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Background: Consumption of alcohol is a risk factor for non-communicable and infectious diseases, mental health problems, and can lead injuries and violence. The aim of this study was to evaluate the prevalence of alcohol-involved deaths among decedents who died of external causes and underwent autopsy in Lithuania., Methods: Study includes age persons of any age (from 0 to 110 years) who died and were autopsied in Lithuania from 1 January 2017 to 31 December 2020. Data were obtained from the Lithuanian State Register of Deaths and Their Causes., Results: Among external causes of death, the presence of alcohol was detected in 55.0% of cases. Male decedents had a significantly higher number of positive BAC level recorded, at 46.6%, compared with female decedents (32.1%; P < 0.001). The highest incidence of deaths where the alcohol was detected in the deceased's blood was found when the decedent was listed as being in the victims of assault group (71.5%, 95% CI 65.4-77.2). However, the highest median BAC score was found for those in the accidents group (59.7%, 95% CI: 58.2-61.2, BAC 2.42 ‰, IQR 1.86)., Conclusions: The findings of this study suggest that alcohol use may be a contributing factor in a wide range of fatal incidents, including accidents, injuries, and cases of violent intent. Inequalities between males and females were identified, with a higher proportion of males with alcohol detected in blood at the time of death., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Public Health Association.)

The epithelium of the gastrointestinal (GI) tract has been extensively characterized using advanced histological and RNA sequencing techniques, which has revealed great cellular diversity. Pathogens, such as viruses and bacteria, are highly adapted to their host and often exhibit not only species-specificity but also a preference or tropism for specific GI segments or even cell types-some of these preferences are so specific, that these pathogens still cannot be cultured invitro. Organoid technology now provides a tool to generate human cell types, which enables the study of host cell tropism. Focussing on the GI tract, we provide an overview about cellular differentiation in vivo and in organoids and how differentiation in organoids and their derived models is used to advance our understanding of viral, bacterial, and parasitic infection. We emphasize that it is central to understand the composition of the model, as the alteration of culture conditions yields different cell types which affects infection. We examine future directions for wider application of cellular heterogeneity and potential advanced model systems for GI tract infection studies., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Postoperative complications after colorectal cancer surgery have been linked to the gut microbiome. However, the impact of mechanical bowel preparation using oral preparation agents or rectal enema on postoperative infections remains poorly understood. This study aimed to compare the impact of oral preparation and rectal enema on the gut microbiome and postoperative complications., Methods: This open-label pilot RCT was conducted at the National Cancer Institute, Vilnius, Lithuania. Patients with left-side colorectal cancer scheduled for elective resection with primary anastomosis were randomized 1 : 1 to preoperative mechanical bowel preparation with either oral preparation or rectal enema. Stool samples were collected before surgery, and on postoperative day 6 and 30 for 16S rRNA gene sequencing analysis. The primary outcome was difference in β-diversity between groups on postoperative day 6., Results: Forty participants were randomized to oral preparation (20) or rectal enema (20). The two groups had similar changes in microbiome composition, and there was no difference in β-diversity on postoperative day 6. Postoperative infections occurred in 12 patients (32%), without differences between the study groups. Patients with infections had an increased abundance of bacteria from the Actinomycetaceae family, Actinomyces genus, Sutterella uncultured species, and Enterococcus faecalis species., Conclusion: Mechanical bowel preparation with oral preparation or rectal enema resulted in similar dysbiosis. Patients who experienced postoperative infections exhibited distinct gut microbiome compositions on postoperative day 6, characterized by an increased abundance of bacteria from the Actinomycetaceae family, Actinomyces genus, Sutterella uncultured species, and Enterococcus faecalis species., Registration Number: NCT04013841 (http://www.clinicaltrials.gov)., (© The Author(s) 2024. Published by Oxford University Press on behalf of BJS Foundation Ltd.)

Background: High-risk human papillomavirus (HR-HPV) is a known cause of cervical cancer (CC). Latvia has a high incidence of CC compared with the average incidence in the European Union. This study aims to fill the data gap on the HR-HPV burden in Latvia, providing information on its prevalence and associated factors., Methods: The cross-sectional study was conducted from February 2021 to April 2022. Participants 25-70 years old visiting a general practitioner (general population) or those referred to a colposcopy clinic with changes in their cervical cytology (colposcopy population) collected vaginal self-sample and completed a paper-based questionnaire. Samples were analyzed with Cobas 6800 System (Roche) for HPV16, HPV18 and other HR-HPV (HPV31/33/35/39/45/51/52/56/58/59/66/68). Descriptive statistics for categorical variables were performed. The Chi-square test was used to determine for the statistical significance of differences in the proportions of the dependent variable between subgroups of the independent variable. Univariate and multivariate binary logistic regression were used to identify factors associated with positive HR-HPV status. Results were considered statistically significant at P < 0.05., Results: A total of 1274 participants provided a valid sample. The prevalence of any HR-HPV infection was 66.8% in the colposcopy group and 11.0% in the general population. Factors associated with positive HR-HPV status were marital status single/divorced/widowed (vs. married/cohabiting) [adjusted OR (aOR) 2.6; P = 0.003], higher number of lifetime sex partners [aOR 5.1 (P < 0.001) and 4.0 (P = 0.001)] for six or more and three to five partners in the general population; in the colposcopy group, the statistical significance remained only for Latvian ethnicity (vs. other) (aOR 1.8; P = 0.008) and current smoking (vs. never) (aOR 1.9; P = 0.01)., Conclusion: We documented a comparison to European Union HR-HPV infection burden in Latvia. Any HR-HPV positivity was significantly associated with sexual and other health behavior., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Public Health Association.)

Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (∼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Evidence for the routine use of robotic technology and its impact on short-term outcomes in colon cancer surgery is lacking. The aim of this study was to compare the surgically induced systemic stress response and clinical and patient-reported outcomes for patients undergoing robot-assisted or laparoscopic colon cancer surgery., Methods: In this double-blinded superiority RCT completed between August 2021 and March 2023, patients with stage 1-3 colon cancer were randomized in a 1 : 1 ratio to undergo either robot-assisted or laparoscopic colon cancer surgery. The primary outcome was changes in the systemic stress response, characterized by C-reactive protein expression in the first three postoperative days. Secondary outcomes were intraoperative and postoperative complications and patient-reported outcomes. The latter included quality of recovery-15 and pain intensity using a visual analogue scale., Results: In total, 128 patients were screened for potential inclusion in this study; 50 patients (25 in the robot-assisted group and 25 in the laparoscopic group) were included in the final follow-up and analysis. The postoperative C-reactive protein response was higher on the first postoperative day in the laparoscopic group (mean difference = 19.88 mg/l, 95% c.i. 3.89-35.86; P = 0.045). No statistically significant differences were noted for C-reactive protein expression on the second and third postoperative days., Conclusion: Adopting robot-assisted surgery for stage 1-3 colon cancer is associated with a reduction in the surgical stress response., Registration Number: NCT04687384 (http://www.clinicaltrials.gov)., (© The Author(s) 2024. Published by Oxford University Press on behalf of BJS Foundation Ltd. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The aim of the study was to estimate unrecorded alcohol consumption in Lithuania for the period 2000-2021 using an indirect method for modelling consumption based on official consumption data and indicators of alcohol-related harm. Methodology employed for estimating the unrecorded alcohol consumption was proposed by Norström and was based on the country's 2019 European Health Interview Survey and indicators of fully alcohol-attributable mortality. The proportion of unrecorded alcohol consumption was estimated as 8.30% (95% CI 7.7-8.9%) for 2019 in Lithuania. The estimated total (recorded and unrecorded) alcohol per capita consumption among individuals 15 years of age and older in 2019 was 12.2 L of pure alcohol, 1.01 (95% CI 0.94-1.09%) L of which is likely unrecorded. The lowest unrecorded alcohol level was estimated for 2009 and 2014, while 2018 had the highest level (i.e. 9.33% of total alcohol per capita consumption). Unrecorded alcohol consumption in Lithuania is likely to be modest when compared to recorded alcohol consumption, the latter of which still remains a major challenge to public health., (© The Author(s) 2023. Medical Council on Alcohol and Oxford University Press.)

Background: The impact of method of anastomosis and minimally invasive surgical technique on surgical and clinical outcomes after right hemicolectomy is uncertain. The aim of the MIRCAST study was to compare intracorporeal and extracorporeal anastomosis (ICA and ECA respectively), each using either a laparoscopic approach or robot-assisted surgery during right hemicolectomies for benign or malignant tumours., Methods: This was an international, multicentre, prospective, observational, monitored, non-randomized, parallel, four-cohort study (laparoscopic ECA; laparoscopic ICA; robot-assisted ECA; robot-assisted ICA). High-volume surgeons (at least 30 minimally invasive right colectomy procedures/year) from 59 hospitals across 12 European countries treated patients over a 3-year interval The primary composite endpoint was 30-day success, defined by two measures of efficacy-absence of surgical wound infection and of any major complication within the first 30 days after surgery. Secondary outcomes were: overall complications, conversion rate, duration of operation, and number of lymph nodes harvested. Propensity score analysis was used for comparison of ICA with ECA, and robot-assisted surgery with laparoscopy., Results: Some 1320 patients were included in an intention-to-treat analysis (laparoscopic ECA, 555; laparoscopic ICA, 356; robot-assisted ECA, 88; robot-assisted ICA, 321). No differences in the co-primary endpoint at 30 days after surgery were observed between cohorts (7.2 and 7.6 per cent in ECA and ICA groups respectively; 7.8 and 6.6 per cent in laparoscopic and robot-assisted groups). Lower overall complication rates were observed after ICA, specifically less ileus, and nausea and vomiting after robot-assisted procedures., Conclusion: No difference in the composite outcome of surgical wound infections and severe postoperative complications was found between intracorporeal versus extracorporeal anastomosis or laparoscopy versus robot-assisted surgery., (© The Author(s) 2023. Published by Oxford University Press on behalf of BJS Society Ltd.)

Introduction: For decades in Lithuania, the threat of illicit trade has been used to weaken evidence-based tobacco-control policies and to undermine efforts to reduce smoking prevalence and its attributable burden, while also depriving the government of much-needed tax revenue. The aim of this study is to estimate the size of the illicit cigarette market in Lithuania using data from a nationally representative discarded pack collection., Aims and Methods: The study employed a two-stage cluster design by first randomly selecting 65 well-defined population settlements (30 cities and 35 townships), representing both urban and rural areas, in all 10 counties in Lithuania. Next, we randomly selected 358 polling districts within these settlements. Each polling district had one route along which discarded packs were collected between September 2019 and 2020., Results: In total, 28.9% (95% CIs = 27.7 to 30.1) of discarded cigarette packs were classified as illicit. The vast majority (90.1%) of illicit packs originated from Belarus with most (86.9%) packs produced in the Grodno Tobacco Factory Neman. Tax stamps were present on 93.6% of legal packs and also on 76% of illegal packs., Conclusions: Data from this study suggest that the illicit cigarette trade in Lithuania is more widespread than indicated by other methods and primarily supplied by the neighboring Belarus state-owned tobacco factory in Grodno. This signals the need to adopt Belarus-specific border control and security measures., Implications: This study presents data from the first national industry-independent study on illicit tobacco trade in Lithuania using discarded cigarette pack collection method. As customs seizure data show, our results also indicate that the illicit cigarette market is primarily supplied by Belarus state-owned Grodno Tobacco Factory Neman known for filling Europe with cheap cigarettes. An estimate derived from this study is higher than both the industry-independent estimate obtained by the survey method and the estimates offered by the tobacco industry. This adds to the evidence that the difference in estimates obtained by different methods reflects the strengths and weaknesses of each. The study also demonstrates the impact of a rogue neighbor on the illicit market in an adjacent country and offers suggestions on how to address it., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco.)

