In this study, we aim to identify key climatic factors that are associated with the transmission of Japanese encephalitis virus in areas located near the Three Gorges Dam, between 1997 and 2008.We identified three geographical regions of Chongqing, based on their distance from the Three Gorges Dam. Collectively, the three regions consisted of 12 districts from which study information was collected. Zero-Inflated Poisson Regression models were run to identify key climatic factors of the transmission of Japanese encephalitis virus for both the whole study area and for each individual region; linear regression models were conducted to examine the fluctuation of climatic variables over time during the construction of the Three Gorges Dam.Between 1997 and 2008, the incidence of Japanese encephalitis decreased throughout the entire city of Chongqing, with noticeable variations taking place in 2000, 2001 and 2006. The eastern region, which is closest to the Three Gorges Dam, suffered the highest incidence of Japanese encephalitis, while the western region experienced the lowest incidence. Linear regression models revealed that there were seasonal fluctuations of climatic variables during this period. Zero-Inflated Poisson Regression models indicated a significant positive association between temperature (with a lag of 1 and 3 months) and Japanese encephalitis incidence, and a significant negative association between rainfall (with a lag of 0 and 4 months) and Japanese encephalitis incidence.The spatial and temporal trends of Japanese encephalitis incidence that occurred in the City of Chongqing were associated with temperature and rainfall. Seasonal fluctuations of climatic variables during this period were also observed. Additional studies that focus on long-term data collection are needed to validate the findings of this study and to further explore the effects of the Three Gorges Dam on Japanese encephalitis and other related diseases. Japanese encephalitis (JE), which is caused by the Japanese encephalitis virus (JEV), is a mosquito-borne infectious disease that is prevalent throughout Southeast Asia and the Asian Pacific Rim The incidence of JE has been reported annually in China since the establishment of a case reporting system in 1951. Since that time, two major JE epidemics have occurred: first, in 1966 when the annual incidence of JE rose to greater than 15/100,000 nationwide; and second, in 1971 when it rose again to 20.9/100,000 nationwide Nonetheless, JE continues to be a risk for human health. With the exception of the Xinjiang, Qinghai, and Tibet provinces, human JE cases have been reported throughout all of China. There are several reasons for this: first, residents have a low level of awareness of infectious diseases and therefore fail to get vaccinated Culex. tritaeniorhynchus in Asia, while the main vector is Culex. annulirostris in Australia (in the South Pacific area) The emergence of JE involves a complicated interplay of hosts, vectors, climatic variables and anthropogenic factors Chongqing is located on the upper section of the Yangtze and is one of five provinces in China with a mean JE incidence rate higher than 1/100,000. Chongqing, along with Sichuan, Henan, Yunnan, and Guizhou has accounted for more than 50% of the total JE cases, in spite of the fact that they comprise only 26% of the total population of China The Three Gorges Dam (TGD) is a hydroelectric dam that spans the Yangtze River at Sandouping Island built in 2012. The construction of TGD has resulted in substantial changes to the depth and the flow pattern of the Yangtze River and has also led to the substantial development of farmland alongside the river path In this study, we aim to identify the key climatic drivers of JEV transmission in Chongqing from 1997 to 2008. We will also explore the spatial and temporal patterns of JE incidence, as well as the trends of climatic variables in Chongqing.This study was carried out in the following districts, all of which are located along the Yangtze River : Banan, JE is an infectious disease that is routinely surveyed by the Center of Disease Control and Prevention (CDC), in China. The monthly reported cases from 1997 to 2008 for each district were new cases and were obtained from the CDC. The diagnosis of Japanese encephalitis is based on a combination of epidemiological data, clinical symptoms and signs, and specialized laboratory tests of blood, spinal fluid or Cerebrospinal fluid (CSF). These tests typically detect antibodies that respond to the viral infection. However, all confirmed cases have relied on antibody tests or pathogen isolation instead, and for this reason we decided to use the same approach of utilizing antibody tests and pathogen isolation in this study The CDC in China adopted several measures to ensure the quality of data obtained during JE surveillance. First, it fully designed the surveillance scheme and provided training and technical guidance to its local branches on surveillance, data collection and data analysis. Second, the Chinese CDC directly supervised the quality of reported JE cases. If abnormal values were obtained, they were confirmed directly by the CDC – first by the local branch, and if necessary, by the main government branch. Third, a review of reported JE cases was regularly conducted to guarantee the integrity of the data.National Climate Center. As participants of the National Basic Research Program of China (973 Program) (Grant No. 2012CB955500) lead by Professor Qiyong Liu, the Chinese CDC established a sharing platform(http://124.127.202.241/Share/index.jsp)in 2012 to provide a variety of data services for all of the study participants.The Chinese CDC also provided the monthly meteorological data, including average temperature (T), relative humidity (RH), rainfall, and sunshine duration (SD). The meteorological variables were recorded by the The values of missing JE cases were 5.55%. Most of these values had occurred during the winters of 2004, 2006 and 2007. To account for this, we used the “expand” procedure with the “step” option of SAS to compute the missing values. To evaluate the difference among the regions of Chongqing, and to get more convincing data, we combined the data from 12 districts into three geographic regions  by summing the count of JE and averaging the data of climatic variables .To explore the spatial pattern of JEV transmission, the average annual JE incidence was separately calculated for each district, each of the three geographic regions, and the total area from 1997 to 2008. The monthly JE incidence was calculated for each district and each geographic region of Chongqing city, and the epidemic curves for three geographic regions were plotted to explore the temporal pattern of JEV transmission from 1997 to 2008. The monthly cases of JE occurred in the three regions were plotted to explore the seasonal trend of JE occurrence. In this study, the incidence rate was the number of new JE cases per population in a given time period. The annual population data was also obtained from the sharing platform created by the Chinese CDC. The incidences for each district, each of the three geographic regions, and the total area were calculated using the population of the corresponding area at certain time period. The denominator was given as 100,000 persons per year or per month.A simple linear regression model was conducted to examine the trend of climatic variables over time from 1997 to 2008 in all three regions. To remove the seasonal effect, the regression was run using one month per year over the 10-year period and it was done for all 12 months separately.For the main analysis, the response variable is the monthly JE incidence rate. Since it had a certain frequency of zero, a Zero-Inflated Poisson Regression (ZIPR) model was conducted to examine the association between the monthly JE incidence and the multiple climatic variables. In ZIPR, the count of disease is assumed to follow a mixture distribution of point-mass zero and a Poisson distribution, and the probability of the component and the mean parameter of Poisson distribution component are regressed on the covariates. Autocorrelation plots showed that the current incidence was significantly correlated with the incidence with a lag of 1, 11 and 12 months, and regressing the JE incidence on merely climatic variables resulted in highly auto-correlated residual terms. Therefore, we kept the JE incidence with a lag of 1, 11 and 12 months as covariates when studying the association between the incidence and the climatic variables.t denote the count of JE at time t. The ZIPR assumed that yt  = 0 with probability pt, and yt followed a Poisson distribution with mean µt with probability 1-pt. Instead of modeling the mean µt, we modeled the incidence rate at time t ratet  = µt/Nt, where Nt denoted the population of a district at time t. Let ratet-1, ratet-11 and ratet-12 respectively denote the incidence rate with lag 1, 11 and 12 months, gdpt denote the GDP value. The preliminary analysis models for three regions were fitted as below:We hypothesized that JE incidence was partially confounded by the improvement in the economic condition in region. So the annual gross domestic product (GDP) values of Chongqing city were included in the models to control for economic changes over time. We first ran the preliminary analysis to examine whether the GDP impacted the JE incidence, adjusting for the autocorrelation of the series. To do this, we ran the zero-inflated Poisson regression with incidence with a lag of 1, 11 and 12 months together with GDP values as covariates for the area as a whole as well as each of the three regions. Let yWe then did the formal analysis to examine the relationship between JE incidence and climatic variables, adjusting for the autocorrelation of series and GDP. To do this, we checked the possible multi-collinearity among the different climatic variables with the different lags by examining the correlation matrix. It turned out that the temperature and sunshine series are highly correlated, so we just included temperature series as our predictor. In the final models, we included temperature and rainfall data due to their significant impact on the response.t-1, ratet-11 and ratet-12 respectively denote the incidence with lag 1, 11 and 12 months, gdpt denote the GDP series, tempt-1, tempt-3 denote the temperature series with lag 1 and 3 months, raint, raint-4 denote rain series with lag 0 and 4 months. The models for the whole area are shown as following:Then we constructed the final model with following covariates: JE = incidence (denoted as “rate”) with lag = 1, 11 and 12; GDP series; Temperature (denoted as “temp”) and rainfall (denoted as “rain”) with possible lags. To determine lags, we used a forward model selection method, keeping the incidence with a lag of 1, 11 and 12 months together with the GDP values in the model and sequentially adding the lags of temperature or rain in the model; when each new term was added, we checked VIF to avoid the multicollinearity, and examined to make sure that the coefficients of climatic variables in the model