-cDNA kit (Life Technologies) in a final reaction volume of 12 μL containing random hexamer primers. The incubation conditions for reverse transcription (RT) were: 37 °C for 60 min, 95 °C for 10 min and hold at 4 °C indefinitely. RT-negative controls (reactions with no reverse transcriptase) of two RNA samples were prepared for testing DNA contamination. The cDNA was kept at −20 °C until used for quantitative real-time PCR (qPCR).The qPCR was performed using SYBR Green PCR master mix (Life Technologies) in MicroAmp optical 384-well reaction plates (Life Technologies) on an Applied Biosystems 7900HT Fast Real-Time PCR System. The primers for qPCR were designed using AlleleID 7.6 (PREMIER Biosoft) to span at least one exon-exon junction and to cover all the transcript variants found in Entrez Gene Table . Each qP-∆∆Ct method was used for the qPCR data analysis [GAPDH and HPRT1 were chosen as the reference genes for normalization, as they showed smaller SD of Ct values compared with two other putative housekeeping genes RNA18S5 and GUSB that were assessed in our pilot study. The ∆Ct for the gene of interest was calculated by subtracting the average of Ct for GAPDH and HPRT1 from the Ct for the gene of interest [-∆Ct) were skewed, thus the normalized and log2-transformed data, i.e. -∆Ct, was used for data analysis [The 2analysis . GAPDH ainterest . The relanalysis .Log2-transformed and normalized mRNA expression data was used for all statistical analyses. The Wilcoxon signed-rank test was used to test for differences in the mRNA levels between tumours and matched normal tissues. The Spearman’s rank correlation analysis was employed to evaluate the correlations between the mRNA levels of pairwise genes. The Wilcoxon rank-sum test and Kruskal-Wallis test were employed to test for differences in the mRNA levels in subgroups stratified by tumour characteristics, namely, tumour diameter, regional lymph nodes involvement, distant metastasis, ER status, PR status, HER2 status, triple-negative, histological type, TNM stage, tumour grade and PAM50 subtype.TGFB1, TGFB2, TGFB3, TGFBR1 and TGFBR2 mRNA and patients’ overall and relapse-free survival in the TCGA and GEO cohorts. Patient’s survival time was calculated from the date of diagnosis to the date of event (death or relapse) or censored at the last follow-up or last known alive time whichever was longer. To evaluate if the tumour mRNA levels had different associations with patients’ clinical outcomes in the early and late years after patients were diagnosed with breast cancer, the Cox analyses were performed at different periods of follow-up time from 1 to 10 years. For each Cox model, the proportional hazards assumption was evaluated, and the results indicated that some of the gene expression variables were time-dependent. Thus extended Cox models using a Heaviside function approach [The Cox proportional hazards regression model was used to evaluate the associations between tumour levels of approach  were appTo evaluate if stratifying patients by the tumour mRNA levels would predict patients’ survival, the patients of each cohort were divided into two groups according to their tumour mRNA levels of each gene, and then the relevant hazard ratios for different periods of follow-up time (from 1 to 10 years) were calculated using Cox proportional hazards regression model. To avoid arbitrary grouping, the cut point was set to include 10–90 % of the patients with the lowest expression in each cohort. Representative Kaplan-Meier curves were plotted for the cut points that produced the lowest Wald test p-values for HR. The Likelihood-ratio test was used to assess statistical significance of the calculated Cox regression coefficients (β) and hazard ratios (HR), where β = ln(HR). The 95 % confidence interval (CI) of HR was calculated based on the Wald test. The Log-rank test was used to assess significant differences between the Kaplan-Meier estimates.All the statistical analyses were performed using R . The following R packages were used: qvalue, GEOquery, rms, heatmap.plus, ggplot2, gplots, gridExtra and reshapes2. All the p-values (P) presented were two-sided, and the significance level was set at 0.05. Bonferroni correction for multiple testing was applied where appropriate and corrected p-values were given the symbol P’. For comparison, Storey’s false discovery rate (FDR) procedure, a less stringent multiple testing correction method, was also applied where appropriate .P = 3.6 × 10−6), in which the tumours from TCGA were overall larger than the tumours from the local tissue banks.The bio-specimens from the TCGA, GEO and RT-qPCR cohorts were all collected from newly diagnosed patients with breast tumours that had received no prior treatment, such as chemotherapy or radiotherapy. The clinical, pathological and molecular characteristics of the breast cancer specimens were summarised in Table TGFB1 and TGFB3 mRNA levels and lower TGFBR2 mRNA levels were observed in the primary breast tumours compared with their matched normal tissues  and TGFB3  were observed in the tumours (n = 59).Significantly higher function . InteresTGFB1, TGFB2, TGFB3, TGFBR1 and TGFBR2 and the tumour characteristics displayed in Table TGFB3 was significantly lower in the ER-negative, PR-negative and triple-negative tumours  with HR = 0.3, 95 % CI: 0.18 – 0.51  tumours , rather than TGFB3 expression was down-regulated in malignant tumour cells. The up-regulated TGFB1 expression in malignant tumour cells and the up-regulated TGFB3 expression in premalignant tumour cells were very likely to have different, even opposite effects on the corresponding tumours, which also implicated the specific biology of different TGFβ isoforms.The of TβRII . The resTGFB1, TGFB2, TGFB3, TGFBR1 and TGFBR2 and patients’ clinical outcomes such as overall and relapse-free survival in each group using Cox proportional hazards regression model, then we should expect negative log hazard ratios for type 1 tumours, log HRs of zero for type 2 tumours and positive log HRs for type 3 tumours, respectively. Obviously, the HRs for different genes should be different. Unfortunately, we are still not able to separate the three types of tumours. However, for a cohort composed of patients with mixed types of tumours, a significant negative log HR implicates that there are more patients with type 1 tumours, a significant positive log HR implicates that there are more patients with type 3 tumours, and a non-significant HR implicates that there are more patients with type 2 tumours, the patients are heterogeneous or the population is too small. Thus the association between the expression levels and patients’ clinical outcomes may serve as an indicator of the proportion of the three types of tumours. The conflicting results such as that were reported for the associations between the protein levels of TGFβ1 and TβRII and the patients’ clinical outcomes [Assuming that we can group breast cancer patients according to the tumour cells’ responses to TGFβ, and evaluate the associations between the expression levels of outcomes –11, wereTGFB2, TGFB3 and TGFBR2 mRNA and clinical outcomes in patients diagnosed with lymph node-negative diseases implicated that the majority of the tumours from those patients still responded to the suppressive effect of TGFβ signalling, and the tumours with lower TGFB2, TGFB3 and TGFBR2 expression were more advantageous in tumour progression. In patients diagnosed with small tumours, those with higher TGFBR1 mRNA levels in the tumours had much poorer prognosis for the first 3 years. One of the rationales for anti-cancer drugs targeting the TGFβ pathway is to block the TGFβ signalling for type 3 tumours but not type 1 or type 2 tumours. Our data implicated that in the patients with small breast tumours, there was a high proportion of patients with type 3 tumours that might be more likely to benefit from the drugs targeting TβRI.The significant positive associations observed between tumour levels of TGFBR2 expression in tumours and the differentially regulated TGFB1 and TGFB3 expression by malignant and premalignant tumour cells may also have potential clinical implications that need to be further explored.In conclusion, the mRNA levels of the TGFβ isoforms and receptors in breast tumours were differentially associated with patients’ overall and relapse-free survival in patients stratified by different tumour characteristics. Before we can develop techniques to precisely classify breast tumours into different types according to their responses to TGFβ, the associations between the tumour mRNA levels and patients’ clinical outcomes may not only provide prognostic value for patients but also assist in classifying tumours according to their potential responses to TGFβ and selecting patients for the TGFβ signalling pathway targeted therapies that are under development . The mec


