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Supplementary data are available online at: www.aob.oxfordjournals.org and consist of the following. Table S1: soil physico-chemical properties during the second growing season in the eight plantation sites; Figure S1: total height growth of two white spruce seed sources at the end of the second growing season (H2) plotted against site mean July temperature (MJT), soil total nitrogen and soil C:N ratio. Figure S2: Nmass and SLA of plants of two white spruce seed sources growing at eight plantation sites; Figure S3: Aopt and Rd10 plotted against Nmass for the two white spruce seed sources; raw data on An response to Ci and temperature.
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| 99.94 |
The ultrafast dynamics and relaxation of hot carriers and phonons in a variety of materials investigated by pump-probe techniques has attracted a lot of attention in the last decade123456789101112131415. Rapid development of pulsed lasers combined with increased utilization of optical absorbers161718 stimulated spectroscopic investigations of materials on picosecond and sub-picosecond timescales under optically driven non-equilibrium conditions. Time-resolved experiments use intense laser pulses to create non-equilibrium states in materials and track their relaxation in real time. They provide information about unoccupied electronic states, electron-electron (e-e) interactions146, electron-phonon (e-ph) coupling91419, phonon-phonon (ph-ph) coupling2021, etc. Here we show that combining time-resolved photoemission with time-resolved Raman (TRR)22 scattering provides insights that are much more difficult to obtain otherwise. Angle-resolved photoemission spectroscopy (ARPES) views the process from electron perspective tracking electron-hole (e–h) excited state occupation as a function of time. Raman scattering used as a probe reveals the phonon perspective providing vibration frequency, lifetime, and occupation number of Raman-active phonons. The fundamental physical law of detailed balance allows extracting the phonon occupation number n from each Raman-active phonon directly from the ratio of the energy loss (Stokes) and energy gain (anti-Stokes) one-phonon Raman scattering intensities23. The anti-Stokes side, where the phonon intensities are proportional to n, is especially sensitive.
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Up to now the data have been described in terms of the two-temperature (2T) model where hot electrons thermalize with a few “hot” phonons relatively quickly (few hundred fs) via strong e-ph coupling21324. In this description, hot electrons and hot phonons are characterized by their temperatures. Other e-ph and ph-ph interactions are assumed to be much weaker resulting in the whole system reaching the final thermal equilibrium at much longer picosecond timescales.
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Our investigation focused on graphite as a model electron-phonon system. TRR as well as trARPES has been previously applied to graphene, graphite, and carbon nanotubes123202521262728, but they have never been applied to one compound simultaneously as we have done in this paper. G-phonons in graphite and related materials are believed to play the role of “hot” phonons in the 2T description due to strong coupling to electrons. We observed G-phonon population (data points in Fig. 1(a)) as a function of time in the sub-picosecond region by Raman scattering and electron-hole population (black points in Fig. 1(b)) by ARPES with nearly the same time-resolution and pump fluence. The unexpected result is that G-phonon generation is delayed by about 65 fs from the prediction of the 2T model (green dashed line in Fig. 1(a)). Furthermore, the ARPES measurements show that the e-h pairs decay faster than expected from direct coupling to the G-phonon, and the G-phonon generation time constant is reduced with a higher pump power in contradiction with the 2T model. This suggests that hot carriers couple to G-phonons not directly but via an intermediate excitation that is not Raman active (red lines in Fig. 1(a,b)). We argue that K-phonons play this role (Fig. 1(c)). Our results agree with LDA calculation that predicts that e-h pairs predominantly decay into K-phonons29.
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The Raman spectrum of graphite measured with TRR setup shown in Fig. 2(a) has two main features: the G peak at ~1580 cm−1 and the 2D peak at ~2700 cm−1 (Fig. 2(b)). The G peak is the zone center E2g phonon. The 2D peak is due to the two-phonon scattering at K points called double resonance303132. These peaks measured by the 395 nm ultrafast probe pulse sit on a luminescence background and have large linewidths due to the finite bandwidth of the pulse. The absence of the defect-induced D peak (~1350 cm−1) indicates good quality of our sample. Figure 2(c) displays the anti-Stokes spectra at various delay times. We fit the phonon spectrum at each time delay with a Gaussian (Fig. 2(d)) and then extracted the integrated intensity as a function of time (Fig. 2(e) blue dots and inset).
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The time evolution of the G-phonon can be fit with two exponential functions −exp[−(t − t0)/τgrowth] + exp[−(t − t0)/τdecay] convolved with the instrument response. The time constant τgrowth is 220 ± 20 fs for the quick buildup of G-phonons and τdecay is 2400 ± 83 fs for the slow decay (Fig. 2(e) inset). The fitting parameter t0 = 2 ± 15 fs. The G-phonon temperature is determined from the G peak intensity ratio on the Stokes and anti-Stokes sides IS/IAS = exp(ħΩ/kBTG), where ħΩ is the G-phonon energy, kB is the Boltzmann’s constant, and TG is the phonon temperature (Fig. 1(a)). TG is over 3000 K (over 1650 K for pump fluence 0.15 mJ/cm2 as shown in Fig. 1(a)) around t = 400 fs. It decreases to room temperature with a time constant of 2.4 ps, which agrees with previous results2021. The slow decay has been attributed to the energy transfer from G-phonons to other phonon modes via anharmonic coupling2533. The apparent oscillation around t = 600 fs in Fig. 3d might be due to imperfect laser power stability. We did not observe this oscillation in other datasets.
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Figure 3 shows electronic dynamics probed by trARPES. Electronic temperature Tel was determined by fitting the slope of the high energy tail in the momentum-integrated ARPES spectra (Fig. 3 inset). Tel increases rapidly after an optical excitation and reaches 3400 K at t = 100 fs. Tel fits well to a bi-exponential decay with decay times of τfast = 0.35 ps and τslow = 3.5 ps. The decay of Tel has a similar timescale to the one observed by previous photoemission experiments125. We evaluated the ratio of the electronic heat capacity to G-phonon heat capacity (see supplementary) from the electronic temperature and G-phonon temperature. It is one of the parameters in our theoretical model as will be discussed later. The 2D Raman peak also reflects electronic temperature, though it is less precise than trARPES results due to self-pumping and greater statistical error (see Supplementary).
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| 100.0 |
Figure 4 shows G-phonon dynamics with pump fluence 2.1 mJ/cm2 and 0.15 mJ/cm2 respectively. The phonon dynamics are similar for these two pump powers. Maximum phonon temperature decreases with lower pump fluence as expected. The generation of G phonons is slower with low pump fluence (τgrowth = 210 fs) than for the high pump fluence (τgrowth = 100 fs).
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We now briefly discuss the electronic background in the Raman spectra, which has been assigned to hot carrier luminescence34. Figure 2(d) inset shows that the background starts to increase at t = −200 fs and reaches its maximum at t ~ 0 fs, when the G-phonons begin to appear. The background then decreases to the initial level at t = 300 fs, while the phonon intensity keeps increasing from t = 0 fs to t = 300 fs. This behavior reflects changes in electronic temperature and is consistent with previous reports34353637. However, it is not possible to read out instantaneous electronic temperatures from the observed intensities.
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Our ultrafast pump pulse generates hot electrons and holes, which quickly thermalize via e-e interaction on femtosecond timescales12. These e-h pairs decay into phonons with strong e-ph coupling on longer timescales11131415; The hot phonons in turn slowly decay into other phonons, which carry energy out of the system20. Here we focused on graphite where there are no experimental complications due to the substrate and sample size issues. Our results also apply to graphene since its high energy electronic structure is similar. The states with energy less than the hot-phonon frequency are less relevant for the phonon dynamics but are relevant for other physics such as high electric and thermal conductivity resulting from massless Dirac fermions.
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Previous experiments on graphite and graphene reported that hot carriers transfer most of their energy to hot optical phonons within 500 fs713143839. They observed a quick buildup of G-phonon occupation through e-ph coupling320, but they were not able to clearly resolve the short-time delay region and identify which transition is dominant. Due to finite time resolution, the G-phonons generated directly from e-h recombination are supposed to appear at negative times. Given our 90 fs time resolution, the increase of phonon population would be detected at ~−35 fs (see the green dashed line in Fig. 1). The abrupt appearance of G-phonons near t = 0 therefore suggests that G-phonon emission occurs with a time delay. The presence of a time delay is also clear when the probe pulse is used without the pump. In this case photons in the early-time part of a probe pulse pump the system, and the photons coming later but still belonging to the same pulse serve as an excitation for Raman scattering (This process is called self-pumping20). Due to the short time duration of the probe pulse (~50 fs), self-pumping integrates the excitations within the first 35 fs, which suggests that G-phonons are emitted more than 35 fs after a pump pulse, not about 10 fs as expected from direct decay of e-h pairs into this phonon.
