Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

Melting of an Anchored Bilayer: Phase Transitions in the Organic-Inorganic Hybrid Pervoskite (CH3NH3)(CH3(CH2)(n)NH3)(2)Pb2I7 (n=11, 13, 15, 17)

The conformation, organization, and phase transitions of alkyl chains in organic-inorganic hybrids based on the double pervoskite-slab lead iodides, (CH3NH3)(CH3(CH2)(n)NH3)(2)Pb2I7 (n = 11, 13, 15, 17) have been investigated by X-ray diffraction, calorimetry, and infrared vibrational spectroscopy. In these hybrid solids, double pervoskite (CH3NH3)Pb2I7 slabs are interleaved with alkyl ammonium chains with the anchored alkyl chains arranged as tilted bilayers and adopting a planar all-trans conformation at room temperature. The (CH3NH3)(CH3(CH2)(n)NH3)(2)Pb2I7 compounds exhibit a single reversible phase transition above room temperature with the associated enthalpy change varying linearly with alkyl chain length. This transition corresponds to the melting in two-dimensions of the alkyl chains of the anchored bilayer and is characterized by increased conformational disorder of the methylene units of the chain and loss of tilt angle coherence leading to an increase in the interslab spacing. By monitoring features in the infrared spectra that are characteristic of the global conformation of the alkyl chains, a quantitative relation between conformational disorder and melting of the anchored bilayer is established. It is found that, irrespective of the alkyl chain length, melting occurs when at least 60% of the chains in the anchored bilayer of (CH3NH3)(CH3(CH2)(n)NH3)(2)Pb2I7 have one or more gauche defects. This concentration is determined by the underlying lattice to which the alkyl chains are anchored.

The Exeter Contemporary flanged cemented acetabular component in primary total hip arthroplasty

Aims: We report on the outcome of the Exeter Contemporary flanged cemented all-polyethylene acetabular component with a mean follow-up of 12 years (10 to 13.9). This study reviewed 203 hips in 194 patients. 129 hips in 122 patients are still in situ; 66 hips in 64 patients were in patients who died before ten years, and eight hips (eight patients) were revised. Clinical outcome scores were available for 108 hips (104 patients) and radiographs for 103 hips (100 patients). Patients and Methods: A retrospective review was undertaken of a consecutive series of 203 routine primary cemented total hip arthroplasties (THA) in 194 patients. Results: There were no acetabular component revisions for aseptic loosening. Acetabular revision was undertaken in eight hips. In four hips revision was necessitated by periprosthetic femoral fractures, in two hips by recurrent dislocation, in one hip for infection and in one hip for unexplained ongoing pain. Oxford and Harris hip scores demonstrated significant clinical improvement (all p < 0.001). Radiolucent lines were present in 37 (36%) of the 103 acetabular components available for radiological evaluation. In 27 of these, the line was confined to zone 1. No component had migrated. Conclusion: Kaplan–Meier survivorship, with revision for aseptic loosening as the endpoint, was 100% at 12.5 years and for all causes was 97.8% (95% confidence interval 95.6 to 100) when 40 components remained at risk. The Exeter Contemporary flanged cemented acetabular component demonstrates excellent survivorship at 12.5 years. Take home message: The Exeter Contemporary flanged cemented acetabular component has excellent clinical outcomes and survivorship when used with the Exeter stem in total hip arthroplasty.

Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials

Background We aimed to assess the effect of afatinib on overall survival of patients with EGFR mutation-positive lung adenocarcinoma through an analysis of data from two open-label, randomised, phase 3 trials. Methods Previously untreated patients with EGFR mutation-positive stage IIIB or IV lung adenocarcinoma were enrolled in LUX-Lung 3 (n=345) and LUX-Lung 6 (n=364). These patients were randomly assigned in a 2:1 ratio to receive afatinib or chemotherapy (pemetrexed-cisplatin [LUX-Lung 3] or gemcitabine-cisplatin [LUX-Lung 6]), stratified by EGFR mutation (exon 19 deletion [del19], Leu858Arg, or other) and ethnic origin (LUX-Lung 3 only). We planned analyses of mature overall survival data in the intention-to-treat population after 209 (LUX-Lung 3) and 237 (LUX-Lung 6) deaths. These ongoing studies are registered with ClinicalTrials.gov, numbers NCT00949650 and NCT01121393. Findings Median follow-up in LUX-Lung 3 was 41 months (IQR 35–44); 213 (62%) of 345 patients had died. Median follow-up in LUX-Lung 6 was 33 months (IQR 31–37); 246 (68%) of 364 patients had died. In LUX-Lung 3, median overall survival was 28·2 months (95% CI 24·6–33·6) in the afatinib group and 28·2 months (20·7–33·2) in the pemetrexed-cisplatin group (HR 0·88, 95% CI 0·66–1·17, p=0·39). In LUX-Lung 6, median overall survival was 23·1 months (95% CI 20·4–27·3) in the afatinib group and 23·5 months (18·0–25·6) in the gemcitabine-cisplatin group (HR 0·93, 95% CI 0·72–1·22, p=0·61). However, in preplanned analyses, overall survival was significantly longer for patients with del19-positive tumours in the afatinib group than in the chemotherapy group in both trials: in LUX-Lung 3, median overall survival was 33·3 months (95% CI 26·8–41·5) in the afatinib group versus 21·1 months (16·3–30·7) in the chemotherapy group (HR 0·54, 95% CI 0·36–0·79, p=0·0015); in LUX-Lung 6, it was 31·4 months (95% CI 24·2–35·3) versus 18·4 months (14·6–25·6), respectively (HR 0·64, 95% CI 0·44–0·94, p=0·023). By contrast, there were no significant differences by treatment group for patients with EGFR Leu858Arg-positive tumours in either trial: in LUX-Lung 3, median overall survival was 27·6 months (19·8–41·7) in the afatinib group versus 40·3 months (24·3–not estimable) in the chemotherapy group (HR 1·30, 95% CI 0·80–2·11, p=0·29); in LUX-Lung 6, it was 19·6 months (95% CI 17·0–22·1) versus 24·3 months (19·0–27·0), respectively (HR 1·22, 95% CI 0·81–1·83, p=0·34). In both trials, the most common afatinib-related grade 3–4 adverse events were rash or acne (37 [16%] of 229 patients in LUX-Lung 3 and 35 [15%] of 239 patients in LUX-Lung 6), diarrhoea (33 [14%] and 13 [5%]), paronychia (26 [11%] in LUX-Lung 3 only), and stomatitis or mucositis (13 [5%] in LUX-Lung 6 only). In LUX-Lung 3, neutropenia (20 [18%] of 111 patients), fatigue (14 [13%]) and leucopenia (nine [8%]) were the most common chemotherapy-related grade 3–4 adverse events, while in LUX-Lung 6, the most common chemotherapy-related grade 3–4 adverse events were neutropenia (30 [27%] of 113 patients), vomiting (22 [19%]), and leucopenia (17 [15%]). Interpretation Although afatinib did not improve overall survival in the whole population of either trial, overall survival was improved with the drug for patients with del19 EGFR mutations. The absence of an effect in patients with Leu858Arg EGFR mutations suggests that EGFR del19-positive disease might be distinct from Leu858Arg-positive disease and that these subgroups should be analysed separately in future trials.