Background: According to the Lithuanian law to prevent the sale of alcohol to customers below the legal minimum purchasing age of 20 years, young adults below 25 years must be asked to show an age-verification document when purchasing alcohol. The aim of this study was to assess whether off-premise outlets comply with the law., Methods: In 2022, mystery-shopping study was carried out in three consecutive phases: (i) in a representative sample (n = 239) of off-premise alcohol outlets covering all Lithuanian district centres, (ii) after lifting the requirement to wear a mask and (iii) after warning the outlets that a mystery-shopping study was ongoing. Phases 2 and 3 were held in two cities. The mystery shopping involved attempts by young, but legally eligible customers to purchase alcohol. Across the three study phases, we compared compliance with the law by measuring overall success of purchase attempts and included situational characteristics (working day or weekend), time of day and number of customers in line as an additional predictor., Results: Out of 239 attempts to purchase alcohol from off-premise outlets in the main phase of the study, 107 (or 44.8%) were considered to be successful (visits in which staff were willing to sell alcohol). There was a significantly higher chance of success to purchase alcohol with no ID request if a mystery shopper was the only customer in a queue and on weekends., Conclusions: The results indicate an insufficient level of age-verification control in Lithuania, and that additional action is needed to increase compliance., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Public Health Association.)

Summary: Sequence homology is a basic concept in protein evolution, structure and function studies. However, there are not many different tools and services for homology searches being sensitive, accurate and fast at the same time. We present a new web server for protein analysis based on COMER2, a sequence alignment and homology search method that exhibits these characteristics. COMER2 has been upgraded since its last publication to improve its alignment quality and ease of use. We demonstrate how the user can benefit from using it by providing examples of extensive annotation of proteins of unknown function. Among the distinctive features of the web server is the user's ability to submit multiple queries with one click of a button. This and other features allow for transparently running homology searches-in a command-line, programmatic or graphical environment-across multiple databases with multiple queries. They also promote extensive simultaneous protein analysis at the sequence, structure and function levels., Availability and Implementation: The COMER web server is available at https://bioinformatics.lt/comer., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press.)

Aims: To determine the effect of an alcohol policy change, which increased the minimum legal drinking age (MLDA) from 18 years of age to 20 years of age on all-cause mortality rates in young adults (18-19 years old) in Lithuania., Methods: An interrupted time series analysis was conducted on a dataset from 2001 to 2019 (n = 228 months). The model tested the effects of the MLDA on all-cause mortality rates (deaths per 100,000 individuals) in three age categories (15-17 years old, 18-19 years old, 20-22 years old) in order to control for general mortality trends in young adults, and to isolate the effects of the MLDA from other alcohol control policies. Additional models that included GDP as a covariate and a taxation policy were tested as well., Results: There was a significant effect of the MLDA on all-cause mortality rates in those 18-19 years old, when modelled alone. Additional analyses controlling for the mortality rate of other age groups showed similar findings. Inclusion of confounding factors (policies on alcohol taxation, GDP) eliminated the effects of MLDA., Conclusions: Although there was a notable decline in all-cause mortality rates among young adults in Lithuania, a direct causal impact of MLDA on all-cause mortality rates in young adults was not definitively found., (© The Author(s) 2021. Medical Council on Alcohol and Oxford University Press. All rights reserved.)

Aims: To examine how standard analytical approaches to model mortality outcomes of alcohol use compare to the true results using the impact of the March 2017 alcohol taxation increase in Lithuania on all-cause mortality as an example., Methods: Four methodologies were used: two direct methodologies: (a) interrupted time-series on mortality and (b) comparing predictions based on time-series modeling with the real number of deaths for the year following the implementation of the tax increase; and two indirect methodologies: (c) combining a regression-based estimate for the impact of taxation on alcohol consumption with attributable-fraction methodology and (d) using price elasticities from meta-analyses to estimate the impact on alcohol consumption before applying attributable-fraction methodology., Results and Conclusions: While all methodologies estimated reductions in all-cause mortality, especially for men, there was substantial variability in the level of mortality reductions predicted. The indirect methodologies had lower predictions as the meta-analyses on elasticities and risk relations seem to underestimate the true values for Lithuania. Directly estimated effects of taxation based on the actual mortalities seem to best represent the true reductions in alcohol-attributable mortality. A significant increase in alcohol excise taxation had a marked impact on all-cause mortality in Lithuania., (© The Author(s) 2022. Medical Council on Alcohol and Oxford University Press. All rights reserved.)

Background: The COVID-19 pandemic might impact substance use behaviours around the globe. In this study, we investigate changes in alcohol and tobacco use in the second half of 2020 in countries of the eastern part of the WHO European Region., Methods: Self-reported changes in alcohol and tobacco use among 11 295 adults from 18 countries in the eastern part of the WHO European Region were collected between August 2020 and January 2021. The non-probabilistic sample was weighted for age, gender and education. For each country, proportions of respondents reporting a decrease, no change or increase in substance use over the past 3 months were examined, and multinomial regression models were used to test associations with age, gender and past-year alcohol use., Results: In most countries, about half of the respondents indicating past-year alcohol or tobacco use reported no change in their substance use. Of those alcohol users who reported changes in their alcohol use, a larger proportion reported a decrease than an increase in most countries. The opposite was true for tobacco use. Women, young adults and past-year harmful alcohol users were identified as being more likely to change their substance use behaviour., Conclusion: We found diverging overall trends for alcohol and tobacco use in the second half of 2020. The patterns of change vary according to age, gender and past-year substance use. Individuals at risk to increase their substance use during the COVID-19 pandemic require most policy considerations., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Public Health Association.)

