Characterization of DNA Strand Transfer Promoted by Mycobacterium smegmatis RecA Reveals Functional Diversity with Mycobacterium tuberculosis RecA†

The RecA-like proteins constitute a group of DNA strand transfer proteins ubiquitous in eubacteria, eukarya, and archaea. However, the functional relationship among RecA proteins is poorly understood. For instance, Mycobacterium tuberculosis RecA is synthesized as a large precursor, which undergoes an unusual protein-splicing reaction to generate an active form. Whereas the precursor was inactive, the active form promoted DNA strand transfer less efficiently compared to EcRecA. Furthermore, gene disruption studies have indicated that the frequencies of allele exchange are relatively lower in Mycobacterium tuberculosis compared to Mycobacterium smegmatis. The mechanistic basis and the factors that contribute to differences in allele exchange remain to be understood. Here, we show that the extent of DNA strand transfer promoted by the M. smegmatis RecA in vitro differs significantly from that of M. tuberculosis RecA. Importantly, M. smegmatis RecA by itself was unable to promote strand transfer, but cognate or noncognate SSBs rendered it efficient even when added prior to RecA. In the presence of SSB, MsRecA or MtRecA catalyzed strand transfer between ssDNA and varying lengths of linear duplex DNA with distinctly different pH profiles. The factors that were able to suppress the formation of DNA networks greatly stimulated strand transfer reactions promoted by MsRecA or MtRecA. Although the rate and pH profiles of dATP hydrolysis catalyzed by MtRecA and MsRecA were similar, only MsRecA was able to couple dATP hydrolysis to DNA strand transfer. Together, these results provide insights into the functional diversity in DNA strand transfer promoted by RecA proteins of pathogenic and nonpathogenic species of mycobacteria.

Functional transfer of Salmonella pathogenicity island 2 to Salmonella bongori and Escherichia coli.

The type III secretion system (T3SS) encoded by the Salmonella pathogenicity island 2 (SPI2) has a central role in systemic infections by Salmonella enterica and for the intracellular phenotype. Intracellular S. enterica uses the SPI2-encoded T3SS to translocate a set of effector proteins into the host cell, which modify host cell functions, enabling intracellular survival and replication of the bacteria. We sought to determine whether specific functions of the SPI2-encoded T3SS can be transferred to heterologous hosts Salmonella bongori and Escherichia coli Mutaflor, species that lack the SPI2 locus and loci encoding effector proteins. The SPI2 virulence locus was cloned and functionally expressed in S. bongori and E. coli. Here, we demonstrate that S. bongori harboring the SPI2 locus is capable of secretion of SPI2 substrate proteins under culture conditions, as well as of translocation of effector proteins under intracellular conditions. An SPI2-mediated cellular phenotype was induced by S. bongori harboring the SPI2 if the sifA locus was cotransferred. An interference with the host cell microtubule cytoskeleton, a novel SPI2-dependent phenotype, was observed in epithelial cells infected with S. bongori harboring SPI2 without additional effector genes. S. bongori harboring SPI2 showed increased intracellular persistence in a cell culture model, but SPI2 transfer was not sufficient to confer to S. bongori systemic pathogenicity in a murine model. Transfer of SPI2 to heterologous hosts offers a new tool for the study of SPI2 functions and the phenotypes of individual effectors.

Functional Imagery Training to reduce snacking: Testing a novel motivational intervention based on Elaborated Intrusion theory

Functional Imagery Training (FIT) is a new theory-based, manualized intervention that trains positive goal imagery. Multisensory episodic imagery of proximal personal goals is elicited and practised, to sustain motivation and compete with less functional cravings. This study tested the impact of a single session of FIT plus a booster phone call on snacking. In a stepped-wedge design, 45 participants who wanted to lose weight or reduce snacking were randomly assigned to receive a session of FIT immediately or after a 2-week delay. High-sugar and high-fat snacks were recorded using timeline follow back for the previous 3 days, at baseline, 2 and 4 weeks. At 2 weeks, snacking was lower in the immediate group than in the delayed group, and the reduction after FIT was replicated in the delayed group between 2 and 4 weeks. Frequencies of motivational thoughts about snack reduction rose following FIT for both groups, and this change correlated with reductions in snacking and weight loss. By showing that FIT can support change in eating behaviours, these findings show its potential as a motivational intervention for weight management.

