WNK1 and WNK4 cause familial hypertension, the Gordon syndrome. WNK1 and WNK4 conserved noncoding regions were targeted to polymorphism screening using DHPLC and DGGE. The scan identified an undescribed polymorphic AluYb8 insertion in WNK1 intron 10. Screening in primates revealed that this Alu-insertion has probably occurred in human lineage. Genotyping in 18 populations from Europe, Asia, and Africa (n = 854) indicated an expansion of the WNK1 AluYb8 bearing chromosomes out of Africa. The allele frequency in Sub-Saharan Africa was ∼3.3 times lower than in other populations . Meta-analysis across three European sample sets  detected significant association of the WNK1 AluYb8 insertion with blood pressure . Gender-stratified analysis revealed that this effect might be female-specific , whereas no statistical support was identified for the association with male BP . In leucocytes, the expressional proportions of the full-length WNK1 transcript and the splice-form skipping exon 11 were significantly shifted in AluYb8 carriers compared to noncarriers. The WNK1 AluYb8 insertion might affect human BP via altering the profile of alternatively spliced transcripts. Hum Mutat 32:1–9, 2011. © 2011 Wiley-Liss, Inc.Mutations in  Essential hypertension is a complex disease promoted by an unfavorable combination of person's life style and heritable factors. It is a significant health risk leading to other cardiovascular and renal diseases. Genetic studies of monogenic, Mendelian forms of hypo- and hypertension have identified ∼20 rare mutations in blood pressure regulating genes with a strong effect on the phenotype  were purchased from Applied Biosystems, Inc. . Primers and probes for the WNK1 transcripts lacking exon 11 (ex−11+12) and both exons 11 and 12 (ex−11−12) were designed using Primer Express version 3.0 (Applied Biosystems Inc.). Oligonucleotide sequences are given in Relative expression analysis of three The real-time RT-PCRs were performed using Applied Biosystems 7900HT Fast Real-Time PCR system in 96 microwell plates. Target region and endogenous control were amplified in the same well. The experimental conditions for the real-time PCR are given in detail in http://www.langsrud.com/fisher.htm). The significance of the associations between the WNK1 AluYb8 insertion and BP  as a quantitative trait was tested using linear regression (additive genetic model) with age and gender as covariates. Additive genetic model assumes a trend per copy of the minor allele to contribute to the trait or disease susceptibility on genotype categories. Association with the diagnosis of hypertension as a binary trait was assessed by logistic regression adjusted for age and sex. Association tests and calculation of LD between SNP pairs (r2) were implemented in the PLINK software, version 1.04 (http://pngu.mgh.harvard.edu/∼purcell/plink/). The Bonferroni threshold for multiple testing correction was estimated 0.05/9 = 5.56 × 10−3, taking into account the number of tested phenotypes (three) and tested study samples (three). Results were combined in a meta-analysis using the inverse-variance method under fixed-effects model using R, version 2.7.2 .Statistical differences in allele frequencies between populations were calculated using the Web-based Fisher's Exact Test calculator (HPRT). For every study subject six replicate values of relative expression per each alternative WNK1 splice form  was calculated. As each of the genotypes  was represented by three individuals, in total 18 data points were collected per transcript within a genotype group. The most outlier Ct value within the respective genotype group was excluded from the statistical testing. Differences of normalized expression values between alternative genotype groups were estimated by Wilcoxon rank sum test implemented in R software.Normalized expression values of target regions were calculated using Microsoft® Excel®-based software Q-Gene [Muller et al., WNK1  and the WNK4 (11 CNRs) genes (WNK4 and six SNPs in the WNK1 gene (DHPLC and/or DGGE assays were designed for screening novel polymorphisms in CNRs of the s) genes . Based oNK1 gene . All butWNK1 CNRs, a novel unreported common indel (∼300 bp) was detected (AluYb8 element (288 bp without flanking T nucleotides) into a poly-T tract within WNK1 intron 10,∼780 bp upstream from exon 11  studied population samples. Frequency of the AluYb8 insertion in human populations differed based on their geographic affiliation  compared to North-African , European , and Asian . On average, the allele frequency of the WNK1 AluYb8 in Sub-Saharan Africa was ∼3.3 times lower than in other studied populations (P = 9.7 × 10−9).The d Africa . The geniliation . The proWNK1 intron 10 in 11 chimpanzees, 1 gorilla, and 1 orangutan revealed that the WNK1 AluYb8 insertion has most probably occurred in human lineage. No AluYb8 insertion was detected in the WNK1 intron 10 of the studied primate genomes (WNK1 genomic fragment (exon10/intron10/exon11) amplified from a chimpanzee and from human wild-type as well as AluYb8 insertion carrying chromosomes revealed high conservation of intron 10 , exon 11 (459 bp), and intron 10 , respectively. Overall substitution rate was 0.7 and 1.3% between WNK1 exons and introns, respectively. The uniqueness of the WNK1 AluYb8 insertion in human was supported by a negative result of the BLAST search among available genome sequences.The analysis of the  genomes . Comparantron 10 . The subWNK1 AluYb8 insertion was tested for association with BP in the Estonian HYPEST cohort subjects . The analysis detected significantly higher SBP  and DBP , whereas no statistical support was found for the association in men . We also observed higher WNK1 AluYb8 frequency among HYPEST essential hypertension patients  compared to normotensive controls , we performed Stage 2 replication testing in two independent European samples: the BRIGHT (the British) and the CADCZ (Czech)  improved significantly the support for the association with BP . The pronounced effect of this Alu-insertion on BP in women was confirmed . Detected associations of the WNK1 AluYb8 insertion with SBP in the full sample and with female SBP and DBP remained significant after stringent correction for multiple testing .To confirm the discovery association between the  (Czech) . Stage 1 (Czech) . Meta-anWNK1 AluYb8 was observed in CADCZ hypertensive patients compared to controls (17.1 vs. 15.3%), but not in the BRIGHT cases representing extreme family based hypertension . However, AluYb8 was in strong LD with three previously genotyped WNK1 SNPs in the BRIGHT resource: rs11064527 , rs12816718 , and rs956868  , the strongest association was detected with AluYb8  . Amino ain human . When ash AluYb8 .WNK1 gene codes for a high number of mRNA transcripts and extensive alternative splicing has been described for exons 9, 11, and 12 [Verissimo and Jordan, WNK1 transcript completely lacks exons 11 and 12 [Delaloy et al., AluYb8 insertion on the expression profile of WNK1 alternative transcripts, we quantified the gene transcripts in mRNA extracted from the leucocytes of nine women with alternative genotypes  and ex−11−12  was upregulated among AluYb8 −homozygous carriers (P = 3.56 × 10−2). We conclude that the carrier status of the AluYb8 insertion may have an impact on the profile in WNK1 transcript in leucocytes.The human enotypes . The stu exon 12 . CompareWNK1 and WNK4 to polymorphism screening in order to identify functional variants potentially contributing to BP determination. We identified a novel human-specific polymorphic AluYb8 insertion in WNK1 intron 10. The AluYb8 insertion belongs to a young Alu subfamily represented with ∼2,200 copies in the human genome compared to only nine insertions detected in chimpanzee [Gibbons et al., WNK1 AluYb8 insertion in chimpanzee, gorilla, and orangutan . The carriers of the WNK1 Alu insertion had a consistent tendency for higher blood pressure  and no association was detected in men. Consistent with our findings, a sex-specific effect on BP determination was recently shown for a WNK1 SNP in intron 1 (rs10774461), which was also associated with BP only in females [Padmanabhan et al., Alu insertion (rs4646994) located in intron 16 of the ACE (angiotensin converting enzyme) gene [Rigat et al., ACE Alu I/D variant is associated with the hypertension risk only in men and not in women [Higaki et al., Our study identified a significant association between pressure . NotablyWNK1 SNP rs765250 [Newhouse et al., AluYb8 and rs765250 in both samples, which may indicate independent effects of WNK1 intron 1 and intron 10 polymorphisms on BP. Interestingly, strong LD (r2>0.8) between AluYb8 and three previously studied WNK1 SNPs in the BRIGHT study alone  positioned AluYb8 on the WNK1 haplotype, was reported to show borderline associations with SBP and DBP (P≤9 × 10−2) and a strong association with 24-hr urine potassium (P<1 × 10−4) [Newhouse et al., P-values for the association with BP were the lowest for the AluYb8 insertion compared to SNPs in LD. Previously, rs956868 has been shown to exhibit suggestive effect on ambulatory SBP in Europeans (P<9 × 10−2) [Tobin et al., P<5 × 10−2) [Osada et al., AluYb8: +/+). Functional assays should bring understanding whether the detected association with BP is driven by one primary variant or by a combinatory effect of the haplotype-forming alleles.Previously, BP variation in the BRIGHT and the HYPEST subjects has been associated with the WNK1 AluYb8 insertion to the risk for developing hypertension. The initially observed but not confirmed higher proportion of AluYb8 carriers among hypertensives may have resulted from nonoptimal selection of HYPEST controls (biased to too young), and/or different recruitment strategies of hypertensive patients among studies .The design of the current study did not allow us to draw any conclusion about the contribution of the Alu elements are considered to be neutral residents of the human genome, an inserted copy of an Alu repeat could interrupt structurally or functionally important genomic regions and consequently affect the expression of a locus [Batzer and Deininger, Alu elements may alter gene expression through modulating alternative splicing, RNA editing, epigenetic regulation, and translation regulation [Cordaux and Batzer, Alu insertions have been identified [Belancio et al., AluYb8 insertion in WNK1 intron 10 on the expressional profile of WNK1 alternative transcripts. Splicing is an incompletely understood process carried out by large macromolecular complex spliceosome and directed by numerous regulatory elements located within exonic and intronic sequence [Black, WNK1 intron 10  is remarkably increased by the ∼300 bp AluYb8 insertion . Thus, we hypothesize that the presence of the AluYb8 insertion may disrupt the spatial intronic structure and/or disarrange the possible splicing regulatory sequences within WNK intron 10. Consequently, it may affect the splicing efficiency of the down-stream exons 11 and 12. As alternative splicing tends to be a tissue and developmental stage specific process [Xu et al., AluYb8 insertion on the expressional profile of WNK1 may vary in different tissues. The current study design was limited to addressing the effect of AluYb8 insertion on WNK1 expressional profile in leucocytes using a small number of samples. Further in vitro and in vivo studies using renal tissues would reveal the potential effect of this Alu-insertion on WNK1 expressional profile in kidneys, where it plays an important role in contributing to the regulation of ion transport.Although a majority of e Black, . The sizAluYb8 insertion in WNK1. This AluYb8 insertion showed significant replicated association with blood pressure and a potential effect on the expressional profile of alternative WNK1 transcripts in leucocytes.In summary, we identified a novel human-specific polymorphic


Optimal oxygenation level during post-cardiac arrest (PCA) care is currently undefined, and studies have suggested harm from hyperoxia exposure . We aime2 levels higher than 40 kPa [We conducted a prospective observational cohort study in 21 ICUs in Finland between 2009 and 2010. The Utstein Guidelines were used for collecting resuscitation and PCA care data, such as initial rhythm and delay to return of spontaneous circulation (ROSC). Measured arterial blood oxygen values during the first 24 hours from admission to the ICU were divided into the following predefined oxygenation categories: low (<10 kPa), normal (10 to 19 kPa), intermediate (20 to 29 kPa), and high (>30 kPa). Exposure to hyperoxia was defined as paO2 measurements during the first 24 hours was eight per patient. A total of 6% of patients experienced paO2 values higher than 40 kPa at any time during the first 24 hours. Average times spent in each time oxygenation category during the first 24 hours were as follows: low 14%, normal 69%, intermediate 14%, and high 2% of the time. Survivors spent less time in the low band (P = 0.029) and more time in the intermediate band (P = 0.029) compared with nonsurvivors. The median paO2 during the first 24 hours was higher in survivors than in nonsurvivors  but there was no difference in lowest  or the highest paO2 values . In separate multivariate models neither time spent in the low or the intermediate categories, or the median, highest or lowest paO2 was found to correlate with mortality.A total of 489 patients were included. The average number of paOIn this multicentre observational study we were unable to define an optimal oxygenation level during PCA care, but hypoxia seemed to be more harmful than hyperoxia. Exposure to hyperoxia was less common than in previous trials, and we were unable to confirm previous findings indicating an association with mortality.