Synechocystis sp. PCC 6803 has a bidirectional [NiFe]-hydrogenase (Hox hydrogenase) which reversibly reduces protons to H2. This enzyme is composed of a hydrogenase domain and a diaphorase moiety, which is distinctly homologous to the NADH input module of mitochondrial respiratory Complex I. Hox hydrogenase physiological function is still unclear, since it is not required for Synechocystis fitness under standard growth conditions. We analyzed the phenotype under prolonged darkness of three Synechocystis knock-out strains, lacking either Hox hydrogenase (ΔHoxE-H) or one of the proteins responsible for the assembly of its NiFe active site (ΔHypA1 and ΔHypB1). We found that Hox hydrogenase is required for Synechocystis growth under this condition, regardless of the functional status of its catalytic site, suggesting an additional role beside hydrogen metabolism. Moreover, quantitative proteomic analyses revealed that the expression levels of several subunits of the respiratory NADPH/plastoquinone oxidoreductase (NDH-1) are reduced when Synechocystis is grown in the dark. Our findings suggest that the Hox hydrogenase could contribute to electron transport regulation when both photosynthetic and respiratory pathways are down-regulated, and provide a possible explanation for the close evolutionary relationship between mitochondrial respiratory Complex I and cyanobacterial [NiFe]-hydrogenases.The cyanobacterium  In particular, the cyanobacterium strain Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) is a commonly used model for both functional and biotechnological applications. Synechocystis can grow in both sunlight (phototrophic growth), through oxygenic photosynthesis, and darkness (heterotrophic growth), through glycolysis and oxidative phosphorylation. Moreover, Synechocystis genome has been completely sequenced3Hydrogen metabolism is one of the most ancient and crucial processes of life. Today, it is at the center of increasing attention in the context of bioenergy production technologies, that will be of critical importance in the near future. In a variety of prokaryotic and eukaryotic microorganisms, hydrogen metabolism is mediated by the hydrogenases, metalloproteins which catalyze the reversible reduction of protons to molecular hydrogen H in condiSynechocystis has only a bidirectional [NiFe]-hydrogenase, the Hox hydrogenase; this heteropentameric enzyme is encoded by the hox operon, which includes the genes hoxE, hoxF, hoxU, hoxY, and hoxH, and three additional open reading frames (ORFs)52 or the reduction of protons, and HoxY contains a [4Fe-4S] cluster, mediating the electron transfer to and from the active site. The subunits HoxF, HoxU, and HoxE also contain FeS clusters and form the diaphorase moiety, a flavoprotein interacting with NAD(P)+/NAD(P)H. As in all [NiFe]-hydrogenases, the assembly of the NiFe active site and the activation of the Hox hydrogenase require a highly coordinated network of accessory proteins, encoded by the hypABCDEF operon and extensively described in Escherichia coliSynechocystis  is still under debate2)591012131415The precise physiological function of the Hox bidirectional hydrogenase in hox and hyp genes are constitutively expressed in Synechocystis under both anaerobic and aerobic conditions17Synechocystis mutant strains, lacking hypA1 or hypB1, or all hox genes. Our results indicate that the Hox hydrogenase could be involved in the adaptive response of Synechocystis to prolonged darkness, a harsh growth condition frequently occurring in natural environments.Interestingly, the All chemicals were of the highest commercially available purity.Synechocystis strains were grown in standard BG11 medium−2 s−1), unless otherwise stated. ΔHypA1 and ΔHypB1 Synechocystis strains (see below) were grown in the same medium in the presence of 5 to 50 μg/ml kanamycin. The hox− strain  was kindly provided by Jens Appel . Liquid cultures were kept under continuous shaking. When grown under anaerobiosis in the dark, cultures were kept in sealed flasks wrapped with aluminium foil. For cultures grown on plates, BG11 medium was supplemented with 1.5% agar and 0.3% sodium thiosulfate. Cell growth was monitored by measuring the culture optical density at 730 nm (O.D.730). Chlorophyll concentration was determined spectrophotometrically as described inE. coli strain XL1-Blue was used for DNA manipulation. E. coli cultures were grown in LB medium (with 50 μg/ml kanamycin when requested). Solid medium was obtained by adding 1.5% agar.For photoheterotrophic growth, all hypA1 (slr1675 gene) and hypB1 (sll1432 gene) knock-out strains  were obtained from wild type Synechocystis by homologous double recombination. A part of each gene was replaced with a kanamycin-resistance cassette (kanr), derived by BamHI digestion from the plasmid pUC4K (Pharmacia). The plasmids used to generate these deletion mutants were based on the pBSKII vector (Agilent Technologies) and contained the Synechocystis chromosome regions flanking the slr1675 and sll1432 genes, respectively. The regions upstream and downstream slr1675 and sll1432 were amplified by a three-steps PCR strategy, using primers designed on the basis of the Synechocystis genome sequence accessible through CyanoBase and containing sequences with restriction sites for cloning purposes  SacIhypA1_for/BamHIhypA1_rev and BamHIhypA1_for/EcoRVhypA1_rev (for hypA1−) or ii) SacIhypB1_for/BamHIhypB1_rev and BamHIhypB1_for/EcoRVhypB1_rev (for hypB1−). In a second step, the products of these PCR-up and PCR-down were amplified using the primers couples i) SacIhypA1_for/EcoRVhypA1_rev (for hypA1−) or ii) SacIhypB1_for/EcoRVhypB1_rev (for hypB1−), thus obtaining an overlapping band. In the last step, the overlapping bands were digested by the restriction enzymes SacI and EcoRV and individually cloned in a pBSKII vector, which was finally digested with BamHI to introduce the kanr cassette. The correctness of the resulting recombinant plasmids pBSKII_hypA1KO_ kanr and pBSKII_hypB1KO_ kanr was confirmed by DNA sequencing . To transform wild type Synechocystis730 : 0.5) and then centrifuged at 3000 × g for 5 minutes at room temperature. The cell pellet was suspended in fresh BG11 medium without glucose at a density of 1 × 1010 cells/ml and mixed with 0.5 μg/ml of exogenous DNA containing pBSKII_hypA1KO_ kanr and pBSKII_hypB1KO_ kanr. The mixture of cells and DNA was incubated for 5 h, and then spread onto a membrane filter resting on a BG11 agar plates. After 24 h, the membrane filter was moved on a BG11 agar plate containing 5 μg/ml kanamycin. Several single recombinant colonies appeared after 10–15 days. Since Synechocystis has multiple genome copies, to obtain the complete segregation of the recombinant chromosomes the colonies were subcloned on plates containing increasing antibiotic concentrations up to 50 μg/ml. The complete segregation was verified by PCR using the primers pairs FLhypA1_for/FLhypA1_rev and FLhypB1_for/FLhypB1_rev for the ΔHypA1 and ΔHypB1 strains respectively  (Perkin Elmer). All steps were performed under anaerobic conditions in a glove-box (MBRAUN MB 200B), using oxygen-free solutions.Hydrogenase activity of whole cell extracts was measured Synechocystis strains were grown until they reached the exponential phase (O.D.730 : 2.5); then, they were diluted 1:10 in fresh medium (O.D.730 at day 0: 0.2). Each experiment was performed in triplicate i) under continuous white light (50 μE m−2 s−1), ii) with 12 h light/12 h dark cycles or iii) complete darkness. After five days, we assessed the cultures capabilities to generate colonies on plates: each liquid culture was diluted to an O.D.730 between 0.3 and 0.4 and quantified using the cell counter Cellometer Auto X4 with a Cellometer SD100 counting chamber (Nexcelom Bioscience LLC.). The cell densities of the diluted cultures ranged between 1 × 106 and 1.5 × 106 cells/ml. Each diluted culture was then plated in three serial dilutions  and incubated at 30 °C in a 20 μE m−2 s−1 light regimen. Colonies, usually appeared after about ten days, were finally counted. Experiments were made in triplicate.Cultures of wild type and mutant Synechocystis pellets were fixed in 3% glutaraldehyde  overnight at 4 °C and then processed for electron microscopy as described inTM F12) operating at 100 kV .Synechocystis wild type and ΔHoxE-H strains were grown for 5 days under continuous light or darkness as described above. Cells were collected by 5000 × g centrifugation for 10 minutes at 4 °C , frozen in liquid nitrogen, and stored at −20 °C. For protein extraction, the cells were washed twice with 50 mM HEPES-KOH, pH 7.5 and then suspended in the presence of 1 mM PMSF. The cells were centrifuged and the pellet was suspended in equal volumes of extraction buffer  and glass beads (150–212 μm diameter). Then the cells were homogenized at 3500 rpm in a Mini Bead Beater (Biospec Products). Intact cells and glass beads were removed by 1000 × g centrifugation for 5 minutes  and the supernatant was incubated in 2% SDS for 30 minutes to facilitate the extraction of membrane proteins. Samples were centrifuged again at 12 000 × g for 20 minutes at 4 °C, and the supernatant was stored at −20 °C until further use. Protein concentration was determined by Bradford method . The proteins were precipitated in ice-cold acetone  at −20 °C overnight and stored at −80 °C.Three biological replicates of Synechocystis (3 wild type and 3 ΔHoxE-H strains grown either under light or in the dark) were suspended in 40 μl Laemmli buffer and loaded into a NuPage 12% precast SDS-PAGE (Invitrogen). The electrophoretic process was carried out for few minutes, just enough to allow all proteins to enter the separating gel and focus in single narrow bands. Proteins were stained using SimplyBlue Safestain (Invitrogen) and each band was manually excised from the gel. The staining was then removed and the proteins were reduced with 10 mM dithiotreitol (DTT) in 25 mM triethylammonium bicarbonate (TEAB) at 55 °C for 1 h. Then, cysteines were alkylated using 55 mM iodoacetamide in 25 mM TEAB for 45 minutes in the darkness. Gel slices were thoroughly washed with 50 mM TEAB and acetonitrile (ACN) and vacuum dried in a SpeedVac system. Samples were incubated with 130 μl of sequencing grade modified trypsin , overnight at 37 °C. Peptides were extracted from the gel slices by two consecutive treatments with 50% ACN  and finally vacuum dried. Each digested sample was dissolved in iTRAQ dissolution buffer and labeled with iTRAQ tags following the manufacturer’s instructions (AB Sciex). To reduce any potential variability introduced by the labeling reaction, samples from the 3 replicates were labeled with a tag-swapping strategy. To verify the labeling efficiency, each sample was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described below. Acquired data were searched with Mascot search engine, setting iTRAQ labeling as variable modification. No unmodified peptides were identified from the search and all the peptides were correctly modified at the N-terminus and at each lysine residue. Finally, the iTRAQ labeled samples were combined in a 1:1 (wild type:ΔHox) ratio and vacuum dried.One hundred μg of protein pellets from each of the 12 preparations of 2PO4, 25% ACN, pH 2.9) and loaded into a SCX cartridge with a syringe-pump system at a 50 μl/min flow rate. Peptides were eluted in an eight-step mode by adding increasing KCl concentrations in equilibration buffer . Each SCX fraction was vacuum dried, suspended in 0.1% formic acid (FA), and desalted using C18 cartridges . Samples were vacuum dried and stored at −20 °C.Strong cation exchange (SCX) was performed using a SCX cartridge (AB Sciex) and following the manufacturer’s protocol. Each iTRAQ labeled sample was suspended in equilibration buffer  coupled online with a Ultimate 3000 nano-HPLC (Dionex - Thermo Fisher Scientific), as previously describedSynechocystis database , concatenated with a database of the most common contaminants found in proteomics experiments. Peptide and fragment tolerances were set to 10 ppm and 0.6 Da respectively, enzyme specificity was set to Trypsin with up to 2 missed cleavages. Carbamidomethylcysteine and 4-plex iTRAQ labels at N-terminus and Lys residues were set as fixed modifications; oxidation of methionine was set as variable modification. Percolator and a search against the corresponding randomized database were used to calculate False Discovery Rates (FDR). Proteins were grouped into families according to the principle of maximum parsimony. Data were first filtered by considering as positive hits the peptides that were identified with medium (95%) or high (99%) confidence, based on the q-value calculated by the Percolator algorithm. To obtain the final list of proteins, data were further filtered by considering as positive hits only the proteins that were identified with at least two unique peptides and quantified with at least two independent peptides. Protein expression levels of wild type and mutant strains, under light or darkness growth conditions, were compared; a fold change of 1.5 or greater in the expression level was considered relevant. Statistical significance was determined with a two-tailed Z Test.Raw MS/MS files were analyzed using Proteome Discoverer 1.4 (Thermo Fisher Scientific) connected to a Mascot Search Engine server version 2.2.4 (Matrix Science), using a MudPIT protocol. Data were filtered to exclude MS/MS spectra containing less than 5 peaks and with a total ion count lower than 50. Spectra were searched against the Uniprot Synechocystis mutant strains lacking one or more of the hox genes from the hoxE-H operon  show no significant differences from the wild type strain. This observation raises the question of what exactly is the role of the Hox hydrogenase. Furthermore, this enzyme is constitutively expressed under both aerobic and anaerobic conditions, suggesting that it likely provides an evolutionary advantage that would manifest under non-standard growth conditions, and that it could have an additional role besides H2 metabolism. Most cyanobacteria are aerobic photoautotrophs, and sunlight-driven oxygenic photosynthesis is their main energy metabolism pathway. However, it is well known that some cyanobacterial communities can be exposed and survive to natural environments where regular prolonged periods of complete darkness occur, for example in lake sediments, soil water or in dense aquatic accumulations produced at the surface. Under these conditions cyanobacteria are believed to shift to an anaerobic fermentation metabolismhox genes are absent in cyanobacterial strains found in open oceans1Synechocystis growth in the darkness, we analyzed the phenotype of ΔHoxE-H, a Synechocystis knock-out strain where the entire hox operon has been deleted.Under standard growth conditions, all hox operon on Synechocystis fitness during prolonged dark periods (up to five days) in the presence of glucose as carbon source, the growth of wild type and ΔHoxE-H strains were compared under three different light conditions: i) continuous white light (50 μE m−2 s−1), ii) complete darkness or iii) 12 h light/12 h dark cycles. This last condition was added as an additional control, since Synechocystis is known to possess a circadian clock system. hox knock-out mutants11131415161718Synechocystis cannot grow under complete darkness unless it is given a daily light pulse (i.e. 40 μmol m−2 s−1 for 5 minutes)Synechocystis strain can grow in the dark without this pulse  ΔHypA1 and ii) ΔHypB1, lacking two accessory proteins required for the assembly of the NiFe active site of the Hox hydrogenase. In both ΔHypA1 and ΔHypB1 the HoxEFU diaphorase activity is preserved whereas the hydrogenase catalytic core is inactivated.It is worth noting that similar results were obtained by incubating wild type and ΔHoxE-H strains in the dark under anaerobic  or in aeSynechocystis has two homologues hypA and hypB genes ; however, only HypA1 and HypB1 are required for the assembly of the Hox hydrogenase, whereas HypA2 and HypB2 are likely to have a role in the maturation of other metalloproteinshypA1 and hypB1 genes with a kanamycin-resistance cassette (kanr)   and thuse (kanr) , confirmnitoring  and chlonitoring , and fouSynechocystis to prolonged darkness, regardless of whether its NiFe active site module is folded in a functional state or not, and open a new perspective to gain clues on the role of this protein under stress conditions.Taken together, these results suggest that the Hox hydrogenase does play a key role in the adaptive response of Synechocystis and other cyanobacteria showed that when exposed to different conditions of environmental stress , they responded with different survival mechanisms3435363738Synechocystis adaptive metabolic response to a prolonged darkness we analyzed and compared the proteomes of both wild type and ΔHoxE-H strains grown either under continuous light or in the dark.Several proteomic studies recently conducted on Synechocystis that were grown under continuous light or prolonged darkness, using isobaric tag for relative and absolute quantification (iTRAQ) technology and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analysis was conducted on three independent biological replicates. Across all replicates a total of 780 distinct protein families were identified and quantified. All relevant information relative to proteins and peptides identification and quantification are reported in Synechocystis theoretical proteomeSynechocystis) are underestimated in our analysis, probably due to the intrinsic limitations in the applied methodology. Nevertheless, we determined that 112 proteins were differentially regulated between light and darkness grown organism , among which 58 showed an increased abundance (i.e. dark/light ratio ≥ 1.50) and 54 a decreased abundance (i.e. dark/light ratio ≤ 0.67). We grouped these proteins into different categories according to their known or predicted functions  complex were also found to have a lower expression level under darkness. Synechocystis contains a respiratory electron transport chain on both the cytoplasmic and thylakoid membranes, working in an interwoven way with the photosynthetic electron transport chainb6f and plastocyanin), whereas other complexes are specific for the respiratory electron flow, including NDH-1, succinate dehydrogenase (SDH), and the terminal oxidase. NDH-1 is a proton-pumping NADPH/plastoquinone oxidoreductase and it is analogous to the mitochondrial Complex I; in Synechocystis, it is composed of 11 subunits encoded by the ndh genes3Synechocystis, respiratory electrons also enter the PQ pool via SDH, an enzyme composed by three subunits  which oxidizes succinate to fumarate, in a reaction similar to that occurring in mitochondria. We identified six NDH-1 subunits, all belonging to the hydrophilic catalytic domain; we found that when wild type Synechocystis was grown under prolonged darkness four subunits  were down-regulated in their expression. Furthermore, our data suggest that the remaining two NDH-1 subunits, Ndh-I (ndhI/sll0520) and Ndh-H (ndhH/slr0261) are affected in the same way, but they have been excluded from the final list of differentially expressed proteins either because the value is slightly higher than the established cutoff  or due to the biological variability among the three replicates (as in the case of Ndh-H). Moreover, collected data indicate that also the expression of the SDH flavoprotein subunit (sdhA/slr1233) is reduced, although it was not included in the final list because it did not completely meet the chosen criteria . Our data also indicate that two antioxidants, the iron superoxide dismutase (sodB/slr1516) and catalase-peroxidase (katG/sll1987), show a trend of increased abundance . However, based on our growth experiments, wild type Synechocystis can adjust better than ΔHoxE-H to a light-deprived environment. Thus, the presence of the Hox hydrogenase seems to give Synechocystis an adaptive advantage under such a stress condition, by contributing to mantaining a proper redox balance of the electron transport chains when both photosynthetic and respiratory machineries are down-regulated.The altered expression of all these proteins, in particular NDH-1 hydrophilic subunits, suggests a scenario where the overall respiration electron flow is somehow affected when Synechocystis grows in the dark require additional investigations to be interpretated, and are currently under way in our laboratory.Further analyses of overall proteome changes and a closer examination of the metabolic pathways that are altered when 2 metabolism. Increasing experimental evidence shows that they could have a key role in regulating electron flow pathways under stress conditions.The highly evolutionary conserved [NiFe]-hydrogenases are no longer considered simply primordial enzymes whose only function is HSynechocystis Hox bidirectional hydrogenase is essential to grow under prolonged darkness (either in anaerobic or aerobic conditions). Interestingly, we found that under this stress condition the expression levels of several hydrophilic NDH-1 subunits in the respiratory complex are reduced. Given the homology between the NAD(P)H oxidoreductase portion of the Hox hydrogenase and the NADH input module of mitochondrial Complex I, we hypothesize that the Hox hydrogenase can compensate for the reduction on the NDH-1 activity and thus allow Synechocystis to grow in the dark.In this work we show that in 2 metabolism, and contribute to explain the close evolutionary relationship between [NiFe]-hydrogenases and Complex I in the aerobic respiratory chain.Taken together, our data point toward an additional role for bidirectional Hox hydrogenase beside HHow to cite this article: De Rosa, E. et al. [NiFe]-hydrogenase is essential for cyanobacterium Synechocystis sp. PCC 6803 aerobic growth in the dark. Sci. Rep.5, 12424; doi: 10.1038/srep12424 (2015).