Pathobiological Aspects of Nonreheumatic Aortic Valve Stenosis

Aortic valve stenosis (AS) is an active disease process akin to atherosclerosis, with chronic inflammation, lipid accumulation, extracellular matrix remodeling, fibrosis, and extensive calcification of the valves being characteristic features of the disease. The detailed mechanisms and pathogenesis of AS are still incompletely understood, however, and pharmacological treatments targeted toward components of the disease are not currently available. In this thesis project, my coworkers and I studied stenotic aortic valves obtained from 86 patients undergoing valve replacement for clinically significant AS. Non-stenotic control valves (n=17) were obtained from patients undergoing cardiac transplantation or from organ donors without cardiac disease. We identified a novel inflammatory factor, namely mast cell, in stenotic aortic valves and present evidence showing that this multipotent inflammatory cell may participate in the pathogenesis of AS. Using immunohistochemistry and double immunofluorescence stainings, we found that a considerable number of mast cells accumulate in stenotic valves and, in contrast to normal valves, the mast cells in diseased valves were in an activated state. Moreover, valvular mast cells contained two effective proteases, chymase and cathepsin G, which may participate in adverse remodeling of the valves either by inducing fibrosis (chymase and cathepsin G) or by degrading elastin fibers in the valves (cathepsin G). As chymase and cathepsin G are both capable of generating the profibrotic peptide angiotensin II, we also studied the expression and activity of angiotensin-converting enzyme (ACE) in the valves. Using RT-PCR, imunohistochemistry, and autoradiography, we observed a significant increase in the expression and activity of ACE in stenotic valves. Besides mast cell-derived cathepsin G, aortic valves contained other elastolytic cathepsins (S, K, and V). Using immunohistochemistry, RT-PCR, and fluorometric microassay, we showed that the expression and activity of these cathepsins were augmented in stenotic valves. Furthermore, in stenotic but not in normal valves, we observed a distinctive pattern of elastin fiber degradation and disorganization. Importantly, this characteristic elastin degradation observed in diseased valves could be mimicked by adding exogenous cathepsins to control valves, which initially contained intact elastin fibers. In stenotic leaflets, the collagen/elastin ratio was increased and correlated positively with smoking, a potent AS-accelerating factor. Indeed, cigarette smoke could also directly activate cultured mast cells and fibroblasts. Next, we analyzed the expression and activity of neutral endopeptidase (NEP), which parallels the actions of ACE in degrading bradykinin (BK) and thus inactivates antifibrotic mechanisms in tissues. Real-time RT-PCR and autoradiography revealed NEP expression and activity to be enhanced in stenotic valves compared to controls. Furthermore, both BK receptors (1 and 2) were present in aortic valves and upregulated in stenotic leaflets. Isolated valve myofibroblasts expressed NEP and BK receptors, and their upregulation occurred in response to inflammation. Finally, we observed that the complement system, a source of several proinflammatory mediators and also a potential activator of valvular mast cells, was activated in stenotic valves. Moreover, receptors for the complement-derived effectors C3a and C5a were expressed in aortic valves and in cultured aortic valve myofibroblasts, in which their expression was induced by inflammation as well as by cigarette smoke. In conclusion, our findings revealed several novel mechanisms of inflammation (mast cells and mast cell-derived mediators, complement activation), fibrosis (ACE, chymase, cathepsin G, NEP), and elastin fiber degradation (cathepsins) in stenotic aortic valves and highlighted these effectors as possible pathogenic contributors to AS. These results support the notion of AS as an active process with inflammation and extracellular matrix remodeling as its key features and identify possible new targets for medical therapy in AS.

