Plasmodium falciparum require expensive materials and equipment, thus limiting the search for new drugs, particularly in developing countries. This study describes an inexpensive procedure that is based on the advantage of a positive correlation between the haemozoin level of infected erythrocytes and parasite load.The spread of drug resistance in malaria parasites and the limited number of effective drugs for treatment indicates the need for new anti-malarial compounds. Current assays evaluating drugs against 50) values of chloroquine, quinine, artemisinin, quinidine and clotrimazole against P. falciparum K1 and 9A strains were determined using the novel assay method.The relationship between parasitaemia and the haemozoin level of infected erythrocytes was investigated after converting haemozoin into monomeric haem. The 50% inhibitory concentration (IC2 = 0.9929). Furthermore, the IC50 values of drugs were within the range of the values previously reported.The haemozoin of parasites was extracted and converted into monomeric haem, allowing the use of a colorimeter to efficiently and rapidly measure the growth of the parasites. There was a strong and direct linear relationship between the absorbance of haem converted from haemozoin and the percentage of the parasite (RThe haemozoin-based colorimetric assay can be considered as an alternative, simple, robust, inexpensive and convenient method, making it applicable in developing countries. Qinghaosu) that has been used in the treatment of fevers for more than a thousand years, is now considered an essential component of artemisinin-based combination therapy against drug-resistant malaria [Malaria is more than just a problem for tropical countries, it also is a major global public health concern. Annually, there are approximately 300 million new malaria infections and millions of deaths worldwide due to malaria ,2. Becau malaria . However malaria -7. The ain vitro by quantifying the uptake of radioactive substrates by a parasite as a measure of growth and viability in the presence of the test drug [in vitro methods exist, the 3H-hypoxanthine method [For decades, the anti-malarial activity of a drug has been measured est drug ,9. Althoe method  is a pope method ,11 and te method ,13 methoe method -16. Howein vitro assay was developed based on the colorimetric quantification of haemozoin in infected red blood cells to evaluate the anti-malarial activity of drugs.During the development and proliferation stage in host erythrocytes, the malaria parasites degrade haemoglobin for use as a major source of amino acids. This is accompanied by the release of free haem. With haem as a prosthetic group of haemoglobin, the iron is in the ferrous state, but free haem loses one electron and assumes the ferric state. This ferric haem could be oxidatively active and toxic to both the host cells and malaria parasites, and can even cause parasite death. Moreover, due to the absence of haem oxygenase, the parasite is unable to cleave haem into an open-chain free haem, which is necessary for cellular excretion . To protPlasmodium falciparum K1 (chloroquine resistant) and 3D7-9A (chloroquine susceptible) strains [Chloroquine diphosphate, quinine sulphate, primaquine, clotrimazole, haemin chloride (haem), RPMI 1640 medium, hypoxanthine, and gentamycin were purchased from Sigma Aldrich Chemical Company . Albumax II (Gibco), and the other chemicals used in the present study were of a high grade.  strains  were proPlasmodium falciparum K1 and 9A strains were maintained in vitro with continuous culture according to a previously described method with a slight modification [3, 11 mM glucose and 0.5% albumax II, and adjusted to a pH of 7.3 to 7.4. The parasite was cultured and maintained in a tissue culture flask with complete culture medium containing 5% human erythrocytes. The parasite density was maintained at about 1.5% parasitaemia under an atmosphere of an AnaeroPack sachet  to create 20% CO2 and remove O2 (<0.1%) 37°C [fication . The cul1%) 37°C . Every tP. falciparum K1 strain was serially diluted with uninfected erythrocytes in complete medium to yield a haematocrit of 5% and parasitaemia ranging from 0 to 10%. The serial culture containing 200 μl was prepared independently in triplicate in microtubes, followed by the addition of 800 μl of 2.5% sodium dodecyl sulphate in 0.1 M sodium bicarbonate pH 8.8, then the samples were mixed at room temperature for 15 min. After centrifugation at 13,000 rpm for 10 min, the supernatant was removed. The pellet was washed twice with 800 μl of 2.5% sodium dodecyl sulphate in 0.1 M sodium bicarbonate (pH 8.8), then 200 μl of 5% sodium dodecyl sulphate was added to 50 mM NaOH to convert the haemozoin into haem. After incubation at room temperature for 30 min, the sample (200 μl) was transferred to a 96-well microplate and scanned at 405/750 nm (A405 nm minus A750 nm) using an IMark microplate reader (Bio-Rad). After the background absorbance of haemozoin was purified of uninfected erythrocytes (5% haematocrit) then subtracted, the amount of haemozoin in the infected erythrocytes was presented as the absorbance at 405/750 nm and then plotted against parasitaemia.A culture of the P. falciparum K1 and 9A strains were used to evaluate the anti-malarial activity of quinine, chloroquine, pyrimethamine, artemisinin and clotrimazole by using the haemozoin-based spectrophotometric method. Stocks of drugs were prepared in dimethyl sulphoxide or phosphate buffer saline (for chloroquine) and were then serially diluted with complete culture medium. To each well of a microplate, 10 μl of serially diluted drug solution was added into 200 μl of final asynchronous parasite culture. Dimethyl sulphoxide or phosphate buffer saline were also tested by adding a similar amount to control wells. The microplates were cultured 72 hr under the conditions described above. The haemozoin of infected erythrocytes was extracted, purified, and quantified, as described above. The 50% inhibitory concentration (IC50) value was calculated by non-linear fitting of the absorbance at 405/750 nm against the logarithm of the drug concentration using the GraphPad Prism 5 . Sigmoidal doses with variable slope models were used with the following equation:The where y is the absorbance at 405/750 nm; x is the logarithm of the drug concentration, min is the absorbance at 405/750 nm measured at time zero (starting point of assay), and max is the maximal absorbance of a particular drug. The Hill slope is the steepness of the curve. The logarithm of the concentration at zero was defined at 2 log lower than the lowest concentration of a particular drug.The relationship between the haemozoin amount in a parasitized erythrocyte and parasitaemia was revealed by measuring the absorbance of the haem content of the haemozoin obtained from the parasitized erythrocytes after degradation to monomer haem. As shown in Figure 50 values of some anti-malarial drugs were determined with a dose-response experiment using a haemozoin-based colorimetric assay. The result showed that increasing the concentration of anti-malarial drugs resulted in a decreased absorbance at 405/750 nm (Figure The ICm Figure . The dat50 using the haemozoin-based colorimetric method. The P. falciparum K1 strain was primarily observed for quinine, chloroquine, clotrimazole, pyrimethamine and artemisinin, with IC50 values of 0.258, 0.873, 0.805, 23.03, and 0.139 μM, respectively. For the P. falciparum 9A strain, pyrimethamine was not evaluated, and the IC50 values were 0.398, 0.132, 1.67 and 0.483 μM in succession for quinine, chloroquine, clotrimazole and artemisinin, respectively. The IC50 values for quinine, chloroquine, clotrimazole, and pyrimethamine were in a range that was similar to those observed in previous reports [50 values for artemisinine were higher than previous reports, probably due to asynchronous cultures, ring stage-specific target of artemisinine, accumulation of released hemozoin in the continuous cultures, or several rounds of continuous cultures and cloning of parasite strains in our laboratories. Therefore, further studies are required to compare the novel assay with recent developed methods to validate the accuracy in the screening new antimalarial compounds [Table  reports ,29. On tompounds . Anotherompounds .In recent years, the number of laboratories, diagnosis centres and research institutes has risen in developing countries. However, most of them lack the modern equipment and expensive chemicals to apply new methods for screening anti-malarial candidates. In addition, some methods have potential risks of toxicity, so it is prudent to wear disposable gloves at all times when proceeding. Another obstacle is that many laboratories lack the facilities to treat toxic contamination before the toxin is discarded in the environment. The novel anti-malarial assay is safe, non-expensive and easy to apply in laboratories.50 values of chloroquine and quinine obtained from the haemozoin-based colorimetric method are similar to other methods. Even though this report describes specific conditions, the current experiment has introduced an assay that is adaptable to a wide range of conditions. The results also show that using this method has several advantages over using current methods. First, the method is fast and is based on a simple technique that uses a microplate reader, which is available in most laboratories. Second, the assay is based on inexpensive chemicals with no requirement of cold storage. Last but not least, the assay of the inhibition of P. falciparum growth using a haemozoin-based colorimetric method is feasible, reproducible, non-toxic, and more convenient than other assays, which makes it particularly useful for developing countries in the screening of novel anti-plasmodials, as a useful high throughput screening method for anti-malarial drug candidates.The standard curve obtained in this study was strongly linear between the absorbance of monomeric haem converted from haemozoin and the percentage of parasitaemia. The ICThe authors declare that they have no competing interests.NTH and KK developed the idea for the project. TTM and NTH conceived and designed the experiments. TTM, NTH and MNS carried out the laboratory work. TTM, NTH, DTXT, KH, and KK analysed and interpreted the data. TTM, NTH, MNS, KH and KK contributed reagents/materials/analysis tools. . TTM, NTH, DTXT and KK wrote the paper. All authors had full access to all data in the study, read and approved the manuscript.