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Another experimental result contradicting the scenario of direct e-h recombination is the slower G-phonon buildup with lower pump fluence. The opposite is expected, because e-ph coupling was previously reported to increase with decreasing electronic temperature3340.
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| 99.94 |
Slow thermalization of carriers cannot explain these results. If hot carriers couple to G-phonons directly, G-phonons can be emitted regardless of whether carriers are thermalized or not as long as hot carriers occupy the states near EF. The inverse Auger process that dominates e-e scattering leads to a rapid accumulation of hot carriers near EF within 30 fs4.
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| 99.94 |
The presence of the time delay and the unusual fluence dependence lead us to propose an alternative relaxation pathway where e-h pairs decay primarily into K-point optical phonons. These in turn decay into the G-phonon via strong ph-ph coupling (Fig. 1(c)). Consistent with this scenario, lifetimes calculated from e-ph coupling for K-phonons are 176 fs, 3 times faster than G-phonons4142. Furthermore, strong 4-phonon scattering at high temperature has been reported4344 with the inferred lifetimes for the decay of K-phonons into G-phonons of about 132 fs (latter based on the G-phonon linewidth of over 40 cm−1 at T > 2500 K44. The inverse process where K phonons decay to G phonons has the same time constant.). Thus the overall timescale for this indirect process, 300 fs, is consistent with the observed phonon buildup (~220 fs). The fact that 2D peak is more intense than 2D′ peak (double resonance for Γ phonons) also suggests a stronger e-ph coupling for K-phonons312945.
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In order to understand how K-phonons may be responsible for the late appearance of G-phonons, we simulate the optical phonon temperature based on the above scenario where anharmonic hot phonons are involved. Our model assumes that carriers thermalize rapidly into a Fermi-Dirac distribution with a well-defined temperature and that G phonons and K phonons thermalize among themselves. The electronic system, G-phonons, and K-phonons are characterized by their temperatures Tel, TG, and TK and are linked by e-ph coupling, Γe−ph(Tel, TG/K), and ph-ph coupling between G and K-phonons, Γph−ph(TG, TK). This leads to three coupled differential equations:
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where I(t) is the laser irradiance, Cel and CG/K are the heat capacity of electrons and G/K-phonons respectively, T0 is room temperature, and τ = 2.4 ps is the measured decay rate of optical phonons into low-energy phonons through anharmonicity. I(t), Γe−ph(Tel, TG/K), and Γph−ph(TG, TK) contain adjustable parameters to fit experimental results (see supplementary for details). Combining ARPES and Raman results constrains the fits so that the same adjustable parameters have to describe the ARPES and the Raman data. Specifically, having to obtain the ARPES simulation close to the experiment constrains Cel.
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The simulation without coupling between G and K phonons (Γph−ph = 0) is equivalent to the 2T model. It always gives a curve that is shifted from the Raman data to earlier times (Fig. 1(a)) because the 2T model predicts that the G-phonons are generated starting at t ~ −35 fs due to the finite width of the pulse. Note that the coupling constant λG in Γe−ph only determines the time constant of the G-phonon generation, but it has no influence on when TG starts to rise. As a result, the phonon dynamics in the early stage of relaxation is clearly not consistent with the 2T model.
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We call the simulation with nonzero Γph−ph the anharmonic hot phonon model (AHP model), because it includes anharmonic couling between the G and K phonons. Due to energy and momentum conservation, the two K-phonons can decay into one G-phonon and one Γ phonon in the branch of lower energy, or decay into one G-phonon and one K-phonon in the branch of lower energy (Fig. 5). It agrees much better (Fig. 1(a,b)) with the data. Within this picture, pump of lower power (0.15 mJ/cm2) gives a slower G-phonon buildup (see Fig. 4) consistent with decreased anharmonicity at lower phonon temperature. So the inclusion of ph-ph scattering is essential for explaining the observed time delay.
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Some previous experiments and calculations in the literature agree with out result. They are not consistent with the dominance of e-h channel in the G-phonon population buildup. For example, phonons are still emitted efficiently in the early stage even when doping puts EF well below or above half of G-phonon energy28. As the interband transition that contributes to G-phonon emission is suppressed with doping, the G-phonons would not be generated as fast as in the undoped samples by direct e-h recombinations, which suggests that G-phonons are emitted by other processes. However, all these results are consistent with our scenario.
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To conclude, we applied TRR and trARPES to investigate e-ph relaxation in graphite within a few hundred femtoseconds. Our simulations combined with experimental results show that instead of electrons coupling to G-phonons directly, K-phonons are emitted via strong e-ph scattering and then transfer energy to G-phonons via ph-ph scattering. This relaxation pathway is also consistent with previously published experiments and calculations28294441, though the mechanism was not explicitly discussed.
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Our findings have a broader implication for understanding of e–h relaxation processes in other materials such as light absorbers where maximizing photoexcited carrier lifetime is an important engineering challenge. For example in graphite and graphene, carrier recombination could be slowed down significantly by reducing the coupling between the K-point phonons and G-phonons. Thus it is essential to understand and control ph-ph interactions in addition to e-ph ones in order to design better optical absorbers and optoelectronic devices.
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| 99.5 |
Our TRR setup (Fig. 2(a)) uses a 20 kHz 790 nm (1.57 eV) laser pulse from an amplified mode-locked Ti:sapphire laser as the pump pulse. The pulse width is about 50 fs. Second harmonic generation at 395 nm (3.14 eV) was used as the probe light source for Raman scattering. The cross-correlation measurement of the pump and probe pulses gave the time resolution of 90 fs. The scattered light was collected by a pair of parabolic mirrors and then analyzed on a McPherson custom triple spectrometer equipped with a water-cooled CCD detector. The spectral resolution is limited by the time-energy Heisenberg uncertainty principle for the probe pulse. The experiment was performed in air at room temperature. The fluence of the pump was from 0.15 mJ/cm2 to 5 mJ/cm2, and the probe fluence was 500 μJ/cm2. TrARPES used the same laser system with 1.57 eV for the pump with a fluence of 130 μJ/cm2 and 6.3 eV for the probe (Fig. 2(a)). The time resolution for trARPES based on a cross-correlation measurement was 110 fs.
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Time zero in TRR was determined by measuring the sum frequency generation (SFG) intensity of pump and probe pulses on graphite. SFG that originates from the weak nonlinear dielectric polarization of graphite is greatly enhanced when pump and probe pulses arrive at graphite at the same time, which allows us to determine time zero accurately. The SFG intensity at various delay time fits well to a Gaussian function that gives an accuracy of ±4 fs in the determination of time zero. The 0.1 μm translation resolution of the delay stage only adds 0.67 fs to the time uncertainty. Therefore the total uncertainty of time zero is less than 5 fs.
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| 100.0 |
In 2015, after the massive influx of refugees to Europe and the change of the political option, the attitude of Polish society began to transform and become more nationalistic and xenophobic. Similar changes occurred in Hungary, the Czech Republic and Slovakia. These specific transformations are perceived and noted in other EU countries. Feedback causes further effects.
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The question arises whether the described changes have an impact on public mental health [1–4] According to so-called ‘positive psychology’ and ‘positive psychiatry’ , such transformations have an impact on the average state of health. A xenophobic attitude is accompanied by a higher incidence of anxiety, disputes by reason of the polarization of opinions, a sense of embarrassment and contradictions with so-called ‘Christian values’, adverse demographic perspectives, depression and reduced life satisfaction . A xenophobic attitude is usually the result of deeper social changes caused by economic crisis, loss of confidence in the existing political elite and an increase in populist argumentation.
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| 99.8 |
Therefore, we intend to discuss first the so-called ‘sequence of adverse events’ that led to the xenophobic attitudes, and then characterize the current state of public mental health and propose actions that could counteract the consequences of that transformation.
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| 99.9 |
Since the end of the 1980s, there has been an ideological dispute in Poland between two alternative camps in society. One of them is the vision of the society which is mono-ethnic, nationalistic, Catholic, conservative, as was formulated by Roman Dmowski, co-founder of the II Polish Republic. The second vision comes from the times of the creators of the Constitution of May 3, which assumes the existence of a multi-ethnic, multi–religious, civil state – a concept supplemented later by the liberal ideas of freedom, the market economy and democratic organization of the state. The violent ethnic events during the Second World War and the decisions of Stalin’s regime turned Poland into a monolithic state in terms of ethnicity. A quite similar transformation occurred in Czechoslovakia, divided later on into the more monolithic states of the Czech Republic and Slovakia, as well as the territorially truncated Hungary.
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According to Timothy Snyder, the nationalist contemporary stance arises from the belief that prosperity and a just organization of society is most easily obtained by completely independent, fully sovereign national states . He writes in a recent, very well-known article as follows:I do not understand how people can interpret the interwar times as an object of nostalgia, and so this period is presented by nationalistic politicians promoting populism. It is a great illusion that then … nation states were durable and prospered well. It was the opposite. This period was short and it ended awfully… a nation is necessary but insufficient … history is established by greater entities, organisms, empires … If a government avoids any integration, it is wrong … There are two ways to deal with the effects of globalization. The first is to build a regional organism, which is like a roof over its head, which defends. The second way is the defense of national identity, focusing on the opposition, we - them…
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| 99.94 |
I do not understand how people can interpret the interwar times as an object of nostalgia, and so this period is presented by nationalistic politicians promoting populism. It is a great illusion that then … nation states were durable and prospered well. It was the opposite. This period was short and it ended awfully… a nation is necessary but insufficient … history is established by greater entities, organisms, empires … If a government avoids any integration, it is wrong … There are two ways to deal with the effects of globalization. The first is to build a regional organism, which is like a roof over its head, which defends. The second way is the defense of national identity, focusing on the opposition, we - them…
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| 99.94 |
Poland, the Czech Republic and Hungary are countries that did not participate in colonial conquests. Therefore, they did not bring to their territories larger groups of people of different racial, national and religious identity. The ways of living together in one country with citizens of different races, nationalities and religious beliefs are not known here from personal experiences of the last 70 years. Opinions on the possibility of co-existence and integration of so-called ‘strangers’ are based only on the opinions presented by publicists and politicians. As is known, the political and military events that occurred in the last few years in the countries of North Africa and the Middle East have led to the exodus of refugees, the emergence of the so-called Islamic State and acts of terror . These events, of course, are widely commented on by Eastern European publicists and politicians. They also comment on the average beliefs and attitudes of Muslims and analyze in particular the possibility of integrating refugees in Western societies, especially the phenomenon of so-called ghettos.
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Many citizens of European countries consider that the administration of the EU and even present democratic national governments are not able to solve this new growing problem of the influx of refugees, and in particular to end the military conflicts generating this wave of migration.
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| 99.94 |
Some observers of the social transformations in Europe postulate that the trend towards a more nationalistic and xenophobic policy in Europe started already with the economic crisis in 2008 (troubles of Greece and problems for Euro currency) [10, 11]. The refugee influx from 2015 only amplified this effect. This point of view can be justified. The citizens of many countries assume, in these circumstances, that the current ruling political elite is also responsible for the growing inequality, and high unemployment in some European countries . For this reason the populist programs find approbation. Nevertheless, populist politicians always try to identify the guilty. Therefore a part of populist programs consists usually in accusing and prosecuting foreigners, newcomers.
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It should be noted, however, that the xenophobic attitudes significantly increase always from the moment of the winning of a populist coup. The group of people who won in this way the power increases and consistently supports the nationalist and xenophobic sentiments.
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| 99.94 |
It can be exemplified by the facts observed in Poland. Here the significance of the impact of the economic crisis of years 2008 – 2012 was not great, because our country as an exception during this period was in a good economic condition [13, 14]. The GDP growth was here permanently above 3 %, therefore our country was called “the green economic island”.
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The intensification of nationalistic attitudes that have occurred recently in Western countries (Brexit, Marine Le Pen’s movement in France) have also had an impact on the convictions of citizens of Eastern European countries. The events and opinions recalled here are used for the current political struggle. As a result, the nationalist and xenophobic attitude is intensified.
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| 99.94 |
There are centers carrying out objective assessments of the most important parameters, defining the state of public mental health in Eastern European countries. A very useful tool constitutes the annual report of the so-called ‘World Justice Project’ (rule of Law Index) . This annual report provides a summary of indicators and graphic illustration of the situation in 102 countries of the world on objective findings concerning: 1. Compliance with the standards of democracy, 2. The level of corruption, 3. Transparency in governance, 4. The degree of respect for human rights, 6. Order and Security 7. The effectiveness of legislation, 8. Civil (social) justice, 9. Judiciary (criminal) justice. We propose looking at the graphs, for example, of Poland, the Czech Republic, Germany, Russia and Turkey, and to draw conclusions.
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| 99.9 |
Among the parameters included in the ‘World Justice Project’ the features relating most to the problem of intensifying nationalistic and xenophobic attitudes are: freedom of expression, religious freedom, the right to privacy, the right to association and demonstration, labor rights, as well as the parameters for the senses of social and criminal justice. The new data, which will be announced after the observations made in 2016, may reflect changes which have occurred since the intensified inflow of refugees in the second half of 2015.
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The project “Social Diagnosis” supplements indicators, which are presented annually by the Polish Central Statistical Office. The authors of this project derive data for their own questionnaires, which since 13 years are yearly distributed among 22 thousand people living in selected households. The collected data concern the conditions and quality of life of citizens as well as their attitudes, state of mind and behaviors.
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| 99.9 |
There are taken into account all the important aspects of both economic and non-economic factors such as income, material affluence, savings, loans and indices of quality of education, medical care and ways to deal with problems, signs of stress, mental well-being, lifestyle, pathological behaviors, participation in culture, the use of modern communication technologies and many others.
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| 99.9 |
The last available report is for the year 2015. The results of the survey conducted in 2015 are puzzling. Contrary to popular opinion expressed in casual conversations and most of publications in mass media - the authors of the project “Social Diagnosis” noted the improvements in a number of indicators relating to the conditions and quality of life. They write inter alia that the percentage of Poles who consider themselves as very or quite happy to be 81.5 %. This proportion increased by 3.1 % compared to 2013. The real income of households increased in comparison to 2013 by more than 12 % and the personal income by 10 %. There has been an increase in satisfaction in most aspects of life, especially in the evaluation of satisfaction with the situation in the country, the prospects for the future and the financial situation of the family. The economic stratification of Polish society diminished. Inequality of income distribution of households measured by the Gini coefficient falling in the last six years. The value of this ratio in 2009 was 0.318; in 2011 was 0.307; in 2013 was 0.305 and in 2015 was 0.285 (far below the average for the 27 countries of the European Union). Also the dissection of personal income fell from 0.373 in 2009 to 0.330 Gini coefficient in 2015. Income of the poorest households grew faster than the richest. The authors found that below the limit of extreme poverty in Poland in June 2015 lived 3.3 percent of households (1.8 % less than two years earlier, the least in the whole study period).
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| 99.9 |
According to the authors of the project, there is in Poland little sign of development of civil society. Compared with previous studies, the percentage of people who trust other people increases from 12 % in 2013 to 15 per cent in 2015. Also slightly increased the sensibility to violations of the common good, but still almost half of the population is indifferent to acts of violating the public good.
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| 99.9 |
Probably, the subjective explanation formulated by those who have a negative opinion on the national-populist upheaval, which happen in Poland will be reflected in the objective data that will provide the authors of the project “Social Diagnosis” in the report for 2016.
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| 99.94 |
It may turn out, however, that the mentioned parameters are not able to reflect the nature of new social trends. Many publicists emphasize that authoritarian populist politicians promise “to restore order, a sense of dignity, social discipline and to control the market and augment social support.” This brings to mind yet another possibility for estimating changes in mental health. Aaron Antonovski, in the times of relative social balance in the 1980s defined the concept of coherence and proposed to estimate such a state of the mind by means of his SOC-29 test . Coherence is composed of: (1) the ability to understand events (comprehensibility), which allows one to perceive them as less stressful, (2) a sense of meaningfulness, consisting in the belief that it is worth being involved and creating one’s own life, (3) a sense of resourcefulness (manageability) .
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The concept of coherence has become very well known. Hundreds of authors have published papers relating to the measurements of the level of coherence and proposed even a modification of its individual components . The method of prevention and health promotion based on the concepts of coherence is called ‘salutogenesis’ .
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It should be noted that the sociological, practical problem is two-fold. It is necessary to consider the willingness and ability of immigrants to integrate in the society to which they came. In addition, there are variable degrees of difficulty to assimilate immigrants by the host communities.
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| 99.94 |
Antonovsky’s concepts are highly relevant to the problem of xenophobia because he came from a family of immigrants and he was personally an immigrant, first in the United States, and then in Israel (an ‘immigrant’ country), where he also conducted extensive research on the relationship between well-being and health, depending on the degree of integration of newcomers .
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| 99.94 |
He considered the so-called ‘pro-health quality of the receiving culture’.As the author of salutogenesis, he stresses that the receiving culture should promote conditions enabling citizens to experience life as comprehensible, manageable and meaningful. Comprehensibility arises from a stable culture that sends consistent messages to people, manageability is created by a culture that gives people the tools to live up to norms set by the culture, and meaningfulness is supported by a culture that values the role of people and gives them a place in the world. His original statement on this topic is as follows: “Clearly, if one has a high intelligence, lots of money, or a clear ego identity or lives in a stable, integrated culture – to mention some ‘generalized resistance resources’– there will be consequences not only for the emergence of a strong SOC, and therefore health, but for other areas of well-being as well” .
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| 99.9 |
To assess the state of public mental health, it is worth yet quoting a recognized, widespread general definition of mental health. Manwell et al., promotes the formulation made by the Public Health Agency of Canada, which states that: “Mental health is the capacity of each and all of us to feel, think, and act in ways that enhance our ability to enjoy life and deal with the challenges we face. It is a positive sense of emotional and spiritual well-being that respects the importance of culture, equity, social justice, interconnections and personal dignity” .
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| 99.9 |
In light of this definition, is easy to see that every citizen of Poland is troubled by the consequences of polarization of attitudes and views on the values which have been promoted during the past 25 years. Those with the convictions to being liberal, libertarian, tolerant are now in the defensive. It is difficult to achieve in this situation the determinants of good mental health, which require citizens to “enjoy life in the sense of emotional and spiritual well-being”. Yet other consequences of the intensification of the xenophobic attitude have occurred. The most obvious is the threat to public finances due to the slowdown of investment. A more subtle, but very unfavorable change consists in decreased “social capital”, i.e. the ability of citizens to cooperate with others [21–27]. Deepening the isolation of the country also diminishes the opportunities to improve the educational process and creativity.
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| 99.9 |
Repealing from adopting even a very limited number of refugees is squandering a chance to force the development of professional, mental, cultural and linguistic skills of many institutions. The need to integrate people from different cultures is a challenge which stimulates different intellectual processes. The lack of implementing such efforts is detrimental to the development of practical skills of representatives of many professions.
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| 99.94 |
The question arises if there are any possible actions to mitigate the xenophobic attitude, even in very unfavorable conditions, when those ruling the country are not interested. It seems to us that such actions are possible. They result from the methods of ‘positive psychology’ and ‘positive psychiatry’ [5, 6], the principles of strengthening the local social capital [28–35] and the acquired knowledge of psychological regularities related to the formation of xenophobic attitudes, especially in children and adolescents [36–42]. These include: (1) specific publicist and cultural interventions (promotion of specific novels, movies) [43, 44] and (2) the promotion of certain already-developed software tools and social media e.g.: Viacharacter – a survey on character strength [45, 46], Sense of Coherence – Orientation to Life Questionnaire or Learning to live together . The efforts made in other European countries should be noted. An example is the recently announced program of implementations of new ways of intensifying the integration of refugees announced in Germany .
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other
| 99.56 |
Readers of this article may feel the need to be more aware of what the methods are resulting from so-called ‘positive psychology and ‘positive psychiatry’ and the principles of ‘strengthening the local social capital’. It is quite easy to make a review of these methods because one can indicate relevant publications available on-line [43–47].
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| 99.4 |
Counteracting a xenophobic attitude boils down to eliminating prejudices. Citizens do away with prejudices when open-mindedness, the recognition of the rights of others to have a different opinion, appreciation of diversity, tolerance and the freedom to make choices become values for them. Hence, Hershberger, one of the authors representing the domain of ‘positive psychology’ proposes the use of such methods of personality transformations as ‘Three good things’, ‘The interpersonal sharing of good news’, ‘Sacrificing unnecessary effort and considering whether it is good enough’, ‘Realization the strengths and virtues of one’s own character’, ‘Learned optimism (not always, not everything)’ .
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other
| 99.94 |
‘Three Good Things’ intervention simply requires an individual to write down three positive occurrences that happened during the day every night for 1 week and for each occurrence an answer to the question of why the good thing happened. ‘Realizing the strengths and virtues of own character’ can be supported by utilizing the website Viacharacter ,
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other
| 99.94 |
An example of another approach of positive psychology can be the postulate of augmenting so-called ‘psychological capital’ [50–54]. Psychological capital is defined as a higher-order core psychological construct and includes four states: psychological resources of self-efficacy, optimism, hope and resiliency. All these four factors can be measured and enhanced.
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other
| 99.75 |
The targets of the enhancing self-efficacy, optimism, hope and resilience can be treated as tasks of supportive psychotherapy. We tried to explain in one of our previous papers, accessible on–line, how time–consuming behavioral psychotherapy can be supported by determining so-called ‘therapeutic tasks’ .
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other
| 99.9 |
It should be noted, however, that having tools that help to eliminate prejudices which are prevalent in the population affected by xenophobia is not tantamount to effective influence. Actions are impeded by the possibilities to reach the public, especially in the situation of the unfavorable attitude of the current government.
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other
| 99.94 |
The key feature, essential for enhancement of social capital is the trust to other people. It should be noted that the xenophobic attitude lies precisely in the lack of trust in others. So, all the method of counteracting xenophobia through the strengthening of social capital consists of augmentation of the trust to others.
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other
| 99.94 |
Strengthening social capital is easier when the country is organized on the basis of local administration autonomy and when many non–governmental organizations exist. In other words, it is easier to strengthen social capital when the civil society is under development [55–58].
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other
| 99.94 |
The important factors enabling the leveling of xenophobic attitudes consist in appropriate changes in education and an appropriate cultural atmosphere . Unfortunately, the populist regime interferes negatively in the organization of education and promotes its specific ‘cultural policy’.It follows that overcoming xenophobic attitudes is associated with social and even political struggles.
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other
| 99.94 |
It is important to promote the equal rights of men and women, and even a 50 % parity in different bodies, because the attitude of women is more moderate and pacifistic. Efforts should be made to widely disseminate newly acquired knowledge on the reverse relationship between the capacity for friendship and attitudes of tolerance and xenophobia [39–42] and the overwhelming influence of mothers (parents) on the xenophobic attitudes of their children [36–38]. Research on transnational identity is also useful [59–62]. The experiences from the integration of foreigners should be gathered in communities and schools of all types [63–66]. One should promote the training of people willing to undertake counseling and stimulate integration with foreigners .
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other
| 99.7 |
Today there are tools to objectively determine whether the recent resurgence of nationalist and xenophobic attitudes in many countries of the world is evidenced in an objective manner, and whether it causes adverse consequences in the state of public mental health. The estimation of such changes can be made, for instance, by tracking changes that are recorded in the annual reports of the ‘World Justice Project’ (rule of Law Index) .It is possible to formulate remedies to counteract the deterioration of public mental health.Although the reasons for the intensification of nationalist and xenophobic attitudes are very complex, the counteractions for these changes in social consciousness can be underaken not only by enlightened publicists and politicians but also by people who are concerned about the state of public health. They can refer to the methods of so-called positive psychology and positive psychiatry and widely known famous and reputable cultural products (movies, novels, mass media performances).Development and implementation of the methods and tools influencing the whole population, which counteract spreading xenophobia is a new challenge for the people who care about the state of public health.
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other
| 99.9 |
Today there are tools to objectively determine whether the recent resurgence of nationalist and xenophobic attitudes in many countries of the world is evidenced in an objective manner, and whether it causes adverse consequences in the state of public mental health. The estimation of such changes can be made, for instance, by tracking changes that are recorded in the annual reports of the ‘World Justice Project’ (rule of Law Index) .
|
other
| 99.94 |
Although the reasons for the intensification of nationalist and xenophobic attitudes are very complex, the counteractions for these changes in social consciousness can be underaken not only by enlightened publicists and politicians but also by people who are concerned about the state of public health. They can refer to the methods of so-called positive psychology and positive psychiatry and widely known famous and reputable cultural products (movies, novels, mass media performances).
|
other
| 99.94 |
Proteomic techniques using mass spectrometry (MS) provide a platform for the comprehensive analysis of proteins, thereby facilitating the implementation of systems biology approaches and circumventing the limitations of a traditional, reductionist approach adopted by techniques like western blotting that are based on a priori assumptions of the proteins to be investigated. Furthermore, proteomics is without the constraints of antibody-dependent protein detection and has the capability of detecting post-translational modifications (PTMs), which is beyond the means of gene expression platforms.1
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review
| 99.7 |
Tissue fibrosis is a hallmark of most cases of cardiovascular disease (CVD) and includes modification and deposition of extracellular matrix (ECM). However, detailed studies on the cardiovascular ECM have been sparse due to the lack of analytical tools that facilitate comprehensive characterization of its components. In recent years, proteomics has been successfully applied to study the ECM, providing unprecedented insights into its biology and pathological remodelling.2–5 In the present review, we describe the utility of ECM proteomics as applied to cardiovascular research and the potential pitfalls. In addition, we highlight the means to overcome common proteomic challenges and present translational applications of proteomic datasets.
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review
| 99.9 |
The ECM not only confers mechanical stability, but is also a reservoir for bioactive molecules. Remodelling of the ECM, including quantitative but also qualitative changes in composition, is a hallmark of CVD. Numerous studies have demonstrated that structural, but also non-structural ECM proteins play crucial roles during disease progression and normal cardiac physiology.
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review
| 99.1 |
Table1 summarizes important findings in clinical studies as well as in animal models of cardiac disease.5–42 Additional studies reported ECM proteins as potential biomarkers for cardiac pathologies43; these have been intentionally omitted from the table, the focus of which are ECM and ECM-associated proteins (i.e. extracellular proteases and non-structural proteins that bind to or regulate ECM) from a functional perspective. Most proteins included in the table were individually studied using antibodies and loss-of-function models in order to assign their relevance to disease. Proteomics can quantify most of these ECM proteins in a single experiment, leading to the identification of previously unreported links between ECM components in disease.2–4 For example, in a recent study we demonstrated that genetic deletion of biglycan was accompanied by an unexpected rise of aggrecan in murine aortas.44 Table 1Role of ECM and ECM-associated proteins in cardiac diseaseProteinClinical contextMain findingsAdiponectinCardiac remodelling (m)Induces cell migration, MMP activation, and collagen remodelling via APPL1-AMPK signalling6ADAMTS9Developmental defects (m)Haploinsufficiency leads to reduced versican cleavage, associated with cardiac anomalies7BiglycanMI (m)Required for adaptive remodelling8Cathepsin-KAF (h, rb)Increased levels and activity accompanied atrial changes linked to the AngII/ATR1R signalling pathway9Cathepsin-SMI (m)Mediates fibroblast transdifferentiation during remodelling10Collagen IDilated cardiomyopathy (m)Point mutation induces cardiomyopathy11Collagen VIMI (m)Absence improves cardiac function, structure, and remodelling12Collagen XIVDevelopmental defects (m)Important for growth and structural integrity of the myocardium13Collagen XVHypertension (m)Necessary for remodelling. Deficiency predisposes to cardiomyopathy14Connective tissue growth factorPressure overload (m)Inhibition attenuates left ventricular remodelling and dysfunction15DecorinLeft ventricular assist device implantation (h)Ameliorates adverse remodelling by mediating TGF-beta inhibition16MI (m)Absence leads to abnormal scar tissue formation17FibronectinMI (m, h)Essential for progenitor cell response during cardiac repair18MI (m)Lack of EDA domain promotes survival and prevents adverse remodelling19Fibulin-2MI (m)Loss protects against progressive ventricular dysfunction20Laminin alpha-4Dilated cardiomyopathy (h, z)Mutations cause human cardiomyopathy via defects in cardiomyocytes and endothelial cells21LumicanHypertrophy (m)Deficiency results in cardiomyocyte hypertrophy with altered collagen assembly22MimecanMI (m, h)Prevents cardiac dilatation and dysfunction via collagen strengthening23MMP-14Pressure overload (m)Mediates pro-fibrotic signalling, leading to alterations in interstitial fibrosis and diastolic function24MMP-28MI (m)Deletion exacerbates cardiac dysfunction and rupture by inhibiting M2 macrophage activation25TIMP-2Pressure overload (m)Loss leads to exacerbated left ventricular dysfunction and adverse ECM remodeling26MMP-9AF (p, h)Increased gelatinase activity contributes to atrial ECM remodelling27,28MI (h,m)Crucial for generation of bioactive collagen I fragments that promote scar formation after MI5MI (m)Deletion leads to decreased collagen accumulation and left ventricular enlargement29MMP-2MI (m, r)Contributes to ischemia-reperfusion injury, and deletion/inhibition prevents cardiac rupture30,31OsteopontinMI (m)Deletion leads to left ventricular dilatation and reduced collagen deposition after MI32PeriostinMI (r)Blockade of Exon 17 preserves cardiac performance33Pressure overload (m)Deletion results in less fibrosis and hypertrophy34PerlecanDevelopmental defects (m)Perlecan is critical for heart stability35SPARCMI (m)Mediates early ECM remodeling36Tenascin-CPressure overload (m)Accelerates fibrosis by activating macrophages via the integrin αVβ3/nuclear factor-κB/interleukin-6 axis37MI (m)May aggravate left ventricular remodelling and function38Thrombospondin-1Pressure overload (m)Protects myocardium by modulating fibroblast phenotype and ECM metabolism39MI (d, m)Role in preventing expansion of healing myocardial infarcts40Thrombospondin-4Pressure overload (m)Regulates myocardial fibrosis and remodelling41VersicanDevelopmental defects (m)Associated with chamber specification, septation, and valvulogenesis in the developing heart42MMP, matrix metalloproteinase; ADAMTS, a disintegrin and metalloproteinase with thrombospondin domains; TIMP, tissue inhibitor of metalloproteinases; SPARC, secreted protein acidic and rich in cysteine; MI, myocardial infarction; AF, atrial fibrillation; m, mouse; h, human; rb, rabbit; z, zebrafish; r, rat; p, pig; d, dog.
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review
| 99.8 |
MMP, matrix metalloproteinase; ADAMTS, a disintegrin and metalloproteinase with thrombospondin domains; TIMP, tissue inhibitor of metalloproteinases; SPARC, secreted protein acidic and rich in cysteine; MI, myocardial infarction; AF, atrial fibrillation; m, mouse; h, human; rb, rabbit; z, zebrafish; r, rat; p, pig; d, dog.
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other
| 99.94 |
Until recently, the identification of proteins in a tissue or a protein lysate has been limited by the availability of antibodies that recognise certain regions (epitopes) of a protein of interest. Antibodies have been and continue to be an important component of the armamentarium for protein research but they are not without limitations. While antibody arrays overcome the restriction to only one protein, ECM proteins tend to be under-represented in arrays. The main issues of antibody-based protein quantification, however, remain the same: (i) Usually only a small portion of the protein (epitope) is recognised by an antibody. Protein detection by antibodies relies on the presence of unmodified epitopes. In ECM proteins, however, common PTMs include hydroxylation, glycosylation or fragmentation. Due to epitope masking, ECM proteins may not be detectable by antibodies. (ii) Antibodies are often not commercially developed to target proteins in species beyond the commonly used mice and rats such as canine and porcine models for myocardial infarction,2,45,46 rabbit and goat models for studies involving atrial fibrillation9,47–49 and sheep as models of dilated cardiomyopathy.50 While some anti-human or anti-mouse antibodies will cross-react, many others will not recognize their target protein in different species or will display a high degree of non-specific binding. Vice versa, proteins in the bovine serum supplements of cell cultures can be detected not only in the conditioned media but also in the cell lysates.51
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review
| 99.9 |
Contrary to antibodies, proteomics does not rely on recognition of one specific epitope and can be applied across species. Moreover, the use of MS allows for determination of changes that occur at the protein level (i.e. amino acid modifications) (Figure 1). For example, we demonstrated using MS that the C-terminus of decorin, a small leucine-rich proteoglycan, is often cleaved in the left atrium but not in the ventricle.52 MS data provided an explanation why the use of different antibodies for the same target protein yielded very different results (Figure 2). Figure 1Antibody limitations. Detection by antibodies relies on binding to specific regions (epitopes) of the target protein. PTMs such as glycosylation or fragmentation may hinder epitope accessibility. Antibody A recognises non-glycosylated regions and always yields detection independently of sugar removal (left panel). Antibody B recognises epitopes in the vicinity of glycosylated regions. Therefore, recognition is only achieved after deglycosylation. Similarly, if protein fragmentation occurs, only antibody C, which recognises an intact portion, reveals a degradation pattern. Antibody D targets a region affected by fragmentation and can only detect the intact epitope. Consequently, information about degradation is missed. Proteomics interrogates peptides across the whole sequence and allows for consideration of variable modifications at the amino acid level. Different protein forms can therefore be identified and quantified. GAG, glycosaminoglycan; Pan-DG, pan-deglycosylation. Figure 2MS to explain discrepancies between different antibodies. An antibody against a C-terminal epitope (green on left panels, red on right panels) results in less intense staining for decorin (DCN) in the atrium compared to the left ventricle. An antibody against a different epitope (green on right panel) shows no such difference in staining intensities. This may be explained by cleavage of decorin at the C-terminus, which was detected in the atrium using MS.
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study
| 100.0 |
Antibody limitations. Detection by antibodies relies on binding to specific regions (epitopes) of the target protein. PTMs such as glycosylation or fragmentation may hinder epitope accessibility. Antibody A recognises non-glycosylated regions and always yields detection independently of sugar removal (left panel). Antibody B recognises epitopes in the vicinity of glycosylated regions. Therefore, recognition is only achieved after deglycosylation. Similarly, if protein fragmentation occurs, only antibody C, which recognises an intact portion, reveals a degradation pattern. Antibody D targets a region affected by fragmentation and can only detect the intact epitope. Consequently, information about degradation is missed. Proteomics interrogates peptides across the whole sequence and allows for consideration of variable modifications at the amino acid level. Different protein forms can therefore be identified and quantified. GAG, glycosaminoglycan; Pan-DG, pan-deglycosylation.
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study
| 95.9 |
MS to explain discrepancies between different antibodies. An antibody against a C-terminal epitope (green on left panels, red on right panels) results in less intense staining for decorin (DCN) in the atrium compared to the left ventricle. An antibody against a different epitope (green on right panel) shows no such difference in staining intensities. This may be explained by cleavage of decorin at the C-terminus, which was detected in the atrium using MS.
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study
| 100.0 |
Proteomics is the study of the complete protein component of a living organism, tissue or cell and yields unbiased data without a priori knowledge. The workhorse of modern proteomics is the mass spectrometer and although it is not a new technology per se, it was for a long time confined to areas outside the biological sciences. However, it was the advent of matrix-assisted laser desorption ionization (MALDI)53 and in particular of electrospray ionization (ESI)54—which enables liquid chromatography (LC) systems to be interfaced directly to mass spectrometers—that MS branched from analytical chemistry into biology.
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review
| 57.12 |
The gold standard for contemporary proteomics is LC-tandem MS (LC-MS/MS). Briefly, the LC column separates the peptides (typically generated by digesting proteins with trypsin) in the analyte prior to ionisation and subsequent MS analysis. In addition to recording the mass of the peptide ions, MS/MS technologies induce the subsequent fragmentation of these precursor ions. The masses of these fragment ions can therefore be used to delineate the amino acid sequence of the peptide. The availability of annotated protein sequence databases and algorithms that match the observed MS/MS spectra to protein entries have been crucial for the biomedical application of MS to study proteins.55,56 MS data can also be aligned to databases generated using DNA or RNA sequences to infer amino acid sequences. Current MS technologies now allow for the characterization of the ECM composition and turnover in CVD in unprecedented detail that is not possible using other techniques.2–5,52,57–59
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review
| 99.75 |
Over the past years our group has focused on ECM remodelling in cardiac2,52,60,61 and vascular tissues3,4,57,59 using proteomics. In a previous review, we highlighted the potential of proteomics when applied to systems biology.62 A recent review by Chang et al.63 has focused on clinical applications of ECM proteomics (i.e. biomarker discovery and tissue engineering). In this review, we discuss how MS can be used to assess ECM composition in CVD.
|
review
| 99.9 |
Contemporary mass spectrometers have exceptional sensitivity, providing detection at attomole concentrations.64 However, such sensitivity is largely confined to pure solutions and not yet achievable in complex biological samples. For example, the dynamic range of proteins present in plasma spans 10 to 11 orders of magnitude (e.g. 4 × 1010 pg/ml for albumin compared to few pg/ml for some interleukins).65 Current MS instrumentation can only resolve 4–6 orders of magnitude. While proteomics offers a comprehensive analysis of high abundant proteins it has not yet overcome the difficulties of analysing low abundant proteins in complex samples. Unlike PCR or antibody-based techniques, proteomics lacks the ability to amplify low abundant proteins to aid detection and instead relies on enriching the target proteome.
|
review
| 99.9 |
For instance, cardiac ECM proteins are markedly less abundant than cytosolic and mitochondrial proteins.46 Thus, studies that use whole tissue lysates for proteomic analysis inevitably mask the identification of the less abundant ECM constituents. With cardiac tissue this is exacerbated due to the higher cellular content.2 Accordingly, methods that enrich for ECM proteins have received considerable interest of late and principally focus on removing plasma contaminants and soluble cellular proteins.57,66
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review
| 99.8 |
While the inherent insolubility of many ECM proteins lends itself to effective enrichment by decellularization, subsequent proteomic analysis requires all proteins to be solubilized. Standard lysis buffers are not effective for ECM solubilisation. Instead, we implemented a stepwise extraction of vascular ECM proteins.57 This involves treating vascular tissues with sodium chloride (NaCl) to remove plasma proteins and extract loosely bound extracellular proteins before decellularizing the tissue with sodium dodecyl sulfate (SDS). Each incubation step takes 4 h. Solubilisation of mature ECM proteins is finally achieved by treatment with guanidine hydrochloride (GuHCl) which destabilizes the ionic, disulfide-dependent protein conformations in large aggregating proteoglycans (versican, aggrecan, etc.), small proteoglycans (decorin, biglycan, etc.), cell-attachment glycoproteins such as type VI collagen, fibronectins, laminins, and basement membrane components.67 The method was later adapted for the use in porcine cardiac tissue by reducing the incubation time for NaCl and prolonging the SDS treatment2 (Figure 3A). In smaller animal models (i.e. mouse, rat) cardiac cellularity is proportionally higher compared to that of larger animal models such as pig or goat (Figure 3B). With increased cellularity, decellularization is more difficult to achieve and may require additional enrichment steps, i.e. for glycoproteins or glycopeptides.52 Figure 3Enrichment of cardiac ECM proteins. (A) Our previously published 3-step ECM extraction method for cardiac tissue is based on decellularization and ensures enrichment and detection of ECM proteins. The image shows a decellularized heart after prolonged SDS perfusion. The ECM is solubilized by GuHCl and analysed using proteomics. (B) Smaller species display higher levels of cardiac cellularity as measured by the ratio of 3 members of different ECM protein classes and the cardiac-specific troponin T (TNNT2, y-axis). (C) Proteins identified in murine hearts using the Texas 3-step66 extraction method compared to those identified by our previously published method (see Drozdov et al.61). Most proteins are identified by both methods. Unlike the Texas 3-step method, our ‘English Quickstep’ method did not include an analysis of the remaining pellet after GuHCl extraction.
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study
| 99.94 |
Enrichment of cardiac ECM proteins. (A) Our previously published 3-step ECM extraction method for cardiac tissue is based on decellularization and ensures enrichment and detection of ECM proteins. The image shows a decellularized heart after prolonged SDS perfusion. The ECM is solubilized by GuHCl and analysed using proteomics. (B) Smaller species display higher levels of cardiac cellularity as measured by the ratio of 3 members of different ECM protein classes and the cardiac-specific troponin T (TNNT2, y-axis). (C) Proteins identified in murine hearts using the Texas 3-step66 extraction method compared to those identified by our previously published method (see Drozdov et al.61). Most proteins are identified by both methods. Unlike the Texas 3-step method, our ‘English Quickstep’ method did not include an analysis of the remaining pellet after GuHCl extraction.
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study
| 100.0 |
Others have adopted similar workflows to extract ECM proteins in a number of tissues.66,68–70 Of note is the Texas 3-step extraction method by Lindsey’s group.66 In their method, applied to mouse hearts, a similar sequential extraction consisting of NaCl and GuHCl extraction steps as well as the SDS decellularization2,4,57 are performed. In addition, the Texas 3-step method includes further extraction of the insoluble protein pellet after incubation in GuHCl for 48 h. Notably, the vast majority of ECM proteins are identified in the GuHCl fraction. The pellet, however, contains few polymerized proteins, which are not extracted by our ‘English Quickstep’ method (Figure 3C).
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study
| 100.0 |
In a recent proteomics study, Johnson et al.71 studied the human cardiac ECM from cadaveric donor hearts. Decellularization was achieved after perfusion with high SDS concentration (i.e. 10 times greater than that used in our protocol) for more than 3 days. This yields a simplified ECM, but the ECM proteins will be denatured and ECM-associated proteins will be lost during prolonged incubation with such a high concentration of detergents. The study of ECM using MS approaches described below, requires a gentler extraction method from snap-frozen tissues that strikes a balance between removal of cellular components while preserving ECM-associated proteins.
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study
| 99.94 |
Discovery proteomics refers to the use of proteomics as a hypothesis-free tool to globally profile the proteome of a given sample. In discovery proteomics, bottom-up or shotgun proteomics is based on the analysis of peptides generated after enzymatic digestion of a protein mixture. Digested peptides are separated by LC before MS/MS analysis. In comparison to gene expression analysis ECM proteomics offers certain advantages. First, many diseases manifest over years. Therefore, although transcript levels provide a window into cellular activity at the time of harvest, they merely provide an indirect assessment of protein synthesis at a single time point. When studying dynamic entities such as the ECM, transcript levels become more extraneous, particularly as nascent ECM proteins are incorporated into the existing matrix, and actual ECM protein abundance is determined by the balance of protein synthesis, deposition, and degradation.
|
review
| 99.44 |
There are multiple MS approaches that can be applied to yield accurate quantitation. However, each approach comes with distinct trade-offs.62 Label-free methods can provide relative quantification in simple mixtures. In complex mixtures, isotopic labelling should be employed, which allows multiplexing of samples. For instance, stable isotope labelling with amino acids in cell culture (SILAC) is based on metabolic labelling of proteins in vitro with amino acids containing heavy (e.g. 13C) stable isotopes. Fully labelled SILAC mice have also been generated.72 Methods for protein labelling are based on the use of isobaric tags, such as isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tag (TMT).73 Isobaric tags have the same chemical structure but different isotope substitutions. When samples are labelled with different tags, they can be subsequently mixed in equal portions, and the protein abundance from the different samples can be assessed by comparing the abundance of peptides labelled with the different tags in a single LC-MS/MS run. Although these tagging methods overcome issues such as technical reproducibility of LC-MS/MS runs, labelling is only introduced after protein digestion and therefore, unlike SILAC, isobaric tags do not allow for in vivo or in vitro labelling but have been used for quantitative comparisons using tissue samples.74,75
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review
| 99.9 |
The discovery proteomics approach is largely limited by the scan speed as peptides are selected for fragmentation based on abundance. This stochastic process results in a bias towards the more abundant proteins.62 In contrast to discovery proteomics, targeted proteomics focuses on a predetermined group of proteins of interest (e.g. ECM proteins). Proteotypic peptides unique to these proteins are quantified in what is known as selected reaction monitoring (SRM) or multiple reaction monitoring (MRM).76 The targeted approach increases selectivity, sensitivity, and accuracy and enables simultaneous measurement of hundreds of transitions in a single LC-MS/MS run.76 The transitions for proteotypic peptides will be interrogated as a surrogate of total protein levels, but peptides not included in the search (e.g. non-annotated PTMs) are not detected.55 This approach is particularly useful to detect CVD biomarkers, as Domanski et al.77 demonstrated in a study that also included ECM biomarkers of fibrosis. Moreover, targeted proteomics constitutes a robust method to validate findings obtained from discovery experiments (Figure 4).3 Figure 4MS strategies for ECM characterisation. Untargeted proteomics is appropriate for discovery experiments where no a priori information is available. When a delimited number of targets of interest are known a priori, targeted proteomics offers a robust method for detection and quantification. Novel MS methods such as a combination of higher energy collision dissociation (HCD) and electron transfer dissociation (ETD) allow for characterisation of complex PTMs including glycosylation. ZIC-HILIC, zwitterionic hydrophilic interaction LC; Pd, product-dependent; Alt, alternating.
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review
| 99.9 |
MS strategies for ECM characterisation. Untargeted proteomics is appropriate for discovery experiments where no a priori information is available. When a delimited number of targets of interest are known a priori, targeted proteomics offers a robust method for detection and quantification. Novel MS methods such as a combination of higher energy collision dissociation (HCD) and electron transfer dissociation (ETD) allow for characterisation of complex PTMs including glycosylation. ZIC-HILIC, zwitterionic hydrophilic interaction LC; Pd, product-dependent; Alt, alternating.
|
review
| 98.44 |
Collagens are the major fibril-forming proteins in the ECM and they consist of a basic triple-helical conformation. The triple helix increases molecular stability and provides resistance to tensile stress. Although many types of collagen exist, a consistent pattern can be observed for amino acid sequences of all collagens; each chain contains enriched triplet repeats consisting of the sequence Xaa-Pro-Gly, where Xaa is any amino acid, Pro is proline, and Gly is glycine. Prolines within these domains become hydroxylated under the action of prolyl-hydroxylases.78 Hydroxyprolines provide the substrate for the formation of hydrogen bonds between the adjacent collagen alpha chains. Prolyl-4-hydroxylases and prolyl-3-hydroxylase catalyse the hydroxylation of specific proline residues. The former enzyme reacts on proline with the minimum sequence Xaa-Pro-Gly and the latter appears to require a Pro-4Hyp-Gly (Hyp is hydroxyproline) sequence.78,79 Hydroxylation is a stable, non-reversible PTM that adds +15.99 Da (i.e. an oxygen atom) to proline. Xaa-Pro-Gly domains are rare in ECM proteins other than collagens, and this confers specificity to the acquisition of this PTM.
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study
| 100.0 |
Similar to prolyl-hydroxylases, lysyl-hydroxylases catalyse the hydroxylation of lysine, which is critical for collagen stability. The specifics of lysine hydroxylation are beyond the scope of this review, and are discussed elsewhere.80 Adding hydroxylation as a variable modification, improves identification and quantification of collagen levels in disease.3,68 Ascorbic acid (vitamin C) is a key cofactor for prolyl-4-hydroxylase, and its deficiency causes defects in collagen assembly.81 Inhibition of this enzyme has been shown to affect left ventricular remodelling after myocardial infarction in rats.82 In this study only proline:hydroxyproline ratios were assessed. MS provides assessment of hydroxylation with concomitant assignment to specific collagen types.
|
review
| 99.9 |
Glycosylation is an enzymatic process through which a glycan is covalently attached to a second biomolecule. Glycosylation is a very common form of PTM of ECM proteins. Attached glycans affect ECM protein structure and function by influencing its folding, solubility, aggregation, and/or degradation behaviour.83 Indeed, aberrant glycoforms are already approved as biomarkers for cancer.84 In cardiac tissue, Montpetit et al.85 showed that aberrant glycosylation of extracellular domains alters ion channel activity.
|
review
| 87.1 |
There are two main glycosylation types in mammals: N-glycosylation occurs at the carboxamido nitrogen on asparagine residues (Asn) of secreted/membrane proteins within the consensus sequence Asn-Xaa-Thr/Ser, where Xaa is any amino acid except for proline.86 The second main type of glycosylation is O-glycosylation, in which sugar residues attach to serine and threonine residues (Ser, Thr) or, to a much lesser extent to hydroxyproline and hydroxylysine.87 The latter two are particularly abundant in collagens and add an additional level of regulation to collagen biosynthesis. If both present, O-glycosylation occurs after N-glycosylation. Moreover, O-glycosylation is not restricted to secreted proteins and to date, no consensus sequences have been identified for this PTM.88 ECM proteins may be extensively modified by addition of N- and O-linked large and repetitive glycosaminoglycans (GAGs) and shorter and diverse N- and O-linked oligosaccharides. Aberrant glycosylation can lead to pathological abnormalities and disease. In the last decade, proteomics has emerged as a powerful platform to characterize glycosylation profiles of ECM proteins, including the cardiovascular field.52,58,89 There are two major strategies that can be used to study glycoproteins by MS (Figure 4).
|
review
| 99.9 |
A common large-scale strategy utilizes a glycopeptide or glycoprotein enrichment step followed by glycan removal. Glycopeptides are usually enriched using lectins, hydrophilic interaction LC, hydrazide or graphite. The method of choice will determine the type of glycopeptides that will ultimately be enriched.90,91 After enrichment, PNGase-F is used to enzymatically remove the glycan moiety from asparagine residues, serving two purposes: Firstly, the core peptide can be analysed without interference from sugars during MS/MS and secondly, PNGase-F via a deamidation reaction converts the asparagine to aspartic acid. This conversion is characterized by a 0.984 Da mass shift that can be detected using MS. Moreover, if the reaction is performed in the presence of H218O, it instead leads to a 2.99 Da mass shift, indicative for the presence of glycosylation at that position. Using this methodology in rat hearts, Parker et al.58 identified 1556 N-linked glycosites from 972 protein groups. This study provided information on the changes in glycosylation following ischemia and reperfusion. Enzymatic deglycosylation allows for the separate analysis of the core protein and glycan,92 but the link between the glycans and peptides is lost.
|
study
| 99.94 |
The combined analysis of the glycan motif (glycomics) and the protein (proteomics) forms the field of glycoproteomics. For such analysis, proteins in the sample are first digested into peptides, followed by glycopeptide enrichment using zwitterionic hydrophilic interaction LC (ZIC-HILIC)93 or alternative approaches.91,94 Recently, the combination of higher energy collision dissociation (HCD) and electron transfer dissociation (ETD) have facilitated direct MS analysis of glycopeptides. HCD fragmentation breaks glycosidic bonds, whereas ETD preserves the glycan attachment and fragments the peptide backbone, providing peptide sequence information.89 Direct analysis of intact glycopeptides has rarely been applied in the cardiovascular context. Our study by Yin et al.89 characterised the glycopeptides of secretomes from human endothelial cells. More recently, we have characterized the glycosylation profile of human cardiac ECM proteins.52
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study
| 99.94 |
Glycosylation and hydroxylation are among the most common PTMs in ECM proteins. Importantly, they constitute non-reversible modifications, but reversible PTMs such as phosphorylation and sulfonation also occur. For example, the transmembrane collagen XVII can be phosphorylated and this mechanism regulates shedding of its ectodomain.95 Similarly, phosphorylation of osteopontin inhibits vascular calcification.96 In a study by Lundby et al.97 proteomics was used to identify phosphosites on 14 different rat tissues including hearts. Phosphopeptides were enriched using titanium dioxide. Notably, many previously unrecognized phosphosites were reported in ECM proteins including several collagens and non-collagenous ECM proteins such as laminins, fibronectin, versican, and decorin to name a few. This methodology was effective on fresh animal tissues, but has yet to be applied to the context of cardiac disease. Challenges to its application will include preservation of short-lived PTMs in patient samples and during sample preparation.