Pelon ja toivon välissä : Pätkätyöläisen urakerronta

This study analyzes the forming of the occupational identity of the well-educated fixed-term employees. Fixed-term employment contracts amongst the well-educated labour force are exceptionally common in Finland as compared to other European countries. Two groups of modern fixed-term employees are distinguished. The first comprises well-educated women employed in the public sector whose fixed-term employment often consists of successive periods as temporary substitutes. The other group comprises well-educated, upper white-collar men aged over 40, whose fixed-term employment careers often consist of jobs of project nature or posts that are filled for a fixed period only. Method of the study For the empirical data I interviewed 35 persons (26 women and 9 men) in 33 interviews, one of which was conducted by e-mail and one was a group interview. All the interviews were electronically recorded and coded. All the interviewees have two things in common: fixed-term employment and formal high education. Thirteen (13) of them are researchers, four nurses, four midwives, four journalists, and ten project experts. I used the snowball method to get in touch the interviewees. The first interviewees were those who were recommended by the trade unions and by my personal acquaintances. These interviewees, in turn, recommended other potential interviewees. In addition, announcements on the internet pages of the trade unions were used to reach other interviewees. In analysing process I read the research material several times to find the turning points in the narrative the interviewees told. I also searched for the most meaningful stories told and the meaning the interviewees gave to these stories and to the whole narrative. In addition to that I paid attention to co-production of the narrative with the interviewees and analyzed the narrative as performance to be able to search for the preferred identities the interviewees perform. (Riesman 2001, 698-701). I do not pay much attention to the question of truth of a narrative in the sense of its correspondence with facts; rather I think a working life narrative has two tasks: On the one hand one has to tell the facts and on the other hand, he/she has to describe the meaning of these facts to herself/himself. To emphasize the double nature of the narrative about one’s working life I analyzed the empirical data both by categorizing it according to the cultural models of storytelling (heroic story, comedy, irony and tragedy) and by studying the themes most of the interviewees talked about. Ethics of the study I chose to use narrative within qualitative interviews on the grounds that in my opinion is more ethical and more empowering than the more traditional structured interview methods. During the research process I carefully followed the ethical rules of a qualitative research. The purpose of the interviews and the research was told to the interviewees by giving them a written description of the study. Oral permission to use the interview in this research was obtained from the interviewees. The names and places, which are mentioned in the study, are changed to conceal the actual identity of the interviewees. I shared the analysis with the interviewees by sending each of them the first analysis of their personal interview. This way I asked them to make sure that the identity was hidden well enough and hoped to give interviewees a chance to look at their narratives, to instigate new actions and sustain the present one (Smith 2001, 721). Also I hoped to enjoy a new possibility of joint authorship. Main results As a result of the study I introduce six models of telling a story. The four typical western cultural models that guide the telling are: heroic story, comedy, tragedy and satirical story (Hänninen 1999). In addition to these models I found two ways of telling a career filled with fixed-term employments that differ significantly from traditional career story telling. However, the story models in which the interviewees pour their experience locates the fixed term employers work career in an imagined life trajectory and reveals the meaning they give to it. I analyze the many sided heroic story that Liisa tells as an example of the strength of the fear of failing or losing the job the fixed term employee feels. By this structure it is also possible to show that success is felt to be entirely a matter of chance. Tragedy, the failure in one’s trial to get something, is a model I introduce with the help of Vilppu’s story. This narrative gets its meaning both from the sorrow of the failure in the past and the rise of something new the teller has found. Aino tells her story as a comedy. By introducing her narrative, I suggest that the purpose of the comedy, a stronger social consensus, gets deeper and darker shade by fixed-term employment: one who works as a fixed term employee has to take his/her place in his/her work community by him/herself without the support the community gives to those in permanent position. By studying the satiric model Rauno uses, I argue that using irony both turns the power structures to a carnival and builds free space to the teller of the story and to the listener. Irony also helps in building a consensus, mutual understanding, between the teller and the listener and it shows the distance the teller tells to exist between him and others. Irony, however, demands some kind of success in one’s occupational career but also at least a minor disappointment in the progress of it. Helmi tells her story merely as a detective story. By introducing Helmi’s narrative, I argue that this story model strengthens the trust in fairness of the society the teller and the listener share. The analysis also emphasizes the central position of identity work, which is caused by fixed-term employment. Most of the interviewees talked about getting along in working life. I introduced Sari’s narrative as an example of this. In both of these latter narratives one’s personal character and habits are lifted as permanent parts of the actual professional expertise, which in turn varies according to different situations. By introducing these models, I reveal that the fixed-term employees have different strategies to cope with their job situations and these strategies vary according to their personal motives and situations and the actual purpose of the interview. However, I argue that they feel the space between their hopes and fears narrow and unsecure. In the research report I also introduce pieces of the stories – themes – that the interviewees use to build these survival strategies. They use their personal curriculum vitae or portfolio, their position in work community and their work morals to build their professional identity. Professional identity is flexible and varies in time and place, but even then it offers a tool to fix one’s identity work into something. It offers a viewpoint to society and a tool to measure one’s position in surrounding social nets. As one result of the study I analyze the position the fixed-term employees share on the edge of their job communities. I summarize the hopes and fears the interviewees have concerning employers, trade unions, educational institutions and the whole society. In their opinion, the solidarity between people has been weakened by the short-sighted power of the economy. The impact the fixed-term employment has on one’s professional identity and social capital is a many-sided and versatile process. Fixed-term employment both strengthens and weakens the professional identity, social capital and the building of trust. Fixed-term employment also affects one’s day-to-day life by excluding her/him from the norm and by one’s difficulty in making long-term plans (Jokinen 2005). Regardless of the nature of the job contract, the workers themselves are experts in making the best of their sometimes less than satisfying work life and they also build their professional identity by using creatively their education, work experiences and interpersonal relations. However, a long career of short fixed-term employments may seriously change the perception of employee about his/her role. He/she may start concentrating only in coping in his/her unsatisfactory situation and leaves the active improvement of the lousy working conditions to other people. Keywords: narrative, fixed-tem employment, occupational identity, work, story model, social capital, career  