In the section titled, "Overall survival and Progression free survival" within the Results Section the value of overall survival of patients at 3 years was incorrectly described as 68.7%. The correct value is 40.6%.


To assess the effectiveness and functional capacity with rheumatoid artritis treated with biologic after dose reduction.A retrospective study on a cohort of 13 patients diagnosed rheumatoid artritis treated with anti-TNF  and clinical remission (DAS28 < 2.6) in the last 6 mounths, in which reducing the dosing. Clinical activity was evaluated by clinical activity index (DAS28), for functional capacity was used HAQ . Others variables studied was visual analog scale (VAS). Analyzing before starting dose reduction, at 3 and 6 months.The study includes 13 patients, 12 female, with RF in 90% and erosions in 40%, treated with etanercept 10 and adalimumab 3. More of them (85%) had recieved at least 2 FAMES before biological. Dose reduction in the etanercept group was 2 weekly injections to one injection every 5 days. In the case of adalimumab the pattern used was an injection every 20 days.Of the 10 patients treated with etanercept 4 returned to their usual pattern, as well as 2 of the 3 patients with Adalimumab.The reduction of etanercept and adalimumab doses in patients with rheumatoid arthritis in clinical remission (DAS28 <2.6) appears to be effective, since it manages to keep the DAS28 and the VAS. Not so the HAQ in which if there are significant differences that support that after the patient expresses reduce doses higher score on the test. Although the results 6 of 13 patients (46.2%) return to your regular schedule by mutual self-referred as subjectively feel worse and increase in seizures of exacerbation.


Sprouty2, a negative modulator of RAS/ERK pathway, is important for regulating Fgf8 morphogenetic signal activity by controlling Fgf8-induced signaling pathways and positional information during early brain development.Early brain patterning depends on proper arrangement of positional information. This information is given by gradients of secreted signaling molecules (morphogens) detected by individual cells within the responding tissue, leading to specific fate decisions. Here we report that the morphogen FGF8 exerts initially a differential signal activity along the E9.5 mouse neural tube. We demonstrate that this polarizing activity codes by RAS-regulated ERK1/2 signaling and depends on the topographical location of the secondary organizers: the isthmic organizer (IsO) and the anterior neural ridge (anr) but not on zona limitans intrathalamica (zli). Our results suggest that  Proper embryonic development requires an accurately orchestrated complex network of interactions between signaling and transcription factors. Secreted signaling molecules (morphogens) organize fields of surrounding cells into molecular patterns and are tightly associated to the concept of positional information. This concept implies that a cell reads its position and determines its developmental fate/response according to a concentration gradient of these extracellular factors Fgf8 is expressed preferentially at the so-called secondary organizers The process of neurulation in vertebrates implies a major morphogenetic step for the initiation of brain regionalization. Localized signaling centers along the tube  and the morphogens emanating from them have a key role in refining the subdivisions of the embryonic brain. Among other morphogens, Fibroblast Growth Factors (FGFs) are a family of structurally related polypeptides with pleiotropic activities and are involved in a signaling system conserved from insects to humans Fgf8 transcription at early neural plate stages causes death of the entire mesencephalic and cerebellar primordia revealing a requirement for FGF8 signal in survival of neural progenitors Sprouty2  did not change. Surprisingly, at this developmental stage we still found a small portion at the most dorsal area of the isthmic constriction where Fgf8, Sef and En2 transcripts were expressed  and negative modulators of Fgf8 signaling such Mkp3 (n = 4/4) and Sprouty2 were not longer expressed at caudal mesencephalon . In these ablation assays the main signaling receptor for FGF8, FGF receptor 1  or Fgf8 negative feedback modulators Mkp3 (n = 7/7) or Sprouty2 (n = 3/3) before any treatment  showed no expression of reatment . The ablerritory . After Berritory ’. The stCre/+; FgfR1 flox/flox; Finally, as an attempt to exclude any sensitive receptor mechanism underlying this initial polarizing activity exerted by FGF8b signaling we conducted FGF8b bead implantation assays on ONTCs of mutant embryos where FgfR1 was conditionally inactivated in the midbrain-rhombomere 1 region  during these initial planar instructions of FGF8 signaling. We decided to deprive pharmacologically the E9.5 mouse neural tube from any endogenous secreted molecule to the extracellular space (including FGF8 protein). Brefeldin A (BFA) was chosen for the ability to inhibit protein secretion in mammalian and other eukaryotic cells by interfering with the function of the Golgi apparatus, resulting in dysregulation of membrane traffic in vivo by Immunohistochemistry in whole-mount E9.5 mice with specific antibody against FGF8. The results revealed FGF8 staining either at the neuroepithelial intracellular or at extracellular levels in cryostat sections transverse to the IsO constriction  in the mouse IsO, secrete the morphogen near the lumen of the neural tube but the gradient of secreted Fgf8 protein forms a gradient along the basement membrane.We therefore analyzed the contribution of the FGF8 feedback modulators . Indeed, this signal would probably come from the caudal part of the mesencephalon or the isthmus, needed for normal antero-posterior polarity of the epithelium and therefore would not be directly related to an FGF8 signal.The ability of a rapidly internalized receptor to signal after endocytosis is important to ensure the sufficient duration and intensity of signaling. However, this capacity requires receptors to remain active in endosomes and therefore able to di-phosphorylate ERK FgfR1 in the mesencephalon was not affected during the time of the experiments . However, this powerful approach faces the difficulty of dissecting the function of each modulator because of their redundancy in FGF8 signal modulation Mkp3 expression was concomitantly downregulated in the same domain where phosphorylated ERK1/2 was not immunodetected. On the contrary, expression of Sprouty1 and especially Sprouty2 was maintained. Moreover, during BFA treatment FGF8-bead implantation on caudal mesencephalon maintained ERK1/2 polarized activation, indicating that Mkp3 and probably Sef were not required in the specification of FGF8 differential positional planar induction activity in the mesencephalon. It has been proposed that SPROUTY 1/2 and SEF function synergistically to regulate Gbx2 expression in the anterior hindbrain , caused a mirror-like cerebellar tissue induction rostrally to the ectopic implanted source. Within our results we can conclude that those results were due to the first polarized ERK1/2 activity driven by the FGF8b signal Sprouty1/2) at both sides of the IsO epithelium would maintain basic FGF8 intracellular activity to extend and to equilibrate the long-range distribution of active ERK1/2 along the A-P axis Then, what molecular mechanisms are behind this unbalanced activation of ERK1/2? Mutant mice have been used to understand the function of FGF8 negative feedback modulators in the mouse brain  are predominantly expressed at the ventricular zone in E11.5 mouse embryos Sprouty2 in the isthmus independently of ERK phosphorylation Sprouty2 gene expression pattern was maintained in the absence of ERK1/2 activity provide new horizons of FGF8 function. In conclusion, FGF8 may exert distinct signal responses depending on its cellular localization. These differential planar instructions may allow the segregation of neurogenic and proliferation signaling mechanisms or alternatively facilitate diffusion of FGF8-related activity through the basal lamina during vertebrate neural tube patterning Finally, the immunodetection of FGF8 protein assays in embryos and ONTCs revealed staining domains at the ventricular side and at the basal lamina see also . Interesgion see . In embrTimed pregnant mice were sacrificed by cervical dislocation and embryos were dissected in ice-cold phosphate-buffered saline . The embryonic day (E) 0.5 was the noon of the day of the vaginal plug. The embryonic age was determined more precisely by counting the somites in which at E9.5 ranged between 21 and 29 somite pairs neo/null  background and genotyped as previously described by Trokovic et al., All animal manipulation and experimental procedures were performed accordingly to the directives of the Spanish and European Union governments (Council Directive 86/609/EEC) and approved by the Animal Experimentation Committee of the Institute of Neuroscience UMH-CSIC. Mice from ICR strain were used as wild type. The transgenic mouse strain Fgf8Heparin acrylic beads (Sigma-Aldrich H-5263) were rinsed in PBS and soaked in FGF8b solution  for 1 h at 4°C. FGF8b-soaked beads were rinsed three times in PBS and thereafter implanted in the neural tube explants cultures as previously described Bafilomycin A1  was used for blocking the lysosomal pathway and so, preventing FGF8 degradation after endocytosis Brefeldin A  was used for blocking release of exocytic vesicles content , dehydrated in an ascending ethanol series, and stored in 70% ethanol at 20°C before processing. Whole-mount ISH was performed according to Garda et al. 2001 protocol In the case of IHC procedure whole mount embryos where dissected in ice cold PBS and fixed in 4% PFA with phosphatase inhibitor tablets (Roche Diagnostics 04906837001) following companies protocol for 2–4 hours before starting procedure. In some cases and after ISH or immunostaining procedure, embryos or ONTCs were immersed in ascending sucrose to 30% concentration and then cut at 12 µm thick sections ONTCs in a cryostat at -26°C (Microm-ThermoFischer Scientific) for a cellular analysis.2O2) at 3% for 30 minutes to inactivate the endogenous peroxidase activity. Then after 3 washes in PBS-T, they were blocked with goat serum at 5% and bovine serum albumin  at 2% in PBS-T. Incubation of Rabbit anti-dpERK  was done overnight at room temperature (RT). For the immunodetection in toto of the