Objectives: The European Society of Thoracic Surgeons Lung Transplantation Working Group promoted a survey to evaluate overall survival in a large cohort of patients receiving lung transplants for rare pulmonary diseases., Methods: We conducted a retrospective multicentre study. The primary end point was overall survival; secondary end points were survival of patients with the most common diagnoses in the context of rare pulmonary diseases and chronic lung allograft dysfunction (CLAD)-free survival. Finally, we analysed risk factors for overall survival and CLAD-free survival., Results: Clinical records of 674 patients were extracted and collected from 13 lung transplant centres; diagnoses included 46 rare pulmonary diseases. Patients were followed for a median of 3.1 years. The median survival after a lung transplant was 8.5 years. The median CLAD-free survival was 8 years. The multivariable analysis for mortality identified CLAD as a strong negative predictor [hazard ratio (HR) 6.73)], whereas induction therapy was a protective factor (HR 0.68). The multivariable analysis for CLAD occurrence identified induction therapy as a protective factor (HR 0.51). When we stratified patients by CLAD occurrence in a Kaplan-Meier plot, the survival curves diverged significantly (log-rank test: P < 0.001). Patients with rare diseases who received transplants had chronic rejection rates similar to those of the general population who received transplants., Conclusions: We observed that overall survival and CLAD-free survival were excellent. We support the practice of allocating lungs to patients with rare pulmonary diseases because a lung transplant is both effective and ethically acceptable., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Summary: Searching for homology in the vast amount of sequence data has a particular emphasis on its speed. We present a completely rewritten version of the sensitive homology search method COMER based on alignment of protein sequence profiles, which is capable of searching big databases even on a lightweight laptop. By harnessing the power of CUDA-enabled graphics processing units, it is up to 20 times faster than HHsearch, a state-of-the-art method using vectorized instructions on modern CPUs., Availability and Implementation: COMER2 is cross-platform open-source software available at https://sourceforge.net/projects/comer2 and https://github.com/minmarg/comer2. It can be easily installed from source code or using stand-alone installers., Contact: mindaugas.margelevicius@bti.vu.lt., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press.)

Objectives: Acute type A aortic dissection is an emergency associated with up to 30% of hospital mortality. It has been established that outcomes are improved with specialist aortic team care in high-volume centres. Most centres are limited to a small number of aortic specialists, thus making it logistically impractical to have a dedicated 24/7 single-centre service. In 2011, a rotational 24/7 service between 3 centres covering a geographical location was introduced including 24/7 access to a dissection 'Hotline'., Methods: We analysed data since 2003 from a prospectively collected database. A total of 227 patients underwent surgery for acute aortic syndrome between 2003 and 2017. The results on outcomes were compared before and after the initiation of the dissection hotline and 24/7 dedicated service., Results: We identified 128 patients from the pre-rotational group and 99 patients from the post-rotational group. Both groups were well matched in terms of demographics and comorbidities. In the post-rotational group, there was an increase in arch surgery (11.8% vs 20.2%, P: 0.07). The introduction of the rotational service reduced 30-day mortality (20% vs 8%, P: 0.010). The introduction of the service improved the overall long-term survival [P: 0.04, hazard ratio 1.86; confidence interval (1.03-3.38)] in the multivariable analysis. There was no difference between the groups in postoperative complications. There was an increase in the median length of hospital stay in the post-rotational group (13 days vs 20 days, P: 0.014)., Conclusions: A streamlined aortic dissection service allows for centralized care. This provides the referring centres with 24/7 access to an experienced aortic team and may improve patient outcomes., (© The Author(s) 2018. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Motivation: Protein sequence alignment forms the basis for comparative modeling, the most reliable approach to protein structure prediction, among many other applications. Alignment between sequence families, or profile-profile alignment, represents one of the most, if not the most, sensitive means for homology detection but still necessitates improvement. We aim at improving the quality of profile-profile alignments and the sensitivity induced by them by refining profile-profile substitution scores., Results: We have developed a new score that represents an additional component of profile-profile substitution scores. A comprehensive evaluation shows that the new add-on score statistically significantly improves both the sensitivity and the alignment quality of the COMER method. We discuss why the score leads to the improvement and its almost optimal computational complexity that makes it easily implementable in any profile-profile alignment method., Availability and Implementation: An implementation of the add-on score in the open-source COMER software and data are available at https://sourceforge.net/projects/comer. The COMER software is also available on Github at https://github.com/minmarg/comer and as a Docker image (minmar/comer)., Supplementary Information: Supplementary data are available at Bioinformatics online.

CglI is a restriction endonuclease from Corynebacterium glutamicum that forms a complex between: two R-subunits that have site specific-recognition and nuclease domains; and two H-subunits, with Superfamily 2 helicase-like DEAD domains, and uncharacterized Z1 and C-terminal domains. ATP hydrolysis by the H-subunits catalyses dsDNA translocation that is necessary for long-range movement along DNA that activates nuclease activity. Here, we provide biochemical and molecular modelling evidence that shows that Z1 has a fold distantly-related to RecA, and that the DEAD-Z1 domains together form an ATP binding interface and are the prototype of a previously undescribed monomeric helicase-like motor. The DEAD-Z1 motor has unusual Walker A and Motif VI sequences those nonetheless have their expected functions. Additionally, it contains DEAD-Z1-specific features: an H/H motif and a loop (aa 163-aa 172), that both play a role in the coupling of ATP hydrolysis to DNA cleavage. We also solved the crystal structure of the C-terminal domain which has a unique fold, and demonstrate that the Z1-C domains are the principal DNA binding interface of the H-subunit. Finally, we use small angle X-ray scattering to provide a model for how the H-subunit domains are arranged in a dimeric complex.