The genetic diversity and structure of horses raised in France were investigated using 11 microsatellite markers and 1679 animals belonging to 34 breeds. Between-breed differences explained about ten per cent of the total genetic diversity (Fst = 0.099). Values of expected heterozygosity ranged from 0.43 to 0.79 depending on the breed. According to genetic relationships, multivariate and structure analyses, breeds could be classified into four genetic differentiated groups: warm-blooded, draught, Nordic and pony breeds. Using complementary maximisation of diversity and aggregate diversity approaches, we conclude that particular efforts should be made to conserve five local breeds, namely the Boulonnais, Landais, Merens, Poitevin and Pottok breeds. Therefore, it would be useful to identify priorities among conservation purposes and this requires characterising diversity and genetic relations between breeds [During the twentieth century, horse breeding has undergone large changes in Europe. Previously considered as an agricultural, industrial and war tool, horse is now essentially bred for hobby riding. Draught horses, in particular, have been less and less used as utility horses, and many draught breeds have undergone a dramatic decrease in population size: according to the Haras Nationaux, out of the nine French draught breeds, six have annual births below 1000. Measures for During the last fifteen years, microsatellite markers have frequently been used to evaluate genetic distances and to characterise local breeds, -10. SomeWith about 800 000 animals belonging to 50 different breeds (source: Haras Nationaux), France shows a large diversity of horse populations. Among these breeds, 21 have a French origin or have been bred in France for at least a century. According to the FAO, at least 15 populations have disappeared during the last 50 years, and eight indigenous breeds are still considered as endangered or endangered-maintained. Among those breeds, the majority are draught breeds, namely the Ardennais, Auxois, Boulonnais, Poitevin and Trait du Nord breeds, the other ones being the Merens warm-blooded breed and the Landais and Pottock pony breeds. Information on the genetic diversity of French endangered breeds could help breeders and providers, decide where they should place more emphasis.In the present study, we first analysed the genetic diversity of 39 horse populations reared in France: within-breed diversity, breed relationship and population structure were investigated, using microsatellite data. Then, we focussed on 19 breeds of French origin or having been raised in France for at least a century, and evaluated the conservation priorities between these populations, using different approaches to evaluate within, between and total diversity.French nomenclature divides horse breeds into three groups: warm-blooded, draught horses and ponies. In this study, 39 populations were considered Table . These 3For each of the 39 populations, 23 to 50 animals born between 1996 and 2005, were sampled amounting to 1679 animals. Except for the Przewalski horse, where no pedigree data was available, the sampled animals were known to have no common parents. For the conservation approach, the study focussed on 19 populations, either of French origin, or having been bred in France for at least 100 years . In this approach, 50 animals were randomly sampled among the four and three AA and SF subpopulations, respectively, to constitute two populations.AHT4, AHT5, ASB2, HMS1, HMS3, HMS6, HMS7, HTG4, HTG6, HTG10, VHL20), with all but two (HMS1 and HTG6) being recommended by the International Society of Animal Genetics for parentage testing and used in similar studies (except HMS1) [Eleven microsatellite markers were used to perform the analysis (pt HMS1) ,10. For (MNA), observed (Ho) and non-biased expected heterozygosity (He), were calculated using GENETIX [Allele frequencies, mean number of alleles  GENETIX . Wright  GENETIX  was used GENETIX  and depa GENETIX  on loci. GENETIX .DR [http://bioinformatics.org/~tryphon/populations/). Regarding the DR distance, a NeighborNet tree was drawn using SPLITSTREE 4.8 [K) between 1 and 22, with a burn-in period of 20 000 followed by 100 000 MCMC repetitions. Pairwise similarities (G) between runs were computed using CLUMPP [The matrix of Reynolds unweighted distances DR  was compTREE 4.8 . A factoTREE 4.8 ): using g CLUMPP .CB) is evaluated, based on the Weitzman [Vk of diversity when the population k is removed from the whole set of breeds (in this study we used DR distance). Then, the within-breed contribution (CW) is defined as:To evaluate the conservation priorities in a set of populations, taking into account contributions to within-population and between-population genetic diversity, Ollivier and Foulley  have proWeitzman  loss Vk H(S) is the average internal heterozygosity of the whole set S and H(S/k) the average internal heterozygosity of the set when k is removed. Finally, the aggregate diversity D of a population is defined as:where CP) could be computed as the product between the breed contribution (CB) and the probability of extinction (Pex) of the breed, assumed to be directly proportional to the inbreeding rate (ΔF). Following Simianer et al. [Pex can be approximated asThe cryopreservation potential .where ort term , we consGDT can be considered as the exact sum of the gene diversity within population GDWS and the gene diversity between populations GDBS considering the following equations:Caballero and Toro  have devn is the number of populations, fij is the average coancestry between populations I and j, and Dij is the Nei minimum distance between populations I and j. The contribution of a population to the diversity is evaluated by computing the loss or gain of diversity ΔGD when the population is removed.where ci) of each population, which can maximise the total diversity at the next generation, using the following equation:The authors have also proposed to evaluate the contributions (GDTN in equation (7), with the following restrictions: for each population i, ci ≥ 0 and Σi ci = 1.The contributions can be computed by maximising HTG4 and HMS1) to 15 (locus ASB2). Some rare alleles in the whole data set were found with a high frequency in the PRW population: for instance, with the HTG6 loci, the two most frequent alleles in the PRW population (70%) were seldom found in other breeds (less than 1%). Heterozygosities, mean number of alleles (MNA) and allelic richness (AR) are presented in Table MNA and AR were highly correlated, . He ranged from 0.43 in the FRI breed to 0.79 in the PFS breed, while Fis per breed ranged from -0.08 (TDN breed) to 0.11 (PRE breed).One hundred and nine alleles were found over all populations and all markers. The average number of alleles per locus was 9.8 ranging from seven (locus locus ASB. Some raHMS1). Using global tests, five populations  and two markers (HMS3 and HTG10) showed significant deficit in heterozygotes (P < 0.01). Other studies have shown similar results for these two markers [Some significant heterozygote deficits after corrections were found, for different loci and populations , showing similar results to previous figures  or 17 (0.81) than for K = 15 (0.83), the results are shown for this last value. Figure K, using runs having the highest pair-wise similarity coefficients.Breed assignment to clusters provides complementary information on genetic relationships between populations. As K = 2, there was a clear separation between draught and warm-blooded horses, with other populations showing intermediate results. When K reached 3, Nordic/primitive breeds, ponies, and some warm-blooded horses segregated more or less clearly from the two other clusters. As K increases to 4 and 5, the five clusters were constituted of Nordic/primitive breeds, draught horses, ponies, warm-blooded populations close to the AR breed and warm-blooded populations close to the PS breed. Some breeds were shared among the last three clusters, such as LAND between ponies and AR groups, and APPAL among the three clusters. When K reached 6, depending on the runs, FRI or PRW populations were alternately isolated, which led to a decrease of similarity across runs and explains the low similarity coefficient (0.562) in comparison with other K. When K = 7, these two populations were isolated. The different runs highlight some differences among sub-populations of AA and SF breeds, underlining a more important proportion of AR genes in AA6, AA5 and respectively SFA97 and SF98 groups. Some warm-blooded  and pony breeds (HAF) were classified with draught horses, while the CAM warm-blooded breed was clustered with ponies. As K reached 15, most breeds were shared among different clusters. The ARD, AUX and TDN breed constituted a single cluster while FJ/IS and LUS/PRE constituted two others. In a few cases, a single cluster was essentially associated to a single breed .For GDWS of 0.685, a gene diversity between populations GDBS of 0.073, and a total gene diversity GDT of 0.758. Table CW and ΔGDWS ranged from -0.48 to 0.50 and from -0.0055 to 0.0069 respectively. In both cases, the POIT breed showed a particularly low within-breed diversity. CW and ΔGDWS were negatively correlated . For between-breed diversity, CB and ΔGDBS ranged from 0.85 to 12.60 and from -0.0041 to 0.0024, respectively. Here, the POIT breed showed a particularly high contribution to the between-breed diversity. The correlation between CB and ΔGDBS was not significant. D and ΔGDT, accounting for total diversity, were negatively correlated . They ranged from -0.32 to 1.25 and from -0.0042 to 0.0039, respectively. In both cases, the ARD and PS breeds showed a particularly low and high diversity, respectively.In the set of the 19 French breeds, we found a gene diversity within population Considering contributions to the between-breed diversity and probabilities of extinction, the BOUL, LAND and POIT breeds showed the highest cryopreservation potentials .GDT are given in Table GDT of 0.79. Besides, to maximise the total gene diversity, seven of the 19 breeds should be maintained, namely the BOUL, COBND, LAND, PFS, POT, PS and SF breeds.Contributions of each population for an optimal  i.e. close to the value found by Luis et al. [et al. [He were slightly lower  for the eight breeds shared between their study and the present one. Differences on the within-breed diversity among studies using microsatellites can be explained, on the one hand, by the loci used and, on the other hand, by the populations analysed, incidentally belonging to similar breeds but having different recent histories. In the AR breed, we found a He value of (0.72) with a significant deficit of heterozygotes, which can be explained by the fact that this is an international breed in which mating between close relatives is common [et al. [et al. [et al. [He = 0.78), in comparison with the French PER population (He = 0.68). Because PER populations have been bred in America since the end of the 19th century, such results should be interpreted bearing in mind that the French PER population has probably suffered from recent bottlenecks due to several modifications of the selection aims.Differences between breeds explained 10% of the total genetic variation, which is quite similar to other analyses, where values ranged from 8% to 15% -4,9. Accs et al. . Plante s common . Plante  [et al.  have fouue of 0.7 with a sThe three approaches based on genetic relationships  gave similar results. The populations considered in the present study can be classified into four more or less differentiated clusters: warm-blooded, draught, Nordic and pony breeds. Similar