were significant under level 0.05. We repeated this process until the log-likelihood of the fitted model, which provided a summary of model fitting, became stabilized. Once we determined the model for the whole area, we applied the model to three regions respectively. From the process of model selection, in addition to the GDP term and the incidence with lag 1, 11 and 12 months, we decided to include temperature of lag 1 and 3 months together with rain of lag 0 and 4 months in the model. Let rateAutocorrelation plot and normal probability plot of the residuals were used to examine goodness-of-fit of the ZIPR models after each model was fixed. The Pearson residuals were expected to be uncorrelated and approximately normally distributed.A total of 2,552 cases of JE were reported from 1997 to 2008 in all 12 districts of Chongqing that we studied. The cumulative number of JE cases in each district from 1997 to 2008 ranged from 23 to 510 with a median of 151. The number of cases that occurred in eastern, middle and western parts of Chongqing city was 1456, 739 and 357 respectively. The spatial trend of JE cases decreased gradually from east to west .In the study area, the annual JE incidence varied from 2.2/100,000 in 1997 to 0.7/100,000 in 2008 with a median of 1.8/100,000. The annual incidence peaked in 2000, reaching 3.6/100,000. The spatial variation in JE incidences over the three geographic regions showed that the annual incidence ranged from 0.4/100,000 (western in 2007) to 5.7/100,000 (eastern in 2000) with a median of 1.6/100,000 from 1997 to 2008. The JE incidence peaked in 2000 in the eastern region  and in 2001 in the middle region . In 2000, the annual incidence rapidly increased from 2.8/100,000 to 5.7/100,000 in eastern region, and from 1.5/100,000 to 3.4/100,000 in middle region. In 2006, the annual incidence reached the second peak from 2.9/100,000 to 4.1/100,000 in the eastern region, and from 1.4/100,000 to 3.2/100,000 in the middle region. Nevertheless, the annual incidence remained at relatively low levels in the western region, with a mean of 0.8 ± 0.4/100,000 (mean ± standard deviation) from 1997 to 2008 .The occurrence of JE had a clear seasonal trend in all regions of Chongqing. In total, the number of JE cases in the first half year (January to June) remained at quite low levels and even bottomed out at zero for some time. Beginning in June, the reported number of JE cases rapidly increased, peaking in August before decreasing again in September. By October or November in some districts, the reported cases rapidly decreased back to the low levels . The casIn the three regions, the monthly temperature ranged approximately from 5 to 8.5°C in January, then increased gradually by up to 27.7–31.9°C in July or August and decreased gradually from September to the next January . The annTo assess the impact of GDP on JE incidence, we first ran a preliminary ZIPR analysis including GDP as the predictor. We observed a significant positive association between GDP and the zero point-mass component of incident rate in the middle district (p<0.05), as well as a marginally significant negative association between GDP and the incidence of the disease for the Poisson component in the east district (p>0.05) . These rA final ZIPR model was run to determine the association between climatic variables and JE incidence across different geographical regions, controlling for GDP  indicate that it takes 3 months for temperature to influence the occurrence of JE in Chongqing.It has been suggested that the temperature and rainfall were crucial to JE virus incubation and transmission However, our discovery that the rainfall with a lag of 0 and 4 months has a negative association with JE incidence is contrary to other publications that showed positive association between rainfall and JE incidence In our study, the number of JE cases that occurred between July and September accounts for 91.41% of the total number; the number of JE emerging only in August accounts for 53.44% of the total number from 1997 to 2008. This seasonal trend could be explained by interplay of temperature with a lag of 1 month with rainfall with a lag of 4 months, which may affect the density of mosquitoes and pigs breeding as well as other vectors required for the cycle of the JE virus.Comparing with the whole study area, there were similar results in the association between temperature and rainfall and JE incidence in the three regions of Chongqing. Except for temperature with a lag of 3 months, in both the eastern and middle regions temperature with a lag of 1 month was also positively related to JE incidence. This finding indicated that temperature affected the JE incidence through not only a long-term control mechanism but also a short-term direct mechanism. However, there seemed to be no significant association between the temperature of the current month and the JE incidence in this study.JE cases appeared mostly in the eastern and middle regions of Chongqing from 1997 to 2008, probably due to different economic status among these regions. Interestingly, the incidence of JE gradually increased with increased proximity to the TGD. Even though some negative effects of TGD on ecosystem have been emphasized Although the JE incidence tended to decrease in both Chongqing and individual regions, there were still two evident peaks that emerged in 2000, 2001, and 2006 in both the eastern and middle regions of Chongqing. The emergence of JE involved a complicated interplay of hosts, vectors, climatic variables and anthropogenic factors Our study had limitations involving the collecting of data, spatial analysis of JE incidence, the fitting of the model to individual regions, and weak evidence linking the TGD and JE incidence.In this study we didn’t take into consideration other potential factors that may affect JE incidence. For example, JEV is transmitted to human by mosquitoes, and domestic pigs. Birds can also facilitate the spreading of JEV. The effect of these vector and hosts in the study area were not included into our model. Rice field proportion, which may have been changed by the construction of the TGD, is also directly associated with the incidence of JE because these fields provide breeding sites for mosquitoes, which we did not take into account. The relocation of infected residents may also lead to the reoccurrence of JE incidence. And last, the GDP data we obtained was only related to Chongqing and not to all regions. Nonetheless, we have no evidence of any systematic biases in our analysis that were likely to lead us to the wrong conclusion.In addition, it is important to point out that we separated the study area into three regions in order to observe spatial trends in JE incidence. Theoretically, different results could be obtained by reorganizing these regions, although similar results would be expected.We also only focused on data from 1997 to 2008-the time during which the TGD was being constructed. Pre- and post-construction data were not available, so we could not analyze the impact of TGD on climatic variables or JEV transmission. Future analysis could be performed by including more complete data over a longer time period.In conclusion, temperature with a lag of 1 and 3 months and rainfall with a lag of 0 and 4 months were key climatic variables that were associated with JE incidence in Chongqing from 1997 to 2008. The spatial and temporal trend of JE incidence occurring in Chongqing was associated with temperature and rainfall with different lags. The reasons for seasonal fluctuations of climatic variables are not well understood and needed to be further investigated. Future study is required to validate the findings of this study and further explore the effect of TGD on JE incidence.Figure S1The diagnostic plots in the fitted model. The plots shown here are for the fitted model of the study area. Those for the three regions are similar (not shown).(TIFF)Click here for additional data file.


Previous Analysis of polymorphism of genes associated with the development of coronary heart disease (CHD) reveals that the frequency distribution of genotypes and alleles depends on the ethnic characteristics of the populations under study. Further impetus is derived from the well -established links between alcoholism  and cardiovascular disorders.The purpose of this study was to examine a number of apolipoprotein gene polymorphisms and correlate these alleles with changes of lipid profile in CHD patients of Kazakh and Uyghur nationalities.Four-Hundred Forty Eight (448) males of Kazakh and Uyghur nationalities residing in Kazakhstan were evaluated and genotyped. The age range of these subjects was 30–55 years which included both afflicted and controls. Specifically, 161- Kazakhs suffered from myocardial infarction compared to 112 health controls; 80- Uyghurs suffered from CHD compared to 95 health controls. Blood lipid profiles were examined in the total cohort. Genotyping was performed by polymerase chain reaction (PCR) using oligonucleotide primers identifying; ApoB; ApoC111; and APOE gene polymorphisms.Initial screening revealed a significant inter-ethnic difference on the frequency of alleles associated with both the ApoB and APOE genes. We found that the X1 ApoB gene polymorphism is overrepresented in healthy Kazakhs relative to Uyghurs [86.4% in Kazakhs vs. 69.4% in Uyghurs]. Moreover, we found that the E4APOE allele was also overrepresented in healthy Kazakhs relative to Uyghurs [16.8% in Kazakhs vs. 9.5% in Uyghurs]. There was a significant relationship of polymorphisms of APOE such as ApoB and ApoC 111 with the value of lipid indices in Kazakhs. Additionally, we found that the E4 allele of the APOE gene also correlated with the value of lipid indices in Kazakhs. Further evaluation showed that the X2 allele of the ApoB and the S2 allele of the ApoCIII gene significantly associated with the lipid indices of Uyghurs.This systematic investigation confirms the association of various alleles of Apolipoprotein gene polymorphisms and contribution to aberrant lipid metabolism. Putatively at least in our population we are proposing that certain gene polymorphisms of Apolipoprotein genes such as ApoB; ApoC111; APOE ; X2 of ApoB; and S2 of ApoCIII differentially represented in either Kazakhs or Uyghurs are genetic markers of hypertriglyceridemia. There is ample evidence linking high alcohol intake and cardiovascular disease throughout the world. Importantly, the prevalence rate of alcoholism in the Kazakhstan region and subsequent suicide is rather high and provides a rationale to evaluate genetic antecedents for coronary heart disease (CHD) . It is kBased on many reviews and controversy related to the importance of genetic testing to determine predisposition of CHD risk , we hypoFour-Hundred Forty Eight (448) males of Kazakh and Uyghur nationalities residing in Kazakhstan were evaluated and genotyped. The age range of these subjects was 30–55 years which included both afflicted and controls. These participants were all living in Kazakhstan, and unrelated by ties of kinship. Each subjects was selected by a by random sampling