Few studies have evaluated the impact of lifetime abuse on quality of life (QoL) among older adults. By using a multinational study authors aimed to assess the subjective perception of QoL among people who have reported abuse during the course of their lifetime. The respondents (N = 4,467; 2,559 women) were between the ages of 60 and 84 years and living in seven European countries (Germany, Greece, Italy, Lithuania, Portugal, Spain, and Sweden). Lifetime abuse was assessed by using a structured questionnaire that allowed to assess lifetime experiences of abuse. QoL was assessed with the World Health Organization Quality of Life-Old module. After adjustment for potential confounders, authors found that to have had any abusive experience decreased the score of sensory abilities. Psychological abuse was associated with lower autonomy and past, present, and future activities. Physical abuse with injuries significantly decreased social participation. Intimacy was also negatively associated with psychological abuse, physical abuse with injury, and sexual abuse. The results of this study provide evidence that older people exposed to abuse during their lifetime have a significant reduction in QoL, with several QoL domains being negatively affected., (© 2017 National Association of Social Workers.)

The restriction endonuclease CglI from Corynebacterium glutamicum recognizes an asymmetric 5'-GCCGC-3' site and cleaves the DNA 7 and 6/7 nucleotides downstream on the top and bottom DNA strands, respectively, in an NTP-hydrolysis dependent reaction. CglI is composed of two different proteins: an endonuclease (R.CglI) and a DEAD-family helicase-like ATPase (H.CglI). These subunits form a heterotetrameric complex with R2H2 stoichiometry. However, the R2H2·CglI complex has only one nuclease active site sufficient to cut one DNA strand suggesting that two complexes are required to introduce a double strand break. Here, we report studies to evaluate the DNA cleavage mechanism of CglI. Using one- and two-site circular DNA substrates we show that CglI does not require two sites on the same DNA for optimal catalytic activity. However, one-site linear DNA is a poor substrate, supporting a mechanism where CglI complexes must communicate along the one-dimensional DNA contour before cleavage is activated. Based on experimental data, we propose that adenosine triphosphate (ATP) hydrolysis by CglI produces translocation on DNA preferentially in a downstream direction from the target, although upstream translocation is also possible. Our results are consistent with a mechanism of CglI action that is distinct from that of other ATP-dependent restriction-modification enzymes., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Being a victim of abuse during one's life course may affect social relations in later life. The aims of this study were to: (i) examine the association between lifetime abuse and perceived social support and (ii) identify correlates of perceived social support among older persons living in seven European countries., Methods: A sample of 4467 women and men aged 60-84 years living in Germany, Greece, Italy, Lithuania, Portugal, Spain and Sweden was collected through a cross-sectional population-based study. Abuse (psychological, physical, sexual, financial and injury) was assessed through interviews or interviews/self-response questionnaire based on the Conflict Tactics Scale-2 and the UK study on elder abuse. Perceived social support was assessed by the Multidimensional Scale of Perceived Social Support., Results: Victims of lifetime abuse perceived poorer social support in later life. Multivariate analyses showed that high levels of perceived social support were associated with being from Greece and Lithuania (compared to Germany), being female, not living alone, consuming alcohol and physical activity. Poorer perceived social support was associated with being from Portugal, being old, having social benefits as the main source of income, experiencing financial strain and being exposed to lifetime psychological abuse and injuries., Conclusions: Our findings showed that exposure to psychological abuse and injuries across the lifespan were associated with low levels of perceived social support, emphasizing the importance of detection and appropriate treatment of victims of abuse during their life course. Future research should focus on coping strategies buffering the negative effects of abuse on social relationships., (© The Author 2017. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: High mortality rates from smoking related diseases are a significant public health issue in Lithuania. Study aims to estimate the number of smoking attributable deaths (SADs) and years of potential life lost in Lithuania in 2013., Methods: Gender, age and disease specific mortality was calculated by applying the smoking attributable fractions (SAFs) to prevalence estimates of current and former smokers among Lithuanian adults aged ≥35 years that are based on the 2005 Lithuanian Health Interview Survey. Mortality data were obtained from the Institute of Hygiene Health Information Centre. Eight years lag was assumed between smoking rates and subsequent mortality. Sensitivity analysis was used to calculate SAFs applying smoking impact ratio method., Results: In 2013, 13.9% of total mortality or 5771 deaths in Lithuania were attributable to smoking (5181 men and 590 women). The two leading causes of SADs were ischaemic heart disease (2861) and lung cancer (1054) that accounted for 67.8% of the smoking attributable mortality. In the same year, smoking accounted for 39 279 years of potential life lost (34 663 years for men and 4615 years for women)., Conclusions: Smoking causes a considerable mortality burden in Lithuania, killing nine times more males than females. Therefore reduction of smoking prevalence is an urgent public health need, which calls for implementation of effective and comprehensive tobacco control measures consistent with the World Health Organization Framework Convention on Tobacco Control Articles and Protocols and The Tobacco Products Directive., (© The Author 2017. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Objectives: Thoracoscopy is an effective treatment method for pleural empyema; however, it is still not well defined as to which patient subgroups could benefit from it the most. The aim of the study was to identify preoperative factors that could facilitate selecting appropriate surgical intervention and to evaluate early postoperative period., Methods: Seventy-one patients were prospectively included in the study, which was conducted from January 2011 to June 2014. Thoracoscopic surgery for Stage II/III pleural empyema was performed in all patients. Thoracoscopy failed in 18 (25.4%) patients, requiring conversion to thoracotomy. The preoperative factors that could possibly predict conversion were analysed., Results: Obliterated pleural space (12 patients) and failure to achieve lung re-expansion (6 patients) were the main reasons for conversion. Multivariable logistic regression analysis demonstrated that each day of illness [odds ratio 1.1 (95% confidence interval 1.0-1.2], P  = 0.004] and frank pus [odds ratio 4.4 (95% confidence interval 1.2-15.3), P  = 0.021] were independent predictors of conversion. Using receiver-operating characteristic analysis, it was determined that the duration of illness had a high predictive value for conversion [area under the curve 0.8 (95% confidence interval 0.7-0.9), P  < 0.001]. The cut-off value for duration of illness was 16 days (sensitivity 