Bufadienolides. 8. 12(13→14)abeo skeletal rearrangements

Several methods were developed for converting isodigitoxigenin (2a) into methyl acetals 4b and 4c. Of these, methanolysis (followed by acetylation) of isodigitoxigenin in the presence of p-toluenesulfonic acid proved most useful. Each isomer reached an equilibrium corresponding to ca. 3:1 acetal 4c to 4b within 15 min in benzene containing p-toluenesulfonic acid. Addition of dihydropyran to the equilibrium mixture resulted in excellent conversion into vinyl ether 5a. Heating either acetal 4b or 4c in benzene containing p-toluenesulfonic acid led to a skeletal rearrangement culminating in formation of C-norcardenolide 6. In addition to results of physical measurements, the structure of spiran 6 was confirmed by degradation to methyl ketone 8. Similar rearrangement of isodigitoxigenin gave spiran 9 accompanied by C-norcardenolide 6. Treating lactone 9 with p-toluenesulfonic acid in methanol-water provided acetals 10a and 10b, which on further contact with p-toluenesulfonic acid in refluxing benzene gave lactone 9 and cardenolide 6. Evidence underlying the stereochemical assignments noted for structures 4, 9, and 10 was also discussed.

Generation of Cationic Two-Coordinate Group-13 Ligand Systems by Spontaneous Halide Ejection: Remarkably Nucleophile-Resistant (Dimethylamino)borylene Complexes

Spontaneous ejection of chloride from a three-coordinate boron Lewis acid can be effected by employing very electron rich metal substituents and leads to the formation of a sterically unprotected terminal (dimethylamino)borylene complex that has a short metal-boron bond and remarkable resistance to attack by nucleophilic and protic reagents.

Solution cation-binding properties of segmented polyethylene oxide-A C-13 NMR spectroscopic study

Two segmented polyethylene oxides, SPEO-3 and SPEO-4, were prepared using a novel transetherification methodology. Their structures were confirmed by H-1 and C-13 NMR spectroscopy. The complexation of these SPEO's with alkali-metal ions in solution was investigated by C-13 NMR spectroscopy. The mole-fraction method was used to determine the complexation ratio of SPEO with LIClO4 at 25 degrees C, which showed that these formed 1:1 (polymer repeat unit/salt) complexes. The association constant, K, for the complex formation was calculated from the variation of the chemical shift values with salt concentration, using a standard nonlinear least-square fitting procedure. The maximum change in chemical shift (Delta delta) and the K values suggest that both SPEO-3 and SPEO-4 formed stronger complexes with lithium salts than with sodium salts. Unexpectedly, the K values were found to be different, when the variation of delta of different carbons was used in the fitting procedure. This suggests that several possible complexed species may be in equilibrium with the uncomplexed one. Structurally similar model compounds were also prepared and their complexation studies indicated that all of them also formed 1:1 complexes with Li salts. Interestingly, it was observed that the polymers gave higher K values suggesting the formation of more stable complexes in polymers when compared to the model analogues. (C) 2000 John Wiley & Sons, Inc.

Biosorption of iron(III) from aqueous solutions using the husk of Cicer arientinum

Iron is a major pollutant released as a by-product during several industrial operations especially during acid mining of metal ores. In this paper, the use of Bengal gram husk (husk of channa dal, Cicer arientinum) in the biosorption of Fe(III) from aqueous solutions is discussed. Parameters like agitation time, adsorbent dosage and pH were studied at different Fe(Ill) concentrations. The adsorption data fit well with Langmuir and Freundlich isotherm models. The adsorption capacity (q(max)) calculated from the Langmuir isotherm was 72.16 mg of Fe(III)/g of the biosorbent at an initial pH of 2.5. Desorption Studies were performed at different concentrations of hydrochloric acid showing that quantitative recovery of the metal ion is possible. The infrared spectra of the biomass before and after treatment with Fe(III), revealed that hydroxyl, carboxyl and amide bonds are involved in the uptake of Fe(III) ions.