di-phosphorylated form of ERK1/2, the primary antibody was incubated for three nights at 4°C. In the case of mouse anti-FGF8b  for 2 nights at 4°C. Then, several washes in PBS-T were done before 1 hour incubation with Anti-rabbit or Anti-mouse biotinylated secondary antibodies at 1∶300 . Afterwards, Avidin-Biotin Complex was added at 1∶300 for 1 hour and washed in PBS-T . Colorimetric detection in embryos, ONTCs and tissue sections were incubated with 3,3′-Diaminobenzidine  and 0,003% H2O2. In some cases we used combined protocols of ISH and IHC within the same tissue. Finally for immunofluorescence detection of mouse monoclonal anti-FGF8b in cryostat sections an anti-mouse Alexa-Fluor-594; (1∶500: Molecular Probes A-11032) was used for 1 hour at RT. DAPI staining  was used to visualize the nuclei of the cells. After all colorimetric detection, embryos ONTCs were washed several times in PBT. All images were photographed with Leica stereoscope (Leica MZ16FA) or an upright microscope (Leica DM6000B) for the cryostat sections, using a Leica DC500 camera or DCF350 camera for fluorescence images. All pictures were taken using Leica LAS AF software.Whole mounts embryos, ONTCs and cryostat tissue sections were rinsed 3 times in PBS 1× with 0,1% Triton (PBS-T) and then incubated with hydrogen peroxide (Hneo/null (Fgf8 hypomorph mice in this paper). We also thank Martineźs Laboratory Staff for technical support.We are grateful to Dr. Constantino Sotelo for insightful and helpful suggestions during preparation of the manuscript. We would like to thank specially Drs. Laura Lahti and Joan Galcerán for technical suggestions. We thank Dr. Gail Martin form kindly providing the mouse strain Fgf8Figure S1Maintenance of molecular isthmic organizer signal activity in mouse organotypic tissue cultures (ONTCs). Gene expression profile in mouse isthmic organizer by in situ hybridizations in mouse E9.5 ONTCs (A’-D’) in comparison to in toto mice of same age (A-D) after 6 hours of incubation. Mouse brain subdivisions at E9.5 ONTCs are described with the expression of Meis2 in blue compared to Fgf8 in red (A’) genes and one half of the explant. The transversal black dashed lines illustrate the boundaries depicted by the genes on the mouse brain tissue. B-D) FGF8 negative feedback modulators, Sprouty2 (B), Mkp3 (C), and Sef (D). Note the similarities of these genes with respect to that of Fgf8 expression but the wider territory occupancy of their signals when compared to that of Fgf8, arguing indirectly the long range of FGF8 signal activity through the neuroepithelium from organizer centers.(TIF)Click here for additional data file.Figure S2Expression pattern profile of Fgf8 mRNA versus FGF8 protein in mouse E9.5 embryo. An anti-FGF8b immunohistochemistry was made onto 12µm cryostat longitudinal sections to the isthmus (see drawing) to visualize the intracellular and extracellular FGF8b protein (see arrows for the expansion of the protein in C) and compared with the Fgf8 mRNA domain . Note that the FGF8b protein can detected either at the ventricular side and at the pial side Click here for additional data file.


TGFB1) T29C and TGF β receptor type 1 (TGFBR1) 6A/9A polymorphisms have been implicated in the modulation of risk for breast cancer in Caucasian women. We analyzed these polymorphisms and combinations of their genotypes, in pre menopausal breast cancer patients (N = 182) and healthy women (N = 236) from western India as well as in breast cancer patients and healthy women from the Parsi community . Western Indian women were characterized by a higher frequency of TGFB1*C allele of the TGF β T29C polymorphism (0.48 vs 0.44) and a significantly lower frequency of TGFBR1*6A allele of the TGFBR1 6A/9A polymorphism  as compared to healthy Parsi women. A strong protective effect of TGFB1*29C allele was seen in younger western Indian women . Compared to healthy women, the strikingly higher frequencies of low or intermediate TGF β signalers in patients suggested a strong influence of the combination of these genotypes on the risk for breast cancer in Parsi women . The frequency of low signalers in Parsi healthy women, while comparable to that reported in Europeans and Americans, was three times higher than that in healthy women from western India . These observations, in conjunction with the low incidence rate of breast cancer in Indian women compared to White women, raise a possibility that the higher frequency of TGFB1*29C allele and lower frequency of TGFBR1*6A allele may represent important genetic determinants that together contribute to a lower risk of breast cancer in western Indian women.Transforming growth factor β1 ( Germline mutations in various cancer susceptibility genes can account for only around 10% of all breast cancer cases TGFB1 T29C  and TGFBR1 6A/9A remain the two most extensively studied polymorphisms. In TGFB1 T29C polymorphism, a replacement of Leucine by Proline at position 29 (TGFB1 codon10 T>C) has been shown to result in an increased secretion of the cytokine TGFBR1 gene, giving rise to the TGFBR1*6A allele TGFBR1*9A allele In breast cancer patients, TGFB129*C allele that is associated with higher production of the cytokine, would provide protection in the initial stages by virtue of its anti-proliferative action on mammary epithelial cells but would enhance the risk at later stages by promoting invasion as well as spread of the disease TGFB1*CC genotype at a higher risk of the disease TGFB1*CC genotype in older American women and pre menopausal Japanese women TGBR1 6A/9A polymorphism on the risk for breast cancer TGFBR1*6A allele et alWith regard to the risk for breast cancer, it has been proposed that the presence of TGFBR1 6A/9A gene polymorphisms in pre menopausal women from western India. To that effect the study was restricted to Marathi-speaking (Maharashtrian) subjects from Western India, living within 200 km radius around the state-capital city of Mumbai (who constitute 80% of the population of the state of Maharashtra). In parallel, we have studied these polymorphisms in breast cancer patients and healthy women from the Parsi (Zoroastrian) community. Parsis in India represent a geo-ethnically isolated community Population based data regarding these polymorphisms in Indian women remains limited TGFB1*CC) on the risk for breast cancer, our results reveal trends that are opposite to those reported in Caucasian women. The basis for these observations and their implications has been discussed with due supporting literature.The comparison of genotypes in healthy subjects from the two ethnically distinct communities studied, and the comparisons between patients and healthy subjects within each of the groups have revealed novel trends with significant implications. Further, in terms of the influence of high TGF beta1 producing genotype  pre menopausal women with confirmed diagnosis of breast cancer treated at the Tata Memorial Hospital during 1999–2005 were recruited within twelve to eighteen months of diagnosis. Lack of family history for malignant disorders could be confirmed for 75% of the cases. Two hundred and thirty six unrelated, healthy premenopausal Maharashtrian women without family history for cancer were recruited from the community. Parsi women with breast cancer (N = 48) comprised of those treated in various hospitals in the city. Mean post diagnosis duration for these patients was 4.8±4.2 yrs (Median  = 3), with sixty two percent of the patients recruited within five years of diagnosis. Healthy, unrelated Parsi women (N = 171) were recruited from the community. Eight Parsi patients as well as ten healthy women volunteers reported a strong family history for cancer.The mean age was lower in healthy Maharashtrian controls than in patients . The available data indicated comparable age at menarche and age at first child birth in the two groups . In ParsTGFB1 T29C polymorphism was performed by PCR–SSP based method, using primers described by Perrey et alInternational Histocompatibility Workshop Group (IHWG) reference panel were used to validate the PCR conditions and as controls in each experiment. Results were further confirmed by DNA sequencing in representative samples. Genomic DNA was amplified using primers, GAGGCCCTCCTACCTTTTG (F) and GCAGCTTGGACAGGATCT (R) as described in the SNP500 cancer database http://snp500cancer.nci.nih.gov/sequencing_assays.cfm?snp_id=TGFB1-01. The amplified products were sequenced by the standard method using big dye terminator kit (ABI) on 3100AVANT Genetic Analyzer (Applied Biosystems).Isolation of DNA for genotyping was carried out as described in our earlier report TGFBR1 6A/9A genotyping, exon 1 of TGFBR1 was amplified using primers described by Kaklamani et al2+, 10% DMSO, 0.1% BSA and 0.5 units of Taq polymerase (Fermentas) along with 1.0 µM primers. PCR conditions were as follows −95°C for 5 min, 35 cycles of 94°C for 30 s, 65°C for 30 s, 72°C for 30 s, followed by extension at 72°C for 5 min. PCR products (256/247 bp) were analyzed on 2% agarose gel stained with ethidium bromide. The genotypes were also confirmed by running the products on 10% PAGE and by sequencing of representative samples.For TGFB1*29T and TGFBR1*9A, were taken as reference for the respective polymorphisms. For Maharashtrian subjects, the risk was determined after adjusting for age, and was expressed as odds ratio with 95% confidence interval. In each case, the odds ratios were computed for dominant, additive and recessive models. Further, various combinations of genotypes of these two polymorphisms were categorized into high, intermediate and low signalers TGFB1*CC and TGFBR1*9A/9A genotype combination were identified as high signalers in accordance with the high TGF β levels and strong TGFBR1 activity associated with these genotypes. Individuals homozygous for the allele associated with either higher TGF β levels or with strong signaling (TGFB1*CC or TGFBR1*9A/9A) were grouped as intermediate signalers, and subjects with the remaining combinations constituted low signalers. The risk was estimated for the latter two groups with high signalers as reference. SPSS software (version 15.0) was used for statistical analysis. Power calculations were performed using Quanto Chi-square test was used to find out if the genotype frequencies in controls are in Hardy-Weinberg equilibrium and also to determine the significance of difference in genotype frequencies between different groups. For various comparisons, two sided p values were calculated