94.4%, specificity 54.7%). The conversion group had a significantly greater need for postoperative intensive care unit stay ( P  = 0.022) but a lower rate of reoperations ( P  = 0.105)., Conclusions: Duration of illness and frank pus discovered during thoracocentesis can help in selecting the patient for appropriate intervention. Earlier surgery for pleural empyema can reduce the rate of conversion and reoperation., (© The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Although all Type II restriction endonucleases catalyze phosphodiester bond hydrolysis within or close to their DNA target sites, they form different oligomeric assemblies ranging from monomers, dimers, tetramers to higher order oligomers to generate a double strand break in DNA. Type IIP restriction endonuclease AgeI recognizes a palindromic sequence 5΄-A/CCGGT-3΄ and cuts it ('/' denotes the cleavage site) producing staggered DNA ends. Here, we present crystal structures of AgeI in apo and DNA-bound forms. The structure of AgeI is similar to the restriction enzymes that share in their target sites a conserved CCGG tetranucleotide and a cleavage pattern. Structure analysis and biochemical data indicate, that AgeI is a monomer in the apo-form both in the crystal and in solution, however, it binds and cleaves the palindromic target site as a dimer. DNA cleavage mechanism of AgeI is novel among Type IIP restriction endonucleases., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Summary: The PPI3D web server is focused on searching and analyzing the structural data on protein-protein interactions. Reducing the data redundancy by clustering and analyzing the properties of interaction interfaces using Voronoi tessellation makes this software a highly effective tool for addressing different questions related to protein interactions., Availability and Implementation: The server is freely accessible at http://bioinformatics.lt/software/ppi3d/ ., Contact: ceslovas.venclovas@bti.vu.lt., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Motivation: Wide application of modeling of three-dimensional protein structures in biomedical research motivates developing protein sequence alignment computer tools featuring high alignment accuracy and sensitivity to remotely homologous proteins. In this paper, we aim at improving the quality of alignments between sequence profiles, encoded multiple sequence alignments. Modeling profile contexts, fixed-length profile fragments, is engaged to achieve this goal., Results: We develop a hierarchical Dirichlet process mixture model to describe the distribution of profile contexts, which is able to capture dependencies between amino acids in each context position. The model represents an attempt at modeling profile fragments at several hierarchical levels, within the profile and among profiles. Even modeling unit-length contexts leads to greater improvements than processing 13-length contexts previously. We develop a new profile comparison method, called COMER, integrating the model. A benchmark with three other profile-to-profile comparison methods shows an increase in both sensitivity and alignment quality., Availability and Implementation: COMER is open-source software licensed under the GNU GPLv3, available at https://sourceforge.net/projects/comer, Contact: mindaugas.margelevicius@bti.vu.lt, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5'-W/CCGGW-3' (W stands for A or T, '/' denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an 'open' configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The stress-sensitive restriction-modification (RM) system CglI from Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease (R.CglI and R.NgoAVII, or R-proteins) and a predicted DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R2N2 stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (∼170 ATP/s/monomer) are required for site-specific DNA cleavage by R-proteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6-7 nucleotides) downstream of the asymmetric recognition sequence 5'-GCCGC-3'. Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The restriction endonuclease (REase) NgoAVII is composed of two proteins, R.NgoAVII and N.NgoAVII, and shares features of both Type II restriction enzymes and Type I/III ATP-dependent restriction enzymes (see accompanying paper Zaremba et al., 2014). Here we present crystal structures of the R.NgoAVII apo-protein and the R.NgoAVII C-terminal domain bound to a specific DNA. R.NgoAVII is composed of two domains: an N-terminal nucleolytic PLD domain; and a C-terminal B3-like DNA-binding domain identified previously in BfiI and EcoRII REases, and in plant transcription factors. Structural comparison of the B3-like domains of R.NgoAVII, EcoRII, BfiI and the plant transcription factors revealed a conserved DNA-binding surface comprised of N- and C-arms that together grip the DNA. The C-arms of R.NgoAVII, EcoRII, BfiI and plant B3 domains are similar in size, but the R.NgoAVII N-arm which makes the majority of the contacts to the target site is much longer. The overall structures of R.NgoAVII and BfiI are similar; however, whilst BfiI has stand-alone catalytic activity, R.NgoAVII requires an auxiliary cognate N.NgoAVII protein and ATP hydrolysis in order to cleave DNA at the target site. The structures we present will help formulate future experiments to explore the molecular mechanisms of intersubunit crosstalk that control DNA cleavage by R.NgoAVII and related endonucleases., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Public health needs to adapt to the complex context of 21st century Europe. Unquestionably, leaders for health require new skills to face a myriad of wicked problems and challenges that are at a critical juncture for potential improvements. Public health curricula are traditionally oriented around core educational disciplines, and there is little room for developing students' leadership capabilities within the context of public health. The aim is to present the meaning of contemporary public health leadership based on qualitative research and propose a curriculum model for contemporary public health leadership., Methods: A series of in-depth semi-structured interviews were carried out with six European public health leaders from a variety of countries and professional backgrounds. The interviews recorded and transcribed. A thematic content analysis was undertaken to identify themes within the data., Results: Five common themes that help to inform future leadership capacity arose from the interviews: the inner path of leadership, the essence of leadership, new types of leadership, future leaders' imperatives functioning within a complex and uncertain European public health context., Conclusion: The leadership thematic model makes an important contribution to defining public health leadership in Europe and can help to guide the content development of public health leadership curricula. The authors assert that a new 'integrative inquiry-based learning model', with leadership as a central component, will allow schools and departments of public health across Europe to be able to ensure that tomorrow's public health leaders are adequately trained and prepared for the challenges they will face., (© The Author 2014. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5'-CCTGG-3'). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5'-ACTGGG-3') complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C-DNA and EcoRII-N-DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C-DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs.