The relative biological effectiveness (RBE) was calculated from the initial slope of the dose–response curve for chromosome exchanges with respect to low dose and low dose-rate γ-rays (denoted as RBEmax), and relative to acute doses of γ-rays (denoted as RBEγAcute). The linear dose–response term was similar for all energies of protons, suggesting that the decrease in LET with increasing proton energy was balanced by the increase in dose from the production of nuclear secondaries. Secondary particles increase slowly above energies of a few hundred megaelectronvolts. Additional studies of 50 g/cm2 aluminum shielded high-energy proton beams showed minor differences compared to the unshielded protons and lower RBE values found for shielded in comparison to unshielded beams of 2 or 2.5 GeV. All energies of protons produced a much higher percentage of complex-type chromosome exchanges when compared to acute doses of γ-rays. The implications of these results for space radiation protection and proton therapy are discussed.We have investigated chromosome exchanges induced in human cells by seven different energies of protons (5–2500 MeV) with LET values ranging from 0.2 to 8 keV/μm. Human lymphocytes were irradiated  The study of the biological effectiveness of accelerated proton exposures is of interest for clinical treatment plans and for assessing normal tissue damage from protons of various energies that are generated outside of the Bragg peak during proton therapy –4. Protomax values for protons of energies above 10 MeV vary from <1 to about 2 in comparison to X-rays or γ-rays, whereas lower energy protons (<10 MeV) were significantly higher than unity and the values were LET dependent  and/or organization(s).2 of aluminum and 50 g/cm2 of aluminum plus 10 cm of polyethylene. At these proton energies, the dose increases as the protons pass through the shielding due to secondary radiation, and doses were normalized using BNL dosimetry to generate the same total dose to the sample as the unshielded studies.Whole blood was collected from healthy volunteers and was irradiated with accelerated protons using the NASA Space Radiation Laboratory (NSRL) facility at Brookhaven National Laboratory (BNL). The same volunteer donated the blood samples for each experiment. All samples were exposed in the plateau portion of the Bragg curves and dose rates were between 0.2 and 0.5 Gy/min, depending on the dose delivered. Doses were measured at the target using ionization chambers. Samples were exposed at room temperature. Each sample received at least three pulses and no exposure lasted more than 10 min. The beam uniformity was checked using a digital beam imager and dose did not vary more than 5% over the target area. For the 2.5 and 2 GeV protons exposures, the target areas was shielded, respectively, with 50 g/cmImmediately after exposure, whole-blood cultures in RPMI 1640 medium (Gibco BRL) supplemented with 20% calf serum and 1% phytohemagglutinin  were incubated at 37°C for 48–50 h. Chemically induced PPC were collected using the method described by Durante et al. , which rin situ with a combination of fluorescence whole-chromosome probes for chromosomes 1, 2, and 4, or chromosome 1, 2, and 5 (Rainbow Scientific) using the procedures recommended by the manufacturer. Chromosome 1 was painted with a Texas red fluorophore, chromosome 2 was painted with FTIC, and chromosome 4 (or 5) was painted with a 1:1 combination of Texas Red and FITC that appeared yellow under the triple-band-pass filter set. Unlabeled chromosomes were always counterstained with 4′,6-diamidino-2-phenylindole (DAPI).Chromosomes were dropped onto clean microscope slides and hybridized Chromosomes were analyzed on a Zeiss Axioplan fluorescence microscope. The images of all damaged cells were captured electronically using a Sensys charge-coupled device (CCD) camera  and the Cytovision computer software. The number of cells analyzed for each sample varied, exact numbers are listed in Table The frequency of chromosomal aberrations in the painted chromosomes was evaluated as the ratio between aberrations scored and total cells analyzed. Several studies have indicated that the distribution of radiation damage among chromosomes is random, and the yield of exchanges measured within the first division after exposure is proportional to the DNA content of the chromosome analyzed, with some fluctuation of data . TherefoStandard errors for aberration frequencies were calculated assuming Poisson statistics. Error bars in each figure represent SEs of the mean values. The data were modeled assuming binomial errors per number of chromosomes analyzed with the frequencies of aberrations of various types extrapolated to whole-genome equivalents as described above.γAcute, and from the ratio of initial slopes for γ-rays using our previous data  from the same volunteer used for the proton experiments. For complex exchanges, the low dose and dose-rate γ-rays, complex exchanges were rare and RBEmax estimates could not be made.A weighted linear-quadratic (LQ) or linear (L) regression model was used to fit dose–responses for each proton energy, and the γ-ray dose–responses. Using the maximum likelihood method, the linear and quadratic coefficients α and β inresponse , denotedous data –30 of loTables γAcute and RBE max models, respectively. However, a much higher frequency of complex exchanges was observed for each proton beam compared to γ-rays resulting in RBEs for complex exchanges varied from 2.1 to 4.1, and this led to a modest increase the RBEs for total exchanges. A trend toward increasing RBEmax values for proton energies of 1 GeV and higher was found for simple and total exchanges.The linear (α) coefficients from the dose–response data Tables –5 are si2 of aluminum, and the 2 GeV protons were shielded with 50 g/cm2 of aluminum plus 10 cm polyethylene. The doses represent the values measures at the target. A comparison of shielded and unshielded data shown in Figure max values were reduced with shielding. For example, RBE values for total exchanges induced by unshielded and shielded 2 GeV protons were 2.09 ± 0.34 and 1.26 ± 0.11, respectively, and values were 1.91 ± 0.67 and 1.53 ± 0.14 for unshielded and shielded 2.5 GeV protons, respectively.Data for the yield of chromosome exchanges in the shielded samples are listed in Table 2 for materials of interest. Because the nuclear absorption cross sections are similar, secondary particles and target fragmentation spectrum produced by protons and neutrons are nearly identical for energies above a few hundred megaelectronvolts. Thus, high-energy protons are biologically more effective than neutrons of the same energy per unit fluence because of the proton charge state, while high-energy neutrons will have higher effectiveness per unit dose.The similarity in frequency of simple and complex exchanges over a wide range of proton energies found in our experiments suggests that decreases in LET with increasing proton energy is balanced by the increase in doses from secondary radiation, most notably secondary protons and neutrons , 31, 32.2 of aluminum equivalent shielding. The additional shielding used in our experiments had a minor influence on the biological effectiveness when comparing unshielded high-energy proton beams because the secondary radiation produced behind the shielding will be of similar biological effectiveness as the primary beam, while similar numbers of low energy target fragments of high-LET produced for both the primary and secondary protons and neutrons will be produced for the shielded and unshielded beams.The NSRL beam-line and the sample holders provide a minimum of 1.2 g/cmThe α values for acute and LDR γ-rays fits indicate a dose-rate modification factor of 1.83 and 1.74 for total exchanges and simple exchanges, respectively. These values are similar to those reported for dose-rate reduction factors found by Peng et al. , dose anIn the present study, we used three-color combinations of fluorescence in situ hybridization (FISH) chromosome painting probes  to analyze our data. Presumably complex exchanges would be underestimated with this method due to the presence of some pseudosimple-type exchanges . AlthouIn conclusion, our study of the proton energy dependence of chromosome exchanges in human lymphocytes suggests that biological effects are similar over a wide range of proton energies (5–2500 MeV) with RBE values for total exchanges are close to unity when measured against acute γ rays, and approach 2 when measured against low dose rate γ rays due to the increased number of complex exchanges at all proton energies compared to γ-rays.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.