using Fisher’s exact test. To estimate the risk for breast cancer, the frequencies of subjects homozygous for the wild type alleles, namely Two hundred and twenty four healthy controls (95%) and one hundred and sixty (88%) premenopausal breast cancer patients from the Maharashtrian community could be genotyped for the two polymorphisms studied. Similarly, one hundred and sixty (94%) healthy Parsis and forty three Parsi patients (95%) could be genotyped and included in the analysis. The genotype frequencies for the two polymorphisms studied were in agreement with Hardy-Weinberg equilibrium in healthy subjects from both the communities.TGFB1*29C) in healthy Maharashtrian women was higher compared to that in healthy Parsi women .The frequency of the variant allele  was noted in Maharashtrian subjects for the dominant model only  and a protective influence of the TGFB1*CC genotype recessively, were not statistically significant  as reported by two groups In Maharashtrian subjects, the observed frequencies for TGFBR1*6A allele in Maharashtrian women compared to that seen in Parsis [2.0% vs 6.8%, respectively; p<0.01; power-67%) is also in contrast to data from Whites, where the frequency of this allele varies between six and eleven percent TGFBR1*6A allele observed in Maharashtrian women in the present study is the lowest reported so far.The strikingly lower frequency of TGFB1*29C allele (45% power), and a reduced risk for the disease especially in the younger age group . These observations are in direct contrast with those from the Breast Cancer Association Consortium TGFBR1*6A allele to the observed effect of the TGFB1*29C allele on the risk for the disease in Maharashtrian women cannot be ruled out.Our findings in Maharashtrian premenopausal women suggest an association between the presence of TGFB1 T29C genotypes with hormone receptor status in Maharashtrian patients. Of the one hundred and twenty two patients where the information was available, the hormone receptor negative tumor bearing subjects were characterized by a strikingly lower frequency of TGFB1*CC genotype compared to the hormone receptor positive group  with adequate power, sixteen thousand (dominant model) and forty six thousand (recessive model) patients and controls would have to be studied TGFB1*T29C polymorphism with the observed allele frequencies in the western Indian population, to detect a 15% change in the risk for breast cancer, nearly four thousand patients and an equal number of healthy controls would be needed to achieve a power of 80% (at α = 0.05). Similar computations for TGFBR1*6A allele indicate that for a fifteen percent increment in the risk for breast cancer The associations seen in our study however must be viewed with caution in view of the inadequate power of our study  & 4. TheTGFB1*29C allele and lower frequency of TGFBR1*6A allele in healthy Maharashtrian women was revealed when combinations of these genotypes were analyzed. Categorization of individuals into low, moderate and high TGF β1 signalers based on an approach described by Kaklamani et alvs 10.6%; p<0.01). A comparison of the frequencies of high, intermediate and low signalers among healthy subjects reported in literature  for breast cancer indicated for low and intermediate  in western Indian women suggested by our study and in White women by others In summary, the present study on Figure S1Analysis of association of TGFB1 T29C genotypes with hormone receptor status in Maharashtrian subjects. OR – Age adjusted odds ratios with 95% CI; n = 224; **p<0.01; *p = 0.05 for TGFB1*CC genotype.(TIF)Click here for additional data file.Table S1Distribution of various TGFB1 T29C genotypes with respect to ER/PR status.(DOC)Click here for additional data file.


Split-hand/foot malformation type 1 is an autosomal dominant condition with reduced penetrance and variable expression. We report three individuals from two families with split-hand/split-foot malformation (SHFM) in whom next generation sequencing was performed to investigate the cause of their phenotype.DYNC1I1 exons recently identified as limb enhancers in mouse studies from their target genes, DLX5 and DLX6. In the second family, X-linked recessive inheritance was suspected and exome sequencing was performed to search for a mutation in the affected proband and his uncle. No coding mutation was found within the SHFM2 locus at Xq26 or elsewhere in the exome, but a 106 kb deletion within the SHFM1 locus was detected through copy number analysis. Genome sequencing of the deletion breakpoints showed that the DLX5 and DLX6 genes are disomic but the putative DYNC1I1 exon 15 and 17 enhancers are deleted.The first proband has a de novo balanced translocation t identified by karyotyping. Whole genome sequencing showed that the chromosome 7 breakpoint is situated within the SHFM1 locus on chromosome 7q21.3. This separates the DYNC1I1 exonic enhancers in normal limb formation in humans.Exome sequencing identified a 106 kb deletion that narrows the SHFM1 critical region from 0.9 to 0.1 Mb and confirms a key role of  DLX5/6); double-knockout mice exhibit severe skeletal abnormalities and die shortly after birth,DLX5 has recently been reported to cause split-hand/foot malformation and hearing loss in a consanguineous family.DLX5/6 coding mutations leading to SHFM1 have been reported to date. Several chromosomal abnormalities have been reported in this region that lead to SHFM1; some do not affect the DLX5/6 genes and are thought to disrupt one or more regulatory elements, or result in a physical separation of such elements from the most likely target genes, DLX5/6. The smallest reported deletion is of 880 kb encompassing SLC25A13 and part of DYNC1I1, but leaving DLX5 and DLX6 intact.Dlx5 and Dlx6 are thought to be regulated by the transcription factor tumour protein p63TP63 mutations cause split-hand/split-foot malformation 4 . An enhancer element with a p63 binding site has been reported within the SHFM1 locus 300 kb proximal to Dlx5/6.3Split-hand/split-foot malformation is a congenital limb abnormality characterised by the absence of one or more median rays or digits that results in cone-shaped clefts of hands and/or feet. It is often accompanied by other limb anomalies including monodactyly, syndactyly, and aplasia or hypoplasia of the phalanges, metacarpals, and metatarsals. The malformation can be isolated or syndromic , OMIM #220600), and the severity can be variable between patients as well as between different limbs of the same individual. The condition is genetically heterogeneous with six loci defined, including the SHFM1 locus 7q21 (OMIM #183600) and SHFM2 locus Xq26 (OMIM #313350). The 7q21 locus contains the candidate SHFM genes distal-less homeobox 5 and 6 , a gene that is not expressed during limb development. DYNC1I1 eExon (exonic enhancer) 15 is marked by an enhancer chromatin signature and physically interacts with the Dlx5/6 promoter regions 900 kb distal to DYNC1I1, specifically in the limb.Slc25a13 (solute carrier family 25 member 13), driving the expression of Dlx5/6 in otic vesicle, forebrain, branchial arch and limb—a pattern that correlates with some but not all SHFM1 phenotypes observed in humans.6Recent studies in mouse and zebrafish models identified novel tissue-specific enhancers that correlate with limb, craniofacial and hearing phenotypes observed in individuals with chromosomal rearrangements.supplementary figure 1A) which showed that the chromosome 7 breakpoint is within 7q21.3, not 7q22. The precise breakpoints were confirmed by Sanger sequencing of PCR products spanning the breakpoints (see online supplementary figure 1B), and the translocation was defined as t. The chromosome 2 breakpoint is located within intron 1 of the ornithine decarboxylase 1 (ODC1) gene encoding the rate-limiting enzyme of the polyamine biosynthesis pathway. A polymorphism in this gene has been associated with colon cancer risk,DLX5/6 genes and 500 kb downstream of the DYNC1I1 exons 15 and 17 identified as exonic enhancers in mice and zebrafish (figure 2).We studied three patients with SHFM from two families. Informed consent was obtained from all participants or their parents. Proband 1 had split-hand/split-foot malformation A affecti tp2.1;q21.3. Biallelic mutations in SLC25A13 cause citrin deficiency, an adult- or neonatal-onset metabolic disorder, while homozygous Slc25a13 knock-out mice show no skeletal defectsDYNC1I1 gene encodes the intermediate chain 1 subunit of the cytoplasmic dynein motor protein complex, the primary motor protein responsible for retrograde axonal transport in neurons. Since this gene is not expressed in the developing limb a partial gene deletion is not predicted to cause split-hand/foot malformation. However, the deleted DYNC1I1 exons include the enhancers identified by Birnbaum et al.supplementary figure 1D). Sanger sequencing of a PCR product spanning the breakpoints confirmed a 105 935bp deletion; NC_000007.13:g.95704812_95810747del (see online supplementary figure 1E). The presence of the junction fragment was confirmed in the two affected individuals and also in four non-penetrant unaffected family members , and genital tissues, and the position of these functional enhancers compared to SHFM1-associated chromosome rearrangements suggests that their disruption might, in some cases, explain the additional clinical phenotypes such as hearing loss and craniofacial defects.SLC25A13 is an enhancer with activity in the otic vesicle , it is possible to accurately resolve translocation or deletion breakpoints. For proband 1 the genome sequencing was more accurate than metaphase chromosome analysis and resulted in a reassignment of the 7q breakpoint to the neighbouring chromosome band. The application of next generation sequencing for precise characterisation of breakpoints is likely to find clinical utility as it can show whether the coding region of a candidate disease gene is disrupted or, as in this study, identify the spatial relationship between regulatory elements and their target genes.et al21The use of paired-end whole genome sequencing to map rearrangement breakpoints to the exact nucleotide has previously been described for just seven patients.In summary, by using exome sequencing copy number analysis and whole genome sequencing to map deletion and translocation breakpoints, our study demonstrates that exonic enhancers recently discovered through mouse and zebrafish models are also critical for limb development in humans.


This phenotype has precluded analysis of Bmp7 function in the later stage of nephrogenesis. In this study, utilization of conditional null allele of Bmp7 in combination with systemic inducible Cre deleter mice enabled us to analyze Bmp7 function at desired time points during kidney development, and to discover the novel function of Bmp7 to inhibit the precocious differentiation of the progenitor cells to nephron. Systemic knockout of Bmp7 in vivo after the initiation of kidney development results in the precocious differentiation of the kidney progenitor cells to nephron, in addition to the prominent apoptosis of progenitor cells. We also confirmed that in vitro knockout of Bmp7 in kidney explant culture results in the accelerated differentiation of progenitor population. Finally we utilized colony-forming assays and demonstrated that Bmp7 inhibits epithelialization and differentiation of the kidney progenitor cells. These results indicate that the function of Bmp7 to inhibit the precocious differentiation of the progenitor cells together with its anti-apoptotic effect on progenitor cells coordinately maintains renal progenitor pool in undifferentiated status, and determines the nephron number at birth.The number of nephrons, the functional units of the kidney, varies among individuals. A low nephron number at birth is associated with a risk of hypertension and the progression of renal insufficiency. The molecular mechanisms determining nephron number during embryogenesis have not yet been clarified. Germline knockout of  Over the last decade, the impacts of prenatal conditions on health and disease in later life have been subjects of intense research The embryonic kidney is derived from the intermediate mesoderm and formed by a reciprocal induction between the cap mesenchyme and ureteric bud Bmp7 knockout mice exhibited aplastic kidneys with a few nephrons, leading to perinatal death Bone morphogenetic protein 7 (Bmp7) is a morphogen expressed in the kidney. Germline Bmp7 in the developing kidney were analyzed utilizing +/LacZBmp7 mice Bmp7 is widely expressed in cap mesenchyme, ureteric buds, and some derivatives of cap mesenchyme, podocytes and distal tubules. Given that various types of cells in the developing kidney express Bmp7, coupled with the fact that Bmp7 is a secreted factor, we surmised that the deletion of Bmp7 in any specific cell type might not result in a prominent phenotype. We therefore opted for ubiquitous global ablation of Bmp7 at desired time points as a strategy to define its role in kidney maturation.The expression domains of Bmp7 in combination with systemic inducible Cre deleter mice enabled us to analyze Bmp7 function at different time points during kidney development. While the knockout of Bmp7 before the initiation of kidney development recapitulates germline Bmp7 knockout mice , the knockout of Bmp7 after the initiation of kidney development allowed us to demonstrate the novel phenotype, precocious differentiation of progenitor cells. We also confirmed that Bmp7 inhibits differentiation of renal progenitor cells utilizing kidney explant culture and colony-forming assay. Taken together, we demonstrated for the first time that Bmp7 maintains nephron progenitor cells in the undifferentiated state, thereby influencing the number of nephrons at birth.In our study, the utilization of the conditional null allele of Bmp7 conditional knockout mice Cre (CreERT2Gt(ROSA)26Sor) mice. We bred +/LacZBmp7;CreERT2Gt(ROSA)26Sor mice with fl/flBmp7 mice to yield LacZ/flBmp7;CreERT2Gt(ROSA)26Sor (Bmp7 knockout) and +/flBmp7;CreERT2Gt(ROSA)26Sor (control) embryos. We knocked down Bmp7 expression through the administration of tamoxifen to pregnant mothers bearing both types of embryos. Because of the toxicity of the systemic expression of CreERT2 on hematological tissues in CreERT2Gt(ROSA)26Sor mice Bmp7 knockout and control embryos carry CreERT2.To analyze the role of Bmp7 in kidney development, we utilized Bmp7 in the kidney was approximately 80% (2). While the proliferation of metanephric mesenchyme was similar between the two genotypes (Bmp7 knockout kidneys (We administered tamoxifen to pregnant mice at embryonic day 12.5 (E12.5), a time when the epithelialization of cap mesenchyme has begun, and analyzed the embryos at E14.5 . Histological analysis revealed a significant reduction in the volume of cap mesenchyme in knockout kidneys (2), which was also confirmed by immunostaining of WT1, a marker for cap mesenchyme (and podocytes) (2). The proliferation of knockout kidneys appeared to be decreased 26SorCreERT2Bmp7 embryos at E12.5 and cultured for 72h in the presence or absence of 4-hydroxytamoxifen (4-OHT). The phenotypes of the explants treated with 4-OHT (knockout explants) were compared to those of contralateral explants treated with a vehicle (ethanol) (control explants). The administration of 4-OHT to wild-type kidney explants did not cause any noticeable changes (data not shown). The knockout efficiency of Bmp7 was approximately 90% in knockout explants .The epithelialization and subsequent differentiation of cap mensenchyme is the first step of nephrogenesis. We hypothesized that the precocious maturation of nephron segments in explants Figure 3We also investigated the effect of Bmp7 on the epithelialization of cap mesenchyme using a colony-forming assay, in which the coculture of single cells from cap mesenchymes of E11.5 kidneys with Wnt4 expressing feeder cells (3T3Wnt4) The addition of Bmp7 to this culture system significantly inhibited colony formation in a dose-dependent manner  of metanephric mesenchyme caused by the reduction of endogenous Bmp7, which is also supported by the reduction of Pax2. What we observed in conditional knockout mice, kidney explants and colony assay in this manuscript is the inhibitory effect of Bmp7 on the epithelialization of the induced metanephric mesenchyme, which is the next step after the induction of mesenchyme.Bmp7 expression at E12.5 permits the initial expansion of progenitor cells and enables us to observe the precocious differentiation of progenitor cells in the absence of Bmp7. This phenotype is novel, and could not be observed in germline Bmp7 null mice due to massive apoptosis of metanephric mesenchyme.The phenotype of germline knockout mice is the massive apoptosis of renal progenitor cells, which precludes the analysis of Bmp7 function on progenitor cell differentiation Bmp7 knockout kidneys is “earlier” or “faster” remains unproven. However, the results of kidney explants and colony-forming assay showed Bmp7 inhibits epithelialization of mesenchyme, which is the early step of differentiation, and hence, at least, the differentiation in Bmp7 knockout kidneys seems to occur “earlier”, while “faster” differentiation cannot be denied.Whether the differentiation in During tissue development, tissue-specific progenitor cells divide and populate the progenitor pool. The size of the progenitor pool as well as the final size of the tissues is determined by the balance between proliferation, apoptosis and differentiation of progenitor cells. Both massive apoptosis and precocious differentiation of progenitor cells causes depletion of the progenitor cells and reduces the final size of the tissues , indicating the possible contribution of Bmp7-Smad signaling pathway in the maintenance of renal progenitors. More recently, the maintenance of germ line stem cells by Bmp7 has been demonstrated in mouse embryos as well Interestingly, the maintenance of stem/progenitor cells by Bmp may be an evolutionally conserved mechanism in different tissue contexts. It is widely accepted that orthologues of Bmps, Decapentaplegic (Dpp) and Glass bottom boat (Gbb) maintain germ line stem cells via the phosphorylation of Mad, a homologue of Smad proteins in drosophila ovary and testis Bmp7 knockout mice: maternal, extrarenal, and intrarenal Bmp7. Previous reports demonstrated that maternal Bmp7 traverses the placental barrier and localizes in embryonic kidneys until day 14 of gestation +/LacZ;Gt(ROSA)26SorCreERT2Bmp7or fl/flBmp7, such that the amount of Bmp7 originating from the mother is unaltered by the administration of tamoxifen, excluding the possibility that the fluctuation of maternal Bmp7 levels contributes to the phenotypes in Bmp7 knockout embryos. Given the nature of systemic inducible CreERT2Gt(ROSA)26Sormice, the expression of Bmp7 in other tissues was also reduced after tamoxifen treatment, so that the circulating level of Bmp7 (if present) should be lowered. This might contribute to the phenotypes in Bmp7 knockout embryos to some extent. However, the accelerated differentiation of cap mesenchyme was observed in kidney explant culture, in which the ablation of Bmp7 occurs intrinsically within the kidney, indicating that intrarenal Bmp7 is primarily responsible for the phenotypes.There are several possible sources of Bmp7 that contribute to the phenotype in Bmp7 in distinct cell types manifests different spectrums of defects in the kidney. Thus, the loss of Bmp7 from podocytes results in the reduction of proximal tubules in postnatal kidneys Bmp7 knockout mice (this study) exhibits the accelerated maturation of nephrons. These comparisons indicate that there is a broad range of functions for Bmp7 in different cell types at different stages, and raise the possibility that Bmp7 secreted from one cell population functions locally in an autocrine or paracrine fashion to exert a unique function. This hypothesis is strengthened by a previous report indicating that the prodomain of Bmp7 tethers Bmp7 to the extracellular matrix (ECM) near the site of Bmp7 production and inhibits its passive diffusion Interestingly, the inactivation of Finally, our analysis demonstrates for the first time that Bmp7 plays a critical role in the maintenance of the renal progenitor population in the developing kidney, and thereby determines the final number of nephrons. Further analysis will elucidate the mechanism how intrauterine conditions affect the pathway, and provide a rationale for a therapeutic approach to prevent congenital deficit in nephron number.All animal studies were approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University (permission number: MedKyo 12522), and were strictly in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were sacrificed by cervical dislocation by well-trained researchers.Bmp7 conditional allele CreERT2Gt(ROSA)26Sor allele were generated at Regeneron Pharmaceuticals using Velocigene™ technology as described elsewhere +/LacZBmp7 knock-in allele has been described previously +/LacZ;Gt(ROSA)26SorCreERT2Bmp7 mice with fl/flBmp7 mice to yield LacZ/fl;Gt(ROSA)26SorCreERT2Bmp7 (Bmp7 knockout) and +/fl;Gt(ROSA)26SorCreERT2Bmp7 (control) embryos. Due to the hematological toxicity of CreERT2Gt(ROSA)26Sor mice CreERT2 allele.The LacZ/fl;Gt(ROSA)26SorCreERT2Bmp7 and +/fl;Gt(ROSA)26SorCreERT2Bmp7 embryos.Tamoxifen  was dissolved in a sunflower oil/ethanol (9:1) mixture at 30 mg/ml. For activation of CreERT2, the tamoxifen solution was administered at a concentration of 150 mg/kg by intraperitoneal injection to pregnant mothers bearing The kidneys were fixed in Carnoy’s Solution, and embedded in paraffin. Sections (2 µm thick) were sliced at every 100 µm (the diameter of any glomeruli was shorter than 100 µm) and stained with periodic acid-Schiff (PAS) for routine histological examination. Frozen sections of the kidneys were immunostained as previously described Maturation of glomeruli was analyzed in five (E14.5) or three (E18.5) serial sections  at every 100 µm of the embryonic kidneys for 7 control embryos and 5 knockout embryos at E14.5, and 4 control embryos and 6 knockout embryos at E18.5.The criteria for the assessment of glomerular maturation are based on the previous publications We counted the number of glomeruli in each stage in the serial sections and showed the sum number in Scoring of proximal tubule cross-sections was performed according to the previous publication Real-time reverse transcription (RT) polymerase chain reaction (PCR) was performed as described previously GAPDH, CCAGAACATCATCCCTGCATC; CCTGCTTCACCACCTTCTTGA,Bmp7, TGTGGCAGAAAACAGCAGCA; TCAGGTGCAATGATCCAGTCCGAPDH gene.Serially diluted cDNA was used to generate the standard curve for each primer, and the PCR conditions were as follows: 50°C for 2 min, 95°C for 10 min, then 95°C for 15 sec, and 60°C for 1 min for 40 cycles. The results were normalized with the amount of 2.Kidney explant culture was performed as previously described Bmp7 in vitro, one side of E12.5 embryonic kidneys from LacZ/fl;Gt(ROSA)26SorCreERT2Bmp7 embryos was treated with 1 µM 4-OHT (Sigma) dissolved in ethanol, while contralateral kidney was treated with vehicle (ethanol), and cultured for 72 h. Neither LacZ/fl;Gt(ROSA)26SorCreERT2Bmp7 explants treated with the vehicle or +/fl;Gt(ROSA)26SorCreERT2Bmp7 explants treated with 4-OHT exhibited any morphological changes. To cancel the effect of individual difference between embryos, the results of LacZ/fl;Gt(ROSA)26SorCreERT2Bmp7kidneys treated with the vehicle were shown as controls in comparison with contralateral kidneys from the same embryos treated with 4-OHT. For immunostaining, cultured kidney explants were fixed in 4% paraformaldehyde, quenched with 50 mM NH4Cl, permeabilized by 0.1% triton X, and stained as previously described To knockout To inhibit Smad signaling in kidney explants, one side of E12.5 embryonic kidneys from wild-type mice were treated with 0.5 µM dorsomorphin  dissolved in DMSO, while contralateral kidney was treated with vehicle (DMSO), and cultured for 48 h.Immunoblotting was performed as described previously 3 cells/well in 12-well plates and treated with recombinant Bmp7  with or without recombinant Noggin-Fc chimera (R&D systems Inc.) 24 h later. After 7 days of culture, the number of colonies was counted.A colony-forming assay was performed as described previously t-test for two group comparisons, and the ANOVA for more than three.All assays were performed at least three times. Data are presented as the mean ± standard deviation (SD). Statistical significance was assessed by Student’s Figure S1Systemic knockout of Bmp7 at E10.5 recapitulates the phenotypes of germline Bmp7 knockout mice. Pregnant mothers bearing both Bmp7+/fl;Gt(ROSA)26SorCreERT2 and Bmp7LacZ/fl;Gt(ROSA)26SorCreERT2 embryos were administered tamoxifen at E10.5, and sacrificed at E16.5. The Bmp7LacZ/fl;Gt(ROSA)26SorCreERT2 (Bmp7 knockout) kidney was smaller and exhibited severe reduction of cap mesenchyme. Scale bars: 100 µm.(TIF)Click here for additional data file.Figure S2Volume of kidneys and mesenchyme is still maintained in Bmp7 knockout embryos at E14.5, but reduces at E18.5 (related to  and ). The maximum horizontal sectional area of kidneys and the thickness of nephrogenic zone, WT1-positive area were not different between Bmp7 knockout embryos (white column) and controls (black column) at E14.5, but significantly reduced in knockout embryos at E18.5. The mean of the values ± SD is presented in the graphs  n.s.: not significant.(TIF)Click here for additional data file.Figure S3Accerelated maturation of distal tubules in Bmp7 knockout kidneys at E18.5 (related to). (A) Kidneys were stained with nephrin (green) and THP (red) to label glomeruli and distaltubules, respectively. The volume of THP-positive distal tubule sections in Bmp7 knockout kidneys was comparable to the control kidneys, whereas the number of glomeruli was significantly reduced. (B) The number of THP+ distal tubule cross sections normalized by the number of nephrin+ glomeruli tended to increase in knockout kidneys (white column) compared to control kidneys (black column). Data are represented as mean ± SD. Three sections were stained for each kidney. The sum of the number of THP-positive distal tubule cross sections was divided by the sum of the number of nephrin-positive glomeruli. The mean of the values from five (control) or six (knockout) embryos is presented in the graph. Scale bars: 100 µm. n.s.: not significant.(TIF)Click here for additional data file.Figure S4The branching of ureteric buds tends to decrease in Bmp7 knockout kidneys (related to ). Kidney explants were taken from Bmp7+/fl;Gt(ROSA)26SorCreERT2 and Bmp7LacZ/fl;Gt(ROSA)26SorCreERT2 embryos at E11.5 and cultured for 48 h with or without 4-OHT. In Bmp7+/fl;Gt(ROSA)26SorCreERT2 embryos, the number of ureteric bud tips of tamoxifen-treated explants was almost equal to vehicle-treated explants. In Bmp7LacZ/fl;Gt(ROSA)26SorCreERT2 embryos, although the difference was not significant, the number of ureteric bud tips of tamoxifen-treated (Bmp7 knockout) explants tended to decrease compared to vehicle-treated explants. Data are represented as mean ± SD (n = 4). n.s.: not significant.(TIF)Click here for additional data file.Figure S5Smad signaling inhibits the differentiation of cap mesenchyme in the kidney explant culture (related to). Kidney explants were taken from wild-type mice at E12.5 and cultured for 48 h in the presence or absence of a Smad1/5/8 inhibitor, dorsomorphin. In explants treated with dorsomorphin, Jagged1-positive regions were significantly expanded. Scale bars: 100 µm. Data are represented as mean ± SD (n = 8). Immunoblotting of the lysates of kidney explants demonstrated the phosphorylation of Smad1/5/8 was decreased in dorsomorphin-treated explants. Ten micrograms of kidney explants lysate was loaded in each lane. As a positive control, primary kidney cells were stimulated with 100 ng/ml Bmp7 for 1 h. GAPDH was used as a loading control. pSmad1/5/8 denotes phospho-Smad1/5/8.(TIF)Click here for additional data file.


Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates.Although rapid diagnostic tests (RDTs) for Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle.Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed.pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs.The levels of transcription of  The ability to accurately diagnose malaria infections is critical to the control and elimination of this disease Plasmodium falciparum histidine-rich protein 2 (PfHRP2) that circulates in the bloodstream of the patient Many of the RDTs marketed to diagnose malaria target Several factors may contribute to the variable sensitivity reported for PfHRP2-detecting RDTs. These include genetic variation of PfHRP2 between parasites. We have previously shown that while genetic polymorphism in PfHRP2 is extensive, it does not appear to affect RDT detection sensitivity at levels > 200 P/uL P. falciparum. It is encoded by pfhrp2, a subtelomeric gene located on chromosome 7 PfHRP2 is a 60-105 kDa water-soluble protein specific to s-tRNA syn or tRNA) and MAL13P1.209 60S ribosomal subunit protein L18 , are transcribed at a relatively constant rate across the asexual lifecycle of the 3D7 falciparum strain The parasite expresses most of its genes as it invades and develops within the erythrocyte, with at least 60% of the genome transcriptionally active during the intraerythrocytic asexual cycle pfhrp2 and pfhrp3 across the asexual cycle A plausible hypothesis is that the amount of PfHRP produced by the parasite varies between different isolates, and as a consequence different amounts of this target protein would be available for detection by RDTs. PfHPR2 production and release has been characterised in a limited number of strains during the intraerythrocytic life cycle pfhrp2 and pfhrp3, and of abundance of PfHRP protein, in different parasite strains, at several time points over the blood stage life cycle. Further, the potential impact that the variation in protein expression has on RDT detection sensitivity is investigated. Defining reasons for RDT failure is of significant public health importance and will contribute to improving diagnostics for falciparum malaria.In this paper, we report the pattern of transcription of in vitro® Separation Column (Miltenyi Biotec USA) pfhrp2 and pfhrp3 through the erythrocytic life cycle  in RNase-free tubes then frozen at −80°C. For protein isolation, culture supernatant was removed and red cell pellets were frozen at −80°C until protein extraction.Cryopreserved parasites originating from varied geographic areas  were culfe cycle , where sfe cycle  were useFor each sample, the number and proportion of ring-stage parasites was determined by microscopically counting 500 infected erythrocytes. No gametocytes were observed in these cultures.Total RNA was isolated from each parasite pellet using the NucleoSpin® RNA II Kit (Macherey-Nagel Germany), following the manufacturer's instructions. A second elution step was added to maximise yield as previously described pfhrp2 fragment alignment of P. falciparum 3D7 (GenBank accession number BM275665), FCBR (GenBank accession X69922) and ITG2 (GenBank accession U69551). The forward primers for both pfhrp2 and pfhrp3 were designed to span both exon 1 and part of exon 2, with the reverse primer binding in exon 2. Primers for pfhrp3 were designed using an alignment of ITG2 (GenBank accession U69552) and FCC1/HN (GenBank accession AF202093). Primer sequences are shown in Total RNA was reverse-transcribed into cDNA using SuperScript™ III Reverse Transcriptase (Invitrogen USA) and random hexamers. The real-time PCR primers were designed based on a pfhrp2 and pfhrp3 were optimised using P. falciparum 3D7 cDNA. The use of 150 nM primers per reaction and a 15 minute enzyme activation period on the MX4000 (Stratagene USA) gave the best reproducibility, and were used in all real-time PCR experiments for this study. Two housekeeping genes, PF07_0073 seryl tRNA synthase (s-tRNA syn or trna) gene and MAL13P1.209 , the 60S ribosomal subunit protein L18 were used as endogenous control transcripts to normalize mRNA levels Real-time PCR conditions for pfhrp2 gave a melting temperature at 76.7°C while for pfhrp3 it was 77.8°C. Experiments where non-specific peaks in relation to the dissociation curves were observed were repeated. The mean Ct was determined and used in the ΔCt method pfhrp2 and pfhrp3 relative to s-tRNA syn and mal13.All samples were run in triplicate using the ABsolute QPCR SYBR Green Mix (ABGene United Kingdom) according to the manufacturer's instructions. The following cycling conditions were used: 15 minutes at 95°C for initial denaturation and enzyme activation, followed by 40 cycles of 95°C for 30 seconds, 57°C for 40 seconds, and 72°C for 40 seconds, followed by a final extension of 57°C for 1 minute. The dissociation curve for Pelleted infected red blood cells were thawed and resuspended in 1% Triton X-100 diluted with phosphate-buffered saline (PBS) and 1/1000 volume of a cocktail of proteinase inhibitors (Thermo Scientific USA). Samples were then frozen (−80°C) and thawed three times, with vortexing every 15 minutes. After protein solubilization the solution was spun at 15 000 rpm for 30 minutes at 4°C and the supernatant removed for use in protein experiments.A 40 µL supernatant aliquot was diluted 1∶5 with PBS-T  to reduce interference from residual hemoglobin. One third volume of 3×SDS sample loading buffer was added to the extracts, and boiled for 10 minutes prior to loading on an SDS-page gel , run for 1 hour at 60 V then 1.5 hour at 150 V. Proteins were then transferred from the SDS-page gel to a 0.45 µm PVDF membrane (Invitrogen USA) at 100 V for 1.5 hours. Following transfer, the membrane was blocked overnight in 5% skim milk powder dissolved in 100mL PBS-T. The membrane was then incubated with the primary antibody, PTL3  at 1∶5000 dilution. Following washing in PBS-T, the membrane was incubated with an anti-mouse polyvalent immunoglobulin-alkaline phosphatase conjugate (Sigma USA) at 1∶5000 dilution. The signal was detected by CDP Star chemiluminescent substrate (Roche Germany) and exposed to imaging films.A PfHRP2 quantitative antigen-capture ELISA  was used according to the manufacturer's instructions. Positive and negative control wells were included to quantitate protein levels for all samples. 