We have developed an in vitro evolution method for the selection for catalytic activity under the conditions of free intermolecular interaction between the enzyme and a substrate. The destabilized ternary enzyme-mRNA-ribosome complexes generated by a ribosome display of the mutant library are compartmentalized in vitro by forming a water-in-oil emulsion in such a way, that every droplet would on average contain no more than a single complex. After the complex dissociates within the droplet, the released enzyme molecule is free to interact with a substrate under the selection pressure on all its enzymatic properties (substrate binding, product formation, rate acceleration and turnover) simultaneously-an opportunity for the most efficient selection for catalytic activity. By using the M-MuLV reverse transcriptase as a model, we demonstrated the high efficiency of the method selecting for mutants synthesizing cDNA at increased temperature. A slightly modified compartmentalized ribosome display (CRD) could be used for the selection of other enzymes activities (e.g. DNA polymerase, RNA or DNA ligase terminal nucleotidyl transferase activity). Employment of microfluidics technique could broaden the scope of CRD technique furthermore providing an opportunity to select almost any enzyme at single molecule level under desired conditions.

Type II restriction endonucleases (REases) are one of the basic tools of recombinant DNA technology. They also serve as models for elucidation of mechanisms for both site-specific DNA recognition and cleavage by proteins. However, isolation of catalytically active mutants from their libraries is challenging due to the toxicity of REases in the absence of protecting methylation, and techniques explored so far had limited success. Here, we present an improved SOS induction-based approach for in vivo screening of active REases, which we used to isolate a set of active variants of the catalytic mutant, Cfr10I(E204Q). Detailed characterization of plasmids from 64 colonies screened from the library of ∼200,000 transformants revealed 29 variants of cfr10IR gene at the level of nucleotide sequence and 15 variants at the level of amino acid sequence, all of which were able to induce SOS response. Specific activity measurements of affinity-purified mutants revealed >200-fold variance among them, ranging from 100% (wild-type isolates) to 0.5% (S188C mutant), suggesting that the technique is equally suited for screening of mutants possessing high or low activity and confirming that it may be applied for identification of residues playing a role in catalysis.

In vitro synthesis of cDNA is one of the most important techniques in present molecular biology. Faithful synthesis of long cDNA on highly structured RNA templates requires thermostable and processive reverse transcriptases. In a recent attempt to increase the thermostability of the wt Moloney Murine leukemia virus reverse transcriptase (M-MuLV RT), we have employed the compartmentalized ribosome display (CRD) evolution in vitro technique and identified a large set of previously unknown mutations that enabled cDNA synthesis at elevated temperatures. In this study, we have characterized a group of the M-MuLV RT variants (28 novel amino acid positions, 84 point mutants) carrying the individual mutations. The performance of point mutants (thermal inactivation rate, substrate-binding affinity and processivity) correlated remarkably well with the mutation selection frequency in the CRD experiment. By combining the best-performing mutations D200N, L603W, T330P, L139P and E607K, we have generated highly processive and thermostable multiply-mutated M-MuLV RT variants. The processivity of the best-performing multiple mutant increased to 1500 nt (65-fold improvement in comparison to the wt enzyme), and the maximum temperature of the full-length 7.5-kb cDNA synthesis was raised to 62°C (17° higher in comparison with the wt enzyme).

Background: Violence against and abuse of older persons (VAO) aged>60 years has become a prominent public health issue. From January 2009-July 2009, we conducted the cross-sectional European study 'Abuse of the elderly in the European region' (ABUEL) among community-dwelling elderly populations aged 60-84 years in Germany, Greece, Italy, Lithuania, Portugal, Spain and Sweden. We describe the cooperation, completion and response rates; the modes of recruitment and administration; and analyse differences in response rates between countries., Methods: We calculated the population fraction (respondents in each age/sex group divided by the population in the same age/sex group) and the population fraction ratio (PFR) to describe and analyse heterogeneity between countries. To analyse associations between methods and response rates we conducted cross tabulations and logistic regression analyses., Results: The response rates ranged from 18.9% in Germany to 87.4% in Portugal. Men were underrepresented in all countries (PFR<1). Cluster- and cohort-based sampling produced the highest overall response rates., Conclusion: More European and international studies investigating response behaviour in VAO research systematically are needed to gain further knowledge about the internal and external validity of research on VAO.

Restriction endonuclease Bse634I recognizes and cleaves the degenerate DNA sequence 5'-R/CCGGY-3' (R stands for A or G; Y for T or C, '/' indicates a cleavage position). Here, we report the crystal structures of the Bse634I R226A mutant complexed with cognate oligoduplexes containing ACCGGT and GCCGGC sites, respectively. In the crystal, all potential H-bond donor and acceptor atoms on the base edges of the conserved CCGG core are engaged in the interactions with Bse634I amino acid residues located on the α6 helix. In contrast, direct contacts between the protein and outer base pairs are limited to van der Waals contact between the purine nucleobase and Pro203 residue in the major groove and a single H-bond between the O2 atom of the outer pyrimidine and the side chain of the Asn73 residue in the minor groove. Structural data coupled with biochemical experiments suggest that both van der Waals interactions and indirect readout contribute to the discrimination of the degenerate base pair by Bse634I. Structure comparison between related enzymes Bse634I (R/CCGGY), NgoMIV (G/CCGGC) and SgrAI (CR/CCGGYG) reveals how different specificities are achieved within a conserved structural core.