|
review
| 94.75 |
Proteolytic fragmentation of ECM proteins by secreted proteases controls their localisation, activation, and interaction, adding an additional layer of regulation for tissue processes. Using experimental data, databases/algorithms such as MEROPS and PROSPER have been created to calculate probability matrices for target protease sequences.98–100 This is a valuable resource for research and is particularly useful when used in conjunction with proteomics. Standard proteomics pipelines work with digested protein mixtures (i.e. trypsin digestion) to screen for the abundance of proteins in a tissue. Trypsin cleaves C-terminally to lysine (Lys, K) or arginine (Arg, R) residues. However, proteases other than trypsin can be present in samples and the endogenous proteolytic activity may give rise to non-tryptic peptides. In a study from Stegemann et al.59, we described a number of potential proteolytic targets for various MMPs in the vasculature after addition of these proteases to human vascular tissue explants.
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study
| 99.94 |
When searching for protease targets, appropriate controls are needed (i.e. healthy or non-digested tissues) in order to avoid reporting artefactual cleavages that may arise from experimental processing, or identifying those that are part of normal physiological turnover. Moreover, the addition of broad-spectrum protease inhibitors during extraction reduces the chance of producing artefactual fragmentation. More sophisticated methods include free C- or N-terminal labelling of endogenous protease-generated fragments prior to digestion for MS analysis.101 For example, the TAILS proteomics approach (isotope-based N-terminal labelling) has been successfully applied by Prudova et al.102 to analyse the degradome of MMP-2 and MMP-9. The same authors used a similar methodology to characterize the degradation of proteolytic fragments in human platelets.103 Ultimately, after identification of cleavage sites, targeted proteomics can be used to study the abundance of ECM fragments in clinical samples.
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review
| 99.75 |
Specific biological activities have been attributed to certain ECM proteolytic fragments (Figure 5).5,19,52,104–122 The term matrikines has been proposed for these fragments. This should not be confused with the term matricryptins, which is more accurately applied to ECM protein domains that are unexposed (and therefore inactive) unless the protein is subject to fragmentation-derived conformational changes. For example, C-terminal cleavage of collagens XV and XVIII, generates restin and endostatin, respectively. Both fragments exert anti-angiogenic activity in vivo.119 Other collagen types also generate biologically active fragments, e.g. collagens IV and VI, which are highly expressed in the cardiac ECM2, as reviewed elsewhere.123 The large proteoglycan versican is cleaved by proteases from the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families.124 Versikine is generated by N-terminal cleavage of versican by ADAMTS-1/4, and influences cell proliferation and apoptosis locally.113 Endorepellin, a C-terminal peptide from perlecan exerts anti-angiogenic effects.120 Last, non-structural ECM proteins also release fragments, e.g. the small leucine-rich proteoglycan decorin releases decorunt and other fragments, that exert local regulatory roles over cytokines and growth factors.52,107,117 Recently, we demonstrated that decorin is fragmented in the cardiac ECM. We detected C- and N-terminal non-tryptic cleavage sites on decorin by MS. The resulting cleavage products may regulate growth factor availability.52 Using similar approaches, the Lindsey group identified cleavage products derived from collagen I that promote scar formation after MI.5 Figure 5Biological activity of ECM fragments. Fragments derived from a variety of ECM proteins (i.e. matrikines) exert functions that regulate diverse cellular and tissue processes. Proteomics offers a tool for the analysis of known ECM fragments as well as the discovery of previously unknown fragments with functions potentially important for cardiac physiology and putative therapeutic targets. *Indicates putative fragments with activities only characterised after exogenous administration. CO1A1, collagen alpha-1(I) chain; FINC, fibronectin; EDA, extra domain A; TENA, tenascin; FN3, fibronectin type III domain; CO6A3, collagen alpha-3(VI) chain; ELN, elastin; PGS2, decorin; CO4A1, collagen alpha-1(IV) chain; OSTP, osteopontin; LAM332, laminin 332; VTNC, vitronectin; CSPG2, versican; EGF, epidermal growth factor-like domain; HGF, hepatocyte growth factor; COIA1, collagen alpha-1(XVIII) chain; PGBM, perlecan; COFA1, collagen alpha-1(XV) chain; POST, periostin; FAS1, fasciclin-like domain; PGCA, aggrecan; HPLN1, hyaluronan and proteoglycan link protein 1.
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study
| 99.94 |
Biological activity of ECM fragments. Fragments derived from a variety of ECM proteins (i.e. matrikines) exert functions that regulate diverse cellular and tissue processes. Proteomics offers a tool for the analysis of known ECM fragments as well as the discovery of previously unknown fragments with functions potentially important for cardiac physiology and putative therapeutic targets. *Indicates putative fragments with activities only characterised after exogenous administration. CO1A1, collagen alpha-1(I) chain; FINC, fibronectin; EDA, extra domain A; TENA, tenascin; FN3, fibronectin type III domain; CO6A3, collagen alpha-3(VI) chain; ELN, elastin; PGS2, decorin; CO4A1, collagen alpha-1(IV) chain; OSTP, osteopontin; LAM332, laminin 332; VTNC, vitronectin; CSPG2, versican; EGF, epidermal growth factor-like domain; HGF, hepatocyte growth factor; COIA1, collagen alpha-1(XVIII) chain; PGBM, perlecan; COFA1, collagen alpha-1(XV) chain; POST, periostin; FAS1, fasciclin-like domain; PGCA, aggrecan; HPLN1, hyaluronan and proteoglycan link protein 1.
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review
| 91.4 |
The application of MS constitutes one of the biggest technological advances introduced to protein research. It offers an unbiased platform to analyse global protein expression and holds potential in facilitating novel insights. As recently highlighted in a scientific statement of the American Heart Association55; it is anticipated that proteomics research will further our understanding of mechanisms of CVD with one important aspect being the elucidation of ECM composition in healthy and diseased cardiovascular tissues. To achieve this goal, bioinformatics approaches should be applied for interpreting the protein datasets and extract the biologically relevant information. Undoubtedly, the amount of data generated by proteomics represent an analytical challenge. In this regard, special attention should be paid to ECM fragments as they hold potential for two purposes: from a diagnostic perspective, they leak from tissues and when released into the blood stream can be used as biomarkers for CVD. Secondly, since many ECM fragments are biologically active, they not only hold potential as therapeutic targets but also as modifiable therapeutic agents—to date an underexplored avenue of cardiovascular medicine.
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review
| 99.9 |
Prostate cancer (PCa) is one of the leading causes of cancer death in men globally . Factors responsible for aggressive or indolent phenotypes of PCa are poorly understood. The chances of developing PCa significantly increases after the age of 40 years . However, even without any form of therapy, PCa often runs a protracted natural history and many men die with it rather than from the disease . Over a million new cases are reported annually according to the GLOBOCAN/ IARC 2012 databases; PCa is the fifth leading cause of cancer death in men globally, accounting for up to 307,000 deaths annually . Among cancers in Africa, PCa was reported to have the highest incidence (59,493), mortality (42,802) and 5 year prevalence rate (155,028).
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review
| 99.9 |
This high incidence, mortality and 5-year prevalence trend is similar in sub-Saharan Africa, Southern Africa, or the Republic of South Africa. This clearly contrasts with the situation in the developed world, where high incidence and low mortality reflect the impact of early diagnosis and prompt treatment. In most situations, cases are diagnosed at advanced stages in Africa and the mortality and incidence rates are almost at par with the situation in the Western, Middle, and Eastern Africa (Figure 1A) . Owing to a relatively higher level of development and infrastructure in the Republic of South Africa, PCa incidence is quite high compared to the rest of Africa. However, mortality rates in this region are high as well. The fact that South Africa has a high incidence rates similar to those in North America, Western Europe and Australia but a high mortality rate comparable to most other sub-Saharan African countries (Figure 1B & 1C) suggests that despite a relatively better diagnostic infrastructure compared with other parts of Africa, limited manpower and resources has complicated the management of the huge burden of diagnosed PCa cases .
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study
| 87.7 |
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