Aging response and its effect on the functional properties of Cu–Al–Ni shape memory alloys

The β-phase aging response of Cu–Al–Ni single crystal shape memory alloys (SMAs) within the temperature range of 473–573 K has been investigated. Alloys in austenitic (Cu–14.1Al–4Ni wt.%, alloy A) and martensitic (Cu–13.4Al–4Ni wt.%, alloy M) conditions at room temperature were considered. Aged samples show presence of β1′ and γ1′ martensites in both the alloys and formation of γ2 precipitates in the alloy A. The differential scanning calorimetry (DSC) thermograms of the aged samples show increase in transformation temperatures as well as transformation hysteresis with aging. Dynamic mechanical analysis (DMA) was conducted on both the alloys to ascertain the role of precipitates and martensitic transition on tan δ, which characterizes the damping behaviour of the material. With aging, a steady decrease in tan δ value was observed in both the alloys, which was attributed to the decrease in the number of interfaces per unit area with increasing aging temperature. Moreover, in alloy A, as the volume fraction of precipitate increases with aging, the movement of martensitic interfaces is restricted causing a decreased tan δ.

Atrial remodeling in atrial fibrillation : Epidemiological, clinical and magnetocardiographic aspects

Atrial fibrillation is the most common arrhythmia requiring treatment. This Thesis investigated atrial fibrillation (AF) with a specific emphasis on atrial remodeling which was analysed from epidemiological, clinical and magnetocardiographic (MCG) perspectives. In the first study we evaluated in real-life clinical practice a population-based cohort of AF patients referred for their first elective cardioversion (CV). 183 consecutive patients were included of whom in 153 (84%) sinus rhythm (SR) was restored. Only 39 (25%) of those maintained SR for one year. Shorter duration of AF and the use of sotalol were the only characteristics associated with better restoration and maintenance of SR. During the one-year follow-up 40% of the patients ended up in permanent AF. Female gender and older age were associated with the acceptance of permanent AF. The LIFE-trial was a prospective, randomised, double-blinded study that evaluated losartan and atenolol in patients with hypertension and left ventricular hypertrophy (LVH). Of the 8,851 patients with SR at baseline and without a history of AF 371 patients developed new-onset AF during the study. Patients with new-onset AF had an increased risk of cardiac events, stroke, and increased rate of hospitalisation for heart failure. Younger age, female gender, lower systolic blood pressure, lesser LVH in ECG and randomisation to losartan therapy were independently associated with lower frequency of new-onset AF. The impact of AF on morbidity and mortality was evaluated in a post-hoc analysis of the OPTIMAAL trial that compared losartan with captopril in patients with acute myocardial infarction (AMI) and evidence of LV dysfunction. Of the 5,477 randomised patients 655 had AF at baseline, and 345 patients developed new AF during the follow-up period, median 3.0 years. Older patients and patients with signs of more serious heart disease had and developed AF more often. Patients with AF at baseline had an increased risk of mortality (hazard ratio (HR) of 1.32) and stroke (HR 1.77). New-onset AF was associated with increased mortality (HR 1.82) and stroke (HR of 2.29). In the fourth study we assessed the reproducibility of our MCG method. This method was used in the fifth study where 26 patients with persistent AF had immediately after the CV longer P-wave duration and higher energy of the last portion of atrial signal (RMS40) in MCG, increased P-wave dispersion in SAECG and decreased pump function of the atria as well as enlarged atrial diameter in echocardiography compared to age- and disease-matched controls. After one month in SR, P-wave duration in MCG still remained longer and left atrial (LA) diameter greater compared to the controls, while the other measurements had returned to the same level as in the control group. In conclusion is not a rare condition in either general population or patients with hypertension or AMI, and it is associated with increased risk of morbidity and mortality. Therefore, atrial remodeling that increases the likelihood of AF and also seems to be relatively stable has to be identified and prevented. MCG was found to be an encouraging new method to study electrical atrial remodeling and reverse remodeling. RAAS-suppressing medications appear to be the most promising method to prevent atrial remodeling and AF.

Position preference and diffusion path of an oxygen ion in apatite-type lanthanum silicate La9.33Si6O26: A density functional study

Using density functional theory, we investigated the position preference and diffusion mechanisms of interstitial oxygen ions in lanthanum silicate La9.33Si6O26, which is an apatite-structured oxide and a promising candidate electrolyte material for solid oxide fuel cells. The reported lanthanum vacancies were explicitly taken into account by theoretically determining their arrangement with a supercell model. The most stable structures and the formation energies of oxygen interstitials were determined for each charged state. It was found that the double-negatively charged state is stable over a wide range of the Fermi level, and that the excess oxygen ions form split interstitials with the original oxygen ions, while the neutral and the single-negatively charged states preferably form molecular oxygen. These species were found near the lanthanum vacancy site. The theoretically determined migration pathway along the c-axis essentially follows an interstitialcy mechanism. The obtained migration barrier is sensitive to the charge state, and is also affected by the lanthanum vacancy. The barrier height of the double-negatively charged state was calculated to be 0.58 eV for the model structure, which is consistent with the measured activation energy.

Depressive Disorders in Primary Health Care

The Vantaa Primary Care Depression Study (PC-VDS) is a naturalistic and prospective cohort study concerning primary care patients with depressive disorders. It forms a collaborative research project between the Department of Mental and Alcohol Research of the National Public Health Institute, and the Primary Health Care Organization of the City of Vantaa. The aim is to obtain a comprehensive view on clinically significant depression in primary care, and to compare depressive patients in primary care and in secondary level psychiatric care in terms of clinical characteristics. Consecutive patients (N=1111) in three primary care health centres were screened for depression with the PRIME-MD, and positive cases interviewed by telephone. Cases with current depressive symptoms were diagnosed face-to-face with the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I/P). A cohort of 137 patients with unipolar depressive disorders, comprising all patients with at least two depressive symptoms and clinically significant distress or disability, was recruited. The Structured Clinical Interview for DSM-IV Axis II Disorders (SCID-II), medical records, rating scales, interview and a retrospective life-chart were used to obtain comprehensive cross-sectional and retrospective longitudinal information. For investigation of suicidal behaviour the Scale for Suicidal Ideation (SSI), patient records and the interview were used. The methodology was designed to be comparable to The Vantaa Depression Study (VDS) conducted in secondary level psychiatric care. Comparison of major depressive disorder (MDD) patients aged 20-59 from primary care in PC-VDS (N=79) was conducted with new psychiatric outpatients (N =223) and inpatients (N =46) in VDS. The PC-VDS cohort was prospectively followed up at 3, 6 and 18 months. Altogether 123 patients (90%) completed the follow-up. Duration of the index episode and the timing of relapses or recurrences were examined using a life-chart. The retrospective investigation revealed current MDD in most (66%), and lifetime MDD in nearly all (90%) cases of clinically significant depressive syndromes. Two thirds of the “subsyndromal” cases had a history of major depressive episode (MDE), although they were currently either in partial remission or a potential prodromal phase. Recurrences and chronicity were common. The picture of depression was complicated by Axis I co-morbidity in 59%, Axis II in 52% and chronic Axis III disorders in 47%; only 12% had no co-morbidity. Within their lifetimes, one third (37%) had seriously considered suicide, and one sixth (17%) had attempted it. Suicidal behaviour clustered in patients with moderate to severe MDD, co-morbidity with personality disorders, and a history of treatment in psychiatric care. The majority had received treatment for depression, but suicidal ideation had mostly remained unrecognised. The comparison of patients with MDD in primary care to those in psychiatric care revealed that the majority of suicidal or psychotic patients were receiving psychiatric treatment, and the patients with the most severe symptoms and functional limitations were hospitalized. In other clinical aspects, patients with MDD in primary care were surprisingly similar to psychiatric outpatients. Mental health contacts earlier in the current MDE were common among primary care patients. The 18-month prospective investigation with a life-chart methodology verified the chronic and recurrent nature of depression in primary care. Only one-quarter of patients with MDD achieved and maintained full remission during the follow-up, while another quarter failed to remit at all. The remaining patients suffered either from residual symptoms or recurrences. While severity of depression was the strongest predictor of recovery, presence of co-morbid substance use disorders, chronic medical illness and cluster C personality disorders all contributed to an adverse outcome. In clinical decision making, beside severity of depression and co-morbidity, history of previous MDD should not be ignored by primary care doctors while depression there is usually severe enough to indicate at least follow-up, and concerning those with residual symptoms, evaluation of their current treatment. Moreover, recognition of suicidal behaviour among depressed patients should also be improved. In order to improve outcome of depression in primary care, the often chronic and recurrent nature of depression should be taken into account in organizing the care. According to literature management programs of a chronic disease, with enhancement of the role of case managers and greater integration of primary and specialist care, have been successful. Optimum ways of allocating resources between treatment providers as well as within health centres should be found.

Microbiological, environmental and proteolytic aspects in chronic rhinosinusitis with nasal polyposis

Chronic rhinosinusitis is one of the most common chronic respiratory tract diseases affecting up to 15% of the adult population in the Western world. It may be perpetuated by factors predisposing to sinus ostial obstruction together with inflammatory changes in the sinus mucosa. Chronic rhinosinusitis is associated with asthma, and it may represent the same disease process. Chronic rhinosinusitis with nasal polyposis (CRSwNP) and asthma share also the characteristic inflammatory features and histopathologic feature of airway remodelling. Remodelling is considered as a key event in the pathogenesis of asthma. It is controlled by a delicate balance between the matrix metalloproteinases (MMPs) and their regulators. The purpose of the present study was to evaluate the microbiological findings, inflammatory features and MMP and tissue inhibitor of metalloproteinases-1 (TIMP-1) expression in CRSwNP. The results were related to the patient history, exposure to moisture and clinical outcome in order to find out possible explanations for the etiology and chronicity of CRSwNP. Bacterial culture results were similar in patients and in controls and do not explain the chronic course of CRSwNP. The presence of fungi seems to be more common in CRSwNP than chronic rhinosinusitis in general, and they should be actively searched for using microbiological as well as histological methods. Typical outdoor fungal species were found in nasal lavage samples taken from controls in the autumn but not in the winter, reflecting environmental exposure. Exposure to moisture was reported by 46% of the CRSwNP patients, which is in accordance to the Finnish general population. Exposed patients did not differ significantly from non-exposed subjects with regards to microbiological findings, tissue eosinophilia and clinical outcome. Significantly elevated levels of collagenase-2 (MMP-8) and interleukin (IL)-8 but not tumour necrosis factor-α were found in CRSwNP patients. In particular, the activation of mesenchymal-type MMP-8 but not polymorphonuclear-type MMP-8 was associated with elevated IL-8 levels. IL-8 and MMP-8 may form an inductive cytokine-proteinase cascade in CRSwNP pathogenesis and provide a target for novel therapies and a diagnostic tool for monitoring CRSwNP treatment. The proteolytic spectrum is different in eosinophilic and non-eosinophilic CRSwNP with the up-regulation of MMP-8 and MMP-9 in non-eosinophilic CRSwNP, suggesting different pathophysiology in these subgroups. The lack of MMP up-regulation was associated with a poor prognostic factor and worse clinical outcome, representing a possible synergic anti-inflammatory function of MMP-8 and MMP-9 in CRSwNP. This study provides new information about possible immunologic mechanisms in the pathogenesis of CRSwNP. The recently discovered anti-inflammatory/ defensive properties of MMP-8 and MMP-9 in animal models are reported for the first time in a clinical setting in human inflammatory diseases.

CancerLectinDB: a database of lectins relevant to cancer

The role of lectins in mediating cancer metastasis, apoptosis as well as various other signaling events has been well established in the past few years. Data on various aspects of the role of lectins in cancer is being accumulated at a rapid pace. The data on lectins available in the literature is so diverse, that it becomes difficult and time-consuming, if not impossible to comprehend the advances in various areas and obtain the maximum benefit. Not only do the lectins vary significantly in their individual functional roles, but they are also diverse in their sequences, structures, binding site architectures, quaternary structures, carbohydrate affinities and specificities as well as their potential applications. An organization of these seemingly independent data into a common framework is essential in order to achieve effective use of all the data towards understanding the roles of different lectins in different aspects of cancer and any resulting applications. An integrated knowledge base (CancerLectinDB) together with appropriate analytical tools has therefore been developed for lectins relevant for any aspect of cancer, by collating and integrating diverse data. This database is unique in terms of providing sequence, structural, and functional annotations for lectins from all known sources in cancer and is expected to be a useful addition to the number of glycan related resources now available to the community. The database has been implemented using MySQL on a Linux platform and web-enabled using Perl-CGI and Java tools. Data for individual lectins pertain to taxonomic, biochemical, domain architecture, molecular sequence and structural details as well as carbohydrate specificities. Extensive links have also been provided for relevant bioinformatics resources and analytical tools. Availability of diverse data integrated into a common framework is expected to be of high value for various studies on lectin cancer biology.