It was clearly confirmed that the investigated XLPE hip and knee implants, which were produced from different orthopaedic grade resins (GUR 1050 and GUR 1020), consisted of two structural regions in the as-received states: the near-surface transitional anisotropic layer (≈100 μm thickness) and the bulk isotropic structural region. These XLPEs exhibited a different crystalline anisotropy and molecular texture within the near-surface layers. In addition, the knee insert showed a slightly higher efficiency of shape recovery against the applied strain over the hip liner owing to a markedly higher percentage of the bulk amorphous phase with intermolecular cross-linking. The quantitative data presented in this study might contribute to construct manufacturing strategies for further rationalized structures as alternative bearings in THA and TKA.The present study investigated effects of processing procedures on morphology of highly cross-linked and re-melted UHMWPE (XLPE) in total hip and knee arthroplasty . The shape recovery behavior was also monitored via uniaxial compression test at room temperature after non-destructive characterizations of the in-depth microstructure by confocal/polarized Raman spectroscopy. The goal of this study was to relate the manufacturing-induced morphology to the  Increased incidence of periprosthetic osteolysis with increasing polyethylene wear has historically been the leading complication of total hip arthroplasty (THA), resulting in the growing necessity of revision surgery due to aseptic loosening of implant devices –3. In thThe kinematics of knee joint represents more anisotropic/constrained motions within small surface area of contact, leading to higher compressive and shear stresses as compared to that of hip joint. Under the applied loading of 3000 N (the entire joint load on the component), the maximum compressive stresses at the surface of UHMWPE components were computed by finite element analysis (FEA) as ≈15 MPa and ≈40 MPa for hip (using 28 mm head) and knee joint, respectively . Moreove in vivo motion of hip and knee joints. From this viewpoint, it would be of tremendous benefits to explore the effects of the manufacturing-induced local strain and plastic deformation on the initial molecular anisotropies in the final products for maximizing the potential wear performances.Although several manufacturing attempts in industry have been made so far to rationalize preferred choices of resin types (GUR 1050 or GUR 1020), consolidation methods , and radiation doses (50~300 kGy), consensus for alternative bearings has not been achieved yet among polymer scientists , 19. A kThe present investigation focuses on quantifying the molecular anisotropy and phase percentages such as crystalline, amorphous, and third intermediate phase along the depths of commercially available XLPE hip and knee joint prostheses by means of high spatial resolution Raman microscopy. In addition, the deformation and shape recovery behaviors from external uniaxial compressive forces were also monitored. The quantitative data obtained in this study might contribute to construct further improved manufacturing strategies for highly rationalized structures as alternative joint bearings for hip and knee arthroplasty applications and also to minimize the time-consuming manufacturing trials. in vivo performance and functionality of each type of prosthesis.The first-generation XLPE hip and knee implants were evaluated through the following quantitative analyses: (i) phase percentages, that is, amorphous, crystalline, and third intermediate phase; (ii) preferred orientation of molecular chains; (iii) degree of crystalline anisotropy; and (iv) deformation and shape recovery behaviors from applied uniaxial compression. We have also built a structural model based on the results of the above morphological characterizations in order to discuss the potentialn = 3 for each prosthesis) were analyzed in this study. The investigated hip and knee bearings are both referred to as the same trade name, XLPE, which were manufactured by Smith & Nephew Orthopedics, Inc. . XLPE has been clinically introduced since 2001 for THA and 2008 for TKA in USA. The thickness of these test components was 10 mm for the hip and 11 mm for the knee in posterior stabilized (PS) designs. A comparison of the processing procedures for each XLPE component was given in The never implanted, first-generation remelted XLPE acetabular liner and tibial insert (αa), orthorhombic crystal (αc), and third intermediate region (αt), nondestructive Raman spectroscopic analyses were conducted. The measured sections for each test sample, where intensive plastic deformation and wear would be expected to occur during the in vivo services [μm, respectively. Individual spectra were typically collected in 15 seconds. The recorded spectra were averaged over three successive measurements. The focal plane was eventually shifted toward the sample subsurface direction in order to nondestructively screen the depth regions of the samples. At each depth of samples, an inplane sampling of 2.5 μm lateral steps was applied within the area of 50 × 50 μm2. A total of 2205 different locations (= 441 points/map × 5 maps) were selected within the sections, and the average value was assumed to be representative of the phase fractions at each selected depth. The computations of these fractions were made according to the following equations [I is the integral intensity of the Raman band whose wavenumber is identified by the subscript. Note also that the sum (αa + αc) might locally be <1, because of the possible presence of an anisotropic intermediate state αa=Id phase” ). The voxlabylabzlab) describing the laboratory frame, a system (xpypzp) to describe the average preferential molecular orientation (as detected by the Raman probe of finite dimensions), and a system (xmolymolzmol) to describe the orientation of individual molecular chains with respect to the mean axes of preferential orientation. Euler angles for the above three Cartesian frames can be defined as a set of angle  describing the rotation of (xmolymolzmol) with respect to (xlabylabzlab), a set of angle  describing the rotation of (xpypzp) with respect to (xlabylabzlab), and a set of angle  describing the rotation of (xmolymolzmol) with respect to (xpypzp). Note that the  determines the preferential molecular orientation in the XLPE components and  determines the spatial distribution angles from preferential orientation . However, taking into consideration the cylindrical symmetries of the long linear polyethylene chains, the numbers of the orientation angles relevant to Raman scattering may be reduced. In such a symmetric case, we can neglect the dependences of any torsional orientation such as φ, φp, α, and γ. In this study, we set the angle θp, that is, the out-of-plane tilt angle, as the preferred molecular orientation. Specifically, θp = 0° indicates the alignment of C–C chains perpendicular to the articulating surfaces, while θp = 90° indicates the alignment of C–C chains parallel to the surfaces. In addition, we assumed the existence of a uniaxial symmetry with respect to the preferential orientation of the molecular chains, which is only dependent on one polar angle, β   can be analytically formulated as follows [f(β) is called orientational probability distribution or orientation distribution function (ODF).For the assessments of three-dimensional molecular orientation of the carbon chains  and the degree of crystalline anisotropy, polarized measurements combined with confocal Raman spectroscopy were conducted in the same sections where the phase percentages were examined. During the measurements, a parallel polarization filter and a half-wave plate were placed between samples and spectrometer and set to pass scattered Raman radiation horizontally to a CCD camera. To express the spatial position and distribution of the molecular orientation, three different Cartesian coordinate systems were defined as a system ∫γ=0χ-axis (inplane angle on the bearing surface) rotation jig and polarized Raman spectra in parallel configuration were collected at 19 different azimuthal angles within the interval 0 ≤ χ ≤ 180°, with sequential rotational steps of 10°  is the most affected by polyethylene orientation [I1130||) for XLPE products is provided by the following general equation [IAg+Bg1|| is the dependence of Raman intensity of a single polyethylene molecular chain in parallel polarization configuration according to its angular dependence on Euler angles, θ and χ (note that θ and χ are functions of θp and χp) [f(β), representative of the molecules oriented in a cone around the orientation axis  are the so-called Lagrange multipliers; that is, P2  (cosβ) = (3cos2β − 1)/2, P4  (cosβ) = (35cos4β − 30cos2β + 3)/8, used in the definition of the principle of maximum information entropy [χ). Setting a computational routine, θp and f(β) can be determined, and the degree of molecular orientation can be calculated using the following equation [P2(cosβ)〉 is referred to as Herman's orientation parameter and represents the degree of molecular orientation. A value 0 for it indicates that the molecular orientation is fully random (isotropic), while a value 1 represents a perfect orientation along a preferential orientation axis. For a partial molecular orientation, the value should be 0 < 〈P2(cosβ)〉 < 1.0.As the experimental procedures, XLPE samples were placed on a  Figures . Particuentation , 28 and equation :4)I113and χp) , and theaxis cf. , will ta entropy . A matheI113and χxis (cf. f(β)=Aexpequation , 31, 32:n = 3 for each liner) were obtained from each surface of these samples where the morphological assessments were performed by means of Raman spectroscopy. Deformation tests (cross-head speed of 0.1 mm/min) were performed using uniaxial compression equipment. The rectangular specimens were obtained from the sections corresponding to the Raman investigations. Particular care was taken in order to smooth down only the four corners of the slightly concave surfaces of the samples. This latter procedure enabled us to flatten the originally concave surfaces to match the compressive surface of the jig while preserving the original microstructure of the samples, thus reproducing as closely as possible the conditions encountered during in vivo loading. One-dimensional stress relaxation tests were also performed. The residual strain in each as-received material was assumed as ε = 0 and an increasing compressive load was applied by a 3% strain (180 μm) step. At each step, the materials were subjected to a compressive strain of a predetermined magnitude, which was kept constant for at least 24 hours in order to allow the full development of internal deformation allowed by the microstructure. The load was then released and the samples allowed recovering of the inelastic strain for at least 24 hours, a time interval sufficient to obtain a nearly full recovery, especially at low and moderate levels of deformation, as in the case of the present investigation [εi and εf, respectively.In the present investigation, a compressive deformation was applied at room temperature (24 ± 2°C) in the samples purposely prepared by cutting the unused XLPE acetabular liner and tibial insert into rectangular prisms 3 × 3 × 6 mm in dimension. These rectangular samples  as detected in XLPE hip liner and knee insert. Confocal Raman analyses confirmed that the phase fractions in both products rapidly change along the subsurface depths within the first 35 μm from the articulating surface, but subsequently show the nearly constant and homogeneous trends. The differences between minimum and maximum values along the αc and αt profiles  was observed in their final products. On the other hand, their bulk profiles exhibited about 9% difference in the αa and αt. XLPE knee showed a markedly higher amorphous phase percentage, that is, a lower third intermediate phase content, in its bulk region, as compared to the hip.Figures  Figures  were larI1130||) of XLPE hip and knee implants depending on the inplane sample rotation angle (χ) were plotted in Figures π or π/2 angular periodicities of I1130|| plots are indicative of a preferred molecular orientation (θp) nearly parallel or perpendicular to the articulating surfaces, respectively, while the constant intensity (no angular periodicity) trend implies the isotropic molecular structure (no preferred orientation).The normalized Raman intensities  within the depth of about 75 μm and the C–C chains gradually rotated toward the anterior-posterior axis with proceeding along the depth.In addition to the tilt angle of hape cf. . Thus, tf(β)) as determined at each depth of the hip and knee samples. The angle β represents orientation of individual molecular chains with respect to the mean preferential orientation (θp). Note that the molecular axis of β = 0 corresponds to the axis of θp. Therefore, a material with a higher anisotropy shows a narrower (shaper) shaped curve of ODF, while a lower anisotropic material represents a broader (flatter) ODF.Figures μm below the surface, while ODFs for the knee start from a much broader distribution at the surface and then rapidly increase up to the subsurface depth of 10 μm. Beyond the depth, both components showed a similar trend of ODFs, which were gradually getting broader with the increase in the subsurface depths.In the superficial layers of both components, quite different ODFs were obtained. In the hip liner, significantly narrow distributions of C–C chain orientation were found within the first 5 P2(cosβ)〉 was plotted in P2(cosβ)〉 takes a range of values from a minimum of 0 (isotropic) to a maximum of 1.0 (perfect orientation). P2(cosβ)〉 value was obtained as 0.68 ± 0.05. However, the knee surface showed a much lower value (〈P2(cosβ)〉 = 0.34 ± 0.03). The maximum values of 〈P2(cosβ)〉 were observed at the depths of 0 and 10 μm for the hip and knee, respectively, and these subsequently decreased down to 0 at the depth of 100 μm. In the subsurface regions from 10 to 100 μm, the knee insert had a slightly higher anisotropic structure.The degree of anisotropy 〈εf) as a function of externally applied strain (εi) for the hip and knee bearings. The full lines represent the fitting curves to the experimental data. The strain behaviors of these components before and after shape recovery were found to phenomenologically obey the quadratic functions as follows:εf) accumulation in the knee insert.μm below their surfaces and the lowest crystallinity (αc) was always detected in the very surfaces. Correspondingly, molecular chain orientation parallel to the articulating surface with high anisotropy was also observed within those regions. The above structural characteristics can be considered the main effects of decrystallization induced by surface finishing performed at the end process of manufacturing, that is, lathe grinding and polishing. According to our past study using Raman spectroscopy, the original bulk αc of GUR 1050 resin was reported as ≈50% in its nonirradiated and nonheated state [αc values detected in the bulk region of XLPE hip liner (αc = 45.04 ± 1.15%), a ≈5 vol.% loss of crystalline phase can be identified as a result of its fabrication processes. The gamma irradiation by itself might possibly destroy the crystalline regions at certain high doses but the 100 kGy irradiation does not substantially influence crystallinity change [αc over the GUR 1050 [αc level to the analyzed hip liner, indicating a total of ≈11 vol.% loss of crystalline phase during its remelting. Spiegelberg et al. [Confocal/polarized Raman spectroscopy allows quantitative and rigorous assessments of the three-phase percentages and crystallographic texture of XLPE hip and knee implants. It was clearly confirmed that all the microstructural parameters investigated in this study showed a transitional behavior from the surface down to the subsurface regions  of the overall thickness of each component. The anisotropic surfaces with the decreased crystallinity are a clear structural evidence for initial existence of plasticity layer formed in their manufacturing stages. It can be considered that the above two phases of the anisotropic surface and isotropic bulk structures are mainly responsible for wear and creep resistance, respectively. The mechanical properties of polyethylene are governed by (i) molecular weight (ii) balance among amorphous, crystalline, and third intermediate phase percentages; (iii) preferred molecular orientation; (iv) degree of crystalline anisotropy; (v) degree of cross-linking; and (vi) presence or absence of additives. The tested two prostheses had no preferential orientation in their bulks and contain no additives such as calcium stearate and antioxidant agent. Although, as explained above, the knee insert can possess a less cross-link density, it showed a slightly higher capacity of shape recovery, that is, a less accumulation of residual strain, against the applied uniaxial compressive load  UHMWPE under lubricated and unlubricated dry sliding conditions , 20, 43.On the other hand, Wang et al.  pointed After the procedure of remelting, a higher loss of crystallinity percentage was observed in the irradiated GUR 1020 resin used for the knee insert, as compared to the irradiated GUR 1050 for the hip liner.μm thickness) and the bulk isotropic structural region.It was confirmed that the studied hip and knee components consisted of two structural regions induced by the manufacturing procedures: the near-surface transitional anisotropic layer (≈100 The knee insert showed a slightly higher capacity of the shape recovery against the applied uniaxial compressive load over the hip liner owing to a markedly higher percentage of the bulk amorphous phase with cross-linking.Our present observations implied the possibility to further maximize their wear performances by the surface rearrangements of crystalline texture as the outcome of different technological regimes for surface machining and polishing during the production processes.The quantitative and nondestructive analyses of confocal/polarized Raman spectroscopy were applied to describe and compare the depth profiles of the three-phase percentages and crystalline anisotropy between XLPE hip and knee replacement implants of the first-generation highly cross-linked ultrahigh molecular weight polyethylene. The impacts of the surface and subsurface morphologies on plastic deformation and wear were explicitly discussed from the viewpoints of the molecular mobility and strain-softening resistance. The main outcomes of our investigation can be summarized as follows.


Tyrosine (Y)-recombinases have evolved to deliver mechanistically different reactions on a variety of substrates, but these evolutionary transitions are poorly understood. Among them, integron integrases are hybrid systems recombining single- and double-stranded DNA partners. These reactions are asymmetric and need a replicative resolution pathway, an exception to the canonical second strand exchange model of Y-recombinases. Integron integrases possess a specific domain for this specialized pathway. Here we show that despite this, integrases are still capable of efficiently operating the ancestral second strand exchange in symmetrical reactions between double-stranded substrates. During these reactions, both strands are reactive and Holliday junction resolution can follow either pathway. A novel deep-sequencing approach allows mapping of the crossover point for the second strand exchange. The persistence of the ancestral activity in integrases illustrates their robustness and shows that innovation towards new recombination substrates and resolution pathways was a smooth evolutionary process. The integron integrases have evolved to perform recombination of single and double stranded DNA. Here the authors show that the ancestral pathway is still functional at double stranded sites, revealing the evolution towards the modern resolution pathway. Tyrosine (Y)-recombinases mediate recombination between two specific DNA sites to deliver a variety of biological functions (for a review see ref. ). They a156attC site in the cassette and the attI site in the integronattC sites. These sites show no sequence conservation, except for the crossover point on the bottom strand (bs) (5′- AAC -3′), that provides microhomology with attI sites, necessary for strand transferattC sites share a conserved palindromic structure, and are only recognized as a folded single strand (ss)attC and the double strand (ds)-attI site forms an asymmetric HJ (aHJ)    that cans (EHBs)  that aressDNA is central to the integron, also controlling integrase expression through the SOS responseattC sites have been extensively studied17192027attI sites. These are more canonical than attC sites, since they are recognized as dsDNAattI sites possess accessory binding domainsWhile attI sites by sequence and ss-attC sites through structural landmarks. From an evolutionary standpoint, this duality could reflect a single specialized activity (the recombination of ss/ds DNA partners) or two distinct ones: the canonical Y-recombinase activity of integrase ancestors on attI, and the functional innovation, that is, the recombination of ssDNA structures, on attC sites. For the latter to be true, both classical and innovative activities must be complete: integrases should be capable of resolving HJs through a second strand exchange.Integrase site recognition underscores a remarkable conformational dynamismattI sites. This is a symmetrical reaction involving exclusively ds partners for which resolution through a second strand exchange is theoretically possible, since it is neither abortive nor impeded by EHBs. We show that the top strand of attI1 is reactive and that HJ resolution can take place through a second strand exchange. We have determined the crossover point through a novel deep-sequencing approach that is applicable to other recombination systems, and validated this approach in vitro. Finally, we prove that attC sites govern the directionality of the reaction. To our knowledge, integrases are the first example among recombinases of a smooth evolutionary transition between substrates and processing pathways. We discuss the possible origins and the evolutionary implications of the dual activity of integrases, as well as the importance of attI1 × attI1 reactions in antibiotic resistance.To unmask the ancestral activity of integrases, we have studied the reaction between two attI1, the integration site of the most widespread and best-studied integron platform, the Class 1 integron.For this work we chose to study attI1 site possesses two direct repeats upstream the L–R boxes that make the site ∼65-bp longattI1 × attI1 HJ, biasing our results. To assess the impact of such host machinery, we have tested the influence of RecA and two well-characterized HJ-processing complexes, RecG33attI1 × attI1 reaction  so that the restriction pattern of cointegrates reveals which strand is transferred during recombination  strain containing an attI-bearing pSU plasmid (p929) as well as a plasmid with the integrase under the control of a PBAD promoter (pBAD-intI1). The mutS− background ensures that the mismatches are not repaired. By selecting for the circle resistance marker, we recovered clones in which these molecules had recombined with the resident pSU plasmid through an integrase-mediated attI1 × attI1 reaction. The mismatch-containing region was PCR amplified from 100 recombinant clones and used for the restriction pattern analysis  and traanalysis , revealiattI1 site could be transferred during the same reaction, we used a modified version of the dapA recombination reporterattI1 sites leads to reconstitution of the essential gene dapA, allowing recombinants to grow on media lacking diaminopimelic acid . Comparison of the number of clones growing with and without DAP yields a recombination frequency for a given reaction. We produced two versions of this reporter system, one with two attI1 sites in direct orientation, and the other with sites in inverted orientation. Both structures were inserted ectopically in single copy in the chromosome of an E. coli MG1655 ΔdapA strain. The rationale of this experiment is that, when both sites are in inverted orientation, viable recombinants can only be recovered if HJ resolution takes place through a second strand exchange that inverts the region between both sites. In this orientation the replicative resolution pathway linearizes the chromosome and is hence abortive  (To avoid expression of ttISTOP) . As a coattC sites, attCaadA7 and attCereA2  in which the central structure was found inverted showed that the site had not been recombined in a double-stranded form. Indeed, the resulting sites were largely conserved, with the presence of moieties of the original sites only visible after the canonical 5′- AAC -3′ cleavage point. Although these inversion events require a second cleavage that could not be elucidated, and therefore seem to be the consequence of illegitimate recombination reactions (possibly between sites on different replicating chromosomes), they are strong proof that attC sites are exclusively recombined in their structured form and confirms previous observationsA similar experiment was performed using two ttCereA2 . When siattI1 site in direct and inverted orientation from 5′- AAC -3′ to 5′- AAA -3′  of pSW-attI1N plasmids to be used in our suicide conjugation assay. The rationale of this experiment is that the pattern of segregation of random bases between the two resulting sites in the cointegrate molecule will reveal the precise crossover point in the attI top strand (L box). Furthermore, since base composition of the L box can influence IntI1 binding, some attI mutants might be poorly recognized. A skew in the proportion of bases in a given position after recombination would underscore the importance of a specific base in the binding process  (CTAAA -3′), in accordance with previous interference data for the central T baseattI1 × attI1 reaction in vitro using purified MBP-IntI1 protein, which is known to be fully active in vivoattI1 fragments bearing a mismatch between the top and the bottom strand in the adenine that is transferred during the reaction   . SequencMethods) . It alsoGTT -3′) . Such a ibrary n= of pSW-aattC site folding, the inverted repeats of the dsDNA molecule hybridize to form the R and L boxes in the hairpin  and only 1 (0.16%) had recombined through the L box with the top strand of attI1  suggesting a role  for the complementary A base in avoiding L-box recombination of ts attC sites. The crossover point in these recombinants could not be determined precisely due to an adenine stretch, but it was not coherent with the cleavage site determined previously for the attI1 L box. This is probably related to conformational differences between attI and attC sites that force the crossover to shift. This need for a different crossover point could be itself at the basis of the low frequency of L-box events in the attI × attC reaction.During  hairpin . These bof attI1 . Since tattI sites at frequencies matching those of the bottom strand and that there is no order in the use of strands during the reaction. The crossover point in the top strand of the attI site has been elucidated using a novel technique that involves deep sequencing. This approach has allowed overcoming technical difficulties found with classical in vitro methods due to the low recombination activity observed. With deep sequencing becoming more accessible, this technique could be useful for others working with recombinases that show low activity or for which optimal conditions in vitro have not been elucidated.In this work we show that integron integrases can perform the second strand exchange in reactions involving exclusively double-stranded sites. We found that integrases recombine the top strand of attI × attI reactions, with broad evolutionary implications that are discussed below. Still, other aspects of the attI–IntI interaction remain unclear. attI sites are recognized for their sequence, as proven by cognate IntI–attI recognitionin vitro experiments a cooperative binding starting at the R box. It is also known that secondary recombination sites contain the 5′- AAC -3′ signature of the R box of all attI sitesattI sites and would not allow integrase discrimination of cognate sites. Also, the sequence of the attI1 bears a 5′- AAC -3′ triplet at the border of the L box, but the crossover point occurs between different bases (A/A) compared with that of the bottom strand (A/C), suggesting that topology is more important than sequence in the cleavage of the top strand. Therefore, the relative influence of the universal crossover triplet and of the rest of the R box in the binding remains a subject of interest that would need further studying.Our work reveals surprising aspects of the dynamics of attC sites is not due to the mere presence of the integrase-specific I2 α-helix, but rather to its interaction with attC sites. The extremely low frequencies at which attCaadA7 recombines through the L box highlights the strong constraints that attC sites impose on the reaction. It is noteworthy that in natural conditions L-box reactions between attC and attI sites would place the attI site downstream of the cassette. Hence, such tight control on directionality likely serves to ensure the correct functioning of the integron by keeping the attI site next to the Pc promoter after consecutive cassette insertions.This work shows that the avoidance of the second cleavage on attI sites has an unclear adaptive meaning. It could be argued that in chromosomal integrons these reactions have deleterious effects on bacterial fitness, since they lead to the formation of chromosomal dimers that have to be further resolved before cell division. This is probably the reason for the 3–4 orders of magnitude lower recombination frequency for attI × attI reactions compared with those involving attC sites. Nevertheless, in the multicopy world of mobile integrons, one cell can contain more than one integron on the same or different replicons4142attI × attI reaction can lead to the en bloc transfer of cassette arrays between integrons. This would alter the expression levels of multiple antimicrobial resistance genes at once, and could have important effects on chemotherapy outcomes. It could be argued that the importance of such rearrangements is probably minor, due to the low recombination frequencies for attI × attI in laboratory conditions. Nevertheless, one aspect of this reaction that has been constantly overlooked is that, while attC sites are normally in their non-recombinogenic form, attI sites are recombinogenic during almost the entire cell cycle, with the brief exception of the passage of the replication fork. Despite this low recombination frequency, it is plausible that attI × attI reactions are more prevalent among mobile integrons than previously envisaged, especially under transient, low-level (or even stochastic) expression of the integrase. These expression pulses typically occur during horizontal gene transfer events36From a functional perspective, the reaction between Vibrio cholerae superintegronThis work shows that despite millions of years of evolution towards a system specialized in structured ssDNA recombination, and the acquisition of a 20-residue-long domain within the catalytic core, integron integrases have conserved the activity of their canonical Y-recombinase ancestors. Many examples of DNA breakage and rejoining enzymes acting on dsDNA (like Y-recombinases) or ssDNA  on the initial site. From this starting point, the streamlining through natural selection, to reach what integrons are today, seems a more straightforward process.From an evolutionary perspective, the functional innovation of integrases towards the recombination of folded ss-Vibrio cholerae. Nevertheless, CTXφ cannot be considered a complete system since it does not encode an integrase but instead hijacks the host XerCD recombinases50To date, the only recombination reaction that shares some similarities with cassette recombination is the integration of phage CTXφ of attI site. Nevertheless, the classical recombination pathway could have been lost through the specialization of integrases towards the single strand exchange pathway, without affecting any of the known aspects of integron dynamics. Therefore, preservation of this pathway after eons of evolutionattI sites occur at similar frequencies  at 37 °C. Growth at 42 °C was used to induce the expression and force the loss of the plasmid bearing lambda phage integrase. Antibiotics were used at the following concentrations: carbenicillin (Ap), 100 μg ml−1, chloramphenicol (Cm), 25 μg ml−1, kanamycin (Km), 25 μg ml−1, spectinomycin (Sp), 40 μg ml−1, Thymidine (Thy) and DAP were supplemented when necessary to a final concentration of 0.3 mM. Glucose and L-arabinose were added at 10 and 2 mg ml−1 final concentration, respectively. Chemicals were obtained from Sigma-Aldrich (France).E. coli DH5α, MG1655 (laboratory collection), П1, and β2163  and MacPymol on templates c2a3vA (VchIntIA) and c1ma7A (Cre). Sequence analysis was performed using Geneious 7.1.attI × attI reaction, we performed a suicide conjugation assay based on that of Biskri et al.et al.attI site provided by conjugation is carried on a suicide vector from the R6K-based pSW family that is known to use the Pir protein to initiate its own replication. This plasmid also contains an RP4 origin of transfer (oriTRP4). The donor strain β2163 carries the transfer functions in its chromosome, requires DAP to grow in rich medium and can sustain pSW replication through the expression of a chromosomally integrated pir gene. The MG1655 recipient strains, which contain the pBAD::intI1 [ApR] (expressing the IntI1 integrase) and the pSU38Δ::attI1 [SpR] (carrying the attI1 site), lacks the pir gene and therefore cannot sustain replication of the incoming attI-containing pSW vector. The only way for this vector to be maintained in the recipient cell is to form a cointegrate by attI × attI recombination. We used the ω10001 strain as the donor and as recipient strains the MG1655 E. coli ωA197 (control), and its derivatives ω9987 (recA-), ω9988 , ω9989  and ω9990  .To test whether RecA, RecG and RuvABC impact the ruvABC-) . The recattC sites in plasmids p7770 and p7771 for attI1 sites, giving rise to plasmids p8669 and p8670  and top strand (oriFd2)). Single strand DNA production was performed using M13K07 Helper Phage as in ref. F′ carrying pir strain cells  to respectively obtain the ω8675 and ω8676 strains. M13 infection and purification of ssDNA were performed according to the manufacturer (M13K07 Helper Phage and QIAprep Spin M13 kit). The complementary single strand DNA molecules were annealed, digested by both EcoRI and MfeI restriction enzymes to eliminate oriFd , and self-ligated.Construction of the p8669 and p8670 phagemid vectors to produce mismatched covalent circles was performed as in ref. attI site into MG1655mutS215 strain containing the pBAD::intI1 (p3938) and the pSU38Δ::attI1 (p929) plasmids (strain ω7994). Circles cannot replicate in this genetic background (ω7994). Note that this assay was first envisaged in a mutS-, recA-strain but transformation efficiency was too low due to a filamenting (sick) phenotype in the double mutant. We hence studied the influence of recA and found it to be responsible for <0.5% of all recombinants in our assay (see below recombination frequencies with and without pBAD::intI1). This justified the use of a recA+ strain neglecting the influence of homologous recombination in the recombination process. Competent cells were prepared in the presence of 0.2% arabinose to allow integrase expression. Selection on chloramphenicol (Cm)-containing plates (the mismatched circles marker) yielded, almost exclusively, attI × attI recombination events. The attI × attI cointegrate formation was checked on 101 clones by PCR with SeqattI and SeqNar primers. These PCR products include the mismatch-containing region, and were analysed for their resistance or sensitivity to both SacII and NarI restriction enzymes.This assay was developed previously in the laboratorymutS215 strain containing the pSU38Δ::attI1 but lacking pBAD::intI1 (strain ωA266). To establish a recombination frequency, we transformed in parallel the same mutS strain lacking both the pSU38Δ::attI1 and pBAD::intI1 plasmids but containing a Pir-expressing plasmid  ensuring the replication of the incoming circle (strain ω7120). Recombination activity corresponds to the ratio of CmR clones obtained in pir− conditions (with and without integrase) to those obtained in pir+ conditions. Note that the efficiency of transformation of each strain was determined beforehand and used to adjust the final ratio and normalize the results. We obtained a recombination frequency of 1.17 × 10−3 in presence of integrase, and of 4.24 × 10−6 in the absence of integrase.As a control, we performed a similar experiment using an MG1655R clones obtained in pir+ context (pir+ competent cells (ω1628). Then, 101 transformants were analysed by SacII and NarI restriction and/or by sequencing (using SeqNarI and SeqSacII primers). The segregation pattern was, as indicated in As a supplementary control, we analysed the Cm context . After pdapA gene allowing for growth in media not supplemented with DAP28in silico and synthesized by GeneART (Life Technologies). To avoid background noise (expression and translation of dapA) before recombination, the right attI site (the one immediately upstream dapA) was modified to include a STOP codon 1 bp upstream of the L box (attI site and the PndmA promoter (registry part J23100) that drives transcription of dapA after recombination. Through XhoI/SmaI double digestion we inverted the left site to obtain the inverted orientation. These constructions were subsequently cloned EcoRI/NruI into the pir-dependent plasmid pA669 (SpR) containing a lambda phage attP site, and transformed in П1 pir+ competent cells (ω1628). A FRT recombination site was added in EcoRI to allow for the resolution of dimers after integration in the chromosome. Sequence was verified and these plasmids were transformed in the ω8488 strain, an MG1655 recA- dapA- E. coli strain containing the replication/expression-thermosensitive plasmid p3153 bearing the lambda phage integrase (ApR). In this strain, pir-dependent plasmids do not replicate and are only maintained through their integration in the lambda attB site of the chromosome. Integration events where selected for using the Sp resistance marker of the plasmid. Loss of the p3153 plasmid after overnight incubation at 42 °C was verified by replica plating on media containing carbenicillin (selecting for the ApR marker of p3153). Monomeric integration was verified through PCR with primers (1329 and 1698). Sequence was verified in all strains produced. Strains ωB36 and ωB37 containing the constructions in direct and inverse orientation were selected for the study and transformed with the pBAD::intI1 (p3938) plasmid giving rise to strains ωB82 and ωB83. To test the recombination rate of the genetic structures, strains were grown overnight in LB supplemented with DAP, carbenicillin, spectinomycin and glucose, then diluted 1:50 into fresh media containing DAP, carbenicillin (to avoid the loss of p3938) and glucose or arabinose to repress or induce the expression of the intI1, and incubated for 3 h at 37 °C with shaking. Cultures were then plated on selective media with and without DAP. The recombination frequency is calculated as the ratio of colonies growing without DAP (recombined) to the total number of colonies growing on DAP-supplemented media. Recombination frequencies correspond to the average of at least three independent assays.In this experiment we used an assay in which recombination between two sites leads to the reconstitution of the essential he L box . The desattI site bore a C to A mutation in the cleavage site, we performed site-directed mutagenesis through overlap extension PCR combining primers attI CxA R and attI GxT F with primers XhoI F and XhoI R and using pA873 as template. The PCR product was cloned in both orientations in plasmid pA873 using XhoI restriction sites, giving rise to plasmids pC131 and pC132. These plasmids were inserted in the chromosome of ω8488 as described above and verified clones (strains ωC139 and ωC140) were further transformed with the pBAD::intI1 (p3938) plasmid to obtain strains ωC162 and ωC163. Recombination rates were measured as above.To obtain constructions in which the left recombination attIN sites in the pir-dependent pSW plasmid we combined primer 2915 with primers 2916–2925, obtaining 10 attIN sites, each with a random base at a given position. These sites were mixed, digested XhoI, PstI, cloned into the pSW plasmid pC066 (SpR) and transformed into П1 pir+ competent cells (ω1628). >5,000 transformants were mixed to establish the library (ωC351). Sequencing of random clones showed that 18 out of 20 had bases other than WT in the expected positions, validating the library , giving rise to pC252. Strain ω4826 (MG1655 recA−) was transformed with pC252 and p3938 (pBAD::intI1) (ωC307). As controls of this experiment we included a strain transformed with p929 instead of pC252 (ωC305) and strains without the p3938 (ωC304 (ω4826/p929) and ωC306 (ω4826/pC252)).To force L-box recombination, the −4 in the presence of arabinose (inducing the expression of the integrase), and of 1.88 × 10−6 in the presence of glucose, confirming the IntI1-mediated recombination of pSW-attIN into pSU-attIAAA. More than 7,000 colonies from the conjugation assay were collected (ωC453 to ωC455) and subjected to plasmid extraction. As a control, conjugation assays with ωC373 and ωC305 (pSU-attI) were performed, showing similar recombination frequencies: 1.64 × 10−4 with arabinose and 2.59 × 10−6 with glucose.The library was delivered through conjugation from ωC373 to ωC307. Recombination frequency was of 2.63 × 10attI sites from the library and the recombination products were PCR amplified using primers 1236-attIN's right (library); and 1236-738 and MRV- attIN's right (recombinant sites). PCR products were used for sequencing on an Illumina MySeq. Resulting sequences were filtered to discard abnormal recombination events and further analyzed using Galaxy  and IGV 2.3 (https://www.broadinstitute.org/igv/home). Base frequency was analysed at every position in the library and the recombinant sites. The expected frequency of bases in each randomized position is of 92.5% of wild-type base and 2.5% of every other base. This is because for a given position only 1 out 10 sites bears a random base (90% WT-10% N) and 25% of random bases are wild type (92.5% WT and 7.5% not WT). The observed base frequency composition in the library before recombination matched the expected distribution. Sequencing of the resulting sites after recombination showed a split distribution of randomized bases in both sites, allowing to infer the crossover point.The Y312F by cloning in the commercial vector pMAL-CX5 and transforming E. coli Top10 strain. Plasmids were further purified and transformed in the BL21 strain for protein production. Colonies from this transformation were collected and grown overnight in 25 ml of LB containing carbenicillin and 0.2% glucose. After incubation, cells were collected by centrifugation at 4 °C, resuspended in 25 ml of fresh media, and inoculated in 3 l of LB containing carbenicillin. When the culture reached an OD= 0.6, IPTG was added at 0.3 mM and the culture was incubated at 14 °C overnight. The cells were then collected by centrifugation and frozen. Cells were thawed to room temperature under cold tap water, and lysed by passage through an Emulsiflex C-5 cell breaker (Avestin Europe GmBH) at 13,000 p.s.i. at 4 °C. The cell lysate was centrifuged at 23,500g for 40 min in an RC-5C using an ss-34 rotor . The IntI1-MBP fusion protein was purified with 5 ml Amylose column  on an AKTA-prime FPLC machine .Translational fusions with a maltose-binding protein were obtained of IntI1 and the catalytically inactive IntI132P gamma ATP for 1 h at 37 °C. Once radiolabelled, top and bottom strands were then annealed (85 °C for 3 min and at room temperature for overnight). The strand transfer assay was performed using conditions derived from refs 2, 100 mM NaCl, 10 mM DTT). Samples were incubated for 2 h at 37 °C. After reaction the products were treated 1 h at 55 °C with proteinase K (Promega) 1 mg ml−1 final concentration and deproteinized using phenol/choloroform/isoamyl-alcohol (24/25/1 v/v/v). Aqueous phase was run on 12% polyacrylamide gel at 1,000 V for 5–6 h. Gel was then autoradiographied and quantified using Image J software  and R′′ (ts) and the corresponding mean deviations were deduced by determining the orientation of cassette integration for at least 20 independent clones for each bs transfer  and for 100 clones in the ts transfer assays. The orientation of cassette integration was determined by performing two PCR reactions: the first reaction with SW23begin/MFD primers produced a product only when the recombination took place in the delivered attC strand ; the second reaction with SW23end/MFD primers produced a product only when the recombination took place in the resynthesized attC strand . In addition, the recombination in the L box was detected by performing two PCR reactions on all clones. The first reaction with RpLp/571 produced a product when the recombination took place in the L′ box (340 bp); the second reaction with RppLpp/571 produced a product when the recombination took place in the L′′ box (340 bp). Recombination through the L′L′′ box of attC sites was only detected when the top strand was delivered (data for bs is hence not shown).This experiment is based on the suicidal conjugation assay explained above. This time the plasmids delivered by conjugation carry the top or bottom strand of the wild type or the ΔT23 Accession codes: The crossover point sequencing data for the library, left site and right site has been deposited at the Sequence Read Archive under the accession code SRP071193.How to cite this article: Escudero, J. A. et al. Unmasking the ancestral activity of integron integrases reveals a smooth evolutionary transition during functional innovation. Nat. Commun. 7:10937 doi: 10.1038/ncomms10937 (2016).Supplementary Figures 1-5, Supplementary Tables 1-3 and Supplementary ReferencesReplicative resolution of the HJ formed by attI1 sites in inverted orientation. In this orientation, the replicative resolution of the HJ after only one strand exchange linearizes the replicon and is abortive.Classical resolution pathway of the HJ formed by attI1 sites in inverted orientation. Recombination between attI1 sites in inverted orientation is productive if resolved through a second strand exchange. The DNA segment between both sites is inverted after recombination, as highlighted by the color code of both strands.