Unlabelled: We present Voroprot, an interactive cross-platform software tool that provides a unique set of capabilities for exploring geometric features of protein structure. Voroprot allows the construction and visualization of the Apollonius diagram (also known as the additively weighted Voronoi diagram), the Apollonius graph, protein alpha shapes, interatomic contact surfaces, solvent accessible surfaces, pockets and cavities inside protein structure., Availability: Voroprot is available for Windows, Linux and Mac OS X operating systems and can be downloaded from http://www.ibt.lt/bioinformatics/voroprot/.

PD-(D/E)XK nucleases, initially represented by only Type II restriction enzymes, now comprise a large and extremely diverse superfamily of proteins. They participate in many different nucleic acids transactions including DNA degradation, recombination, repair and RNA processing. Different PD-(D/E)XK families, although sharing a structurally conserved core, typically display little or no detectable sequence similarity except for the active site motifs. This makes the identification of new superfamily members using standard homology search techniques challenging. To tackle this problem, we developed a method for the detection of PD-(D/E)XK families based on the binary classification of profile-profile alignments using support vector machines (SVMs). Using a number of both superfamily-specific and general features, SVMs were trained to identify true positive alignments of PD-(D/E)XK representatives. With this method we identified several PFAM families of uncharacterized proteins as putative new members of the PD-(D/E)XK superfamily. In addition, we assigned several unclassified restriction enzymes to the PD-(D/E)XK type. Results show that the new method is able to make confident assignments even for alignments that have statistically insignificant scores. We also implemented the method as a freely accessible web server at http://www.ibt.lt/bioinformatics/software/pdexk/.

To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or tetramer represents the functionally important assembly, we generated mutants aimed at disrupting the putative dimer-dimer interface and analysed the functional properties of Ecl18kI and mutant variants. We show by atomic force microscopy that on two-site DNA, Ecl18kI loops out an intervening DNA fragment and forms a tetramer. Using the tethered particle motion technique, we demonstrate that in solution DNA looping is highly dynamic and involves a transient interaction between the two DNA-bound dimers. Furthermore, we show that Ecl18kI cleaves DNA in the synaptic complex much faster than when acting on a single recognition site. Contrary to Ecl18kI, the tetramerization interface mutant R174A binds DNA as a dimer, shows no DNA looping and is virtually inactive. We conclude that Ecl18kI follows the association model for the synaptic complex assembly in which it binds to the target site as a dimer and then associates into a transient tetrameric form to accomplish the cleavage reaction.

Summary: Detection of distant homology is a widely used computational approach for studying protein evolution, structure and function. Here, we report a homology search web server based on sequence profile-profile comparison. The user may perform searches in one of several regularly updated profile databases using either a single sequence or a multiple sequence alignment as an input. The same profile databases can also be downloaded for local use. The capabilities of the server are illustrated with the identification of new members of the highly diverse PD-(D/E)XK nuclease superfamily., Availability: http://www.ibt.lt/bioinformatics/coma/

Metal-dependent nucleases that generate double-strand breaks in DNA often possess two symmetrically-equivalent subunits, arranged so that the active sites from each subunit act on opposite DNA strands. Restriction endonuclease BfiI belongs to the phospholipase D (PLD) superfamily and does not require metal ions for DNA cleavage. It exists as a dimer but has at its subunit interface a single active site that acts sequentially on both DNA strands. The active site contains two identical histidines related by 2-fold symmetry, one from each subunit. This symmetrical arrangement raises two questions: first, what is the role and the contribution to catalysis of each His residue; secondly, how does a nuclease with a single active site cut two DNA strands of opposite polarities to generate a double-strand break. In this study, the roles of active-site histidines in catalysis were dissected by analysing heterodimeric variants of BfiI lacking the histidine in one subunit. These variants revealed a novel mechanism for the scission of double-stranded DNA, one that requires a single active site to not only switch between strands but also to switch its orientation on the DNA.

PspGI is a representative of a group of restriction endonucleases that recognize a pentameric sequence related to CCNGG. Unlike the previously investigated Ecl18kI, which does not have any specificity for the central base pair, PspGI prefers A/T over G/C in its target site. Here, we present a structure of PspGI with target DNA at 1.7 A resolution. In this structure, the bases at the center of the recognition sequence are extruded from the DNA and flipped into pockets of PspGI. The flipped thymine is in the usual anti conformation, but the flipped adenine takes the normally unfavorable syn conformation. The results of this and the accompanying manuscript attribute the preference for A/T pairs over G/C pairs in the flipping position to the intrinsically lower penalty for flipping A/T pairs and to selection of the PspGI pockets against guanine and cytosine. Our data show that flipping can contribute to the discrimination between normal bases. This adds a new role to base flipping in addition to its well-known function in base modification and DNA damage repair.

Restriction endonucleases Ecl18kI and PspGI/catalytic domain of EcoRII recognize CCNGG and CCWGG sequences (W stands for A or T), respectively. The enzymes are structurally similar, interact identically with the palindromic CC:GG parts of their recognition sequences and flip the nucleotides at their centers. Specificity for the central nucleotides could be influenced by the strength/stability of the base pair to be disrupted and/or by direct interactions of the enzymes with the flipped bases. Here, we address the importance of these contributions. We demonstrate that wt Ecl18kI cleaves oligoduplexes containing canonical, mismatched and abasic sites in the central position of its target sequence CCNGG with equal efficiencies. In contrast, substitutions in the binding pocket for the extrahelical base alter the Ecl18kI preference for the target site: the W61Y mutant prefers only certain mismatched substrates, and the W61A variant cuts exclusively at abasic sites, suggesting that pocket interactions play a major role in base discrimination. PspGI and catalytic domain of EcoRII probe the stability of the central base pair and the identity of the flipped bases in the pockets. This 'double check' mechanism explains their extraordinary specificity for an A/T pair in the flipping position.

Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI-DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution.