We then catalogued the 13 filters demonstrating a significant anti-inflammatory effect with edema inhibition percentages of more than 70%. The filters are: diethylhexyl butamido triazone (92%), benzophenone-5 and titanium dioxide (90%), benzophenone-3 (83%), octocrylène and isoamyl p-methoxycinnamate (82%), PEG-25 PABA and homosalate (80%), octyl triazone and phenylbenzimidazole sulfonic acid (78%), octyl dimethyl PABA (75%), bis-ethylhexyloxyphenol methoxyphenyl triazine and diethylamino hydroxybenzoyl hexylbenzoate (70%). These filters were tested at various concentrations, including their maximum authorized dose. We detected a dose-response relationship.The anti-inflammatory effect of a sunscreen ingredient may affect the in vivo SPF value. Sun Protection Factor) and the UVA-PF (UVA-Protection Factor). For many years, these two indicators were determined in vivo using volunteers in vitro methods have been more recently proposed in order to spare humans from excessive sun exposure in vivo values are higher than those obtained for in vitro method. This difference can be explained by the use of certain ingredients having anti-inflammatory properties to formulate these products, such as α-bisabolol and 18 β-glycyrrhetinic acid The effectiveness of sunscreen products is quantifiable using two indicators, the SPF  were purchased from Cooper . Xanthan gum (Rhodicare® T) was obtained from Rhodia . Polymethylmethacrylate (PMMA) plates were purchased from Europlast . The standard (reference) molecules Phorbol 12-Myristate 13-Acetate (PMA), niflumic acid, hydrocortisone 17-butyrate, diclofenac and ketoprofen were purchased from Sigma Aldrich . The filters we tested are presented in ad libitum under standard conditions of temperature and light.Swiss mice, male, weighing 14–16 grams, were purchased from Janvier . The mice were bred in the animal facility of the department of Pharmacology. The mice were caged individually and were fed standard laboratory chow and water An O/W emulsion placebo was prepared in the laboratory as previously described in vitro method according to a previously described protocol . 30 mg of product was weighed and then spread across the entire surface of PMMA plates (25 cm2) using a finger cot. 15 mg remained on the finger cot. Three plates were prepared for each product to be tested and 9 measures were performed on each plate. Transmission measurements between 290 and 400 nm were taken using a spectrophotometer equipped with a xenon arc lamp and an integrating sphere . The standard used was the 8% homosalate standard mandated by the US Food and Drug Administration Sunscreen Monograph. The calculations for either term used the same relationship:λ is the CIE erythemal spectral effectiveness, Sλ is the solar spectral irradiance and Tλ is the spectral transmittance of the sample The SPF of the commercial sunscreen products was determined using an −1) was then applied to each ear, in order to provoke an edema. After three and a half hours, the Oditest® was performed again to determine the thickness of the ears. Ear edema, calculated by subtracting the thickness of the left ear (vehicle) from the thickness of the right ear (PMA), was expressed as an increase in ear thickness. We determined the percentage that the inflammatory reaction was inhibited for each mouse by comparing the ear edema in treated and untreated animals. Five mice were used for each product tested.We determined the anti-inflammatory effects of the emulsions formulated in the laboratory and the commercial sunscreen products using a Phorbol 12-Myristate 13-Acetate (PMA) test. The mouse ear edema was provoked according to the method described by Carlson et al. with some modifications in vitro determination) of the commercially-available sunscreen products are presented in in vitro SPF value inferior to the labeled value.The SPF values  was studied. Note that the large majority of the filters demonstrated a significant anti-inflammatory effect  and 5. TThe activity of homosalate  Table 5Table 5 iThe anti-inflammatory effect of cinnamic acid derivatives  such as We can clearly state that there exists a dose-response relationship , 5 and 6We observed an anti-inflammatory effect with the commercial sunscreen products . For halSP Boots® SPF 15, for example, demonstrate a significant anti-inflammatory effect when compared to Vichy Capital soleil® Lait 50+. Besides, the anti-inflammatory effects can be modified when sunscreen is UV-exposed, as many UV-filters are known to be photo-unstable All of this has consequences in terms of determining the effectiveness of the final product. In Europe, it is difficult to determine whether a product is anti-inflammatory or not simply by reading the ingredients on the label because we do not know at what percentage these filters have been incorporated. The effect is not linked to the indicated protection factor, but to the specific filters and their concentrations. Certain sunscreen products, even with a medium protection level, like La Roche Posay® SPF 20 and Soleiin vivo method of SPF testing takes the anti-inflammatory effect of the products tested into consideration while the in vitro method does not. It remains to be shown how the biological response of the human UV-irradiated skin might vary with inclusion of specific anti-inflammatory ingredients in the sunscreens.It is important to note that the


We present a new tree-ring reconstruction of the Atlantic Multidecadal Oscillation (AMO) spanning 1568–2007 CE from northeast Asia. Comparison of the instrumental AMO index, an existing tree-ring based AMO reconstruction, and this new record show strongly similar annual to multidecadal patterns of variation over the last 440 years. Warm phases of the AMO are related to increases in growth of Scots pine trees and moisture availability in northeast China and central eastern Siberia. Multi-tape method (MTM) and cross-wavelet analyses indicate that robust multidecadal (∼64–128 years) variability is present throughout the new proxy record. Our results have important implications concerning the influence of North Atlantic sea surface temperatures on East Asian climate, and provide support for the possibility of an AMO signature on global multidecadal climate variability. The Atlantic Multidecadal Oscillation (AMO) is a mode of natural climate variability occurring in the North Atlantic Ocean. AMO has a principal expression in mean sea surface temperatures (SST) anomalies north of the equator with a period of 65–80 years and an amplitude of ∼0.4°C on a multidecadal timescale Although AMO is a feature of the North Atlantic Ocean basin, recent studies suggest that it is also related to multidecadal variability of Asian and Indian monsoons However, only two full cycles of the AMO (from warm to cool phases) are represented in instrumental records. Thus, it is difficult to fully understand the low-frequency characteristics of AMO and mechanisms of its longer-term influence on East Asian climate Pinus sylvestris) tree-ring widths from trees growing in central eastern Siberia and northeastern China over the past 440 years (1568–2007 CE). This proxy reconstruction suggests a secular influence of AMO on moisture availability in the region. Perhaps most importantly, it provides support to the potential global nature of multidecadal climate variability related to AMO. The reconstruction is compared with both the instrumental AMO record and an existing multicentennial tree-ring reconstruction of AMO Several high-resolution proxies have been developed and used to assess longer-term annual to multidecadal variability in areas surrounding the North Atlantic, but none have looked at the longer-term  characteristics of AMO in East Asia http://www.ncdc.noaa.gov/paleo/treering.html). Two new chronologies were generated from 72 individual Scots pine trees from Mangui and Mengkeshan in northeastern China. To preserve the low-frequency variability, all raw tree-ring series were standardized by fitting them to negative exponential curves or linear regressions with negative or zero slopes. A nested approach as described in A total of 191 tree-ring width records from moisture-sensitive Scots pine from six localities in northeastern Asia were assembled for this analysis , Table 10 = 6) was used in this analysis and the wavelet power significance was tested at the 95% confidence level against a red-noise background Correlations between the six chronologies and the monthly instrumental AMO index calculated from North Atlantic SST anomalies th century over the last 423 years , moderate (Khotugn and Mengkeshan), and strong  responses of tree growth to long term changes in North Atlantic SST . PC1 of 23 years . The intMTM analysis of the East Asian PC1 series reveals pronounced variability at interannual (2.9–2.1 years) and multidecadal (33 and 73 years) time scales . The exiTree-ring time series from northeast Asia exhibit a coherent, AMO-like pattern of variability over the last 440 years. Although a mechanism for AMO influence on East Asian climate is not fully understood, findings reported here support other observational and modeling studies that have demonstrated multidecadal Asia monsoon variability is related to the AMO The results of correlation analyses between tree-ring chronologies and climatic factors indicate that both temperature and precipitation influence Scots pine growth at northeast Asia (not shown here), while the precipitation has less influence on tree-ring widths than temperature in this area especially for Khotugn and Mengkeshan. Also, the precipitation in the previous autumn and winter is important to tree growth, while the temperature have the similar influence during the previous autumn and current growing season. The cross correlation between the tree-ring record from northeast Asia and Gray et al. (2004) AMO reconstruction indicates that the maximum correlation (0.26) occurred at lag one year . This maFurther research is needed to explore how multidecadal variability in East Asian climate related to the AMO may have contingent effects on interannual to decadal climate variations and ecosystem processes. Recent studies of both Pacific and Atlantic Ocean climate teleconnections in western North America found that warm phases of the AMO synchronized sub-continental-scale patterns in droughts and forest fires across the western U.S This is the first Asian AMO-like proxy series spanning the last four centuries. The proxy series confirms and extends previous observational and modeled analyses in which it was found that the AMO plays a role in the multidecadal variability in East Asian climate. Furthermore, our results support that the AMO continues with its quasi-cycle of roughly 70 (65–80) years as well as shown in other proxy series


Anopheles gambiae is a major vector of malaria in the West African region. Resistance to multiple insecticides has been recorded in An. gambiae S form in the Ahafo region of Ghana. A laboratory population (GAH) established using wild material from this locality has enabled a mechanistic characterization of each resistance phenotype as well as an analysis of another adaptive characteristic - staggered larval time-to-hatch.Individual egg batches obtained from wild caught females collected from Ghana and the Republic of the Congo were monitored for staggered larval time-to-hatch. In addition, early and late larval time-to-hatch sub-colonies were selected from GAH. These selected sub-colonies were cross-mated and their hybrid progeny were subsequently intercrossed and back-crossed to the parental strains. The insecticide susceptibilities of the GAH base colony and the time-to-hatch selected sub-colonies were quantified for four insecticide classes using insecticide bioassays. Resistance phenotypes were mechanistically characterized using insecticide-synergist bioassays and diagnostic molecular assays for known reduced target-site sensitivity mutations.Anopheles gambiae GAH showed varying levels of resistance to all insecticide classes. Metabolic detoxification and reduced target-site sensitivity mechanisms were implicated. Most wild-caught families showed staggered larval time-to-hatch. However, some families were either exclusively early hatching or late hatching. Most GAH larvae hatched early but many egg batches contained a proportion of late hatching larvae. Crosses between the time-to-hatch selected sub-colonies yielded ambiguous results that did not fit any hypothetical models based on single-locus Mendelian inheritance. There was significant variation in the expression of insecticide resistance between the time-to-hatch phenotypes.An adaptive response to the presence of multiple insecticide classes necessarily involves the development of multiple resistance mechanisms whose effectiveness may be enhanced by intra-population variation in the expression of resistance phenotypes. The variation in the expression of insecticide resistance in association with selection for larval time-to-hatch may induce this kind of enhanced adaptive plasticity as a consequence of pleiotropy, whereby mosquitoes are able to complete their aquatic life stages in a variable breeding environment using staggered larval time-to-hatch, giving rise to an adult population with enhanced variation in the expression of insecticide resistance. Malaria is holoendemic in Ghana and is responsible for an estimated 22% of mortality in children under five, as well as 9% of maternal deaths , a situaAnopheles gambiae, Anopheles funestus and Anopheles arabiensis [Anopheles gambiae is the nominal member of the An. gambiae complex and is sub-divided into 5 informally named chromosomal forms [An. gambiae populations, particularly in the West African region [An. gambiae and An. funestus [The major vectors of malaria in the West African region are abiensis . Anophelal forms  and two al forms . Recent n region -9. The ifunestus .An. gambiae generally occurs 2-3 days after oviposition depending on environmental conditions [et al [An. gambiae M and S forms and showed that over 80% hatched within the first three days following oviposition regardless of water type. Between 5% and 16.8% of the total number of eggs hatched over the next four days while between 0.6% and 7.2% hatched after the first week. The S form produced significantly more hatchlings in the 3-7 day post oviposition period than the M form, and the M form showed significantly higher levels of hatching one week post-oviposition than the S form. It was also found that the type of water in which the eggs were kept had a highly significant effect on hatching with the distribution most distinct in puddle water. Yaro et al [Larval hatching in nditions . Howevernditions ,13. Yaros [et al  determinro et al  concludeAn. gambiae.Here the adaptive significance of larval time-to-hatch is further assessed by investigating the possibility of a genetic component controlling time-to-hatch as well as the expression of insecticide resistance in association with time-to-hatch in An. gambiae S form colony originating from Ghana and colonized in 2006 formed the basis of this study. SUA, an insecticide susceptible An. gambiae colony from Liberia, was used as the susceptible reference strain in insecticide bioassays. All mosquitoes were reared in the Botha De Meillon insectary at the National Institute for Communicable Diseases, NHLS, Johannesburg. Conditions were maintained at approximately 25°C with 75-85% relative humidity in a twelve-hour light: dark cycle with 30 min dusk and dawn transitions. Larvae were fed on ground dog biscuits and yeast and adults received three blood meals per week.The GAH An. gambiae were collected from Ahafo, Ghana , in June 2008; Damang  and Tarkwa , Ghana, in January 2009; Pointe Noire, Republic of the Congo , in March 2009. The samples from Ghana were used for various evaluations in conjunction with laboratory-reared material as described below. The material from the Republic of the Congo served as a comparative An. gambiae sample from a different region.Samples of Mosquitoes were collected in Ghana and the Republic of the Congo from inside human dwellings using a torch and aspirator. Mosquitoes were placed in polystyrene cups and were provided with a 10% sugar solution prior to transportation.An. gambiae complex were identified to species using the An. gambiae species-specific PCR assay [An. gambiae sensu stricto samples were further characterized as either M or S molecular form by PCR [Wild-caught mosquito samples were transported to the NICD and were initially sorted using morphological keys ,15. ThosCR assay . All An.m by PCR .GAH colony mosquitoes were selected at the larval stage according to early and late hatch phenotypes. During initial selections larvae from the base colony were allowed to develop for approximately 10 days, at which point all fourth instar larvae were removed and placed into a new bowl labelled GAH Early Hatch. The second and third instar larvae were returned to the baseline colony. All first instar larvae at 10 days as well as remaining unhatched eggs were pooled as GAH Late Hatch. Larvae selected in this way were reared through to adults. This process was repeated using several egg batches until separate Early and Late Hatch adult sub-colonies had been produced. These were maintained and blood-fed according to the standard procedure. Eggs from each sub-colony were harvested and the time-to-hatch selection procedure was repeated. All larvae that hatched within four days of oviposition from the Early Hatch sub-colony were kept as GAH Early Hatch while the remaining eggs were transferred to the baseline colony. The Late Hatch sub-colony was selected by transferring all larvae that hatched within four days of oviposition to the baseline colony so that all larvae that hatched subsequently remained as GAH Late Hatch. These selections continued for at least six generations before cross-mating experiments or insecticide susceptibility bioassays were conducted.In order to determine whether there is a genetic component associated with larval time-to-hatch, cross-mating experiments between the Early and Late Hatch sub-colonies were set up. Early Hatch females were crossed with Late Hatch males and vice versa. The numbers of eggs produced from each cross were quantified. Eggs were monitored for hatching and the numbers of F1 hatchlings from each cross were recorded daily. F1 larvae from each cross were reared to adults and these were either back-crossed to the parental strains or were intercrossed. Eggs and F2 hatchlings from each back-cross or intercross were monitored daily as described above. Egg production and larval hatching in the baseline GAH colony was concurrently monitored as a control.Eggs from each sub-colony, the baseline colony and the cross-mating experiments were collected and placed in marked egg bowls. The date of oviposition was recorded for each egg batch and the numbers of eggs quantified. Bowls were checked every morning and any larvae present were counted and removed. Hatchlings that emerged within four days of oviposition were classified as early hatch and those that emerged four days or more post-oviposition were classified as late hatch. Four days post oviposition was used as the cut-off point between early and late hatch as initial observations on the progeny of wild-caught mosquitoes showed that the majority of eggs hatch within four days while the rest tend to hatch in a staggered fashion for up to 22 days post oviposition.An. gambiae females from Ghana and the Republic of the Congo were individually placed in vials lined with moist filter paper for oviposition. Cotton wool pads soaked in a 10% sugar solution were provided. Vials were monitored daily for eggs. All eggs produced were placed in egg bowls by family and were monitored daily for hatching. Hatchlings were counted and removed as described for the cross-mating and time-to-hatch selection experiments. Those families reared from wild-caught females from Ahafo, Ghana, formed the pilot study from which the four-day cut off point for classifying the early and late hatch phenotypes was determined. This cut off point closely approximates with information from Yaro et al [Blood-fed, wild-caught ro et al .The batches of eggs produced by each wild-caught female that was used in the hatch distribution analysis were also used to determine whether the number of eggs produced by a single female had an effect on the proportion of eggs that hatched, or on the proportion of eggs that hatched early. Data were grouped according to number of eggs laid and analysed using scatter-plots and linear regression in Statistix 7 .An experiment to test the effect of disturbance on egg hatching from the time-to-hatch selected sub-colonies and the baseline GAH colony was performed. Three batches (replicates) of eggs from each group were obtained and divided into two cohorts per batch for different treatments. One cohort was rinsed into a larval bowl that contained a floating plastic ring, but the eggs were not specifically contained within this ring and so could be stranded on the sides of the bowl due to water evaporation. These eggs were sprayed with water (disturbed) daily. The other cohort was also rinsed into a larval bowl so that all the eggs were contained within the floating plastic ring. This ring prevented the eggs from being stranded on the sides of the larval bowl and thus they did not require any disturbance to keep them in contact with the water. The disturbed and undisturbed egg batches were monitored daily for hatchlings, which were counted and removed. Each set of experiments lasted 25 days after which all eggs were disturbed to determine if any unhatched eggs would hatch, and later discarded. The total number of eggs that hatched as well as the proportions of early and late hatching larvae per group was determined and analysed using two-sample t-tests or ANOVA (Statistix 7).An. gambiae GAH colony as well as from the larval time-to-hatch sub-colonies. Susceptibility to four classes of insecticide was assessed .In order to test for associations between insecticide resistance phenotypes and larval time-to-hatch, adult insecticide susceptibility assays were performed on samples drawn from the An. gambiae GAH laboratory colony and the time-to-hatch selected sub-colonies. Enzyme synergists can be employed in this manner because they are recognized as substrates by those enzyme systems implicated in insecticide detoxification [A set of enzyme synergists was used to test for associations between enzyme activity and the expression of insecticide resistance in the fication .The synergists used were 20% diethyl maleate (DEM), 10% triphenylphosphate (TPP) and 4% piperonyl butoxide (PBO). These generally synergize glutathione S-transferase (GST), esterase and monooxygenase enzyme activity respectively. However, the specificity of each synergist is likely to be affected by the metabolic pathways associated with resistance and cross-reaction can occur. Approximately 25 adult female mosquitoes, 2 to 5 days old, were exposed to synergist for one hour immediately followed by exposure to a diagnostic concentration of insecticide for 1 h (2 h for fenitrothion). A similar sample was concurrently exposed to insecticide only for 1 h. Controls included concurrent exposure to synergist only as well as exposure to untreated paper. Mortalities were recorded 24 h post exposure. At least five replicates per synergist for each insecticide were performed. Mosquitoes that survived synergist bioassays, as well as those that died following insecticide exposure only, were collected and stored on silica before undergoing molecular screening for target site mutations associated with insecticide resistance. Final mortalities from the synergized samples were compared to their corresponding unsynergized samples using two sample t-tests (Statistix 7).et al [kdr, R ace-1and Rdl genotypes. Data were analysed using Fisher's Exact test, χ2, or two sample t-tests (Statistix7).DNA was extracted from samples to be used in molecular assays according to the method of Collins et al  or usingkdr and Rdl TaqMan® hydrolysis probe molecular assays [kdr (L1014F and L1014S) and Rdl mutations associated with resistance to pyrethroids and DDT (kdr) and resistance to dieldrin (Rdl). All primers used in the real time experiments were supplied by Inqaba Biotechnical Industries, Hatfield, Pretoria, and probes were supplied by Applied Biosystems Inc, Forster City, CA, USA. Kdr and Rdl homozygous (RR and SS) and heterozygous (RS) positive controls as well as no template controls were included in all assays.The r assays ,22 were R ace-1mutation in acetylcholinesterase 1 has previously been associated with organophosphate and carbamate resistance in An. gambiae [Alu1 restriction enzyme  digest was used to genotype samples for R ace-1[The  gambiae . DNA wasr ace-1R .An. gambiae s.s. Of the Ghana Ahafo families, two (Gahf 33 and 49) were M form whilst the remaining 26 were S form. All Republic of the Congo  and Ghana Damang and Tarkwa  families were S form. The GAH laboratory colony was confirmed as S form.All wild-caught mosquitoes used in the time-to-hatch experiments were identified as Most Ghana Ahafo families (Gahf) showed bimodal hatching. Only one family Gahf 41) was entirely early hatching (3.6%) and ten families  were entirely late hatching (35.7%)  showed bimodal hatching. Four 22.22%) were entirely early hatching and none were entirely late hatching using four days post oviposition as the cut-off point between time-to-hatch phenotypes  families showed bimodal hatching. The proportions of late hatching larvae per family Figure  were genThe overall proportions of F1 and F2 progeny that hatched from the crosses, back-crosses and intercross as well as progeny from the unselected GAH colony and time-to-hatch selected sub-colonies did not vary significantly . Between 400 and 3000 eggs per cross or selected sub-colony were monitored in total. The latest hatch occurred 22 days post oviposition, but in general hatching after 9 days was uncommon. The vast majority of larvae hatched early across all batches  to test for an association between the number of eggs laid per female and the proportion of eggs that hatched, or the number of eggs laid per female and the proportion of eggs that hatched early. No significant trends were detected (P > 0.05) suggesting that the number of eggs laid by a female does not influence the proportion that hatch or the proportions of eggs that hatch early.The total numbers of eggs monitored ranged from 1,600 to 3,200 per experiment, divided between three replicates per experiment. Significantly fewer eggs from the undisturbed experiment hatched compared to the disturbed experiment, indicating that egg disturbance is necessary for optimal hatching  (Table An. gambiae GAH showed various levels of resistance to permethrin and deltamethrin (pyrethroids), bendiocarb and propoxur (carbamates), DDT and dieldrin (organochlorines) and pirimiphos methyl (organophosphate). Resistance to the organophosphates fenitrothion and malathion is also suggested . This effect was not detected in the Early Hatch selected sub-colony as resistance to pyrethroids had diminished by this generation with over 90% of the Early Hatch selected mosquitoes not surviving permethrin exposure. TPP and PBO induced no significant reductions in insecticide resistance expression in the time-to-hatch selected sub-colonies when challenged against the other insecticides listed in table kdr mutation were drawn from the GAH laboratory colony and the larval time-to-hatch selected sub-colonies. Sample sizes ranged from 18 to 30. All mosquitoes assayed were characterized by insecticide exposure bioassay as either insecticide resistant or susceptible. Some were further characterized by their response to insecticide exposure following pre-exposure to PBO. There was significant variation in kdr allele frequency between resistant and susceptible samples  as well as kdr and DDT resistance  were detected. However, several homozygous resistant RR genotypes were recorded in the permethrin susceptible samples and a small proportion of homozygous susceptible SS genotypes were recorded in the permethrin resistant samples. Pre-exposure to PBO significantly increased the kdr frequency amongst permethrin resistant samples in the GAH baseline colony  and the GAH Early Hatch sub-colony  but not in the GAH Late Hatch sub-colony .Samples genotyped for the L1014F s Figure  whereby kdr mutation in 82 permethrin and DDT exposed GAH females. All were genotyped as homozygous susceptible SS.The hydrolysis probe molecular assay  was alsoAn. gambiae GAH females were characterized as either dieldrin resistant or susceptible following exposure to 4% dieldrin. The alanine296-glycine (Rdl) GABA receptor mutation was detected by hydrolysis probe assay [Rdl genotype and response to dieldrin phenotype in both the time-to-hatch selected sub-colonies  as well as the base colony  . Exposure to PBO prior to dieldrin exposure did not increase mortality in GAH (two sample t test: P = 0.81) (Table An. gambiae colony [A sample of be assay  and ther1) Table , as was e colony , suggestAn. gambiae GAH female survivors and 27 bendiocarb exposed females that died following exposure were screened for the R ace-1mutation using the RFLP assay [R ace-1genotype and response to bendiocarb exposure phenotype . The apparent scarcity of the RR (homozygous resistant) genotype may be attributable to an unusually high death rate of RR individuals at pupation as previously described [Twenty-eight bendiocarb exposed LP assay . Three oescribed .An. gambiae populations is a growing concern for malaria vector control [An. gambiae laboratory colony GAH shows resistance to all classes of insecticide currently available for use in public health. Although this colony is likely to show reduced genetic variation relative to the wild population from which it is derived, multiple insecticide resistance in An. gambiae has previously been reported from Ghana [Resistance to multiple classes of insecticides in  control . The An.om Ghana .An. gambiae GAH include monooxygenase and esterase mediated detoxification coupled with the L1014F kdr mutation. The correlation between kdr genotype and pyrethroid resistance phenotype was clear but not absolute, with small numbers of homozygous resistant (RR) genotypes occurring in the phenotypically susceptible samples and vice versa. These discrepancies, coupled with the occurrence of RS heterozygotes in resistant and susceptible mosquitoes as well as an increase in kdr frequency in PBO synergized samples, suggest that enzyme mediated detoxification also plays an important role in the production of a measurable pyrethroid resistance phenotype [kdr. Resistance to dieldrin associated closely with the Rdl mutation while resistance to the carbamate bendiocarb associated closely with the ace-1R mutation. Carbamate resistance and the low level of organophosphate resistance recorded in the An. gambiae GAH colony is therefore likely mediated by the assortment of ace-1R although enzyme mediated detoxification may play a supporting role. High frequencies of R ace-1have been recorded in An. gambiae S form populations in Burkina Faso indicating that caution should be employed when using carbamate and organophosphate insecticides for vector control programs in the West African region [From the data presented here, mechanisms of resistance to pyrethroids in henotype ,27. Resin region .An. gambiae GAH eggs hatched within four days after oviposition. However, the proportion of late hatching eggs increased following selection for this phenotype, suggesting that, in addition to environmental factors [An. gambiae families from Ghana and the Republic of the Congo were exclusively early or late hatching. Importantly, there was also significant variation in the expression of insecticide resistance between the time-to-hatch phenotypes. The variation recorded in the response to insecticide exposure assays also correlates with variation in kdr and Rdl frequencies between the time-to-hatch selected sub-colonies, showing that selection for time-to-hatch inadvertently affected the frequencies of those factors controlling insecticide resistance.The majority of  factors , there mAn. gambiae breeding sites [An. gambiae populations is supported by the quantification of this characteristic in geographically diverse samples from the Republic of the Congo and Ghana.Although the adaptive significance of staggered larval time-to-hatch post oviposition has not been quantified, it is highly likely that this characteristic has evolved in response to the variable nature of preferred ng sites . These sAn. gambiae female. This suggests that delayed larval hatch is not an adaptation to avoid over-crowding or competition for resources. Additionally, water disturbance induces a significantly higher rate of egg hatching in An. gambiae.These data further show that the number of eggs that hatch in total as well as the proportions of eggs that hatch either early or late do not depend on the initial number of eggs laid per The presence of insecticides in fluctuating concentrations significantly alters the chemical environment in which mosquitoes breed and rest. A sufficient adaptive response to the presence of multiple insecticide classes necessarily involves the development of multiple resistance mechanisms whose effectiveness may be enhanced by intra-population variation in the expression of resistant phenotypes. The variation in the expression of insecticide resistance in association with selection for larval time-to-hatch described here may induce this kind of enhanced adaptive plasticity as a consequence of pleiotropy. In this scheme cohorts of mosquitoes are able to complete their aquatic life stages in a variable breeding environment using staggered larval time-to-hatch, giving rise to an adult population with enhanced variation in the expression of insecticide resistance. Enhanced variation inadvertently produced in this way offers a wider platform for the continued development of resistance to insecticides.Successfully managing multiple insecticide resistance in malaria vector control should involve an appraisal of the biological and adaptive variation inherent within target vector populations. Effective malaria control can then be achieved by adopting an evidence-based approach, which incorporates the principles of judicious insecticide use in a broader Integrated Vector Management (IVM) system.The authors declare that they have no competing interests.MLK conducted all the experiments, analysed the results and drafted the manuscript; LLK assisted in the design and analysis of the molecular experiments and drafting of the manuscript; MC and RHH assisted with the design of the project and the drafting of the manuscript; BDB designed the project, assisted with data interpretation and statistical analysis and completed the drafting of the manuscript. All field collections were conducted under guidance of RHH. All authors read and approved the final manuscript.


The direct contact of ultrathin polymer films with a solid substrate may result in thin film rupture caused by dewetting. With crystallisable polymers such as polyethyleneoxide (PEO), molecular self-assembly into partial ordered lamella structures is studied as an additional source of pattern formation. Morphological features in ultrathin PEO films (thickness < 10 nm) result from an interplay between dewetting patterns and diffusion limited growth pattern of ordered lamella growing within the dewetting areas. Besides structure formation of hydrophilic PEO molecules, n-alkylterminated (hydrophobic) PEO oligomers are investigated with respect to self-organization in ultrathin films. Morphological features characteristic for pure PEO are not changed by the presence of the n-alkylgroups. An inteCremophor® S9) the self-assembly process in the presence of aqueous solutions favors a double layer formation of the hydrophobic alkyl groups, increasing the molecular weight of PEO units esterified with the stearic acid ( Myrj® 59 ) does not indicate assembly of alkyl chains. X-ray diffractograms of such materials provide only evidence for crystallization of PEO segments [Esterification of OH end-groups of PEO oligomers with long chain n-alkyl units like stearic acid is used to prepare non-ionic surfactants with PEO as the hydrophilic and the n-alkyl groups as hydrophobic structural entities . Dependisegments .Inspired by the structural variability of PEO, this article addresses the particular self-organization of PEO and stearate terminated PEO molecules in ultrathin films. Strong emphasis will be laid on the influence of dewetting structures on the morphology of self-assembled unilamellar entities.In thin film preparation, dewetting is often regarded as an undesired effect. However the local control of dewetting by chemical  or topognot only related to the surface chemistry but also dependent on the layer structure in multilayer substrates like in oxidized silicon wafers [ca. 3 nm thickness [ca. 7 nm), which has been experimentally confirmed by AFM measurement.It is worth to notice that thin film stability and therefore the critical thickness for the onset of the dewetting process is n wafers . In parthickness . Layer thickness . The cryet al. [As already demonstrated in et al. . The metIn a series of recent papers, detailed studies were published on the morphological appearance of short chain PEO lamella crystals grown at various undercooling within thin melt films. These included transmission electron microscopy (TEM) electron diffraction studies concluding on the relationship between growth habit and crystallographic structure, in particular chain orientation and packing.et al. [In our studies, we like to compare the morphological appearance thus described with that observed on the crystallization of stearate terminated PEO oligomers  that we et al.  demonstret al.  are compA consequence of the difference in the chain folding scheme between the central and the branched parts of the lamella is the change in the density of hydrophobic end-groups to be expected on the surface of the different morphologies. A statistical distribution of hydrophobic end-groups on both sides of the lamella would give a probability of 0.25 for the thicker central part, respectively 0.17 for the thinner dendritic part. In order to prove a possible difference on the surface properties of the different crystal areas, we investigated the condensation behavior with respect to water at the dew point. As demonstrated by Mw 2000 g/mol, Rapp Polymers Tuebingen) and polyethyleneoxide  = 100 Sigma Aldrich) esterified with stearic acid were used. For film formation, solutions of PEO or PEO-stearate in chloroform  were prepared . As substrates, silicon wafers with either a native oxide layer or with a thermal oxidized oxide layer of 300 nm thickness were used .Polyethyleneoxide (v = 6 mm/min) using a commercial film-lift . Prior to coating, wafers were ultrasonicated in absolute alcohol, rinsed and dried under a nitrogen flow. For crystallization experiments, dip-coated films were molten at ambient temperatures and crystallized under temperature control of a heating stage (LINKAM THMS 600). Non-isothermal growth was observed under cooling rates of 1 °C/min. Thin film lamella growth was observed by reflective light microscopy (Zeiss Axiskop) using monochromatic illumination (λmax 435 nm) from a LED source (CoolLED) attached to the microscope. For contrast enhancement, the use of silicon wafer substrates with an oxide layer of 300 nm is essential [Ultrathin films of PEO, respectively PEO-stearate, on cleaned silica wafers were prepared by dip-coating substrates from PEO or PEO-Stearate solutions at constant speed  in order to achieve favorable conditions for surface and thin film imaging at low penetration depth of electrons and to avoid charging while samples were imaged as prepared without additional metal or carbon coating.Scanning electron microscopy (SEM) inspection of samples was done in a Zeiss Gemini SEM operated at low voltages (0.300 keV< Quantification of lamella height was done by AFM measurement using an AFM setup attached to a reflective light microscope (SIS Ultraobjective). This setup allows a pre-selection of ultrathin lamella in the light microscope.http://www.crystalmaker.com/).Zone axis diffraction patterns of simulated electron diffraction data were calculated from atom coordinates published in literature  using a We investigated the dewetting behavior and crystallization in ultrathin PEO films. Dewetted areas are covered with an amorphous film, which can be nucleated to start crystallization into highly branched PEO lamella by a diffusion limited aggregation process. The propagating crystallization front could be imaged by a modified reflective light microscopic method.Modification of oligoethyleneoxides by stearic acid generates similar dendritic lamella to those observed for pure PEO of the same molecular weight. The crystallization of these compounds is obviously characterized by the self-organization of PEO segments and not by self-organization of the long chain alkyl groups into close packed paraffin membranes.


A multifactorial aetiology of coronary artery disease (CAD) has been established in the recent past. Extensive research is now underway to understand the mechanisms responsible for plaque vulnerability. The identification of a novel biomarker that will help in the assessment of plaque status is urgently needed for the purpose of patient stratification and prognostication. The aim of the present study was to evaluate leptin, pregnancy-associated plasma protein A (PAPP-A) and C-reactive protein (CRP) levels in patients with acute coronary syndrome and to assess their diagnostic efficacy in the identification of vulnerable plaques.The study group comprised 105 patients who had chest pain along with ECG changes  and raised cardiac enzyme levels. Sixty-two patients with chest pain and ECG changes but with normal cardiac enzyme profiles were included in the control group. Lipid profiles, and leptin, PAPP-A and CRP levels were assessed in these two groups. Receiver operating characteristics (ROC) curves were plotted to determine the utility of the parameters under study as markers of plaque vulnerability.Significantly higher levels of serum lipoprotein (a), leptin, PAPP-A and high-sensitivity CRP (hs-CRP) were observed in the cases than in the controls. A positive correlation was observed between CRP and PAPP-A levels as well as CRP and leptin concentrations. ROC curve analysis revealed similar efficacies of CRP and PAPP-A levels in their ability to detect unstable plaques with areas under the curve of 0.762 and 0.732, respectively. Multivariate analysis established the superiority of hs-CRP as a predictor of plaque instability.Our study highlights the utility of both CRP and PAPP-A levels as determinants of plaque instability. Our findings necessitate population-based follow-up studies to establish the superiority of either of the two biomarkers in the field of preventive cardiology. Acute coronary syndrome (ACS) accounts for 20% of all medical emergency admissions and has the highest risk for adverse effects and deaths.2Atherosclerosis results due to the complex interplay between environmental, genetic and individual risk factors. Physical inactivity, along with the intake of calorie-dense food account for the energy imbalance that is widely prevalent in developing and developed countries. The metabolic perturbations due to this energy imbalance along with smouldering inflammatory processes and other as yet poorly understood mechanisms initiate and aggravate the atherosclerotic process.4The scientific community is engrossed in extensive research in order to discover biomarkers that will help stratify patients who are more prone to develop the complications consequent to atherosclerosis. The inability of lipid levels to provide an insight into the ongoing atherosclerotic process and impending complications has further necessitated the identification of novel markers that assess the dynamics of atherosclerosis. Adipocytokines (leptin), pregnancy-associated plasma protein A (PAPP-A) and high-sensitivity C-reactive protein (hs-CRP) are the newer emerging biomarkers for assessing plaque stability.5Adipose tissue is now identified as an active endocrine organ and not merely a storage site for fat. The mediators released by adipose tissue are known as adipocytokines or adipokines.10et al. reveal that it may be a potential determinant of plaque instability by degrading the extracellular matrix of the fibrous cap of the atherosclerotic plaque.13PAPP-A is a glycoprotein produced by the placental synctiotrophoblastic cells and was initially discovered in the sera of pregnant women.C-reactive protein, determined by high-sensitivity techniques, is the most widely studied marker of inflammation in the field of atherosclerosis. Its role has been identified in the initiation, progression and final outcome of the atherosclerotic process. It is an acute-phase reactant and is an important player in the inflammatory process observed to be active in atherosclerosis. Studies have revealed that it is not an innocent bystander and indicator of the inflammatory process; rather it is actively involved in the pathogenic mechanisms underlying atherosclerosis.15Lipoprotein (a) is a cardiovascular risk factor highly prevalent in subjects with Asian Indian origin. It has both thrombogenic and atherogenic properties. This is due to its homology to plasminogen and its low-density lipoprotein (LDL)-like properties. Hence it is known as a dual pathogen. It may play an important role in the susceptibility of atherosclerotic plaques to instability.3The present study aimed to evaluate these novel risk factors and to look for interrelationships between them in the setting of acute coronary syndrome.The study was jointly conducted by the Departments of Biochemistry and Medicine, Maulana Azad Medical College and associated hospitals, New Delhi, India. The study involved screening of 379 patients with symptoms of ‘chest pain’, presenting to the medical emergency department during the period between April and October 2010. These patients were then further evaluated by electrocardiography. One hundred and sixty-seven patients demonstrated ECG abnormalities such as ST elevation, ST depression and T inversion. They were enrolled as the study population.Cardiac enzymes such as troponins and CK-MB levels were evaluated in these patients. One hundred and five patients had raised serum cardiac enzyme concentrations and hence depicted the onset of acute coronary syndrome. These patients were classified as cases and they were further evaluated by coronary angiography.Angiography was conducted by cardiologists using standard techniques. The angiograms were done by two different cardiologists who did not have any information regarding patient identity and aetiology, to avoid any subjective bias. Those who had normal cardiac enzyme concentrations were enrolled as controls.• AMI patients diagnosed by typical rise and gradual fall of biochemical markers  of myocardial necrosis with at least one of the following:- ischaemic symptoms- development of pathological Q waves on the ECG reading- ECG changes indicative of ischaemia (ST elevation or depression)- coronary artery intervention (e.g. coronary angioplasty)• Unstable angina: chest discomfort at rest with either ST depression of at least 0.1 mV or T-wave inversion in two or more contiguous ECG leads, CK-MB levels normal and angiographically confirmed coronary artery disease (CAD).The exclusion criteria that were considered during the selection of the study population were:• advanced kidney failure indicated by high serum urea and creatinine• overt heart failure as diagnosed by echocardiography• history of major surgery/trauma within the previous month• suspected systemic inflammatory diseases• pregnancy• chronic stable angina (effort induced) diagnosed as chest pain of at least six months’ duration, accompanied by severe CAD on angiography (70% stenosis in any major artery).The inclusion criteria were:A fasting blood sample (8–10 ml) was drawn prior to angiography and the serum was extracted and stored at –70°C until further testing. Fasting serum leptin, hs-CRP and PAPP-A levels were estimated with ELISA kits provided by DRG International, Germany.t-test was used to compare the values between the cases and controls. Spearman’s correlation analysis was used to find the association between the various parameters of our study. A p-value < 0.05 was accepted as statistically significant. Multivariate regression analysis was carried out to assess the role of the different biomarkers in unstable CAD. All statistical analyses were performed with the program Statistical Package for the Social Science 12.0 .The data were expressed as mean ± standard deviation. Student’s The demographic and risk-factor profiles of the study population are shown in The body mass index and waist circumferences were higher in the cases compared to the controls. The prevalence of smoking and diabetes mellitus was significantly higher in the cases than the the controls. Subjects with none of the above risk factors comprised 16% of the cases and 28% of the controls, respectively.The biochemical profile of the study population is given in p < 0.001). Lipoprotein (a) [lp(a)] levels demonstrated a non-Gaussian distribution pattern with significantly higher levels (34.8 ± 30.mg/dl) in the cases compared to 16.7 ± 13.5 mg/dl in the controls (p < 0.001). Significantly higher levels of serum leptin, PAPP-A and hs-CRP were observed in the cases compared to the controls .High-density lipoprotein (HDL) levels were significantly lower in the cases than the controls , leptin, PAPP-A and hs-CRP levels as indicators of plaque rupture, the receiver operating characteristics (ROC) curves were plotted. According to this, a cut off of 3.1 mg/l for hs-CRP has a sensitivity of 77% and specificity of 73%. Similarly, a watershed value of 11.75 mIU/l for PAPP-A exhibited 68% sensitivity and 60% specificity. An attempt to increase the sensitivity was accompanied by a corresponding reduction in specificity and vice versa. Our study therefore proves the superiority of hs-CRP as a marker of plaque rupture. However, PAPP-A also proved to be a sensitive marker. r = 0.658, p < 0.01) and leptin . However, no such correlations were observed between leptin, PAPP-A and lipid levels. This finding highlights the interaction between hs-CRP and PAPP-A in initiating plaque rupture. Multivariate regression analysis proved the superiority of CRP over all the other parameters, with the order of significance being CRP > CK-MB > HDL > total cholesterol/HDL ratio > LDL/HDL ratio > fasting plasma glucose > lp(a) > total cholesterol > LDL > insulin > triglycerides > PAPP-A > leptin A positive correlation was observed between hs-CRP with PAPP-A levels ], insulin reistance (leptin), inflammation (hs-CRP) and matrix remodelling (PAPP-A), as possible predictors of plaque instability. This holistic view of plaque instability was the cardinal point of our study.in vitro and in vivo studies. These include influences on cytokine signalling mechanisms,26Our study revealed that serum leptin levels were significantly higher in the patient group than in the controls. Hyperleptinaemia has been implicated in the aetiopathogenesis of atherosclerosis in a number of studies.Serum leptin is also responsible for the stimulation of mitochondrial superoxide production28et al. reported a positive correlation between serum leptin levels and intima–media thickness in healthy individuals.et al. evaluated the levels of hs-CRP and leptin in atherosclerotic plaques and found leptin levels to be significantly higher in diabetics than non-diabetics.17Our findings are in accordance with studies carried out by many researchers. Serum leptin levels could predict adverse cardiovascular events, even after adjustment for the traditional risk factors, in the WOSCOPS trial.et al. reported a positive correlation between serum leptin levels and coronary artery calcium scores in healthy women.et al. concluded from their study that plasma leptin levels may represent a marker for adiposity, insulin resistance and vascular dysfunction, leading eventually to atherosclerosis.32Iribarren C-reactive protein level has been established as a determinant of the inflammatory process in atherosclerosis. In fact, hs-CRP levels have been correlated with disease initiation, progression and prognosis.34The present study also highlights the significance of hs-CRP levels in determining CAD. The ability of statins to decrease CRP levels has garnered interest in the recent years. The PROVE IT TIMI 22  study highlighted that the CRP levels after statin therapy were as efficient in predicting CAD as LDL levels.36Pravastatin therapy led to maximal benefit in patients with highest CRP values regardless of their LDL concentrations, according to the Cholesterol And Recurrent Events (CARE) trial.et al. concluded from their study that CRP was independently associated with CAD in Asian Indians after adjustment for Framingham risk factors and anthropometric measurements.41Anand et al., who measured PAPP-A levels in the cells and the extracellular matrix of unstable plaques in eight patients who had sudden death due to cardiac causes.13PAPP-A is an emergent biomarker signifying plaque instability. The role of PAPP-A as a biomarker for unstable atherosclerotic plaques was initially suggested by Bayes-Genis PAPP-A is a member of the metzincin metalloproteinase superfamily and is also produced by non-placental tissue such as fibroblasts, vascular endothelial cells and vascular smooth muscle cells. The circulating form of the protein comprises a heterotetrameric complex formed of two subunits of 200 and 250 kDa, bound by eosinophil major basic protein.Studies have revealed that PAPP-A contributes to plaque instability by promoting degradation of the extracellular matrix of the fibrous cap.et al. concluded that PAPP-A was a good predictor of both ischaemic cardiac events and the need for revascularisation in patients with ACS.et al. concluded from their study on patients with ACS that those with complex coronary lesions had significantly higher PAPP-A levels than patients who were free of such lesions.et al. evaluated PAPP-A levels in 547 patients with angiographically proven ACS. They found that elevated PAPP-A levels indicated an increased risk of plaque rupture, myocardial infarction, revascularisation and cardiovascular death in both troponin T-positive or -negative patients.51We observed significantly higher levels of PAPP-A in the patients compared to the control group. It also emerged as a good indicator of plaque vulnerability, just behind hs-CRP, with an area under curve of 0.732. Our findings corroborate the results of previous studies that have been carried out.Studies in patients with stable angina have proved the superiority of PAPP-A over troponin T and hs-CRP levels.et al.et al.57We also observed a statistically significant correlation between PAPP-A and CRP levels in patients with ACS. It has been reported that pro-inflammatory cytokines such as TNF-α stimulate PAPP-A synthesis by macrophages. The accumulation of activated macrophages at the site of unstable plaques leads to the release of PAPP-A by these cells.A similar correlation was observed between CRP and leptin levels. Obesity is associated with an inflammatory response characterised by high levels of pro-inflammatory cytokines.Coronary artery disease is the root cause of mortalities due to non-infectious aetiology. The initiation of florid plaque rupture and the consequent complications lead to various life-threatening sequelae of atherosclerotic disease. Hence, identification of plaque vulnerability at the incipient stages may help in effective and timely management.Currently, detection of plaque status involves invasive procedures such as coronary angiography and intravascular ultrasound, which have their own shortcomings due to their invasive nature and patient non-compliance, as well as financial constraints. Therefore the identification of a simple blood test that may aid in this endeavour is the need of the hour.We observed hs-CRP and PAPP-A levels to be effective indicators of plaque rupture. Interpretation of CRP data requires consideration of confounding factors such as inflammation.PAPP-A levels are not affected by infections or any underlying inflammation. Therefore assessment of PAPP-A levels may be a very attractive alternative for detecting unstable plaque activity and hence useful in stratification and prognostication of subjects. The ability of PAPP-A to detect plaque function even when the other markers of myocardial injury such as troponins are not elevated, is another justification for evaluation of PAPP-A level as a novel marker of plaque vulnerability. The comparison of PAPP-A and hs-CRP levels with the established biomarker troponin T needs to be evaluated and the results substantiated by studies carried out in large, population-based studies.


MicroRNAs, the non-coding single-stranded RNA of 19–25 nucleotides are emerging as robust players of gene regulation. Plethora of evidences support that the ability of microRNAs to regulate several genes of a pathway or even multiple cross talking pathways have significant impact on a complex regulatory network and ultimately the physiological processes and diseases. Brain being a complex organ with several cell types, expresses more distinct miRNAs than any other tissues. This review aims to discuss about the microRNAs in brain development, function and their dysfunction in brain tumors. We also provide a comprehensive summary of targets of brain specific and brain enriched miRNAs that contribute to the diversity and plasticity of the brain. In particular, we uncover recent findings on miRNA-128, a brain-enriched microRNA that is induced during neuronal differentiation and whose aberrant expression has been reported in several cancers. This review describes the wide spectrum of targets of miRNA-128 that have been identified till date with potential roles in apoptosis, angiogenesis, proliferation, cholesterol metabolism, self renewal, invasion and cancer progression and how this knowledge might be exploited for the development of future miRNA-128 based therapies for the treatment of cancer as well as metabolic diseases. MicroRNAs are small non-coding RNAs of 19–25 nucleotides in length and are known to regulate several protein-coding genes both in plants and animals. The first miRNA, lin-4 that controlled developmental timing in Caenorhabditis elegans was identified by two different groups in 1993-11. TheiMicroRNAs (miRNAs) interfere with target gene expression by binding to the 3′ UTRs of their target mRNAs and act primarily at the level of translation. Complete complementarity between miRNA and 3′UTR of its target leads to the degradation of mRNA targets as shown in plants whereas partial complementarity leads to inhibition of translation as seen in mammals-24. Litehttp://www.mirbase.org/), harbours 24521 entries representing hairpin precursor miRNAs and expressing 30424 mature miRNA products in 193 species. F. F278]. MiRNA-128 is encoded by two distinct genes, viz., R3HDM1 and ARPP21. Interestingly, insilico analysis of transcription factor binding sites of these two genes reveals almost similar pattern of transcription factors (unpublised data). This suggests that these two genes may presumably resulted from a gene duplication event. Until now, neither transcription factors binding proteins of hsa-miRNA-128 gene nor epigenetic factors, have been identified that interact with the regulatory region of this miRNA. However, Monteys et al. have recently suggested dual regulation of miRNA-128-2 by both intronic (pol III) and host gene (Pol II) promoters in acute lymphoblastic leukemia. The facThe authors declare that they have no competing interests.YKA and NS conceived the study. The survey of the literature and the inferences were made by YKA and NS. The bioinformatics analysis was carried out by YKA. The manuscript was drafted by YKA and NS. Both authors have read and approved the final manuscript.


Precise DNA-protein interactions play most important and vital role in maintaining the normal physiological functioning of the cell, as it controls many high fidelity cellular processes. Detailed study of the nature of these interactions has paved the way for understanding the mechanisms behind the biological processes in which they are involved. Earlier in 2000, a systematic classification of DNA-protein complexes based on the structural analysis of the proteins was proposed at two tiers, namely groups and families. With the advancement in the number and resolution of structures of DNA-protein complexes deposited in the Protein Data Bank, it is important to revisit the existing classification.etc.On the basis of the sequence analysis of DNA binding proteins, we have built upon the protein centric, two-tier classification of DNA-protein complexes by adding new members to existing families and making new families and groups. While classifying the new complexes, we also realised the emergence of new groups and families. The new group observed was where β-propeller was seen to interact with DNA. There were 34 SCOP folds which were observed to be present in the complexes of both old and new classifications, whereas 28 folds are present exclusively in the new complexes. Some new families noticed were NarL transcription factor, Z-α DNA binding proteins, Forkhead transcription factor, AP2 protein, Methyl CpG binding protein Our results suggest that with the increasing number of availability of DNA-protein complexes in Protein Data Bank, the number of families in the classification increased by approximately three fold. The folds present exclusively in newly classified complexes is suggestive of inclusion of proteins with new function in new classification, the most populated of which are the folds responsible for DNA damage repair. The proposed re-visited classification can be used to perform genome-wide surveys in the genomes of interest for the presence of DNA-binding proteins. Further analysis of these complexes can aid in developing algorithms for identifying DNA-binding proteins and their family members from mere sequence information. The driving forces for the cell to survive and regulate its various processes are the specific interactions between macromolecules. Protein-nucleic acid interactions are important for many high fidelity cellular processes. Both of these macromolecules are known to be involved in various important mechanisms and processes of systems biology- replication, transcription, translation, recombination, DNA-repair, DNA packaging etc. Therefore, DNA-binding proteins serve as the key players in maintaining cell viability and proliferation.Also, DNA-binding proteins constitute both eukaryotic and prokaryotic proteomes. The interplay between DNA and proteins is most fundamental interaction in biology and also has implications in the field of medicine, pharmacology and biotechnology. The diverse function of DNA-binding proteins is accompanied by the diversity in their sequences and structures.Therefore, to elucidate and understand the mechanism of any of the biological processes involving DNA-binding proteins, it is necessary and useful to study the nature of these nucleic acid-protein complexes formed in order to accomplish the specific function . There hetc., families having <50 members were subjected to Jack-knifing but if the members in family are >50, first clustering was performed and representative “seeds” from cluster(s) were chosen and individually as well as in combination from different clusters, were assessed for their coverage.Structures of DNA-protein complexes solved using X-Ray crystallography and resolution better than 3 Å were obtained from PDB. From this dataset, the protein DNA complexes having ssDNA or quadruple DNA was excluded from this classification (see Methods). Some of the complexes were ternary ones having two proteins and DNA molecule; these were split into individual chains and then considered for classification. Thornton and coworkers classified 230 complexes in 2000 and now approximately a four-fold increase in the number of complexes which needs classification was observed (1009 complexes).As of February 2010, 1354 protein DNA complexes were retrieved from PDB. Already classified complexes (241 (including ternary complexes), ..List of Click here for fileTernary protein-DNA complexes.Click here for fileThe number of complexes in both old and new classification possessing each of the common 34 folds.Click here for fileNew vs. Old representatives. 38/51* cases where old representative was still observed to have 100%coverage on the family even after addition of new members. *The number of old families here is 54 but here it is taken as 51 as the three polymerase families  were split in the new classification.Click here for fileRepresentatives for new families. New families with their selected representatives .Click here for filePairwise RMSD values obtained using MATT for all members of Homeodomain family belonging to HTH group.Click here for fileRPS-BLAST alignment of new member of Homeodomain family (1JGG) with Homeodomain representatives (1HDD and 1FJL).Click here for fileStructural superposition using MATT. Structural alignment for 1JGG (Homeodomain new member) with 1TRR (Trp Repressor representative), 1TC3 (Tc3 transposase representative) and 6PAX (Homeodomain representative)].Click here for fileUnique hits obtained by profile-based methods. Some examples of DNA-binding proteins identified using only profile-based searches are observed during genome-wide survey in Arabidopsis thaliana.Click here for file


In patients with Type 1 Diabetes (T1D) who develop microalbuminuria, progressive decline in glomerular filtration rate (GFR) may be initiated by leakage into the urine of toxic proteins (txUPs). This study tested this hypothesis.After archiving baseline urine, we followed T1D patients with microalbuminuria for 8–12 years to distinguish those in whom GFR declined (Decliners) and those in whom it remained stable (Non-decliners). Human proximal tubular cells (HK-2 cells) were grown in serum-free medium enriched with pooled urines from Decliners or Non-decliners. We determined genome-wide expression profiles in extracted mRNA.molecular functions of the 119 up-regulated genes were enriched for protein binding and peptidase inhibitor activities. Their biologic processes were enriched for defense response, responses to other organisms, regulation of cellular processes, or response to stress or stimulus, and programmed cell death. The 195 down-regulated genes were disproportionately represented in molecular functions of cation binding, hydrolase activity, and DNA binding. They were disproportionately represented in biological processes for regulation of metabolic processes, nucleic acid metabolic processes, cellular response to stress and macromolecule biosynthesis. The set of up-regulated genes in HK-2 cells overlaps significantly with sets of over-expressed genes in tubular and interstitial compartments of kidney biopsies from patients with advanced DN . The overlap included genes encoding chemokines and cytokines. Overlap of down-regulated genes was no more than expected by chance.The two pooled urines induced differential expression of 312 genes. In terms of gene ontology, in vitro by exposure to urine from patients with minimal microalbuminuria who subsequently developed progressive renal function decline, presumably due to putative txUPs.Molecular processes in tubules and interstitium seen in advanced diabetic nephropathy can be induced  Moderate elevation of urinary albumin excretion, referred to as microalbuminuria (MA), is the earliest indicator of diabetic nephropathy (DN) in Type 1 diabetes (T1D) serumInvestigation of systemic factors has identified several that contribute to the risk of early GFR loss, such as older age, elevated HbA1c, elevated blood pressure, high normal values of serum uric acid and elevated levels of circulating TNF-Rs Plausible, explanations for the different concentrations of these chemokines in the urine but not in the blood include differences in the rate of clearance of these chemokinases between Decliners and Non-decliners, as well as differences in sensitivity and detectability of the assays used. While the first possibility needs further study, the second possibility is unlikely considering the performance of the assays used in our study.An alternative explanation for the findings reported by Wolkow et al. in vitro study of the effects of urine on gene expression profiles in human proximal tubular cells (HK-2 cells). We postulate that exposure of HK-2 cells to urine from Decliners would induce a different gene expression profile than that induced by exposure to urine from Non-decliners due to the presence of putative txUPs in the former and their absence (or lower concentration) in the latter. The differentially expressed genes may reveal disease processes taking place in tubules during the development of early GFR decline in T1D.Here we report an in vitro in HK-2 cells. Cells were exposed to urine from patients with severe proteinuria or urine from patients with focal glomerulosclerosis and patients with idiopathic minimal change disease, and the results were compared Earlier expression studies include the analysis of candidate genes or candidate pathways (biased approach) in kidney tissue from diabetic rodent models and diabetic humans Recently, unbiased genome-wide transcriptome approaches have been applied to kidney tissue from patients with DN. Two studies catalogued gene expression profiles in microdissected human renal glomerula and tubules and interstitium obtained from kidneys with advanced DN and individuals who were healthy or diagnosed with minimal change disease. Gene expression was correlated with eGFR in the examined patients The Committee of Human Studies of the Joslin Diabetes Center approved the study, its protocols and informed consent procedures. Informed consent was obtained in writing from all participants involved in the study.in vitro effects of the putative txUPs on gene expression profiles in HK-2 cells using urine specimens obtained from two sets of patients; those who developed early eGFR decline during follow-up (Decliners) and those with stable eGFR during follow-up (Non-decliners).This study examined All patients were Caucasian participants in the follow-up study titled “The Joslin Study on Natural History of Early Nephropathy in Type 1 Diabetes”. The urines for this project were obtained from patients who developed new onset MA and were subsequently followed for 8–12 years. Findings regarding changes in urinary albumin excretion and trajectories of renal function decline in these patients were reported by Perkins et al. and Merchant et al. For the current study we identified 17 patients for whom we had urine specimens that met the following criteria. The specimen was obtained 2–5 years after onset of MA, and at least 10 ml of archived urine remained. Serial measurements of cystatin C in sera obtained during follow-up were used to trace the trajectory of estimated GFR. The patients were classified as Decliners and Non-decliners according to criteria described previously Tests for contamination with endotoxins  eliminated two specimens. From the remainder, we selected five from Decliners and five from Non-decliners whose characteristics were most similar see . These uTo reduce the study cost, we pooled the concentrated urines from Decliners and Non-decliners into two samples, thereby reducing the number of RNA specimens to be processed from 60 to 12. The urine pools were added to serum-free K-SFM medium  to give a final urinary albumin concentration that was 2.0 times that in the original samples. Glucose concentration was 25 mMol in the culture medium, which was changed every other day.Cultured HK-2 cells   were exposed to K-SFM serum free medium combined with pooled urine from either Decliners or Non-decliners. Each experiment consisted of four cell cultures. Two were exposed to pooled urines from Decliners, one for 6 hours and one for 24 hours. Similarly, two were exposed to pooled urines from Non-decliners, one for 6 and one for 24 hours. The experiments were repeated three times several weeks apart.After completion of all experiments, RNA was isolated from the HK-2 cells with RNeasy Micro Kit . The twelve specimens were analyzed for RNA quantity and normalized. cDNA was synthesized and hybridized with the Illumina Sentrix® Beadchip Array Human-6  to obtain gene expression profiles. The chip had 24,000 probes that covered 19,600 genes. Array hybridization, washing, and scanning were performed by the core facility at the Enders Institute, Children's Hospital, Boston, MA according to Illumina-recommended protocols .www.dchip.org). Raw intensity values were normalized with an invariant set normalization method Raw expression data were analyzed with dChip software (http://www.genomatix.de). The report for each annotation included the observed and expected numbers of genes, the p value, and the total number of genes in the annotation. Because proteins frequently have different molecular functions and involvement in multiple biological processes, the same gene may be included in several different GO terms. To reduce redundancy (one gene present in many downstream terms) and gain an overview of general biology, significant GO terms for molecular function (MF) (p<0.01) were retrieved upstream towards its ancestors based on the GO Inferred Tree View  in a manner similar to that described in DAVID: Database for Annotation, Visualization, and Integrated Discovery −6 were selected for analysis. As was done for molecular function terms, BP terms are presented at the fourth level downstream from the start of a biological process ). In this report we considered only GO terms that had 5 or more genes observed.Significantly regulated genes were analyzed for enrichment of gene ontology (GO) terms  using Genomatix pathway system software . After 6-hour exposure 274 genes and after 24-hour exposure 154 genes were differentially expressed. Normalized expression of these genes is illustrated in After normalization of the expression data, we applied pre-defined criteria to identify expression differences: a fold-change (≥1.3 or ≤0.77) and a nominal p-value . Among the differentially expressed genes we found GO terms significantly enriched at level 5 for molecular function and biological processes. As described in Methods, to reduce redundancy (one gene present in many downstream terms) and gain an overview of general biology, the GO terms were retrieved upstream towards their ancestors up to level 4. For this analysis we considered GO terms that were significant at p<0.01 at level 5. Since there were hundreds of terms for biological processes significantly enriched at this level, we restricted analysis to the 20 terms with the lowest P-values (p<10−6).To identify GO terms  that are enriched in HK-2 cells in response to exposure to Decliner urine, we analyzed differentially expressed genes with Genomatix pathway system software .The 10% of genes with the strongest negative correlations with eGFR were deemed up-regulated: 1260 in the data of Woroniecka et al. The up-regulated genes in HK-2 cells that overlap with the biopsy subsets of genes are listed in The risk of early GFR decline in T1D patients with MA is associated with elevated concentrations of several urinary chemokines, such as IL-6, IL-8, MCP-1(CCL2), IP-10 (CXCL10) and MIP-1δ (CCL15) CCL2 (MCP-1), IL-6, and IL-8, and others such as CCL5 (RANTES), CCL20 (MIP-3a), CSF2 (GMCSF), CXCL2 (MIP-2a), and CXCL3 (MIP-2b) that were not examined by Wolkow et al. Over-expression of these genes in HK-2 cells is a coordinated defense response to other organisms or to stress and is part of programmed cell death. It most likely accounts for elevated levels of these chemokines in urine.Results of this study in HK-2 cells support our hypothesis. Following exposure to the urine of Decliners whose renal function later declined, expression of 314 genes differs from their expression following exposure to urine from Non-decliners whose function remained stable. Urine from Decliners led to up-regulation of 119 and down-regulation of 195 genes. In a GO analysis of the up-regulated genes, two molecular function terms are significantly enriched. One includes receptor binding terms, and the second includes peptidase inhibitor activity. With regard to GO terms for biological processes, up-regulated genes are enriched for the following processes: defense response, response to other mechanisms, regulation of response to stress, regulation of response to stimulus and programmed cell death. It is interesting that up-regulated genes include some that encoded for chemokines, including those reported by Wolkow et al. GO analysis of the down-regulated genes assigns them to molecular functions such as cation binding, hydrolase activity acting on ester bonds, and DNA binding. GO terms for biological processes assigns the down-regulated genes to the regulation of metabolic processes, nucleic acid metabolic processes, cellular macromolecule metabolic processes, cellular response to stress and macromolecule biosynthetic processes. Since all these genes were under-expressed one can infer that all the above processes are suppressed in HK-2 cells in response to urines from Decliners.An important goal of our study was to determine the extent of overlap between the expression profiles in HK-2 cells exposed to urine from Decliners and expression profiles in kidney tissue (tubules and interstitium) of patients with advanced DN and significantly impaired renal function in vitro studies, two processes are proposed to initiate tubulointerstitial disease. Enhanced protein intake by proximal tubule cells results in lysosomal rupture and direct tubule toxicity and stimulates synthesis of cytokine and chemokines that enhance the inflammatory response and activate fibrotic processes in the interstitial compartment What is the nature of txUPs? Many authors have investigated whether the putative txUPs might be the high concentration of albumin present in patients with overt proteinuria. Based on In a just published review Bonventre proposes the hypothesis that persistent tubular damage initiates inflammation of tubules and interstitium, loss of blood vessels and interstitial fibrosis and results in glomerulosclerosis that may lead to renal function decline Several chemokines/cytokines are excreted in excess in urine in patients with established DN TNFα is a pleiotropic cytokine that plays an essential role in mediating inflammatory processes in vitro studies was that the cultured cells were exposed to serum-free medium. This reduces exposure of cells to serum growth factors and high concentration of albumin present in serum. Note: the media contained high levels of glucose. Fifth, we used immortalized tubular epithelial cell line HK-2 in our experiments. It is unknown whether the findings would be the same if primary cultures of tubular epithelial cells had been used.Finally, we ought to acknowledge the strengths and possible shortcomings of our study. First, application of global transcriptome analysis, rather than an analysis of selected pathways (biased approach), is a strength. This strength, however, was somewhat compromised in the search for overlap of our set of up- or down-regulated pathways in HK-2 cells with the corresponding sets of genes in tubular/interstitial compartments of kidney biopsies previously reported Table S1List of up-regulated and down regulated genes in HK-2 cells exposed to urines from Decliners and gene differentially expressed in tubular and interstitial compartment of kidney biopsies obtained from patients with advanced diabetic nephropathy and reported recently .(DOC)Click here for additional data file.


In severe mitral regurgitation, a subset of patients who are asymptomatic may develop left ventricular decompensation before changes in echocardiographic parameters become evident. Since N-terminal brain natriuretic peptide (NT-proBNP) is used to detect early heart failure, we hypothesised that NT-proBNP would be activated in patients with mitral regurgitation.Patients submitted to surgery were prospectively evaluated over eight months in the Department of Cardiology at Inkosi Albert Luthuli Central Hospital. Control patients with severe mitral regurgitation were obtained from the outpatient clinic. In order to define their value in identifying left ventricular decompensation, NT-proBNP levels and tissue Doppler imaging (TDI) indices were simultaneously measured and compared with conventional echocardiographic indices at baseline and this was repeated at one week and at six weeks after valve replacement.p = 0.0001). The diastolic E-mitral/E-annulus ratio, measured using TDI, was higher in the study group, indicating higher left ventricular filling pressure present in the study group. NT-proBNP levels increased further at one week after surgery and subsided at the six-week follow-up visit to levels similar to the control group. The TDI diastolic ratio also decreased at one week, and increased slightly again at the six-week follow up. These changes were accompanied by significant reduction in left atrium and left ventricular chamber dimensions with an increase in the ejection fraction from one to six weeks.Mean NT-proBNP levels were markedly elevated pre-operatively in all surgical cases compared to controls (Marked differences in mean NT-proBNP levels and TDI ratios between the study and control groups suggest that using TDI and NT-proBNP assays may detect covert left ventricular decompensation. Chronic organic mitral regurgitation (MR) has a variable course and requires careful monitoring by the clinician. Symptoms often occur late due to the compliance properties of the left atrium that allow it to accommodate large volumes of blood without a significant rise in pressure. As the regurgitation becomes more severe, contractile dysfunction may precede the onset of symptoms associated with disease progression as the ejection fraction (EF) declines but it may still remain in the normal range. An EF less than 60% has been shown to be associated with poorer survival rates after corrective surgery and is likely to indicate covert contractile dysfunction in MR patients.1Although numerous qualitative and quantitative echocardiographic modes have been developed, previous studies have demonstrated that existing measures of severity of MR correlate poorly with clinical signs and symptoms.The development of contractile dysfunction and its relation to the severity of volume overload in MR is not clearly understood. Prolonged contractile dysfunction eventually becomes irreversible even after the MR is corrected and is predictive of both congestive heart failure and death.In this study we evaluated tissue Doppler imaging and NT-proBNP levels in patients with severe chronic MR and determined their time-course patterns following mitral valve replacement.The study population was selected from Inkosi Albert Luthuli Central Hospital, Durban. Informed consent was obtained from each patient, and in the case of minors, from the parent or legal guardian. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki. Ethics approval was given by the Biomedical Research Ethics Committee at the University of KwaZulu-Natal, Nelson R Mandela School of Medicine (Ref No. H112/06).2.The study enrolled patients prospectively from February to September 2007. Patients with severe, chronic isolated MR underwent comprehensive quantitative Doppler echocardiography performed by one trained echocardiographer (RP). Patients were excluded if they had acute MR, MR due to ischaemic heart disease or cardiomyopathy, previous valve surgery and associated aortic or congenital valve disease. Patients with associated mitral stenosis were excluded if the valve area was less than 2.0 cmClinical evaluation and management of the patients were conducted by their independent clinicians. Assessment of symptoms was determined clinically by the New York Heart Association (NYHA) classification, and atrial fibrillation was evaluated by electrocardiography. Doppler echocardiographic recordings and blood samples were collected simultaneously and estimation of the NT-proBNP levels were processed independently.Control subjects with severe MR were selected from the cardiology outpatient follow-up clinic where they were assessed as not requiring surgery in the short term and were receiving medical therapy. Controls were selected as severe MR, but without evidence of ischaemic heart disease, as it is known that NT-proBNP is activated in the presence of ischaemia.3et al.Colour Doppler echocardiography was performed on all patients using a Siemens Sequoia machine . Dimensions and wall thickness were measured according to the American Society of Echocardiography guidelines using the leading-edge method,TDI was performed in the apical four-chamber view. The myocardial systolic wave (Sm) velocity, the diastolic indices, namely early myocardial (Em) and atrial contraction (Am) peak velocities, Em/Am ratio, and early diastolic filling ratios  (Em/Ea ratio) were measured. The ratio of the E wave across the mitral valve to the annulus E wave on tissue Doppler (E/Ea) was used as an estimate of the LV filling pressure. All echocardiographic measurements were performed by a single echocardiographer (RP) blinded to all other measurements. The images and measurements were reviewed off-line by a trained cardiologist (DPN). The intra-observer variability for the measurements of proximal isovelocity surface area (PISA) and TDI was < 5%.For measurement of NT-proBNP, venous blood samples were taken in gel-filled tubes with the patient resting quietly at the time of echocardiography. Additional samples were taken from subjects undergoing mitral valve replacement at one and six weeks post surgery.t-test or chi-square tests. Associations of baseline NT-proBNP were tested with linear and non-parametric regression . The time course of NT-proBNP within the cases was evaluated using paired t-tests. The discriminating capacity of the NT-proBNP for separating surgical cases from controls was assessed by the construction of ROC curves. Bivariate analysis was performed to assess the ability of the different parameters in predicting a favourable outcome using an NT-proBNP level of 50 pmol/l.SPSS for Windows version 15.0 was used for the statistical analysis. Clinical variables were normally distributed and expressed as mean ± SD. NT-proBNP levels were log-transformed for statistical analysis. Group comparisons were performed with ANOVA, A total of 54 patients with severe rheumatic mitral regurgitation were enrolled in the study and surgical cases were followed up for six weeks. Their baseline characteristics are shown in Only one patient in the control group had markedly elevated systolic pressure (63 mmHg) together with NYHA class III symptoms. This patient subsequently had a valve replacement seven months after the echocardiographic and NT-proBNP assessment.p = 0.04). A similar pattern was observed with NT-proBNP level, which was elevated in both groups, but was markedly higher (p < 0.001) in the study group The orifice area was markedly increased in 12 patients in the study group The immediate post-operative period showed a rise in NT-proBNP levels from pre-operative levels (262 ± 224 pmol/l) to a mean of 395 pmol/l at one week, and subsiding thereafter to 94 pmol/l at six weeks Four patients exhibited persistently elevated NT-proBNP levels at six weeks compared to the other study cases. The ejection fraction fell in three of these patients post-operatively, and was accompanied by little change in the end-systolic dimension (ESD) and NT-proBNP values compared to pre-operative levels. There was an increase in the diastolic filling ratios (Em/Ea) and a decrease in the TDI systolic wave. The fourth patient had a large drop in the NT-proBNP level, as well as an increase in the TDI systolic wave, in keeping with the preserved LV contractility. There was a small decrease in the ESD post-operatively A ROC curve was constructed for each variable to assess its discriminating capacity to distinguish between surgical cases and controls. The area under the curve was highest for NT-proBNP; this was the only parameter that approached 90% p = 0.014), tissue Doppler S wave (p = 0.049) and left atrium (LA) size (p = 0.027), all pointing to the interrelationship between severe regurgitation, systolic function and NT-proBNP levels.A second ROC curve This is one of the first studies to use new modalities of measuring left ventricular function with TDI and NT-proBNP assays. NT-proBNP level has been shown to be a marker of left ventricular dysfunction and has been used to predict diastolic abnormalities in patients with normal systolic function,et al.In a study similar to ours, Pillai Two other studies have examined patients with varying degrees of MR, and showed that changes in ventricular function occur early in the disease process, even before they could be detected echocardiographically.The six-week correlation between NT-proBNP levels and chamber dimensions suggests that in MR, changes in volume load may be paralleled by changes in the NT-proBNP level and that the fall in NT-proBNP was related to corrections in volume and removal of the diastolic run-off into the left atrium. These results are also in keeping with those found by the Mayo Clinic,Increased Em/Ea (> 12) ratios and elevated NT-proBNP (> 170 pg/ml) have been shown to be useful parameters to identify patients at increased risk of developing paroxysmal atrial fibrillation (AF) as well as to reflect early left ventricular dysfunction.The challenge in evaluating mitral regurgitation is a determination of what really constitutes normal ventricular function in these patients. The limitations of using the ejection fraction in the timing of surgery become clear in subjects with apparently normal ejection fraction and minimal symptoms.et al.In a study of 84 asymptomatic patients who underwent surgical correction for MR, Agricola Left ventricular contractile dysfunction is present in many patients with severe MR despite a normal ejection fraction and returns to normal after corrective mitral valve surgery in most but not all patients.19In our study, 15 (15/27) of the patients referred for surgery had ejection fractions above 60% and ESD values below 45 mm, indicating that the reason for surgery in these patients was the presence of significant symptoms while on medical treatment. There is an inherent subjectivity in defining significant valve-related symptoms. Indeed, when the second ROC curve was constructed using NYHA class as the determinant, NT-proBNP again emerged as the strongest discriminator of advanced NYHA class.The low specificity of 45% for NT-proBNP indicates a high number of false positive cases, i.e. patients selected by NT-proBNP level as requiring surgery when in fact they were still being followed up in the clinic on medical therapy. We believe that what is considered the high false-positive rate with NT-proBNP actually indicates that many of these control subjects with or without minimal symptoms actually required surgery, rendering them true positives. Waiting for more advanced symptoms or change in dimensions increases the risk of left ventricular dysfunction postoperatively. Two studies, both from the Mayo Clinic, have highlighted the poor outcomes in patients with severe MR who were managed conservatively rather than surgically.21et al.In another study, Pizzaro Most of the subjects in our control group had values above this level, indicating that all our patients were at risk while being followed up at the clinic. We attributed this to the subtle nature of symptoms, or particularly in our study, lack of awareness (on the part of patients or patients’ caregivers) of worsening symptoms and poor referral guidelines from peripheral healthcare centres. This low rate of intervention has also been reported in other centres as well.24In our study, no parameter could discriminate pre-operatively between the four patients who exhibited persistently elevated NT-proBNP levels at six weeks and the rest of the sample. All four of these patients had atrial fibrillation pre-operatively, which persisted at six weeks and at six months. There was no indication of difficulties with myocardial preservation to suggest this as the cause for the decline in postoperative EF by almost 30% from normal pre-operative levels.One of the limitations of this study was that it was performed only in patients with severe MR. The study needs to be repeated in different subgroups with varying degrees of MR in order to determine the time course of NT-proBNP levels in the different stages of MR as well as in patients with ischaemic MR. Finally, we need to determine the association of NT-pro BNP levels in a longer follow-up study with hard end-points such as heart failure and death.et al.We have shown that decision making regarding the timing of surgery in this cohort of rheumatic heart disease patients was determined largely by advanced symptomatology, so that patients are referred to surgery late in the course of severe MR, with a risk of permanent LV decompensation. We propose that NT-proBNP level be an additional marker, particularly in less symptomatic patients, even if the EF is normal. In time, it may prove to be the composite marker for the assessment of LV decompensation. These findings support those of Detaint