patterns of clustering have been found in other studies ,3,9,10. K reached the number of breeds, indicate that the most relevant division is situated at a level superior to that of the breeds [Population differentiation tests and Bayesian approaches indicate clear differences between sub-populations of AA and SF. Such results may be largely explained by differences in the proportion of thoroughbred (PS) origins in the gene pool of these sub-populations. Within the AA breed, AA5 and AA6 populations appeared distinct from AA9 and AA10 populations and close to the PS breed. This was in agreement with the studbook rules: on the basis of pedigree data, AA5, AA6, AA9 and AA10 populations were indeed found to have respectively 94%, 89%, 44% and 59% of genes from PS origin . Within the SF breed, the SF8 (not differentiated from the PS breed) was distinct from SFA97 and SFB98 populations. This result was in agreement with previous results from pedigree data : the SF8e breeds . Such a In the present study, an almost comprehensive sampling of French breeds was achieved. The different approaches used gave an estimation of the contribution of each breed to the whole French horse stock. Petit  has propThe results given by the aggregate diversity and gene diversity approaches were slightly correlated. By definition, breeds with low contributions to aggregate and total diversities should have related breeds in the data set. Thus, ARD, TDN, and AUX breeds, which were genetically highly related, illustrate quite well such a hypothesis.D of BOUL, MER and POIT breeds were quite high, and taking into account population size, CP was the highest for BOUL, LAND and POIT breeds. Using the approach of Caballero and Toro [GDT decreased only when LAND and POT breeds were removed, and those two breeds plus the BOUL breed should have been kept to optimise GDT. The differences can be explained by the methods used in the two approaches, particularly considering the evaluation of the contributions to between-diversity. Using the approach of Caballero and Toro [GDBS, which will not be the case when using the Weitzman approach. However, one advantage of the approach of Caballero and Toro [GDT is the sum of GDWS and GDBS. In fact, our results outline that both approaches should be considered as complementary to identify which breeds have to be taken into account in a context of genetic resource management. Therefore, conservation priorities should concern particularly BOUL, LAND, MER, POIT and POT breeds.According to the approaches of Ollivier and Foulley  and Cabaand Toro , GDT decand Toro , some Weand Toro , were noand Toro  is the fAnother advantage of the method of Caballero and Toro  is the pFinally, several considerations have to be taken into account before taking final conservation decisions , such asBased on this study, horse breeds raised in France can be clustered into four groups. These groups were found to be meaningful according to the use of breeds, morphological characteristics and/or geographical origins. The combined use of different methods allowed us to identify breeds for which conservation efforts should be a priority, in order to preserve the maximum genetic variability. Since several horse studies have used similar panels of markers ,9,10, itThe authors declare that they have no competing interests.JCM carried out the genotyping. AR contributed to the description of the populations and carried out the sampling collection. LC performed the preliminary analysis. GL carried out the computational analysis and prepared the manuscript. XR participated in the computational analysis and preparation of the manuscript. CDB participated in the preparation and the revision of the manuscript. EV participated in the design of the study and the revision of the manuscript. All authors read and approved the final manuscript.Supplementary Figure 1. Neighbour-Net for the 34 horse breeds, based on Reynolds DR distanceClick here for fileSupplementary Figure 2. Correspondence analysis of allele frequencies for 33 of the populations studied (PRW is not included). The projection is shown on the first two axes.Click here for fileSupplementary Figure 3. Evolution of mean ln of likelihood according to K on twenty runs (standard deviation indicated)Click here for file


Late-life depression is an important public health problem because of its devastating consequences. The study aims to investigate the prevalence and associated factors of self-reported symptom-based depression in a national sample of older South Africans who participated in the Study of Global Ageing and Adult Health (SAGE wave 1) in 2008.We conducted a national population-based cross-sectional study with a probability sample of 3,840 individuals aged 50 years or above in South Africa in 2008. The questionnaire included socio-demographic characteristics, health variables, anthropometric and blood pressure measurements as well as questions on depression symptoms in the past 12 months. Multivariable regression analysis was performed to assess the association of socio-demographic factors, health variables, and depression.The overall prevalence of symptom-based depression in the past 12 months was 4.0%. In multivariable analysis, functional disability, lack of quality of life, and chronic conditions  were associated with self-reported depression symptoms in the past 12 months.Self-reported depression in older South Africans seems to be a public health problem calling for appropriate interventions to reduce occurrence. Factors identified to be associated with depression, including functional disability, lack of quality of life, and chronic conditions , can be used to guide interventions. The identified protective and risk factors can help in formulating public health care policies to improve quality of life among older adults. Diagnostic and Statistical Manual of Mental Disorders, third edition (DSM-III)  , has beeDSM-III) . The preDSM-III) , 5–8, anDSM-III) , 9–13. TDSM-III) .Socio-economic  , 15–17;Social  , 18, 19;Unhealthy behaviour [poor dietary habits ; lack ofDisease and chronic conditions, including stroke , 25, carCognitive impairment , 32, 33;Functional disability , 10, 12;Low quality of life , 11, 34.Various factors have been identified to be associated with depression in older adults, including:The study aims to investigate the prevalence and associated factors of self-reported symptom-based depression in a national sample of older South Africans who participated in the Study of Global Ageing and Adults Health (SAGE wave 1) in 2008.We conducted a national population-based cross-sectional study with a probability sample of 3,840 individuals aged 50 years or above in South Africa from January to September in 2008. The SAGE sample design entails a two-stage probability sample that yields national and sub-national estimates to an acceptable precision at provincial level, by locality type  and population group . The individual response rate among those aged 50 years or older was 77%. The Global Study on Ageing (SAGE wave 1) survey was carried out in South Africa in partnership among the World Health Organization (WHO), the National Department of Health, and the Human Sciences Research Council (HSRC). The study was approved by the HSRC Research Ethics Committee and the National Department of Health.http://www.who.int/healthinfo/systems/sage/en/.The questionnaire used for this study is the individual questionnaire from SAGE, found at the study website Symptom-based depression in the past 12 months was assessed based on the World Mental Health Survey version of the Composite International Diagnostic Interview . The diaNocturnal sleep problems were assessed with the question, ‘Overall in the last 30 days, how much of a problem did you have with sleeping, such as falling asleep, waking up frequently during the night, or waking up too early in the morning?’ Response options were none, mild, moderate, severe, and extreme. Responses were collapsed in two categories: none/mild/moderate and severe/extreme .Fruit and vegetable consumption was assessed with the questions ‘How many servings of fruit do you eat on a typical day?’ and ‘How many servings of vegetables do you eat on a typical day?’ Researchers were trained to show all respondents a nutrition risk factor card that indicates both in writing and in pictures general categories, amounts, and examples of fruits and vegetables in an attempt to standardise the serving size and number of servings reported . InsuffiBlood pressure was measured three times on the right arm/wrist of the seated respondent using an automated recording device .Out of three measurements, the average of the last two readings was used. In accordance with the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure, individuals with systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg and/or who reported the current use of antihypertensive medication were considered to be suffering from high blood pressure  any tobacco products such as cigarettes, cigars, pipes, chewing tobacco, or snuff?’ The response options were ‘Yes, daily’, ‘Yes, but not daily’, and ‘No, Not at all’. These questions are based on the WHO Guidelines for Controlling and Monitoring the Tobacco Epidemic .Lifetime alcohol use was assessed with the question ‘Have you ever consumed a drink that contains alcohol ?’ Response options were ‘Yes’ or ‘No, never’. Lifetime alcohol users were asked about current (past month) alcohol use, and current alcohol users were asked ‘During the past 7 days, how many drinks of any alcoholic beverage did you have each day?’ .2). BMI was calculated as weight in kilogram divided by height in metre squared. Obesity was defined as ≥30 BMI and underweight <18.5 BMI.Height and weight were measured, and body mass index (BMI) was used as an indicator of obesity  version 2. The instrument gathered information on physical activity in three domains , as well as time spent on sitting. The questionnaire also assessed vigorous and moderate activities performed at work and on recreational activities.Information on the number of days in a week spent on different activities and time spent in a typical day for each activity was also recorded . For phyHigh physical activity. A person reaching any of the following criteria is classified in this category: vigorous-intensity activity on at least 3 days achieving a minimum of at least 1,500 MET-minutes per week or 7 or more days of any combination of walking, moderate or vigorous intensity activity achieving a minimum of at least 3,000 MET-minutes per week.Moderate physical activity. A person not meeting the criteria for the ‘high’ category but meeting any of the following criteria is classified in this category: 3 or more days of vigorous-intensity activity of at least 20 min per day, 5 or more days of moderate-intensity activity, or walking of at least 30 min per day OR 5 or more days of any combination of walking and moderate or vigorous intensity activity achieving a minimum of at least 600 MET-minutes per week.Low physical activity. A person not meeting any of the aforementioned criteria falls in this category. Physical inactivity was defined as those who had low levels of physical activity; moderate and high levels of physical activity were collapsed in further analysis .The number of days and total physical activity MET minutes per week were used to classify respondents into three categories of low, moderate, and high level of physical activity.Functional ability was measured by the 12-item WHO Disability Assessment Schedule, version 2 (WHODAS-II) , designeSocial cohesion was measured with nine items, starting with the introduction ‘How often in the last 12 months have you…’ e.g. attended any group, club, society, union, or organisational meeting?’ Response options ranged from never = 1 to daily = 5. The scores assigned to each of the items – ‘never’ (1), ‘once or twice a year’ (2), ‘once or twice per month’ (3), ‘once or twice per week’ (4), and ‘daily’ (5) – were summed. These responses were used to create a score of overall social cohesion. Cronbach's alpha for this social cohesion index in this sample was 0.73.Quality of life was assessed with the World Health Organization Quality of Life (WHOQOL)-8 containing eight items that were empirically derived from the WHOQOL-Bref . The sumTo estimate economic or wealth status, a random-effects probit model was used to identify indicator-specific thresholds that represent the point on the wealth scale above which a household is more likely to own a particular asset than not. This enabled an estimation of an asset ladder. These estimates of thresholds, combined with actual assets observed to be owned for any given household, were used to produce an estimate of household-level wealth status. This was used to create wealth Quintiles .Other chronic conditions, such as stroke, angina, diabetes, arthritis, and asthma, were assessed by self-report.Cognitive impairment was defined as those who endorsed the question of having severe or extreme difficulty with concentrating or remembering things in the past 30 days. Cognitive tests included verbal learning  ; verbal http://www.statssa.gov.za/publications/P0302/P03022009.pdf). Weights were not normalised. Chi-square tests of significance were used to compare distribution of socio-demographic and health variables by depression prevalence. Associations between the key outcome of depression and socio-demographic, social, and health variables were evaluated calculating odds ratios (OR). Unconditional multivariable logistic regression was used for the evaluation of the impact of explanatory variables for the outcome of depression (binary dependent variable). All variables statistically significant at the p<0.05 levels in bivariate analyses were included in the multivariable models. In the analysis, weighted percentages are reported. Both the reported 95% confidence intervals and the p-value are adjusted for the multi-stage stratified cluster sample design of the study.The data were entered using CSPro and analysed using STATA Version 10. The data were weighted using post-stratified individual probability weights based on the selection probability at each stage of selection. Individual weights were post-stratified by province, sex, and age groups according to the 2009 Medium Mid Year population estimates from Statistics South Africa , almost half (49.9%) were between 50 and 59 years old. The educational level of most participants (71.6%) was lower than secondary school education and almost two-thirds (64.9%) lived in an urban area. Almost half (46.7%) of older adults were obese and 77.3% had hypertension, 20.4% were daily tobacco users, and 9.2% had diabetes. In addition, 4.0% had had a stroke, 5.2% angina, 4.9% asthma, 24.7% arthritis, and 8.9% a nocturnal sleep problem. More than half (60.5%) engaged in low physical activity, 20.4% were daily tobacco users, a small proportion (3.7%) were hazardous or harmful alcohol users, and 67.7% had insufficient fruit and vegetable intake. The mean functional disability was 20.8  and the mean of quality of life was 47.1 . The overall prevalence of past 12 months depression was 4.0% see . The meaIn univariate analysis functional disability, low quality of life and chronic conditions  were associated with past 12 months depression. None of the identified socio-demographic variables, lifestyle factors, cognitive variables, social cohesion, and chronic conditions  were found to be associated with depression. In multivariable analysis, functional disability, lack of quality of life, and chronic conditions  remained associated with the 12 month depression. Older adults with depression were 7.6 times (95% CI = 3.57–16.35) more likely to have nocturnal sleep problems compared to those without depression, and those with a history of angina were 5.4 times (95% CI = 1.93–10.66) more likely and those with medium functional disability were three times (96% CI = 1.58–5.66) more likely to have depression than those without see .The study reports for the first time the prevalence of 4.0% of past 12 months depression in a national sample of older adults in South Africa. This prevalence seems to be lower or similar to previous surveys –8 and clThe study found in agreement with previous studies that functional disability , 12, 15,Unlike in other studies , 20, 21,In many primary care settings, patients presenting with multiple disorders that include depression often do not get diagnosed, and if they do, treatment is often focused towards other chronic diseases , 52. DepThis study had several limitations. First, the self-report of health variables such as depression symptoms, tobacco or alcohol use should be interpreted with caution; it is possible that measurement errors occurred. In addition, recall bias could have occurred since asking about depressive symptoms may carry social stigma , and surSelf-reported depression in older South Africans seems a public health problem calling for appropriate interventions to reduce the occurrence. Factors identified to be associated with depression, including functional disability, lack of quality of life, and chronic conditions , can be used to guide interventions. The identified protective and risk factors can help in formulating public health care policies to improve quality of life among older adults.


The regulation of cell growth and division occurs in an accurate sequential manner. It is dictated by the accumulation of cyclins (CCNs) and cyclin-dependent kinases (CDKs) complexes and degradation of CCNs. In human tumors, instead, the cell cycle is deregulated, causing absence of differentiation and aberrant cell growth. Oncogenic alterations of CCNs, CDKs, and CDKIs have been reported in more than 90% of human cancers, and the most frequent are those related to the G1 phase. Several molecular mechanisms, including gene overexpression, chromosomal translocations, point mutations, insertions and deletions, missense and frame shift mutation, splicing, or methylation, may be responsible for these alterations. The cell cycle regulators are involved in tumor progression given their association with cancers characterized by higher incidence of relapses and chemotherapy resistance. In the last decade anticancer drug researches focused on new compounds, able to target molecules related to changes in genes associated with tumor status. Recently, the studies have focused on the restoration of cell cycle control modulating molecular targets involved in cancer-cell alterations. This paper aims to correlate alterations of cell cycle regulators with human cancers and therapeutic responsivity. Rb), the tumor suppressor gene TP53, and transcription factors of the E2F family  pyrimidine compound, devoid of ABC transporter interaction, has been identified as a highly suitable drug for further preclinical and clinical evaluation in cancer treatment . Tanshinnd CCNB1 . Treatmend CCNB1 . On the nd CCNB1 . A novelnd CCNB1 . Decorinor cells . CDK inhor cells . A specior cells . dFMGEN,or cells . Studiesor cells . Recentlnd CDKIs . Human tnd CDKIs , 116.All such data clearly show the relevance of such an approach for cancer treatment and the need to identify the appropriate combination with conventional anticancer therapy.Nonsteroidal anti-inflammatory drugs (NSAIDs) are primarily used as analgesics for the relief of pain and to control inflammation . NSAIDs μM, and the cell proliferation rate was significantly reduced in a time- and concentration-dependent manner. Previous studies showed that concentrations of ibuprofen comparable to those used in our studies targeted a wide variety of cellular processes [CDC25, CDKN1A, and TP53. CDC25, one of the key regulators of cell cycle transition [μM dose. CDKN1A gene, instead, is upregulated by more than 2-fold at 24 h, followed by downregulation at 72 h. Its upregulation, tightly controlled by TP53, is necessary to block cells in the G1 phase and allow the repair of damaged DNA before their entry into the subsequent S phase. For such role CDKN1A represents the key effector of p53-dependent G0/G1 phase arrest in response to different stress stimuli [TP53 levels show a similar significant upregulation for the whole ibuprofen treatment period and to all tested doses, with a late upregulation likely associated with activation of apoptotic-related genes. The upregulation of TP53 signaling pathways in the first 24 h following ibuprofen treatment has been also shown by a previous study [TP53 signaling was activated early (24 h) as a result of intracellular modifications, likely associated with oxidative stress, which would justify the ROS increase, we observed. In parallel to TP53 upregulation and cell cycle G0/G1 arrest, several S-related genes are downregulated, particularly those expressing proteins and enzymes involved in DNA replication. Regulation of S-phase-critical genes is, therefore, an important component of G1 progression to S. This coordinated regulation of S-phase genes is principally controlled by the E2F family of transcription factors [GADD45 gene was induced by activation of the TP53-dependent pathway and likely contributed to elicit growth arrest for an effective DNA repair, or alternatively to induce apoptosis. The ineffective repair of cell damage, in fact, prevalently triggered, in our model, an apoptotic program, characterized by upregulation of caspase transcripts in the latter stages of the ibuprofen treatment.The primary purpose of the studies performed in our laboratory was to verify the antitumor effects of ibuprofen, a commonly used NSAID, on the MKN-45 human gastric cancer-cell line , where arocesses , 133–135rocesses –138. Theansition , with pransition , 141, is stimuli . TP53 le factors . TreatmeIn conclusion, this study showed that the early alterations of cell cycle regulatory genes and the later induction of apoptosis are the major mechanisms that account for the antiproliferative effects of ibuprofen on the MKN-45 human gastric cancer-cell line. Treatment with ibuprofen altered the cell cycle phase distribution by inhibiting the G1/S transition. The G0/G1 arrest was associated with a decrease in the expression of cyclins and cyclin-dependent kinases and an increase of TP53 and CDKN1A, along with downregulation of transcripts encoding enzymes involved in DNA precursor synthesis and the DNA replication system. The prolonged ibuprofen treatment and the ineffective repair of damaged cells resulted in the late upregulation of caspase transcripts with the consequent activation of an apoptotic program.Despite the high doses of ibuprofen required to elicit the apoptotic effects reported in our studies, the implicated molecular mechanisms suggest that NSAIDs, such as ibuprofen, may be of benefit in the treatment of cancers, particularly as local treatment.Several molecules and their pathways involved in the regulation of the cell cycle are currently considered as promising prognostic biomarkers and target for cancer treatment. This has been made possible by recent advances in molecular genomic and proteomic technologies. Characterization of the cell cycle pathways present in specific cancers will contribute to improving diagnosis and cancer staging, prognostic evaluation of cancer patients, and optimal combinational approaches for innovative, personalized treatment strategies.