technique and subsequently interviewed. The University ethics committee approved this study as well as approving a consent form which was signed by each subject selected for the study.Specifically, 161- Kazakhs suffered from myocardial infarction compared to 112 health controls; 80-Uyghurs suffered from CHD compared to 95 health controls. The average age of the Kazakhs with CHD was 46.4 ± 1.9 years, of the apparently healthy Kazakhs – 40.7 ± 2.1 years, of the Uighurs with CHD – 47.3 ± 0.92 years, of the apparently healthy Uighurs – 41.2 ± 1.02 years.Standard blood lipid profiles were examined in the total cohort. The blood lipid profiles analyzed is as follows: total cholesterol (TC) and high-density lipoprotein cholesterol (HDLC), low density lipoprotein cholesterol (LDLC), triglycerides (TG). It is noteworthy, that analysis of all blood lipid profiles, were determined by a biochemical analyzer utilizing well-known procedures . In addiGenotyping was performed by polymerase chain reaction (PCR) using oligonucleotide primers identifying; ApoB; ApoC111; and APOE gene polymorphisms. DNA was isolated from blood lymphocytes by a standard technique  to invesFor the genotyping of the Apo C111, the products of polymerase chain reaction (PCR) were digested with SStI, and then analyzed in 10% polyacrylamide gel. Alleles not having the SStI restriction site, were designated as S1, and the site carrying SStI – as S2. The combination of these alleles, form three genotypes: S1S1; S1S2; S2S2.For the genotyping of the Apo B, the resultant material derived from amplification was digested with Xba I. The restriction products were analyzed utilizing electrophoresis in 12% polyacrylamide gel. As a result, two alleles were identified: XI – without a restriction site, X2 – with the restriction site for Xba I.For the genotyping of the Apo E, the amplification product was digested with Hhal utilizing an improved method  and analFollowing plasma measurements (mg/dl) we found that dyslipidemia atherogenicity (DA) is due not only to the buildup of lipid content, but is a function of the apolipoprotein components.We observed a decrease of level of the Apo A1 and increase of level of the Apo B in the Kazakhs as well as Uighurs with CHD, and the indices of the Apo A1 are more prevalent and the indices of the Apo B of blood serum are lower in healthy individuals. While the indices noted for the Kazakhs were found to be significant for Apo A1 (<0.001) and Apo B (<0.05) for the Uighurs we found less significance for Apo A1 (<0.05) than in the Kazakhs but the significance for Apo B (<0.05) was the same for both populations  and 2.As displayed in There is a plethora of population genetics throughout many countries regarding apolipoprotein genetic polymorphisms and structural relationships –14.Following genotyping and analysis of the allelic frequency distribution of the Apo B gene we have found that the X1 allele of the Apo B gene is the most common allele in the Kazakhs and Uighurs . HoweverSince there is a large amount of research devoted to the study of associations of the Xba I polymorphism of the Apo B gene with atherogenic changes in lipid spectrum ,16, we dOur analysis  revealedOn the contrary, in the Uighurs with CHD our analysis (depending on the availability of the restriction site) showed  that in The Apo C111 gene produces a protein product that plays an important role in the metabolism of lipoprotein atherogenic fractions. Seven polymorphisms of the Apo CIII gene are described . The mosBased on our genotyping data most of the surveyed individuals of Kazakh and Uighur ethnic groups are the carriers of the S1 allele of the Apo CIII gene. Among healthy Kazakhs the frequency of this allele is 76.6%, and among healthy Uighurs is 74.5% .When comparing the frequency distribution of the alleles of the Apo CIII gene between the groups of patients with CHD and healthy individuals we have not found any differences among the Kazakhs and the Uighurs.To determine the effect of the Apo CIII gene polymorphism on the levels of the lipid and apolipoprotein blood spectrum in the Kazakhs and Uighurs, we analyzed the average values of these parameters with different genotypes  of the Apo CIII gene  and 8. WWe also found for VLDLC (Very Low Density Lipoprotein Cholesterol): its highest value was determined by the SISI genotype, the smallest – by the S2S2 genotype, the average – by the S1S2 genotype. Significant differences were not found in the Kazakhs compared by genotype with respect to levels of the TC, LDLC, HDLC, AI and levels of the A1, B and E apolipoproteins.In contrast, the increased level of triglycerides was associated with subjects processing the S2 allele in the Uighurs with CHD . SignifiHowever, as depicted in Effect of genetic polymorphism of the Apo E gene on the risk of development of CHD has been extensively studied. However, in terms of our population the prevalence of this gene and associated polymorphisms remains unknown.Thus in our study carriers of the E2/E2 genotype were not found among the Kazakhs and the E4/E4 and E2/E4 genotypes were not found in the group of healthy individuals. However, the E3/E3 genotype was the most common genotype in the Uighurs, the E4/E4 genotype was not found, and the E2/E2 genotype was found in only one patient and as such is a rare variant. Thus a high frequency of the E3 allele was observed in the examined groups of Kazakhs and Uighurs, and the frequency of the E4 allele is greater than or close to the frequency of the E2 allele.It is known that among many of the common polymorphisms the Apo E is an allele polymorphism shown to consistently affect lipids in all populations studied, especially on total cholesterol and triglycerides. The E4 allele is more often found in patients with hypercholesterolemia, whereas the E2 allele – in patients with hypertriglyceridemia. Taking into consideration these data, we conducted a study of the relationship of polymorphisms of this gene with the lipid and apolipoprotein spectrum.We thus observed in Kazakhs with CHD that the increase of TC, LDLC, AI is a function of the E2/E3, E3/E3, E2/E4, E3/E4, E4/E4 genotypes .The average level of total cholesterol  in the Kazakhs having the E4/E4 and E3/E4 genotypes is significantly increased in comparison with this parameter in patients with CHD having the E3/E3 and E2/E3 genotypes  (p < 0.05). The lowest concentrations of LDLC are observed in patients having the E2/E3 genotype  and the highest concentrations – in patients having the E4/E4 genotype , [p < 0.05].Finally, in the Uighurs with CHD the E2/E3 genotype is significantly associated only with hypertriglyceridemia . In the case of other genotypes  of the Apo E gene such an association has not been found .The present work is based on the search of genetic markers of coronary atherosclerosis with the help of analysis of associations of polymorphisms of DNA – with three candidate genes of atherosclerosis.One of the most common polymorphisms of the Apo B gene is the Xba1 restriction endonuclease polymorphism. The appearance of the site for this endonuclease gene in the 26-th exon of the gene is due to nucleotide substitution (T-C) in the third codon position 2488. Due to the results obtained we can conclude that the E2 allele of the Apo E gene is the risk factor for development of hypertriglyceridemia in the Uighurs with CHD. Moreover, the presence of the E4 allele of the Apo E gene is a genetic marker of predisposition to hypercholesterolemia in the Kazakhs with CHD.As described in the literature, the XI allele frequencies are very close in the Caucasian populations and ranged from a minimum - 43.0 % in the Danes to the maximum - 56.0% in the Italians. The XI allele frequency in the Indians is much higher - 75.0 %. This parameter reaches a maximum in the Mongoloids - 90.0 %: in the Chinese - 92.0 %, in the Japanese - 95.0 %, to the total mono-morphism in the Koreans - 100 % [Furthermore, the X1 allele frequency in the Kazakhs is close to the X1 allele frequency in other Mongoloids, while the X1 allele frequency in the Uighurs is close to this parameter in the population of the Hindus (75.0 %). The Uighurs polymorphisms are found in between the Hindus and Kazakhs as for the X1 allele frequency. Probably, such a variety of the distribution of alleles of the Apo B gene reflects not only the region of residence, but to a large extent ethnicity as well.This genetic marker studied globally, has a different degree of association with cardiovascular diseases – based on presence or absence of the X1 and X2 alleles. Specifically, association of the Xba I polymorphism with the risk of CHD  was founFurther confirmation of our present results will establish the fact that the Apo B gene polymorphism is not associated with the development of CHD among the Kazakhs and Uighurs. However, in the Uighurs with CHD the X2 allele of the Apo B gene marks the atherogenic shift of the lipid and apoprotein spectrum, which indicates a significant contribution of the Apo B gene in the determination of abrarrant lipid metabolism.In the literature there are reports on the relationship of allelic frequency of the Apo CIII gene not only with the levels of parameters of blood lipid spectrum, but also with coronary atherosclerosis –22.For eIt is known that the distribution of the S1 and S2 alleles among the Caucasoid ethnic groups is varied. Frequency of the S1 allele varies from 87.0 in the Russians to 94.0% in the British, and frequency of the S2 allele – from 13.0 to 6.0% respectively ,27,29. TSince we have found no differences between any of the analyzed alleles of the Apo CIII gene in the Kazakhs and Uighurs, we are proposing that polymorphisms of this particular gene, is not associated with the development of CHD in this population. Our results are consistent with the results of studies conducted in the world. It is established that the S1 allele identified in the Apo C111 gene can be regarded as a risk genetic marker of predisposition to hypertriglyceridemia which is often linked to both atherosclerosis and CHD .Our finding of an association of the Sst I polymorphism of the Apo CIII gene  with an increased level of triglycerides with the frequency of CHD in Uighurs is consistent with the literature. In fact similar associations have been found in other populations such as England, Canada and Finland, whereas Scottish descendants and some other ethnic groups showed negative associations ,33,41.Thus it is shown that the certain restriction polymorphisms of the Apo CIII gene in patients with CHD living in Kazakhstan are not associated with CHD. However, in particular based on these results, we are proposing that the S1 and S2 alleles of the Apo CIII gene may be considered as genetic markers of predisposition to hypertriglyceridemia in both the Kazakhs and Uighurs with CHD.The largest study of MRFIT demonstrated the association of the Apo E4 allele not only with the increased risk of coronary atherosclerosis, but also with mortality ,42.The fIn general, the E4 allele is associated with high level of the TC, LDLC and the Apo B. The E4 allele correlates more likely than other isoforms with vascular lesions in early atherosclerosis, and is considered to be atherogenic. In contrast, the E2 allele in different populations is associated with the decrease of the Apo B, LDLC, and is considered to be anti-atherogenic .Taking into consideration the universality of the Apo E protein, we must mention the role of the Apo E polymorphism in the development of Alzheimer’s disease. From 34 to 65% of these patients are carriers of the Apo E4 allele, while for the general population it is 24–31%. In all likelihood, this is due to the fact that the Apo E is the major apolipoprotein of human cerebrospinal fluid ,45.The EThus in our study, the Apo E gene polymorphism affects the development of CHD among the Kazakhs through interaction with the lipid metabolism system. It is appears that only the E4 allele is a reliable risk factor for development of hypercholesterolemia, increase of LDLC and AI in Kazakhs with CHD. Additionally, due to the results obtained we can conclude that the E2 allele of the Apo E gene is the risk factor for development of hypertriglyceridemia in the Uighurs with CHD.Thus the presence of the E4 allele of the Apo E gene is a genetic marker of predisposition to hypercholesterolemia in the Kazakhs with CHD. However, the presence of the E2 allele is a genetic marker of predisposition to hypertriglyceridemia in the Uighurs with CHD.The polymorphisms of the Apo B, Apo C111, Apo E genes in the Kazakhs and Uighurs are informative for molecular genetic population and clinical studies. A significant inter-ethnic difference of the frequency distribution of alleles and genotypes of the Apo B, Apo E genes studied in ethnic groups has been found. The X1 allele of the Apo B gene and the E4 allele of the Apo E gene are determined more often in the Kazakhs than in the Uighurs.There are significant association of alleles of the Apo E and Apo C111 genes with the value of lipid parameters in the Kazakhs, which confirms their substantial contribution to the determination of the abarrant lipid metabolism; the E4 allele of the Apo E gene is a leading molecular genetic factor in the development of hypercholesterolemia; the S1 allele of the Apo C111gene predisposes to the development of hypertriglyceridemia.Significant association of alleles of the Apo B, Apo C111 and Apo E genes with the value of lipid parameters have been evaluated in the Uighurs, whereby we confirm the contribution of these genes as risk factors in the development of abarrant lipid metabolism. Carriage of the X2 allele of the Apo B gene significantly predisposes an individual to hypercholesterolemia and increased levels of LDLP and the Apo B.The S2 allele of the Apo C111 gene and the E2 allele of the Apo E gene are also genetic markers of hypertriglyceridemia.Understanding these genetic antecedents in this population as risk factors for CHD may provide the physician with important and relevant facts to persuade both Kazakhs and Uighurs hard drinkers to significantly reduce their alcohol intake . These d


The relationships between the amplitude of the first heart sound (S1) and the rising rate of left ventricular pressure (LVP) concluded in previous studies were not consistent. Some researchers believed the relationship was positively linear; others stated the relationship was only positively correlated. To further investigate this relationship, this study simultaneously sampled the external phonocardiogram, electrocardiogram, and intracardiac pressure in the left ventricle in three anesthetized dogs, while invoking wide hemodynamic changes using various doses of epinephrine. The relationship between the maximum amplitude of S1 and the maximum rising rate of LVP and the relationship between the amplitude of dominant peaks/valleys and the corresponding rising rate of LVP were examined by linear, quadratic, cubic, and exponential models. The results showed that the relationships are best fit by nonlinear exponential models. P–R interval on the intensity of the mitral component of S1 where the P–R interval was measured from the beginning of the P wave to the beginning of the QRS-wave. This interval reflects the time the electrical impulse takes to travel from the sinus node through the atrium–ventricle node where it enters the ventricles. The results showed significant increments in the amplitude of the mitral component of S1 occurred at short P–R intervals; whereas the maximum rising rate of LVP did not change significantly when compared to those at longer P–R intervals. There was no correlation between the amplitude of the mitral component of S1 and the maximum rising rate at a steady hemodynamic state. Overall, past researches commonly indicated that the amplitude of S1 was positively correlated with the rising rate of LVP. However, the relationship's linearity or nonlinearity was not clear in previous studies.The mechanisms that determine the intensity of the first heart sound (S1) have been extensively studied during the last century. However, the conclusions of these studies were not well consistent. Sakamoto et al. , 1966 inThe purpose of this study was to further investigate this relationship in anesthetized dogs using various doses of epinephrine to invoke a wide range of blood pressures. The study not only examined the relationship between the peak amplitude of S1 and the maximum rising rate but also the relationships between the amplitude of dominant peaks/valleys and the corresponding rising rates.This study was approved by the Animal Care Committee of the Chongqing Medical University. Three adult beagle dogs were involved in the experiment with weight between 9 and 11 kg, and with chest wall size between 45 and 50 cm. They were anesthetized with Xylazine (0.2 mL/kg). Additional small doses of Xylazine were given when required, and the dogs were in a supine position during the experiment. A disposable catheter (16G) filled with heparinized solution (500 units/mL) was inserted into the left ventricle via the carotid artery. The catheter was coupled with a pressure transducer , which had been calibrated under standard atmospheric pressure. Various doses of epinephrine were used to invoke cardiac dynamics, and the channel to inject epinephrine was maintained using an intravenous infusion of 0.9% saline. A microphone transducer  was used to record external PCGs at the apex of the heart. The blood pressure, external PCG, and electrocardiogram (ECG) of lead II were simultaneously sampled at 1 KHz . The data collection was divided into three stages.Stage 1: Epinephrine (0.5 μg/kg) was injected. The data collection started 10 sec before the injection and ended when the blood pressure returned to baseline.Stage 2: Epinephrine (1 μg/kg) was injected. The remaining operations were the same as stage 1Stage 3: Epinephrine (2 μg/kg) was injected. The remaining operations were the same as stage 1.The catheter, blood pressure transducer, ECG electrical node, and all electrical lines were fixed during data collection to avoid motion artifacts. The repeated trials for each stage are listed in AmS1. The maximum rising rate of the blood pressure in the left ventricle was defined as dp/dtm. The amplitudes of the first two dominant peaks were defined as Ap1 and Ap2, the amplitudes of the first two dominant valleys were defined as Av1 and Av2, and the rising rates of the blood pressure at the first two peaks and the first two valleys were defined as dp/dtp1, dp/dtp2, dp/dtv1, and dp/dtv2. An illustration of these definitions is shown in AmS1 and dp/dtm, the relationship between Ap1 and dp/dtp1, the relationship between Ap2 and dp/dtp2, the relationship between Av1 and dp/dtv1, and the relationship between Av2 and dp/dtv2.The position of the ECG R-wave was detected using the first differential of the ECG signal and its Hilbert transform , bi, ci, and di were the regression coefficients. The regression coefficients were determined using least square criteria.where R2) was used to evaluate the goodness of fit of the models . The relationship was fit by linear, quadratic, and cubic models, as shown in The relationship between dp/dtm approached zero. The wrong extensions of the two models were obviously inconsistent with the physical meaning of the data. The extension of the exponential model, however, had right extension out the data. The amplitude decreased, but remained positive as dp/dtm approached zero. Student's t-test showed that the exponential model fit the data in the 17 trials.Another principle used to select a fitting model is the extension of the model. As shown in AmS1 and dp/dtm was fitted by the exponential modelConsidering the findings mentioned above, the relationship between The regression coefficients for the 17 trials are listed in p/dtp1, dp/dtp2 and Ap1, Ap2. The solid lines in All the S1s of a record with respect to the R-wave were segmented cycle by cycle. The peak S1s are indicated by circles in p/dtp1, dp/dtp2 and Ap1, Ap2 were fitted by the exponential modelThe relationships between dThe regression coefficients of the two peaks with a significance confidence level of 0.01 are listed in p/dtp1, dp/dtp2 and Ap1, Ap2, the valleys of all the recorded S1s are plotted cycle by cycle in Similar to the analysis performed in Relationships between dp/dtv1, dp/dtv2 and Av1, Av2 were fitted using the exponential modelThe relationships between dPrevious studies commonly concluded that the amplitude of S1 was linearly related to the blood pressure rising rate. However, the results in this study showed with high confidence that the relationship is nonlinear exponential. Analyzing the reasons for this inconsistency is important, and the reasons are as follows.Most of the previous studies were conducted before the 1970s; thus, the signal recording systems used were analog. The first derivative of LVP was calculated using an analog circuit, and the S1 amplitudes and rising rates were measured using a ruler on the trace lines printed on a piece of paper with a paper feed speed of 100 or 200 mm/sec. Measurement and timing errors were unavoidable. Conversely, the signals in this study were sampled using a modern advanced digital system. The amplitudes and timing were accurately obtained by A/D conversion; therefore, the measurement errors in this study are negligible.The frequency band of the S1s being analyzed have an impact on amplitude measurement. For example, Sakamoto et al.  filteredThe ranges of the LVP and rising rate in this study were commonly greater than those of previous studies. The ranges of the LVP and LVP in the study by Sakamoto et al. , which wThe relationship between the amplitude of S1 and the rising rate of LVP was investigated in anesthetized dogs using various doses of epinephrine. Linear, quadratic, cubic, and exponential models were examined to fit the relationship. The results showed that the relationship between the maximum amplitude of S1 and the maximum rising rate of LVP, and the relationships between the amplitude of the dominant peaks/valleys and the corresponding LVP rising rates were all exponential and not linear. The main cause of the inconsistency with previous studies was the large range of data that was used in this study.


Sixty rats  were allocated into the 6 groups (n = 10): CG—control group; EG—nerve exposed; IG—injured nerve without treatment; LG—crushed nerve treated with LLLT; PG—injured nerve treated with P1; and LPG—injured nerve treated with LLLT and P1. After 4 or 8 weeks, the nerve samples were processed for morphological, histological quantification and ultrastructural analysis. After 4 weeks, the myelin density and morphological characteristics improved in groups LG, PG, and LPG compared to IG. After 8 weeks, PG, and LPG were similar to CG and the capillary density was higher in the LG, PG, and LPG. In the ultrastructural analysis the PG and LPG had characteristics that were similar to the CG. The application of LLLT and/or P1 improved the recovery from the nerve crush injury, and in the long term, the P1 protein was the better treatment used, since only the application of LLLT has not reached the same results, and these treatments applied together did not potentiate the recovery.This study analyzed the effects of a low-level laser therapy (LLLT, 15 J/cm Peripheral nerves are extensions from neurons that are located in columns and spinal cord ganglia, and the peripheral portions are dependent on the continuity and integrity of the central portions .Approximately 4.5% of injuries to soft tissue and 5% of open wounds in the extremities that are due to accidents result in an impairment of peripheral nerves that requires surgical repair . The tre2), which is irradiated on the sciatic nerve of rats, treatment restored the myelin sheath and cross-sectional area of the nerve [Many studies that have evaluated the low-level laser therapy (LLLT) as a treatment for peripheral nerve injury have observed several changes that are obtained with this therapy , such ashe nerve . These dhe nerve .Hevea brasiliensis), named P1 protein, was identified at the Laboratory of Neurochemistry of Faculty of Medicine of Ribeirao Preto, University of Sao Paulo [A new protein derived from the rubber tree . Animals were housed in polyethylene boxes with 5 animals each in rooms with controlled temperature (22–24°C) and dark/light cycles of 12 h. They were fed with chow and water ad libitum and were not subjected to unnecessary stress. The study protocol was approved by the local Ethics Committee (Protocol no. 078/2010) according to international laws of animal use.Sixty Wistar rats  were allocated into six groups (2 (0.6 cm diameter) totaling 5.2 MPa. After the injury, the nerve was repositioned, the skin was sutured, and antiseptic, anti-inflammatory, and antibiotic medications were applied to prevent further complications. Animals were anesthetized with ketamine and xylazine at dosages of 75 and 10 mg/kg, respectively, and after the left sciatic nerve was exposed, they were positioned on a stainless steel base coupled to a EMIC universal testing machine . The left sciatic nerve crush was performed according to the method of a previous study . The sit The Twin Laser apparatus  apparatus was used for laser irradiation. The application of LLLT was performed in a transcutaneous way in order to simulate clinical use. Three points of application were chosen following the path of the sciatic nerve for better distribution of energy. The application sites were determined by the scar on the skin related to surgical access to the sciatic nerve. The three points were 1 point proximal to the lesion area, 1 point in the damaged area, taking care to move the scar pulling up the skin, so interference will not occur in the scar tissue absorption and scattering of LLLT, and finally 1 point distal region of the scar. During the laser application, a contention was used to immobilize the animals to avoid the use of local or general anesthesia. The parameters for laser irradiation are listed in Hevea brasiliensis was the hyaluronic acid [μL) in situ after injury and just before the suture. Hyaluronic acid and P1 protein were provided by the Laboratory of Neurochemistry, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo.The vehicle used to deliver the P1 protein from nic acid . The hyan = 10) at four (n = 5) and eight weeks (n = 5) after the injury, and the protocols for each group are described below.  The current study evaluated six groups (Control group (CG) animals were anesthetized and kept lying in a lateral position for 10 minutes. For the exposed group (EG), the sciatic nerve was exposed and positioned on the lesion support for 10 minutes. In the injured group (IG), the sciatic nerve was exposed and crushed. The LLLT group (LG) had the sciatic nerve exposed and crushed, and then the animals were submitted to the LLLT irradiation protocol. For the protein group (PG), the sciatic nerve was exposed and crushed, and then the P1 protein was applied on the lesion site. In the laser and protein group (LPG), the sciatic nerve was exposed and crushed, the P1 protein was applied on the injury site, and then the animals were submitted to the laser protocol. μm), mounted on slides, and stained with neutral red and black Sudan to visualize the myelin sheaths of nerve fibers. The histological analyses were performed using a DM4000 B Leica microscope with the EC3 Leica camera. To process the nerve samples for analysis using transmission electron microscopy, the samples were fixed in 2.5% glutaraldehyde for 2 hours, post-fixed in 1% OsO4 at 4°C for 2 h, dehydrated in an ethanol series and propylene oxide, and embedded in Spurr resin. Finally, ultrathin sections (90 nm) were mounted on 200 mesh grids, counterstained with uranyl acetate and lead citrate, and examined under a JEOL 1010 electron microscope operating at 80 kV at the Institute of Biomedical Sciences, University of Sao Paulo. Four or 8 weeks following the nerve injury, the animals were anesthetized  and subjected to intracardiac perfusion with a solution containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer . To prochttp://rsb.info.nih.gov/ij/plugins/area.html/, not included in original plugin pack of ImageJ) provide the area of the dark region in pixels2 representing myelin; if the program was calibrated to the scale of the microscope used in the capture of the images, it is possible to obtain the same area in μm2. For the realization of area ratio of myelin (myelin area/nerve area) it was also necessary to measure the cross-sectional area of the sciatic nerve, which was obtained from the original images (2) was compared among the groups. Statistical analysis of the quantitative analyses was performed using the SPSS 17.0 software, employing an analysis of variance . The quantitative histological analysis of the myelin consisted of quantifying the number of pixels. In the original images , the extl images  using th Histological analysis after 4 weeks from the start of the experiment revealed normal morphology in the CG  and EG  without Eight weeks after the nerve crush injury, the morphological characteristics of the CG  and EG  were norIn the fourth postoperative week, the CG had no alterations in the sciatic nerve, which contained both myelinated and unmyelinated nerve fibers of different sizes arranged in groups, and most of the fibers had thick myelin sheaths . In the Eight weeks after the injury, the CG had characteristics similar to those observed in the control group that was analyzed after four weeks, as the groups of myelinated and unmyelinated fibers were present in various sizes and shapes and Schwann cells were present surrounding the fibers . The mye2)   from the2)   8 weeks The understanding of axonal regeneration has long been a subject of research in neuroscience and regenerative medicine . The higThe myelinated sciatic nerve had fibers of various diameters , and theAxonal regeneration after nerve crush is slow; however, it is most successful when compared to axonal disconnection (neurotmesis) . Even afThe histological quantification assessed the total area of myelin in relation to the cross-sectional area of the sciatic nerve by quantifying pixels in digital images that were processed and transformed into a binary display (black and white). Density analysis using the quantification of myelin in pixels is unusual in studies of peripheral nerves; however, this type of analysis provided data about the recovery of this type of injury in a rapid, reproducible, and intelligible manner regarding the amount of myelin present in the nerve, which is dependent on the quality and speed of the pulse , 33, 34 Quantification revealed a histological improvement in sciatic nerve recovery after crush injury by employing treatments with LLLT and protein P1. After 4 weeks, the treated groups had improvement; however, it was not possible to choose the best treatment. After 8 weeks of injury, significant improvements were observed in the groups using the P1 protein (PG and LPG) and the results were close to the values of the control group. The morphometrics data also revealed that spontaneous regeneration was not achieved, as noted in the IG at 4 and 8 weeks after nerve damage, confirming the findings of Muratori et al. , who repThe improvement in the morphological and morphometric characteristics following the use of LLLT after nerve crush was marked by a greater number of nerve fibers, myelinated fibers of larger diameter, and a higher amount of Schwann cells , 36. In The present study was the first to assess the ultrastructural and morphological changes that occur in a crushed nerve treated with the P1 protein. Thus, no data about the changes in injured tissue treated with this material were found in previously published studies. Our first observation reveals a very positive perspective on the use of this material for nerve crush injury because there were generally better recovery features obtained from using this protein.The P1 protein showed great angiogenic potential in previous studies –19, 38. In conclusion, treatment with LLLT and the P1 protein resulted in the improvement of peripheral nerve trauma recovery 4 and 8 weeks after injury. At 8 weeks after injury, the characteristics of the treated groups were closer resembled to those observed in the control group. In addition to higher capillary density, this suggests that there is a time-dependent characteristic of this type of recovery. In long-term recovery (8 weeks), the animals that received only the P1 protein generally had better recovery of crushed nerve, given the fact that the only application of LLLT with these proposed parameters not achieved the same morphological and morphometric results, and the association of the two treatments did not potentiate the recovery, suggesting that the improvement observed when the 2 treatments were applied mainly refers to protein P1. More studies are needed to improve the understanding of the recovery in other levels of analysis.


Graphic representation of the results, as contoured 3D coefficient plots, also provides a clue to the reasonable modification of molecules. (1) Substituents with a proper length and size at the C5 position of the thiazole core are required to enhance the potency; (2) A small and electron-withdrawing group at the C2 position linked to the thiazole core is likely to help increase the FBPase inhibition; (3) Substituent groups as hydrogen bond acceptors at the C2 position of the furan ring are favored. In addition, the agreement between 3D-QSAR, molecular docking and molecular dynamics simulation proves the rationality of the developed models. These results, we hope, may be helpful in designing novel and potential FBPase inhibitors.Fructose 1,6-bisphosphatase (FBPase) has been identified as a drug discovery target for lowering glucose in type 2 diabetes mellitus. In this study, a large series of 105 FBPase inhibitors were studied using a combinational method by 3D-QSAR, molecular docking and molecular dynamics simulations for a further improvement in potency. The optimal 3D models exhibit high statistical significance of the results, especially for the CoMFA results with  Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder that results from defects in both insulin secretion and insulin action. Nowadays, T2DM, as a major health hazard, is widespread and sometimes even fatal, which leads to both medical and social challenges. It has been reported  that typetc. However, among these agents, some series show very poor oral bioavailability (OBAV), thus, making the development of novel drugs with acceptable OBAV and more potency required.Most of the work has focused on enzymes in the gluconeogenesis (GNG) pathway, because this pathway is partly responsible for the excessive glucose production in type 2 diabetes mellitus . Out of in silico modeling approaches [in silico method based on 63 FBPase inhibitors [y-randomization check. In addition, the predictive abilities of the obtained models were validated statistically with an external test set of compounds using multiple statistical criteria. In addition, a combined computational approach including the docking analysis and molecular dynamics (MD) simulation was also employed to elucidate the probable binding modes of these antagonists at the binding site of the FBPase. The good concordance between the 3D contour maps and the docking result provides our identification of several key features of the binding mechanism for these inhibitors. We hope the developed models could provide some meaningful clues in the future synthesis of highly potent and orally bioavailable FBPase inhibitors.Nowadays, proaches –23, as aproaches –27 to fahibitors . In the hibitors  and comphibitors . The rel27 is taken as the representative to elucidate this point in detail. As illustrated in 27 locates at the active site of the enzyme and binds primarily through the H-bond and van der Waals interactions. The majority of hydrogen-bond and hydrophobic modes are the same as those observed in the crystal structure of AMP complexed with FBPase [The docking results could illustrate to us the active-site architecture and ligand binding model as well as the network of electrostatic, hydrogen bond and van der Waals interactions associated with ligand binding between the inhibitors and FBPase. In this work, the most potent compound h FBPase , which v27 forms hydrogen bond interactions with the main chain NH group residues of Thr27, Glu29, Leu30, with the OH of Tyr113, Thr27, and the side chain NH3+ group residue of Lys112. In addition, the amino group attached to the thiazole ring forms two other hydrogen bonds with the FBPase residues: one with the carbonyl O of Val17 and the other with the side chain OH of Thr31. Besides these hydrogen bond interactions, the van der Waals interactions also play a central role in the ligand binding as shown in As can be seen from the figure, the phosphate of compound 27 forms more van der Waals interactions with the amino residues of FBPase, which may be one possible reason exhibiting its high activity. Through the investigation of the series of thiazoles and oxazoles, an interesting common characteristic was observed for most of the inhibitors that they all have the phosphate group, as a hydrogen bond acceptor, which forms a hydrogen bond network with the amino residues of FBPase.Compared to the interactions between AMP and FBPase , the mosq2), non-cross-validated correlation coefficient (rncv2), and standard error of estimate (SEE), F-statistic values, predicted correlation coefficient for the test set of compounds (rpred2) as well as the principal components (PCs) were calculated.To measure the predictive capability of a QSAR model, several statistical parameters including especially the cross-validated correlation coefficient  and five 3D descriptors for CoMSIA  donor and acceptor fields) as well as their 31 combinations were applied as the independent variables in the building models.When building a 3D-QSAR model, two important elements impacting greatly on the quality of the model must be taken into account. One is the molecular alignment. In this work, both the ligand- and receptor-based alignment rules were applied, with the purpose to compare and implement the results of them for exploring the receptor-ligand interaction mechanism as really as possible. Another factor is the grid descriptors. Herein, two 3D descriptors for CoMFA  and test set (28 molecules) were obtained, with their statistical results shown in q2 = 0.443, rncv2 = 0.874, SEE = 0.314, F = 80.809) than that of the CoMFA one was observed, with three field descriptors  employed. The rpred2 value for the 28 test set compounds is 0.756. As to the relative contribution, the electrostatic field makes the greatest (0.453), followed by steric field (0.379), and the hydrogen bond acceptor field gives 0.168.During the ligand-based CoMSIA analysis, the model with slightly worse but still acceptable statistical performances  and test (red asterisk) sets for the two 3D-QSAR models. Clearly, good correlationships are observed since the predicted values are almost as accurate as the experimental activities for the whole dataset . For the receptor-based 3D-QSAR studies, it can be observed that no statistically acceptable results were obtained in terms of both internal and external validation criteria . Thus, wq2, rncv2 and SEE, we have noticed the two optimal models improve with the addition of components, that 10 components are needed for the CoMFA model and 6 for the CoMSIA model and that further improvement is not obtained with additional components  and without intercept (r02) are also calculated. rpred2 > 0.5, (2) rtest2 > 0.6, and (3) r02 is close to rtest2, such that the (rtest2 − r02)/rtest2 < 0.1 and 0.85 ≤ k ≤ 1.15 or 0.85 ≤ k′ ≤ 1.15. When the observed values of the test set compounds (X axis) are plotted against the predicted values of the compounds (Y axis) setting intercept to zero, the slope of the fitted line gives the value of k. Interchange of the axes gives the value of k′. As can be seen from the table, both the best CoMFA and CoMSIA models successfully pass those tests.To testify firmly the good performance of the prediction, the squared correlation coefficient values between the actual and predicted values of the test set compounds with intercept  between the observed and predicted values of the test set compounds does not necessarily mean that the predicted values are very near to the observed activities . To better evaluate the external predictive capacity of a model a modified r2 term (rm2) is been defined as follows [A previous report  has illu follows :rtest2 value will be very near to the r02. In the best case rm2 may be equal to rtest2, whereas in the worst case rm2 value could be zero. Herein, the rm2 values of both the two models are larger than the recommended value (0.5). We also noticed that, by comparing the CoMFA with CoMSIA model, the former produces higher predictive power than the latter in terms of both the internal and external exams . Thus, one conclusion can be drawn that a proper size alkyl group is required at the C5 position to increase the inhibitory activity.As shown in 2 and 3 with larger C2-groups such as ethyl and vinyl groups result in loss of potency compared to their counterpart, compound 1 having a methyl group at the C2 position of the thiazole core (15–17) than that of the molecule 1. In addition, an elimination of the C2-Me of compound 1 also leads to a 5-fold weaker compound in thiazole derivative 5, indicating that a suitable C2-group is important for the activity. Our previous investigation of the docking results also finds that the amino group attached to the C2 position of thiazole core forms two hydrogen bonds respectively with the side chain of Thr31 (with a bond length of 1.9 Å) and with the main chain of Val17 (with a bond length of 1.9 Å), which indicates that any larger substitute group might lead to a collision with these residues in the binding pocket, and thus the larger group here is forbidden.Another large green-colored map situates at C2 position of the thiazole core, indicating the favor of a large substituent group for enhancing the inhibitory potency. However, at the back of this green-colored contour, a yellow-colored map is also observed, suggesting the careful selection of groups of proper size in this position. This investigation is also consistent with the experimental investigation, where for instance, compounds ole core . We also27 is shown in 2 group, as a hydrogen bond donor, can interaction with both Thr31 and Val17. In addition, one can also notice that substituent of the hydrogen atom with methyl group (compound 11) leads to a large loss of potency compared to compound 10 probably due to the lack of the hydrogen bond donor atom. In addition, a large red contour map exists around the phosphate group and embeds the cyclohexyl group illustrating that in these positions electro-rich groups are beneficial for increasing the activity. This may be a reason that all compounds of the dataset contain such electronegative groups (phosphate group).The CoMFA electrostatic contour map with the most potent compound The CoMSIA steric and electrostatic contour maps  are simirpred2 and rm2 of 0.805 and 0.887, respectively, while, herein the present model exhibits the rpred2 of 0.902, and rm2 of 0.828. Apparently, both the discussed models present the comparable prediction performance. However, it should be noted that the former model was built based on the 63 compounds, while the latter was developed based on 105 molecules with the high statistical confidence. In addition, similar conclusions for modes of interaction between FBPase and inhibitors based the 3D-QSAR models and docking experiments from Chen’s and ours are drawn depicted as follows: (1) At C5 position of the thiazole core, a substituent group with a proper length and size are beneficial to enhance the potency. (2) At C2 position in the thiazole ring, a small and electron-withdraw group is likely to helpful to increase the FBPase inhibition. (3) Substituent groups (such as phosphate) as hydrogen bond acceptors at the C2 position of the furan ring are favored. (4) Several important amino acid residues  are identified to play a central role in the ligand binding. In summary, the findings made by Chen complement our findings and offer a clue to designing novel FBPase inhibitors.Since the previous report  is the sIn the current investigation, a 7 ns molecular dynamics simulation of the docked complex of FBPase with inhibitor 27 was performed to obtain a dynamic picture of the conformational changes that occur in an aqueous solution, with main emphasis to explore the conformational change that takes place in the inhibitor and the enzyme .27 forms three common H-bonds with residues of Thr27, Lys112 and Tyr113. The amino group attached to the thiazole ring forms one common hydrogen bond with Val17. As for van der Waals interactions, both modes basically keep similar interactions. Furthermore, it can also be noticed that there exists slight differences among docking and MD simulation. For the MD results, the phosphate group presents two another H-bonds with residue of Arg140, and lacks the two H-bonds with residues of Glu29 and Leu30 that can be found in the docking results. In addition, the loss of the hydrogen between the amino group attached to thiazole and the residue of Thr31 presented in the docking is compensated by the newly formed H-bond between –NH2 with residues of Met18, Glu20 and Arg25 during the MD simulation.In order to validate the reliability of the docking results, we have compared the results between the MD and docking simulations in terms of H-bond interactions and van der Waals contacts. By and large, the interaction modes produced between MD and docking simulations share the common features. For example, the phosphate of compound 27 represented as stick and line for the initial complex and the average complex, respectively. It can be noticed that, from this figure, there is no significant difference between the average structure extracted from MD simulations and the docked model of the complex, except the cyclohexyl ring exhibiting 90° rotation. However, although the complex has undergone slight movements during MD simulation, both the binding pocket and the conformation of the ligand are still stable, suggesting the rationality and validity of the docking model.In order to compare the structures from MD simulations and docking, a superimposition of both the structures in the last 1 ns is shown in 50 (−log IC50) values, ranging from 4.870 to 8.000 M, were used as the dependent variables in the QSAR regression analysis to improve the normal distribution of the experimental data points. The whole data set was divided into training (77 molecules) and test (28 molecules) sets, respectively. All structures and the corresponding activity values of the dataset as well as their belongings to the training or test set are listed in 50 values are randomly but uniformly distributed in the range of the values for the whole set, and on the other hand, their structures cover as large diversity as possible of the dataset so that the derived models could represent the real characteristics of all the compounds both from the biological activity and the structures.A large, diverse dataset of 105 inhibitors of FBPase were collected from literatures ,39 publiThe 3D structures of all compounds were constructed by using the sketch molecule modules of SYBYL6.9 package. Partial atomic charges were calculated by Gasteiger-Hückel method, and energy minimizations were performed by using the Tripos force field and the Powell conjugate gradient algorithm with a convergence criterion of 0.005 kcal/mol.27 was chosen as a template to fit the remaining training and test set of compounds. Thereafter, all compounds finally minimized with the lowest energy in the dataset were aligned to a common substructure  by substructure-based alignment method using the “align database” command in SYBYL  of another advanced version of SYBYL package (X 1.1) in this study . The doc3 probe atom with a charge of +1.0 at all intersections of a regularly spaced grid of 2.0 Å in all three dimensions with the defined region. The van der Waals and Coulomb-type potentials representing the steric and electrostatic fields, respectively, were calculated using the Tripos force fields. The grid box dimensions were determined by the created automatically features in the CoMFA module within the SYBYL program. The steric and electrostatic energy values were both truncated to 30.0 kcal/mol.The steric and electrostatic field energies were calculated using a spAF) for a molecule j with atom i at a grid point q were calculated by In CoMSIA, the steric, electrostatic, hydrophobic, hydrogen bond donor and hydrogen bond acceptor potential fields were also calculated at each lattice intersection of a regularly spaced grid of 2.0 Å as that used in CoMFA. A probe atom with radius 1.0 Å and a charge of +1.0 with hydrophobicity of +1.0 and dydrogen bond donor and acceptor properties of +1.0 were used to estimate the steric, electrostatic, hydrophobic, donor and acceptor fields. The attenuation factor α was set to 0.3. CoMSIA similarity indices  as dependent variables in PLS regression analysis for building the 3D-QSAR models. The predictive values of the models were evaluated first by leave-one-out (LOO) cross-validation process [q2, was calculated using In the current study, the CoMFA and CoMSIA descriptors served as independent variables and the active values , respectively for the training set. Herein, the term, PRESS). The optimal number of components obtained from the cross-validation was used to derive the final QSAR model. Then, a non-cross-validation analysis was carried out; and the Pearson coefficient (rncv2), standard error of estimates (SEE) and the F values were calculated. Finally, the CoMFA and CoMSIA results were graphically represented by field contour maps, where the coefficients were generated using the field type “Stdev*Coeff”.where q2 for the training set can exhibit a low predictive ability of a model, the opposite is not necessarily true. That is, the high q2 is necessary, but not sufficient, for a model with the high predictive power. Therefore, the external validation must be estimated to establish a reliable and predictive QSAR model. Several often used statistical criteria are listed as follows:As been reported , althougr2, rpred2 is defined as follow:In SD is the sum of the squared deviations between the actual activity of the compounds in the test set and the mean activity in the training set, and “PRESS” is the sum of the squared deviations between predicted and observed activity for each compound in the test set. In rtest2 is the conventional correlation coefficient between experimental values and model predictions in the test set. The r02 is a quantity characterizing linear regression with the Y-intercept set to zero . The k in where To identify a functionally validated complex from protein docking and the most potent molecule 27, we performed 5 ns molecular dynamics simulations to investigate the conformational changes in the complex induced by the ligand 27. The software AMBER 11  was usedThe whole systems were minimized in three stages to remove bad contacts between the complex and the solvent molecules. Firstly, the water molecules were minimized by restraining the protein; Secondly, water and the side chains of the protein were minimized by restraining the backbone of the protein, and each stage was performed by using the steepest descent minimization of 2500 steps followed by a conjugate gradient minimization of 2500 steps. Thirdly, the entire system was minimized without any restriction by 10,000 steps changing the minimization method from steepest descent to conjugate gradient after 5000 cycles. After 15,000 steps minimization and equilibration for 60 ps, the system was then heated gradually from 0 to 310 K in the NVT ensemble and equilibrated at 310 K for another 60 ps. After the minimization and heating, 5 ns MD simulations were performed at a constant temperature of 310 K and a constant pressure of 1 atm. The Particle-Mesh Ewald (PME) method  was emplq2, rpred2 and rm2 for both the internal and external data sets. In addition, both of the two methods pass the y-randomization check, suggesting the robustness of the built models. Graphical interpretation of the optimal results, provided by the CoMFA and CoMSIA analyses, brings to light the important structural features that could be responsible for the activity of FBPase inhibitors. (i) Substituents with a proper length and size at the C5 position of the thiazole core are required to enhance the potency; (ii) A small and electron-withdraw group at the C2 position linked to the thiazole core is likely to help to increase the FBPase inhibition; (iii) Substituent groups as hydrogen bond acceptors at the C2 position of the furan ring are favored; (iv) Furthermore, the key amino residues have been found, i.e., Leu30, Glu29, Lys113, Lys112 and Thr27 which form the important hydrogen bond network with the phosphate group of FBPase, and Thr31, Val17 also play an important role in the binding between the ligand and the target.Presently, a large dataset of 105 thiazoles and oxazoles derivatives as potent orally bioavailable FBPase inhibitors for lowing glucose in type 2 diabetes mellitus has been estimated for the purpose of developing 3D-QSAR models based on both the ligand- and receptor-based superimpositions. Statistically significant models have been derived with two 3D-QSAR methods of CoMFA and CoMSIA on the basis of the database alignment method. The CoMFA model presents higher predictivity than CoMSIA expressed in terms of several rigorous evaluation criteria such as In addition, a good consistency between the CoMFA and CoMSIA contour maps, molecular docking and molecular dynamics simulations proves the reliability and robustness of the developed models.Overall, in this report, several reliable computation models between thiazole/oxazole analogues and FBPase have been built, which not only exhibit satisfied statistics, but also provide several possible mechanism interpretations from a molecular-level. We hope the models may provide some instructions for further synthesis of highly potent FBPase inhibitors.


The Late Glacial Maximum (LGM), ∼20 thousand years ago (kya), is thought to have forced the people inhabiting vast areas of northern and central Europe to retreat to southern regions characterized by milder climatic conditions. Archaeological records indicate that Franco-Cantabria might have been the major source for the re-peopling of Europe at the beginning of the Holocene (11.5 kya). However, genetic evidence is still scarce and has been the focus of an intense debate.Based on a survey of more than 345,000 partial control region sequences and the analysis of 53 mitochondrial DNA (mtDNA) genomes, we identified an mtDNA lineage, HV4a1a, which most likely arose in the Franco-Cantabrian area about 5.4 kya and remained confined to northern Iberia.The HV4a1a lineage and several of its younger branches reveal for the first time genetic continuity in this region and long-term episodes of isolation. This, in turn, could at least in part explain the unique linguistic and cultural features of the Basque region. In the last decade, numerous studies have evaluated the distribution and extent of variation of mitochondrial DNA (mtDNA) in European populations, and evidence of the overwhelming importance of the Franco-Cantabrian refugium for the re-peopling of much of Western and Northern Europe at the beginning of the Holocene has been obtained by the age estimates and geographic distributions of certain mtDNA haplogroups.In 1998, an early study proposed that haplogroup V was an autochthonous European haplogroup that originated in northern Iberia or southwestern France at about the time of the Younger Dryas Recently, a few (mostly control region) mtDNA sequences belonging to haplogroup HV4 were reported in the literature and databases (mainly GenBank) In this study, to shed some light on the origin of HV4 and its derivatives, we surveyed its distribution in a wide range of European (and neighboring) populations and evaluated its sequence diversity by analyzing 53 HV4 mitochondrial genomes.Written informed consent was obtained from all sample donors. Analysis of mtDNA sequences was approved by the three different institutional review boards involved in sampling recruitment: the Ethical committee of the University of Santiago de Compostela, the Ethics Committee for Clinical Experimentation of the University of Pavia , and the Institutional Review Boards of the University of Pennsylvania and the United States Department of Health and Human Services. Moreover, the study conforms to the Spanish Law for Biomedical Research (Law 14/2007- 3 of July).More than 134,800 control region profiles were compiled from the literature  and searched for the putative diagnostic HV4a1a sequence motif C16221T- C16291T. Only 21 HV4a1a sequences were observed in this database, but most of them (∼62%) were located in the Franco-Cantabrian area, and the remaining mainly among French Canadians and in the USA. In addition, more than 8,700 mitochondrial genomes were recruited from the literature and GenBank http://www.goldensoftware.com). We followed the commonly used inverse-squared distance method for interpolating frequency values; other interpolated methods yielded virtually the same results. The haplogroup frequencies are presented in a regular grid covering Europe.The spatial geographical representation of haplogroup HV4a1a was obtained using Surfer 8.0 software ; most of these genomes were from subjects who lived along the Cantabrian Sea coastline, with frequencies peaks in the Basque Country and in the Department of Cantabria (an area that is bordering with the West frontier of the Basque Country). The coalescence age of HV4a1a was estimated to be 5.4 kya (ΔT = 2.0). As inferred from the phylogeny , HV4a1a Les Parlers Francais D'Acadie) suggests that they came from the Loudunais area in northeastern Poitou (northern section of today's Vienne department) (taken from: http://www.acadian-cajun.com/colorig.htm). Therefore, the Canadian HV4a1a haplotypes, most likely of French origin, indicate a direct connection between the Basque Country and western France. The basal motifs of HV4a1a and the HV4a1a5 clade were also found in the USA (23%), but as the ones observed in Canada, they did not show signs of substantial internal diversity indicating that only a few (even only two) founders might have crossed the Atlantic in Colonial times . The phylogeny of all available control-region segments is represented in see also .The post-glacial debate and the odd distribution of HV4 mtDNAs in worldwide databases inspired this study under the hypothesis that a minor sub-clade of HV4, here referred to as HV4a1a, might have witnessed events of (pre)historical isolation in the Franco-Cantabrian region, thus perhaps explaining the uniqueness of some cultural features of the Basque Country.A tentative reconstruction of the pre-historical and historical movements of HV4 and its sub-lineages across Europe and America is summarized in Table S1Mitochondrial DNA data generated in the present study. References and other data resources used in the present study are also given.(XLS)Click here for additional data file.


The morphology of nanostructures could be controlled by simply adjusting the sputtering power of the Ti target. The reflectance measurements showed that the melon seed-shaped nanoflakes exhibited optimal properties of antireflection in the entire visible and ultraviolet region. In addition, we determined their anisotropic reflectance in the direction parallel to the surface of nanoflakes and perpendicular to it, arising from the anisotropic morphology.Regular arrays of Ti Eg = 3.6 eV at 300 K), SnO2 is well known for its potential applications in gas sensing [2 is also expected to be a competitive candidate for optical devices [Much research has recently been focused on the design of nanoscale semiconductor oxide materials with controlled morphology and novel morphology-dependent physical properties. As an n-type semiconductor oxide with a wide band gap  and slightly different lattice parameters of TiO2 and SnO2, the metal cations can replace each other in a wide concentration region [Numerous techniques have been developed to synthesize SnOn region .xSn1-xO2 nanostructures were grown on patterned Si substrates using GLAD with the simultaneous deposition of DC and RF sputtering sources. The RF sputtering of the SnO2 target at low pressures resulted owing to the DC sputtering of the Ti target. The morphology of nanostructures can be modulated by the regulation of the sputtering power.On the other hand, the morphology of films can be modulated by the glancing angle deposition (GLAD) technique. The GLAD technique, which exploits the shadowing effects, is applied to fabricating a variety of nanostructures, such as columns, helixes, and springs -11. WithxSn1-xO2 nanostructures were prepared on Si(001) substrates that were patterned using 200-nm-diameter hexagonal close-packed polystyrene microspheres in an ultrahigh vacuum magnetron sputter deposition system. TixSn1-xO2 depositions were carried out using a 6-cm-diameter Ti target (99.99% pure) and a 6-cm-diameter SnO2 target (99.9% pure) mounted at an angle of 120° with respect to each other, with the substrate backed against the Ti target, as shown in Figure α, equaling θ1 + θ2 , with their structure identified by X-ray diffraction (XRD) analysis, and the reflectance measured with a spectrophotometer.The TiFigure xSn1-xO2 nanostructures were produced. The regular hexagonal arrays replicate the close-packed pattern of the polystyrene spheres, which indicates that each nanosphere leads to the formation of a nanostructure. It illustrates that substrate patterning is effective in positioning GLAD nanostructures into ordered arrays. Atomic shadowing and adatom diffusion are the dominant growth mechanisms in the process of GLAD. Oblique angle flux incidence enhances atomic shadowing which produces areas that vapor flux cannot directly reach while adatom mobility is too low for surface diffusion to fill the voids [For the deposition step, the power of RF sputtering was fixed, while the power of DC sputtering was regulated at the discharge currents 0.15, 0.20, and 0.25 A, respectively, for the three prepared samples named as 1#, 2#, and 3#. Their structures were identified by XRD, indicating an amorphous state in all the samples. As for the morphology, Figure he voids . NanosphIt is observed that the thickness of the nanoflakes in the shape of melon seeds, shown in Figure 2 target, the direction of the growth front was changed from perpendicular to lateral, leading to an increase of the growth rate in the direction parallel to the SnO2 flux and a decrease in the perpendicular direction [2 flux, and suppressed perpendicular to it, as shown in Figure 2 flux illustrated in Figure 2 flux was gradually overweighed by Ti flux, resulting in the increase of the length-width ratio. Thus, it can be concluded that the sputtering rates of Ti target and SnO2 target have a great influence on the morphology of nanostructures.The effect of the deposition parameter to the morphology of nanostructures was studied. As GLAD is a physical vapor deposition process in which the incident flux impinges the substrate from an oblique angle, causing atomic shadowing and resulting in highly porous nanostructures , the morirection . As a rexSn1-xO2. The thicknesses of the prepared Ti films for the three specimens are 42.3, 74.4, and 91.2 nm, respectively, measured through the SEM micrographs (the resolution of the applied scanning electron microscope is 2.0 nm). Because the thickness of the Ti films is directly proportional to the sputtering rate of the Ti target with other factors being held constant, the question can be transformed to the relationship of the thickness of Ti films with the length-width ratio of nanoflakes, as illustrated in Figure To investigate the relationship of the sputtering rate with the shape of nanostructures, the depositions of Ti films were carried out, with the discharge currents set to 0.15, 0.20, and 0.25 A, respectively, and other conditions consistent with the depositions of TiR||, while the reflectance in the other direction is marked as R⊥. Figure R|| and R⊥ of the samples 1#, 2#, and 3# in the spectral range of 200-750 nm. It indicates that the reflectance rises as the Ti content increases, and that the reflectance of sample 1# is almost wavelength independent, which agrees with the previously reported study on the optical properties of metal-dielectric composite media close to the percolation threshold [R|| is apparently lower than R⊥ for all the samples. This feature can be attributed to the anisotropism of the morphology, which plays a crucial role in the anisotropism of the optical property. It demonstrates that the preparation method we proposed in this article can accomplish an adjustment to the morphology of nanostructures, and ultimately the tuning of the properties.In view of the anisotropic morphology of the nanoflakes, the anisotropism of the optical property was studied. The reflectances of samples 1#, 2#, and 3# were measured using a spectrophotometer. Figure hreshold -17. In ahreshold . Hence, hreshold -21. BecaxSn1-xO2 nanostructures were prepared on patterned Si substrates through the co-sputtering of Ti and SnO2. The shape of TixSn1-xO2 nanostructures, characterized by the length-width ratio which linearly depended on the sputtering rate of Ti target, could be controlled by adjusting the sputtering power of Ti target. Optical properties were studied, and the results confirmed that the melon seed-shaped nanoflakes, which were approximately equivalent to gradient-duty cycle subwavelength nanogratings, possessed very low reflectance. Furthermore, their reflectances were anisotropic, which can be ascribed to the anisotropism of the morphology. Consequently, this study provides the opportunity to design optical devices by creating nanostructures with tailored morphology and unique performance.We have reported a general and an effective process for producing semiconductor oxide materials with controllable morphology and properties by glancing angle sputter deposition. Periodic arrays of TiGLAD: glancing angle deposition; SEM: scanning electron microscopy; XRD: X-ray diffraction.The authors declare that they have no competing interests.SC carried out the studies and drafted the manuscript. ZL participated in the design of the study and helped in revising the manuscript. ZZ participated in the design of the study and gave suggestions on the analysis of results. All the authors read and approved the final manuscript.


Peutz-Jeghers syndrome  is a rare, hereditary predisposition characterized by the occurrence of hamartomatous polyps in the gastrointestinal tract, mucocutaneous pigmentation, and increased risk of cancer in multiple internal organs. The incidence of the syndrome, depending on the studied population, has estimated range from 1:25,000 even up to 1:300,000 births. PJS is an autosomal dominant disease and in most of the cases is caused by mutations in the STK11 gene (MIM # 602216) located on a small arm of chromosome 19 at position 19p3.3.The majority of causative DNA changes identified in patients with PJS are small mutations, therefore, developing a rapid and cost-effective method of their detection is a key aspect in the advancement of genetic diagnostics of patients suffering from PJS. In our study we developed a methodology of detection of small mutations in entire coding sequence of the STK11 gene based on High Resolution Melting analysis (HRM).In our group of 41 families with PJS small mutations of the STK11 gene were detected in 22 families (54%). In the remaining cases, where large rearrangements were not detected using MLPA, all of the coding exons were sequenced. However, this did not allow detection of any additional mutations. Therefore, the developed methodology of searching for small mutations using HRM allowed to detect all the STK11 gene sequence changes occurring in our group of patients. The study was financed by the Ministry of Education and Science, Poland, grant number N402 481537.