Altered thyroid status exerts a major effect on the heart. Individuals with hypo- or hyperthyroidism showed various changes in electrocardiograms. However, little is known about how variations in thyroid hormone levels within the normal range affect electrical activities of the heart in the general population.th, 20th, 40th, 60th, 80th, and 95th percentiles of the weighted population distribution. Electrocardiographic parameters were measured from the standard 12-lead electrocardiogram. We found a positive linear association between serum total T4 level and heart rate in men, and a U-shape association between T4 and PR interval in men and women. TSH level was positively associated with QRS interval in men, while a U-shape association between TSH and QRS was observed in women. No clear graded association between thyroid hormones and corrected QT or JT was found, except that men in the highest category of T4 levels appeared to have longer corrected QT and JT, and men in the lowest category of T4 appeared to have shorter corrected QT and JT.We conducted a cross-sectional analysis of 5,990 men and women from the Third National Health and Nutrition Examination Survey. Serum total T4 was measured by immunoassay and TSH was measured by chemiluminescent assay. We categorized T4 and TSH into 7 groups with cut-offs at the 5Variation in thyroid hormone levels in the general population, even within the normal range, was associated with various ECG changes. Thyroid hormones can exert electrophysiologic and inotropic effects on the heart and may modulate the risk of developing atherosclerosis While ECG abnormalities in patients with hypo- and hyperthyroidism are well established, relatively little is known about how variations in thyroid hormone levels within the normal range affect the electrical activity of the heart in the general population We used data from the Third National Health and Nutrition Examination Survey (NHANES III), a cross-sectional study conducted between 1988 and 1994 that used a multistage stratified clustered probability design to select a representative sample of the civilian non-institutionalized US population. Thyroid hormones were measured in participants 12 years of age and older while 12-lead electrocardiograms (ECGs) were performed on participants 40 years of age and older. Therefore, the present study was restricted to participants 40 years of age and older  who were eligible for both thyroid function tests and ECG measurements. We excluded 272 participants with missing ECG parameters, 728 participants with missing T4 or TSH, 525 participants with QRS ≥120 ms, and 603 participants with self-report goiter or thyroid disease, or on thyroid medications. In addition, 443 participants with overt or subclinical thyroid disease based on TSH levels were also excluded  NHANES III included a standardized questionnaire administered in the home by a trained interviewer and a detailed physical examination at a mobile examination center. Demographic characteristics, smoking status, alcohol consumption, medical history, and medication use were assessed during the interview. QT-prolonging medications were defined according to the Arizona Center for Education and Research on Therapeutics database Serum was frozen (−20°C) and shipped on dry ice to the Endocrine Services Laboratory, University of Southern California,  for analysis of T4 and TSH. T4 was measured using an immunoassay  with a reference  range of 57.9 to 169.9 nmol/L. TSH was measured using a chemiluminescence immunometric assay  with a reference  range of 0.39 to 4.6 mU/L.Standard 12-lead resting ECG recordings were performed using a Marquette MAC 12 electrocardiograph  with signals sampled at 250 Hz per channel. A representative P-QRS-T cycle was then derived by selective averaging using the Dalhousie ECG Analysis Program The PR, QT, and JT intervals were adjusted for heart rate, race, and age using the residual method. These adjusted metrics  were used for subsequent analyses. More specifically, we first regressed the PR, QT, or JT interval, respectively, as dependent variables, on RR interval, race, and age. Adjustment for RR-interval duration was performed using restricted quadratic splines with knots at the 5th, 50th, and 95th percentiles of the overall study population to allow a non-linear relationship of the RR with the PR, QT, or JT intervals. We then obtained model residuals for each participant and centered the residuals at the mean value of the PR, QT, or JT intervals of the study population to facilitate the interpretation of the findings. Residuals are uncorrelated with the regressors  and thus adjusted for them. The residuals represent the component of the PR, QT and JT intervals that are not explained by the regressors.th, 20th, 40th, 60th, 80th, and 95th percentiles of the weighted population distribution.The sampling weights for the ECG component of the survey were used to account for the complex design of NHANES III We used multivariable linear regression models to estimate adjusted differences and 95% confidence intervals (CIs) for ECG parameters associated with each category of T4 and TSH compared with the middle category. The basic models were adjusted for age, race/ethnicity , and RR-interval splines (except for the models for heart rate). The full models were further adjusted for BMI, smoking , alcohol consumption , systolic blood pressure, blood pressure lowing medication, total and HDL cholesterol, diabetes, history of myocardial infarction, history of congestive heart failure, use of QT-prolonging medications, and creatinine-based eGFR. Finally, we further included T4 and TSH simultaneously in the fully adjusted models. Tests for linear trend were computed by including a variable with the median values of T4 or TSH in each category in the linear regression models. Tests for quadratic trend were computed by using quadratic contrast coefficients.For sensitivity analyses, we repeated all analyses after excluding participants with conditions that may affect ECG parameters  or diabetes . All analyses were conducted using SUDAAN .The average age of study participants was 54.8 years in men, and 55.8 years in women . AverageAfter adjusting for age, race/ethnicity and RR-interval, T4 levels were positively associated with heart rate in men and women  . In the We found a U-shaped association between T4 levels and PRrra interval in women . In the fully adjusted model, the average difference in PRrra interval comparing the lowest vs. the middle category of T4 was 5.0 ms (−2.5 to 12.6), and the average difference comparing the highest vs. the middle category was 9.6 ms (2.9 to 16.2). There was a similar U-shaped pattern between T4 levels and PRrra in men, but the p-values for quadratic trend did not reach statistical significance .There was a marginally positive linear association between TSH levels and QRS interval in men , but a U-shaped association in women . In the fully adjusted model, the average difference in QRS duration comparing the highest vs. the lowest category of TSH in men was 2.1 ms (−2.1 to 6.2). In women, the average difference in QRS duration comparing the lowest vs. the middle category of TSH was −4.0 ms (−7.2 to −0.9) and the average difference comparing the highest vs. the middle category was −0.9 ms (−3.4 to 1.6).In models adjusted for age, race/ethnicity and RR-interval, T4 levels were positively associated with QTrra and JTrra intervals in women , but the associations became non-significant in the fully adjusted models . In men, the association between T4 levels with QTrra or JTrra intervals was not significant, although men in the highest category of T4 appeared to have longer QTrra and JTrra intervals and men in the lowest category of T4 appeared to have shorter QTrra and JTrra intervals compard to the rest of the groups. In men, the average difference in QTrra and JTrra intervals comparing the lowest vs. the middle category of T4 in the fully adjusted models were −4.3 ms (−9.8 to 1.3) and −6.0 ms (−11.4 to −0.6), respectively, while the average difference in QTrra and JTrra intervals comparing the highest vs. the middle category of T4 were 5.4 ms (0.8 to 10.0) and 4.4 ms (0.2 to 8.6).Finally, including T4 and TSH in the same model  and exclThis analysis of NHANES III men and women with thyroid hormone levels within the normal range showed a positive linear association between serum T4 levels and heart rate in men and a U-shaped association between T4 levels and PR interval in men and women. In addition, TSH levels were marginally positively associated with QRS interval in men, but showed a U-shaped association with QRS interval in women. There was no consistent association between thyroid hormone levels and QT or JT intervals, except that men in the highest category of T4 levels appeared to have longer QT and JT intervals and men in the lowest category of T4 levels appeared to have shorter QT and JT intervals compared to the middle group.Tachycardia and bradycardia are well known signs of hyper- and hypothyroidism, respectively Less is known, however, about how thyroid hormone levels within the normal range affect heart rate in the general population. The Study of Health in Pomerania  seems to be the only previous population-based study that has investigated this question. In this study, there was no significant association between TSH levels and heart rate KsI, or an increased sympathetic influence on the autonomic cardiovascular system  as shown in some studies The effect of thyroid hormones on the QT interval duration is controversial. A majority of animal and clinical studies have reported a prolonged heart rate-corrected QT interval (QTc) in hypothyroidism Limited data from population-based studies were also inconsistent. The Study of Health in Pomerania showed a positive association between TSH levels and QTc intervals Finally, very few studies have evaluated the association of thyroid hormone levels with other electrocardiographic parameters such as PR interval or QRS duration. Experimental studies in hypothyroid dogs have reported both no changes in PR or QRS duration Several limitations of this study need to be considered. Firstly, the cross-sectional design limited our ability to make statements regarding the causality of the relation between thyroid hormone levels and ECG parameters. Secondly, we only had available measurements of total T4, but not of free T4 or T3 measurements in NHANES III. T3 is much more potent than T4, and we might expect stronger associations if we would have used T3 in the analysis. Furthermore, most of the circulating thyroid hormone in the blood is bound to transport proteins and only a very small fraction is free (unbound) and biologically active. Therefore, the measurement of total T4 levels may not represent the actual level of free (active) T4. Finally, thyroid hormones and ECG measurements were performed only at a single time at baseline, which may result in non-differential measurement error as there might be substantial within person variability. Repeated measurements may be needed to reduce measurement error and better characterize the association between thyroid status and ECG changes in future studies.In conclusion, data from NHANES III suggested that variation in thyroid hormone levels within the normality range in the general population was associated with various ECG changes, including a linear association between serum total T4 level and heart rate in men, a U-shaped association between T4 and PR interval in men and women, a linear association between TSH and QRS duration in men, and a U-shaped association between TSH and QRS in women. In addition, men in the highest category of T4 had longer QT and JT intervals, although there was no clear graded association between thyroid hormone levels and QT or JT intervals. Further research is needed to confirm these findings and to elucidate the underlying mechanisms, especially those related to gender differences in the effect of thyroid hormones on ECG parameters.Appendix S1Multivariate adjusted means (95% CI) of heart rate, PRrra interval, QRS duration, QTrra and JTrra interval by categories of T4 and TSH . Models were adjusted for age, race/ethnicity , RR-interval splines (except for the models of heart rate), BMI, smoking , alcohol consumption , systolic blood pressure, blood pressure lowing medication, total and HDL cholesterol, diabetes, history of myocardial infarction, history of congestive heart failure, use of QT-prolonging medications, creatinine-based eGFR, and T4 (in the models for TSH), and TSH (in the models for T4).(TIF)Click here for additional data file.Appendix S2Multivariate adjusted means (95% CI) of heart rate, PRrra interval, QRS duration, QTrra and JTrra interval by categories of T4 and TSH, excluding participant with diabetes, myocardial infarction, heart failure, or taking QT-prolonging medications . Models were adjusted for for age, race/ethnicity , RR-interval splines (except for the models of heart rate), BMI, smoking , alcohol consumption , systolic blood pressure, blood pressure lowing medication, total and HDL cholesterol, diabetes, history of myocardial infarction, history of congestive heart failure, use of QT-prolonging medications, and creatinine-based eGFR.(TIF)Click here for additional data file.


To determine the prevalence and risk factors associated with idiopathic epiretinal membranes (iERM) in a Chinese population aged 60 years or older in Beixinjing Blocks, Shanghai.This population-based study consisted of 3727 participants (89.7% of the eligible). It was performed to describe the prevalence of iERM and possible demographic, systemic, and ocular factors associated with iERM. Each participant underwent a standardized interview and comprehensive ophthalmic examination. iERM was identified and graded from retinal photographs. Then, a case-control study comparing the participants with vs. without iERM was performed to further study the associations between iERM and blood biochemical test results , ocular biological parameters , and the data of optical coherence tomography.The prevalence of iERM was 1.02%. iERM was significantly associated with diabetes  and a higher level of education . Blood biochemical test results and ocular biological parameters showed no significant differences between the iERM and control groups, whereas the incidence of posterior vitreous detachment in the iERM group was much higher than in the control group (26.5% vs. 8.8%), but this difference was not statistically significant. Moreover, the eyes with iERM had poorer visual acuity than the eyes without iERM (P<0.05).In Beixinjing Blocks, Shanghai, iERM was relatively rare, was associated with diabetes and a higher level of education, and caused a substantial decrease in visual acuity. Epiretinal membrane (ERM) is a retinal disease resulting in a disturbance of macular vision and predisposing to rhegmatogenous retinal detachment Epidemiological studies contribute to clarify the pathogenesis and risk factors of iERM. Population-based studies Previous clinical studies Beixinjing Blocks, located in the northwest of Shanghai, was selected for the study because of its relatively stable population  and representative demographic and socioeconomic characteristics. Beixinjing Blocks has mostly urban residents whose economic conditions could be classified as middle class in China: average per capita annual incomes among urban households are 36,230 yuan ($5728 US) This study protocol was approved by the Human Research and Ethics Committee of the Shanghai First People’s Hospital, affiliated Shanghai Jiaotong University, and adhered to the tenets of the Declaration of Helsinki.Between November 2010 and April 2011, a population-based study of the prevalence of iERM was designed and performed in Beixinjing Blocks. Random cluster sampling was used to select the study sample. Clusters were defined geographically using Residence Administrative Committees (RACs) of approximately 1,000 persons . By using 2000 census information, the residents of RAC that had total populations larger than 1,500 were subdivided, and the residents of RAC smaller than 500 were grouped in defining clusters for sampling. Forty-two clusters were defined for random sampling. With an estimated 18.9% of the Beixinjing Blocks population aged 60 years or older, the typical cluster was estimated to contain approximately 190 study participants.2(p)(1-p)/B2 where p = 0.034; B = 0.034(0.25) = 0.0085 with a 25% error bound; and Z = 1.96 for a 95% confidence interval (CI). It was assumed that a cluster design effect as high as 1.5 might be present, which would increase the calculated sample size from 1,746 to 2,619. With an estimated response rate of 90%, the required sample size became 2,910. Accordingly, 16 clusters  were chosen by simple random sampling from the list of 42 clusters.The required sample size was calculated based on estimating with 95% confidence the prevalence of ERM in the Handan Eye Study (3.4%) Two days before the examinations, RACs telephoned the eligible residents, informing them of the examination date and the purpose of the study. The field examinations were mainly carried out in the nearest community meeting room, while home inspections were used to collect data of physically disabled residents. Near the end of this study, the eligible residents who had not participated in the field examination on the previously scheduled day were notified by phone about another examination date.The research group included one lead ophthalmic doctor who had prior experience organizing large-scale epidemiologic studies, four trained ophthalmologists from Shanghai First People's Hospital, affiliated Shanghai Jiaotong University, and two trained physicians from Bingxinjing Community Hospital. Before formal investigation, the members of the research group had trained for two weeks to understand the purpose of the study, methods, and detailed steps for each variable . Written informed consent was first obtained from all study participants. A detailed interview was conducted to collect information regarding demographics , histories of diagnosis and treatment relating to systemic comorbidities  and ocular diseases . After that, all eligible participants underwent a comprehensive ophthalmic examination. Visual acuity of each eye was measured using the log of the minimum angle of resolution (LogMAR) Early Treatment Diabetic Retinopathy Study (ETDRS) chart at a distance of 4 m, with illumination ≥300 lux. In participants who were wearing glasses in their daily lives, visual acuity was measured with their spectacles. Both types of visual acuity mentioned above are known as the presenting visual acuity The retinal photographs were assessed respectively by two ophthalmologists with retinal subspecialty training for the presence of ERM and its grading. The prevalence-adjusted bias-adjusted kappa statistic was 0.82 for the presence of ERM, and the kappa statistic was 0.70 for its grading. In case of doubt, the lead ophthalmic doctor reassessed the retinal photographs to make the final diagnosis and grading. In our study, ERM was subdivided as idiopathic or secondary. iERM was defined as ERM occurring in eyes without a secondary cause, such as DR , retinal vascular disease, retinal detachment, or history of cataract surgery. Moreover, iERM was graded using the method described by Klein et al. In November 2011, the 34 participants with iERM as the case group and 34 healthy participants randomly selected (using a computer-generated random number table) as the control group from the participants without ERM were further examined in the Beixinjing community health service center. Cases and controls were well matched in age, sex, body mass index (BMI), and the iERM-associated risk factors (diabetes and higher level of education) obtained from Part I. After explaining the purpose of this study, we obtained the written informed consent from all participants.Two ophthalmologists, two optometrists and one retinal specialist performed the following examinations. Blood samples were collected for testing plasma glucose, serum total cholesterol, creatinine, and triglyceride after an overnight fast. The uncorrected distance visual acuity (UCDVA) was measured using the ETDRS chart as described in Part I, and near visual acuity was measured using the LogMAR word reading cards at the participant’s preferred reading distance. The best-corrected distance visual acuity (BCDVA) was measured after objective refraction by an autorefractor . For each eye, IOP by a non-contact tonometry, axial length, K1 (keratometry for flat meridian), K2 (keratometry for steep meridian), and ACD by an IOL-master  were examined at least three times, then the average readings were recorded.The B-mode ultrasound  and OCT  examinations  were conducted on all 68 participants by two ophthalmologists, respectively, who were trained and certified by retinal specialists. Posterior staphyloma and the kinetic movements of both the posterior vitreous and the vitreoretinal traction were observed under biomicroscopy, ophthalmoscopy and B-mode ultrasonography. The PVD before the macular region was confirmed when a complete separation of the posterior hyaloid membrane (a floating continuous thin membrane-like echo in the vitreous cavity under ultrasonography) and an optically or acoustically empty subhyaloid space were both present under ultrasonography, and no vitreoretinal adhesion at the macular region was present under OCT examination.OCT examinations, including detected iERM in OCT images, were performed in both groups Statistical analyses were performed with SPSS statistical software version 13.0 . An alpha level of P<0.05 was chosen as the criterion for significance.Descriptive statistical analyses were performed to characterize demographic data, visual acuity, and clinical characteristics. Age-standardized prevalence was calculated by direct methods using 2000 Chinese national census population. Logistic regression was employed to determine the independence of potential risk factors for iERM, including continuous (age and BMI) and dichotomous variables . Odds ratios (ORs) and 95% CIs were reported. Moreover, the independent-samples t-test and Mantel-Haenszel chi-square test were used to determine the significant differences between the case and control groups.A total of 4,153 residents were determined as eligible, and 3727 residents underwent interviews and clinic examinations, corresponding to a response rate of 89.7%. Of these, gradable retinal photographs for epiretinal membranes were available for 3571 participants . The mean age was 71.08±7.36 years . The other 156 participants (4.2%) had un-gradable retinal photographs due to refractive media opacity, such as cataract and vitreous opacity. 245 of 3571 participants (6.9%) were excluded because of a known secondary cause of the development of ERM. DR was present in 32 participants, retinal vascular disease in 50, a history or signs of retinal detachment in 19, and a history of cataract surgery in 162.Of the resulting 3,326 participants, iERM was detected in 39 eyes of 34 participants according to retinal photographs, for a prevalence of 1.02%. CMR was present in 0.63% (21 eyes of 21 participants) and PMF in 0.39% (14 eyes of 13 participants). The age-specific, gender-specific, and age-standardized  prevalence of CMR, PMF and any iERM are listed in In the case-control study, the demographic characteristics of the 34 participants with iERM and the 34 healthy participants were compared in In this population-based study, we observed an overall iERM prevalence of 1.02% in Beixinjing Blocks, Shanghai, China, which included 0.63% for CMR and 0.39% for PMF. Our study suggests that iERM is less frequent in urban Chinese than reported in samples of Asians from the Handan Eye Study (3.0%) Consistent with previous studies In addition to diabetes, we found that a higher level of education was associated with iERM, which was consistent with the Beijing Eye Study In the subsequent case-control study, we unexpectedly found that serum total cholesterol was negatively associated with iERM. However, hypercholesterolemia has been reported as a possible risk factor for iERM in the Hisayama Study There is controversy The limitations of our study should be stated. First, blood biochemical parameters, such as serum total cholesterol In conclusion, iERM occurs at a relatively low frequency in a population-based sample of Beixinjing Blocks aged 60 years or older. Its prevalence was lower than in Western countries and in Chinese subjects in Handan, and it was associated with diabetes and higher level of education. Furthermore, iERM causes a substantial decrease in visual acuity.


Cerebral palsy is currently one of the major diseases that cause severe paralysis of the nervous system in children; approximately 9–30% of cerebral palsy patients are also visually impaired, for which no effective treatment is available. Bone marrow mesenchymal stem cells (BMSCs) have very strong self-renewal, proliferation, and pluripotent differentiation potentials. Therefore, autologous BMSC transplantation has become a novel method for treating cerebral palsy.An 11-year-old boy had a clear history of dystocia and asphyxia after birth; at the age of 6 months, the family members observed that his gaze roamed and noted that he displayed a lack of attention. A brain MRI examination at the age of 7 years showed that the child had cerebral palsy with visual impairment . The patient was hospitalized for 20 days and was given four infusions of intravenous autologous BMSCs. Before transplantation and 1, 6, and 12 months after transplantation, a visual evoked potential test, an electrocardiogram, routine blood tests, and liver and kidney function tests were performed.The patient did not have any adverse reactions during hospitalization or postoperative follow-up. After discharge, the patient could walk more smoothly than he could before transplantation; furthermore, his vision significantly improved 6 months after transplantation, which was also supported by the electrophysiological examinations.The clinical application of BMSCs is effective for improving vision in a patient with cerebral palsy combined with visual impairment. This case study represents a typical case of cerebral palsy combined with visual impairment. With medical advances, the active treatment of preterm infants, children with dystocia, and children with encephalitis or hypoxic-ischemic encephalopathy results in significant increases in the survival rates of these patients; however, compared to normal infants, these patients have significantly increased incidences of periventricular white matter demyelination. Many regions of the brain play important roles in the generation of vision, such as the cerebral cortex, the white matter, and the basal ganglia -3. DurinPrior to stem cell therapy, a consent form was signed by the guardians of the patient. This study was approved by the ethics committee of our hospital.The patient, an 11-year-old boy, was admitted to our hospital mainly due to “binocular vision disorders that had lasted more than 11 years”. The patient was born full-term with dystocia and had a history of significant asphyxia, which improved after symptomatic treatment. At the age of 6 months, his family members found that he had trouble concentrating and following objects as well as easy irritability and hyperactivity. A brain MRI performed at the age of 7 showed cerebral hypoplasia and optic nerve atrophy. The diagnosis was cerebral palsy with visual impairment. He could not see and had no light perception before he was 8 years old. At the age of 8, the patient started to have light perception, and he could only see objects 20 cm in front of him at the age of 9; after that, his vision did not improve. When admitted to our hospital, the physical examination revealed that the patient presented with stuttering, a right-eye visual acuity of 30 cm, and a left-eye visual acuity of 20 cm. The pupils were equal and round with diameters of 3 mm; the eyes were sensitive to light reflection, the movements of both eyes were normal, strabismus was absent, horizontal nystagmus was present, and eye closure was effective. The bilateral finger muscle strength was grade 4, the right lower limb muscle tension was grade 1, the tendon reflexes were active, and the bilateral Chaddock’s sign was (+).A brain MRI examination revealed bilateral parietal-occipital lobe and corpus callosum dysplasia Figure .Ocular fundus examination: A leopard-spot-like fundus was observed, the boundary of the optic disc was clear, the color of the temporal disc border was light, the C/D was 0.5–0.6 , and the fovea reflex was (−).Flash stimulation tests were performed on both eyes. The results revealed that the waveforms of both eyes were poorly differentiated, the fundus was slightly wider than normal, and the P100 latency was basically normal Figure .Bone marrow was collected from the patient. All tests were normal, including blood leukocytes; lymphocytes; liver function ; renal function ; blood sugar; and blood lipids. The patient did not have syphilis, HIV, hepatitis B, or hepatitis C infection, and there was no history of familial or hereditary diseases.5 cells/ml in α-MEM containing 10% FBS. Cells were passaged when the cell density was above 90%. Cells were collected at the 5th generation and were used for transplantation by intravenous infusion.Bone marrow was collected from the posterior superior iliac spine. Sixty microliters of bone marrow was extracted, and mononuclear cells in the bone marrow were separated using density gradient centrifugation. Cells were then inoculated into 100 ml flasks at a concentration of 3 × 10Prior to clinical application, multiple tests were performed on the stem cells, including (1) cell morphology, (2) cell surface marker detection, (3) bacterial detection, (4) mycoplasma detection, and (5) endotoxin detection.7 cells were intravenously infused. Four infusions were performed in each session, and the interval between two infusions was 7 days. After each time infusion, the patient was monitored for vital signs including blood pressure, heart rate, blood oxygen, and respiration over 24 hours. Immunosuppressants were not administered throughout the treatment period.At each treatment session, the stem cells were fully dispersed and mixed using 100 ml normal saline, and then 4 × 10Blood leukocytes, lymphocytes, liver function, and kidney function were monitored before transplantation and 1, 3, 6, and 12 months following.(1) Cell morphology—The cells showed typical BMSC morphology and strong growth under a phase-contrast microscope. (2) Cell surface marker detection: CD44 and CD29 positive rates were greater than 95%; the CD106 positive rate was greater than 80%; and the CD34, CD45, CD14, and HLA-DR expression rates were less than 5%  revealed that the patient had a right-eye visual acuity of 1 m and a left-eye visual acuity of 80 cm. The right lower limb muscle tension was significantly decreased compared to that before transplantation, and the patient had heel contact with the ground and walked more smoothly than before transplantation. The auxiliary examinations were performed 1 year after transplantation (2011-10-12); the VEP results showed that the waveforms of both eyes were well differentiated, the fundus was slightly wider, and the P100 latency was essentially normal when flash stimulation tests were performed on both eyes Figure .The classic visual pathway contains two parts. The first part is the anterior visual pathway, which refers to the visual pathways below the lateral geniculate nucleus, including receptors, bipolar cells, ganglion cells, the optic nerve, the optic chiasm, the optic tract, and the lateral geniculate body. The second part is the posterior visual pathway, which refers to the visual pathway posterior to the lateral geniculate body, including the optic radiation, the subcortical area, and the visual cortex. The patient in this study had a posterior visual pathway injury, which was based on (1) normal pupillary light reflex; (2) fundus examination, which showed no obvious optic atrophy; (3) eye examination, which showed no positive signs; (4) VEP after flash stimulation of both eyes, which showed poor bilateral waveform differentiation and a wider fundus; and (5) a brain MRI, which showed hypoplasia of the bilateral parietal-occipital lobe and the corpus callosum. Posterior visual pathway injuries are commonly observed in cerebral contusions of the occipital lobe and in diffused brain injuries, such as brain edemas and hernias; these injuries are usually accompanied by signs of nervous system damage. In addition to visual injuries, this patient had limb movement disorders.in vitro, such as bone cells, cartilage cells, fat cells, tendons, and muscle cells. BMSCs have many advantages; they are easy to obtain and amplify in vitro, are unlikely to cause immune rejection after transplantation in vivo, and present no ethical controversy. Recent studies have also indicated that BMSCs can be induced into neuron-like cells and glial cells both in vivo and in vitro[BMSCs have very strong self-renewal, proliferation, and pluripotent differentiation capabilities. Under different induction conditions, BMSCs can differentiate into many types of cells  in vitro-6 and ca in vitro,8. Many  in vitro and trau in vitro. Second, in vitro. Third,  in vitro. Finally in vitro.In this study, the characteristics of surface antigens on cultured stem cells were detected using flow cytometry. The results showed that the expression levels of CD44, CD29, CD54, and CD106 were high in nuclear cells . In contrast, the cells were negative for CD45, CD34, HLA-DR, and CD14 expression (<5% expression). In BMSC studies, CD9, CD29, CD44, CD71, CD90, and CD106 are recognized as important surface markers of BMSCs, while the hematopoietic stem cell marker antigen CD3, the leukocyte marker antigen CD45, and the monocyte/macrophage surface antigen CD14 are not expressed . CD45 isThe mechanism underlying the function of stem cell transplantation in nerve repair is still not very clear. There are two general hypotheses: cell replacement function and neurotrophic effect. Therefore, many researchers are trying different methods to induce the differentiation of BMSCs into essential neuron-like cells for transplantation; they believe that this strategy could enable the transplanted stem cells to fully replace the damaged cells; however, from the viewpoint of neurotrophic hypothesis, pure neuron-like cells might not maximally perform the required function. We believe that the transplanted cells should not be too pure; they should consist of a mixture of a few cell types. Some cells could perform nerve repair through cell replacement, while other cells could secrete different cytokines to assist in nerve repair and regeneration, thus supporting the growth of neural stem cells and inducing differentiation . TherefoOne year after receiving one session of BMSC transplantation, the patient described in this study showed significant improvement based on his symptoms, physical examinations, and electrophysiological studies. The physical examination results revealed that the right-eye visual acuity was 100 cm and the left-eye visual acuity was 80 cm. The right lower limb muscle tension was significantly decreased compared to the pre-surgical measurement, and the patient walked more smoothly than previously; the VEP was close to normal. During the treatment and at regular follow-ups at 3, 6, and 12 months post-transplant, the patient did not exhibit fever, headache, blood pressure fluctuations, arrhythmias, or other symptoms of discomfort without an obvious reason. During the period of stem cell transplantation, the patient did not use any neurotrophic drugs or drugs that improved microcirculation; he also did not receive hyperbaric oxygen therapy or participate in functional rehabilitation exercises. Of course, we cannot completely rule out the possibility that the patient self-recovered; however, it is difficult to attribute these improvements to self-recovery, as his vision did not improve significantly in the two years prior to transplantation but improved rapidly within 1 year after stem cell transplantation. Therefore, we have reason to believe that it was stem cell therapy that promoted the recovery of his vision.The mechanism that BMSCs use to repair the nerve damage may involve the nutritional effect of the neurotrophic factors (such as BDNF and NGF) released by stem cells . ResearcThe reason for the improvement in our patient’s symptoms 1 year after stem cell transplantation is unclear; it is possible that in addition to the short-lasting effects that occurred via the trophic support of MSCs, these cells exhibited long-lasting effects ,25. One In summary, BMSC transplantation can be used to effectively treat cerebral palsy combined with visual impairment. To a certain extent, it can improve limb movement disorders and visual impairment caused by posterior visual pathway injury.The authors declare that they have no competing interests.ML carried out the design of the study and drafted the manuscript. AY and FZ carried out the data collection. GD and HC participated in the design of the study. XW participated in the stem cell clinical register. YA conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.


Monosomy × or 45,X is a cytogenetic characteristic for Turner syndrome. This chromosome anomaly is encountered in around 50% of cases, but wide variations of other anomalies have been found. This report is to describe the cytogenetic characteristics of 45,X individuals. To the best of our knowledge, there were no large series of 45,X cases has been reported from Indonesia.Ninety five cases with 45,X cell line found, of which 60 were detected by karyotyping, 4 by FISH for sex chromosomes, and 31 by both karyotyping and FISH. Using karyotyping 37 out of 91 cases(40.6%) were identified as 45,X individuals, while cases who underwent FISH only 4 out of 35 cases (11.4%) showed 45,X result, resulting in total of 39 45,X cases (41.1%), and the rest 56 (58.9%) cases are mosaic. Among these cases, 21 out of 95 (22.1%) have Y or part of Y as the second or third sex chromosome in their additional cell lines. Result discrepancies revealed in 22 out of 31 cases who underwent both FISH and karyotyping, of which 7 showed normal 46,XX or 46,XY karyotypes, but by FISH, additional monosomy × cell line was found. Most of the cases were referred at the age of puberty (8-13 years old) or after that (14-18 years old), 31 and 21 cases respectively, and there were 14 cases were sent in adulthood.Wide variations of sex chromosome aberrations have been detected using the combination of conventional cytogenetic and FISH, including detection of low level of mosaicism and Y-chromosome fragments. Result discrepancies using both techniques were found in 22/31 cases, and in order to obtain a more details of sex chromosome constitution of individuals with 45,X cell line both FISH and karyotyping should be carried out simultaneously. Monosomy × or 45,X is a cytogenetic characteristic for Turner syndrome (TS). This chromosome anomaly is encountered in around 50% of cases, but a wide variation of other anomalies of × chromosome have been found, including mosaicism, Xp or Xq deletion, dicentric × chromosomes, and isochromosomes of the × long arm -3.Despite short stature, which seems to be the general clinical characteristic of TS, all other clinical stigmata are inconsistent, even in individuals with non-mosaic 45,X. Possible explanation for this fact is that the physical manifestations of TS patients largely depends on the karyotype , althougNinety five cases with 45,X cell line found in our series, of which 60 were detected by karyotyping, 4 by FISH, and 31 by both karyotyping and FISH . Seven out of 31 cases (22.5%), which found to be normal 46,XX or 46,XY by karyotyping, were revealed abnormal with additional monosomy × cell lines by FISH. On the other hand several mosaic monosomy × cases, which can be detected only by its proportion using FISH, can be showed its structural rearrangements using karyotyping technique (Table Most of the cases were referred at the age of puberty (8-13 years old) or beyond (14-18 years old), 31 and 21 cases, respectively  and interphase chromosome-specific multicolor banding (ICS-MCB). The molecular cytogenetic techniques are highly efficient for diagnosis of numerous diseases associated with brain dysfunction ,9.Recently, the significant achievement in the field of molecular cytogenetic has brought evidences that demonstrated a higher incidence of chromosomal mosaicism in diseased individuals, i.e. brain diseases , and addMost of our cases (58.9%) have mosaic 45,X cytogenetic analysis results, which assumed to contribute to the phenotypes diversity found. Many authors believed that mosaicism formations occur during fetal life. Spontaneous somatic chromosomal variations presenting as low level mosaicism can be detected in all somatic cell populations. However, the low-level mosaicism is frequently overlooked, because of unapparent phenotypic effects. On the other hand Yurov et al , showed The use of fluorescence in situ hybridization may increase the prevalence of sex chromosomal mosaicism detection in non-mosaic 45,X cases ,11. By cWith conventional cytogenetic analysis, the possibility of mosaicism cannot be excluded without large numbers of mitoses being examined. For karyotyping, we used to analyze 20 metaphase cells, but learning from this experience, to rule out sex chromosome mosaicism, we should analyze more, at least 30 metaphase cells using conventional cytogenetic as recommended by The American College of Medical Genetics . FurtherThe presence of Y chromosome fragments in patients with TS is known to increase the risk of gonadoblastoma ,6,17. InMany studies reported that these Y chromosome aberrations, especially isodicentric Y chromosome can produce chromosome mosaicism, due to their instability during cell division, and consequently alternate cell lines, generally including 45,X (95% cases) may be generated ,19. We fDespite different level of mosaicism found using karyotyping and FISH, in our report three cases  of 45, × cases detected in infancy and most of the cases were referred for chromosomal evaluation at the age of puberty and beyond Table . InteresDespite the possibility of hidden mosaicism in pure 45,X cases, that might lead to milder phenotypes and therefore were missed, looking at our data, it seems that for most of the cases, the awareness of clinicians or the parents rose after significant clinical signs, such as short stature, primary amenorrhea, lack of secondary pubertal signs, appeared Table . While cIn conclusion, using combination of conventional cytogenetic and FISH, we detected a wide variation of sex chromosome aberrations, including detection of low level of mosaicism and Y-chromosome fragments. Result discrepancies may be produced using both techniques and in order to obtain a more details of sex chromosome constitution of individuals with 45, × cell line, both FISH and karyotyping should be carried out simultaneously. Further studies using combination of molecular cytogenetic methods, including MCB and CGH may provide higher detection rate of mosaicism and the possible explanation of the wide variations of TS phenotypic corresponding to its diversity in somatic chromosomal constitutions, as well as the mechanism of sex chromosome aberrations.Subjects of this study were drawn from all cases referred to our clinic between year 2005 until 2009 and whose cytogenetic results contained monosomy × cell populations. Cytogenetic results and data of the subjects were analyzed. We look for the variations of sex chromosomes abnormalities in individuals with monosomy × cell line and the results differences using conventional cytogenetic technique and Fluorescence in situ hybridization (FISH).Metaphase chromosomes from blood lymphocytes were prepared according to standard procedures . ChromosVysis, USA) for up to 300 interphase cells.FISH was carried out as standard procedures according to probes manufacturer manual, using CEPX(DXZ1)/Y(DYZ3) probes (The authors declare that they have no competing interests.NSM counseled the patients, collected and analyzed the data, drafted, revised, and finalized the manuscript. HWA carried out the karyotyping. LPS participated in data collection, carried out the karyotyping. DDA carried out the FISH analysis. CP performed the karyotyping and FISH analysis, involved in manuscript revision. HK performed the karyotyping, and FISH analysis, helped to draft the manuscript, ABP counseled the patients, involved in analyzing the data, and revising the manuscript. AH supervised the cytogenetic results, involved in revising the manuscript, and gave the final approval of the manuscript. All authors read and approved the final manuscript.