3D7 culture supernatant containing a known concentration of PfHRP2 was used for standard curve construction. All samples were tested as undiluted, 1/100, 1/200 and 1/300 dilutions and run in duplicate. Absorbance was measured at 450 nm and 650 nm using a microplate spectrophotometer (SpectraMax Molecular Devices USA).trna transcription value to account for potential differences in the number of parasites in the sample. The trna values from the transcript dataset for the K1 parasite were then used for this normalisation: normalised PfHRP2sample parasite = PfHRP2sample parasite×(trnasample parasite/trnaK1).The software package SoftMax Pro software (Molecular Devices USA) was employed to quantify the protein levels in ng/mL. Samples whose concentration fell outside the standard curve were further diluted and the test repeated. Calculated PfHRP2 levels were then normalised against the samples own To investigate whether differences in PfHRP protein level results in a difference in the detection limit on RDTs, the ring-stage protein sample (80–100% ring) from parasite strains K1, D6 and Hb3 were tested without dilution, then at 1/100, 1/200, and 1/300 dilutions in 1×PBS, on three RDTs .The experiment was then repeated using the same number of intact parasitized red blood cells of the three parasite lines at seven doubling dilutions  at 50% hematocrit. The RDTs were read using the WHO Colour Intensity chart for RDTs with a scale of 0-4, based on the intensity of the band colour, 4 being the strongest band colour, 3 moderate colour, 2 weak colour, 1 faint colour, and 0 negative .Spearman Rank Correlation using GraphPad PRISM software  was applied to assess the relationship between transcript and parasite stage, protein and parasite stage, and the relationship between transcription and expression.P. falciparum to be characterised in this work have different asexual replication times. To compare levels of transcription of pfhrp2 and pfhrp3 at the well-recognized asexual stages, we grouped parasites based on the proportion that were ring-stage at different times across the lifecycle. For 5 of the 7 lines studied . A significant positive correlation was observed between the proportion of rings and the level of transcription for both pfhrp2 and pfhrp3 (P < 0.01)  . This wanst trna  or againa , K1 and GA3 (1.0 vs 0.3), and moderate between SJ44 and N70 (1.1 vs 0.9), FCQ33 and FCQ41 (0.5 vs 0.3).Levels of ainst K1 . The ratPfhrp3 is absent in the P. falciparum laboratory clone Hb3 pfhrp2 in this parasite line was lower compared to all other strains tested. In other words, no compensatory increase in pfhrp2 transcription was observed when pfhrp3 was absent. Similarly, the parasite line Dd2 lacks pfhrp2pfhrp3 in this line was the lowest of those tested with no compensatory increase to account for the lack of pfhrp2.Monoclonal antibodies raised against PfHRP2 have been shown to cross-react with PfHRP3 pfhrp2 sequence were compared at predominant ring stage (Group 1), a 1–4 fold difference in protein levels was observed between the paired strains . K1 returned strong positive results of 4 for all dilutions except the final dilution where the band intensity was slightly weaker at a score of 3. Hb3 worked only weakly until the second dilution, 10 000 P/µL with a score of 2.pfhrp2 and pfhrp3 genes, and the levels of the corresponding protein PfHRP, vary between different parasite strains, and whether these variations influence the detection sensitivity of malaria RDTs. Our data indicate that pfhrp2 and pfhrp3 transcription is highest at the ring stage of the intraerythrocytic life cycle, a finding that agrees with the transcription profiles for these genes using microarray analysis for the 3D7 strain which is available in the PlasmoDB database pfhrp2 vary widely between geographically variant strains of the parasite, even for strains with the same pfhrp2 sequence and also between strains with different pfhrp2 sequence.The purpose of this study was to investigate whether the transcription of the PlasmodiaControl of transcription is believed to be under the influence of various regulatory systems, many of which are incompletely understood in pfhrp2, or even identical coding sequences may differ in their 5′ UTR and 3′ UTR sequences, resulting in different levels of transcription.It is clear that transcription patterns vary between genetically distinct parasites pfhrp2 and pfhrp3 transcription.Epigenetic mechanisms may also significantly affect transcription pfhrp2 and pfhrp3 transcripts and PfHRP protein measured in this study constitute the amounts of transcript or protein present at that timepoint, reflecting the combined result of transcription and degradation of the target. For transcripts, post-transcriptional control mechanisms including mRNA stability and decay may contribute significantly to the variation in the amount of transcripts present pfhrp3 may play a role in mRNA stability It should be noted that the levels of pfhrp2 transcripts was always higher than that of pfhrp3. This could be a consequence of the promoter for pfhrp2 being stronger, or as a result of a slower decay of pfhrp2 transcripts. A 5′ flanking region of pfhrp3 has been extensively used in transfection studies as a promoter for reporter gene expression, paired with a 3′ flanking region of pfhrp2 as a terminator sequence pfhrp2 promoter for transfection, thus precluding a comparison of the two promoters. In an earlier study, levels of PfHRP2 to PfHRP3 were compared using immunochemical methods, with results indicating that PfHRP3 protein levels were lower than PfHRP2 In all strains tested in this study, we observed that the level of pfhrp2 and pfhrp3 genes are structurally very similar, with regions flanking the tandem repeats, including untranslated regions, showing up to 90% homology pfhrp2 and pfhrp3 genes encode the histidine-rich amino acid repeats beginning 75–90 nucleotides downstream from the start pfhrp2 transcription was lower when pfhrp3 was absent and vice versa. If the products of these two genes have a similar function, it would be expected that one would compensate for the absence of the other. From our data, it may be suggested that these genes have a non-overlapping function. pfhrp2 and pfhrp3 are not linked, occurring as single copy genes on separate chromosomes The The variation in transcription we observed was also reflected by differences in the protein expression levels. Although PfHRP protein expression was more constant throughout the intraerythrocytic life cycle in 5 lines tested, wide variations in the level of protein were observed between 16 strains at ring stage, the stage circulating in patient blood, with up to a 24 fold difference observed between strains. The results are in agreement with RDT results observed using patient samples at 200 P/µL recently reported from Colombia Importantly, we have shown that parasite strains with higher protein levels have a lower detection limit on commercial RDTs compared to those with lower protein levels. The same phenomenon was observed when the number of parasites was controlled. Although the quantity of HRPs in D6 was less than that in K1, RDTs used to test these lines were strongly positive. When compared to the Hb3 result, which had a low protein level and was not detected at low concentrations, a threshold is observed, below which the level of protein affects RDT sensitivity. Parasites with low levels of protein will be difficult to detect at low parasitemia (≤200 P/µL). This provides further evidence that the level of PfHRP protein is a major determinant of the detection sensitivity of RDTs, and therefore that such variations in protein abundance could contribute to variation in the performance of PfHRP2- detecting RDTs. This consideration is particularly important for testing of patients at low parasitemia, where parasites with lower protein levels may return negative results on malaria RDTs. Likewise, low parasite protein expression may also explain why some RDTs return a negative result at relatively high (>1 000 P/µL) parasitemia levels. An increased understanding of the factors influencing RDT performance will likely help to improve the performance and use of such malaria RDTs that are of significant public health importance.in vitro culture conditions. Although neither pfhrp2 nor pfhrp3 genes were deleted during culture adaptation, it is unknown whether levels of transcription of these genes, and expression levels of the encoded proteins were affected during the adaptation. Further work using these methods to quantify transcription and expression on additional parasite isolates, and especially testing of field isolates, to determine the extent of variation, and the proportion of parasite isolates that produce low level HRPs would strengthen this observation. The impact of variation in protein level on RDT detection sensitivity in patients also requires further study. Important experimental constraints impede the measurement of PfHRP in field samples. These include the lack of synchrony and inability to control parasitemia levels.It should be pointed out that the parasite lines used in this study have all been adapted to pfhrp2 and pfhrp3 and expression of PfHRP protein levels varies between laboratory strains of the parasite, and demonstrated that the variation in protein levels results in differences in RDT detection thresholds. The outcome of this study provides a possible explanation for reported variation in sensitivity of PfHRP2-detecting RDTs and will assist research aimed at improving malaria RDTs.We have shown here that transcription of Figure S1Transcription of pfhrp2 (A) and pfhrp3 (B) normalised to mal 13 over the intraerythrocytic life cycle (with varying proportion of ring stage).(TIF)Click here for additional data file.Figure S2Western Blot of PfHRP2 for 5 time points, D6 line.(TIF)Click here for additional data file.Figure S3Plot of PfHRP expression level against the proportion of ring stage parasites.(TIF)Click here for additional data file.


Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both. Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from  Bacillus thuringiensis (Bt) kill some major insect pests, but are harmless to vertebrates and most other organisms  X 100%. Because potency is inversely related to LC50, we calculated the potency ratio for modified toxins relative to native toxins as the LC50 of a native toxin divided by the LC50 for a modified toxin [ We calculated resistance ratios as the LCed toxin . We repo For each insect strain, we used the bioassay method described above to test Cry1AbMod, Cry1Ac, and Cry2Ab singly, in pairs, and in a trio, with an aggregate total of 18 sets of concentrations including the control with untreated diet  and 4. O We evaluated potential antagonism or synergism by testing for deviation from the null hypothesis of simple independent action , which a(ab)EXP = S(a)OBS X S(b)OBS(1) S(ab)EXP is the proportion of larvae expected to survive exposure to a combination of toxins a and b, S(a)OBS is the observed proportion of larvae that survived exposure to toxin a, and S(b)OBS is the observed proportion of larvae that survived exposure to toxin b. We calculated expected mortality for larvae exposed to the combination of toxins a and b as (1 - S(ab)EXP) X 100%. We applied the same approach to test for synergism among three toxins: where S(abc)EXP = S(a)OBS X S(b)OBS X S(c)OBS(2) S(abc)EXP is the proportion of larvae expected to survive exposure to a mixture of toxins a, b, and c.where S To calculate expected survival, we first calculated the observed adjusted survival for each toxin tested singly as survival on treated diet divided by survival on untreated diet (control). Survival on untreated diet ranged from 87 to 93% (mean = 90%). All of the results reported for treated diet are based on adjusted survival. 50 of Cry1AbMod). Accordingly, for this exceptional case, we used the mean adjusted observed survival (55%) from the three trials at this concentration conducted in February, July, and September 2010. This is a conservative approach for evaluating synergism, because the high survival (89%) in April 2010 would yield higher expected survival when Cry1AbMod was tested in combination with one or both of the other two toxins (Cry1Ac and Cry2Ab). By excluding the anomalously high survival estimate, we made it less likely that we would conclude synergism occurred in tests with Cry1AbMod and more likely that we would conclude antagonism occurred with tests with Cry1AbMod. The susceptible strain was tested in February 2010 and both the susceptible and resistant strains were tested simultaneously in April 2010. On each date, the sample size was 30 larvae per concentration set for each strain for each of the 18 sets of concentrations . For the susceptible strain, the results for each of three toxins  tested singly at each of two concentrations were similar between dates  in five of six cases (3 toxins X 2 concentrations per toxin). For these five cases, we pooled the data across the two dates to calculate observed adjusted survival for each toxin tested singly. In the exceptional case, which was the susceptible strain tested at 1 µg Cry1AbMod per ml diet, adjusted survival was 50% in February 2010 vs. 89% in April 2010. The 89% survival is anomalously high, because survival ranged from 50 to 59% (mean = 55%) in the three other independent tests at this concentration . For each of the 22 combinations of toxin concentrations tested (11 combinations tested per strain X 2 strains), we used Fisher's exact test  with 2-tailed probability to determine if a significant difference occurred between the observed and expected numbers of dead and live larvae. For each toxin combination tested against each strain, we also pooled the data across the different concentration sets before performing Fisher's exact test. For example, for BX-R versus Cry1AbMod + Cry1Ac, we pooled the data from the four concentration sets tested (n = 120 for observed response and n = 240 for expected response). This approach increases statistical power and determines if a consistent deviation from independent action occurred across the entire set of toxin concentrations tested for each strain for each toxin combination.


Neutralizing antibodies are an important component of the humoral immune response directed against viral infections So far the few available anti HIV-1 broadly neutralizing antibodies have a limited breadth and potency against clade C viruses. More than 50% of the HIV-1 infections worldwide belong to clade C. Clade C is the most prevalent subtype in India.A human scFv phage display library was constructed from the peripheral blood mononuclear cells of an HIV-1 Clade C infected Indian patient, having a good titre of serum neutralizing antibodies. Diversity of the scFv library was checked by sequencing and DNAfingerprinting analysis of randomly selected clones from the preselected library. One round of biopanning was done against V3 peptide of clade C and clade B. Single chain fragments were checked for their soluble expression in E. coli HB2151 and purfied using Ni+2 affinity colums. Specificity of the soluble scFvs was checked by indirect ELISA. Single chain fragments were checked for their stability in different agents like 30%DMSO. 4M NaCl, pH 2-11.50 clones were randomly selected after biopanning and they were checked for their binding to V3C and V3B peptides. In phage ELISA 15 out of 50 clones showed binding to both the V3 peptides. A 32kDa band was observed in polyacrylamide gel electrophoresis as predicted. The expressed product was confirmed by Western blot analysis using anti His Tag antibody. Specificity of the purified scFvs was confirmed by their binding to V3 peptides and no reactivity against other unrelated peptides. Further these scFvs displayed a stable binding to V3 peptides in different denaturing agents.This is the first study to generate human anti-V3 scFvs against HIV-1 clade C. Further characterization of these scFvs for their neutralization potential will help identify unique and shared epitopes responsible for neutralization of clade C and non clade C viruses.


HCV genotype 3a is the major cause of infection in Pakistani population. One of the major problems of HCV infection especially in the developing countries that limits the limits the antiviral therapy is the long term treatment, high dosage and side effects. Studies of antigenic epitopes of viral sequences of a specific origin can provide an effective way to overcome the mutation rate and to determine the promiscuous binders to be used for epitope based subunit vaccine design. An Immunoinformatic tools were applied for the predictive analysis of HCV 3a antigenic epitopes of Pakistani origin. All the predicted epitopes were then subjected for their conservancy analysis in HCV genotypes 1, 2 and 3 of diverse origin (worldwide). Using freely available web servers, 150 MHC II epitopes were predicted as promiscuous binders against 51 subjected alleles. E2 protein represented the 20% of all the predicted MHC II epitopes. 75.33% of the predicted MHC II epitopes were (77-100%) conserve in genotype 3; 47.33% and 40.66% in genotype 1 and 2 respectively. 69 MHC I epitopes were predicted as promiscuous binders against 47 subjected alleles. NS4b represented 26% of all the MHC I predicted epitopes. Significantly higher epitope conservancy was represented by genotype 3 i.e. 78.26% and 21.05% for genotype 1 and 2.The study revealed comprehensive catalogue of potential HCV derived CTL epitopes from viral proteome of Pakistan origin. A considerable number of predicted epitopes were found to be conserved in different HCV genotype. However, the number of conserved epitopes in HCV genotype 3 was significantly higher in contrast to its conservancy in HCV genotype 1 and 2. Despite of the lower conservancy in genotype 1 and 2, all the predicted epitopes have important implications in diagnostics as well as CTL-based rational vaccine design, effective for most population of the world and especially the Pakistani Population. Flavivirus, Pestivirus, and Hepacivirus. Members of these genera cause various diseases in humans and other animals such as birds, horses and pigs. The only genera Flavivirus contain more than 70 members including Hepatitis C Virus (HCV), Dengue virus, West Nile virus and tick-borne encephalitis virus . The number of individual bases in the genome i.e. the number of adenine; cytosine, guanine and thymine were calculated from DDBJ database. The molecular weight of proteins, percentage of highly repeated amino acid and the least repeated amino acid in the viral protein was calculated by using sequence and search analysis tool at PIR database  × A(2) × C(3) × D(4) × P(5) × G(6) × R(7) × A(8) × A(9)Score = P(1) + A(2) + C(3) + D(4) + P(5) + G(6) + R(7) + A(8) + A(9)Where P (1) is score of P at position 1.Only the promiscuous epitopes with score higher than the chosen threshold score were assigned as predicted epitopes for the selected HLA alleles . For thehttp://tools.immuneepitope.org/tools/conservancy/iedb_input). All the epitopes having 77-100% conservancy were selected while rejecting the epitopes having variation at the anchor residues. The anchor residues in the predicted epitopes were highlighted by making it bold. The epitopes that were 100% conserved in the selected proteins of the 3 viral genotypes 1, 2 and 3 were also fully bold. Epitopes with 88/77% conservancy were with single or double amino acid variation respectively and to highlight them bold format was used in the conservancy column against each genotype.All the predicted epitopes of HCV 3a proteins of Pakistani origin were subjected for worldwide conservancy analysis among HCV genotype 1, 2 and 3. 5 sequences against each HCV protein (used for epitope prediction) were retrieved from NCBI randomly. The predicted epitopes of HCV 3a (Pakistani origin) along with 5 selected sequences of individual genotypes  were submitted to epitope conservancy analysis tool available at IEDB database (Asteric sign (*) indicates that one out of five selected sequences either does not respond to epitope conservancy or have conservancy lower then 77%. Double asteric sign (**) indicates that only one sequence responds for 77-100% conservancy to the selected epitope.The Peptides with single or double amino acid variation were analyzed for their hydropathic characteristics or pI value . The pI HCV 3a genome of Pakistani origin comprises 9474 bp with GC content 2622 and 2700 respectively. The GC contents are 12.35% higher then AT contents. The genome encodes a polyprotein that subsequently get fragmented into structural and non structural protein of obvious molecular weight. The envelope protein E2 comprises highest moleculat weight 38755.3 KDa , I (Isoleucine), L (Leucine), M (Methionine), V , W (Tryptophan) and Y (Tyrosine) were mainly the anchor residues for MHC II predicted epitopes and are nonpolar in nature. Total 150 epitopes were predicted against 51 alleles of MHC II Table . The higTotal 69 epitopes were predicted as promiscuous epitopes for MHC I alleles. The anchor residues in case of MHCI are quite varying both in amino acid residues and also in their nature. Mostly represented anchor residues are neutral nonpolar and neutral polar. However, quite small percentage of anchor residues were also acidic polar and basic polar in nature. The highest number of MHC I binding epitopes were represented by NS4b protein comprising 26% of all MHC I predicted epitopes. NFVSGIQYL epitope of NS4b is the best promiscuous binder of highest binding score. NS4b is followed by NS2, E2 and NS3 proteins representing 20.28% (NS2 epitopes) and 11.59% (for E2 and NS3). In case of NS2, 14 promiscuous epitopes were predicted with varying binding efficiency. GSRDGVILL, DGVILLTSL, WAAAGLKDL and LQVWVPPLL are the good binders both in term of score and the HLA allele coverage . E2 predicted epitopes covers 20 to 28 HLA alleles except the PLLHSTTEL epitope that covers only 11 HLA alleles but with highest binding efficiency. NS3 epitopes covers 8 to 25 HLA alleles and were also ranked on the basis of their binding efficiency predicted by the score. The least represented epitopes were by NS5a_1a. It comprises only one epitope (HVKNGSMRL) as predicted promiscuous binders for 16 MHC I binding alleles. The promiscuous binders of MHC I for other proteins were also predicted and summarized in table Out of total 150 predicted MHC II epitopes, 75.33% were (77-100%) conserve in genotype 3  conserve in genotype 3 (Table The modern technique for control of HCV infection is a vaccine preparation that can specifically induce antibody-mediated immunity. The rapid advancements in the computational methodologies and immunoinformatics/immuno-bioinformatics provide new strategies for the synthesis of antigen specific epitopic vaccine against infectious agents such as viruses and pathogens. Epitopic vaccine against HIV, malaria and tuberculosis provided promising results and supported the defensive and therapeutic uses of these vaccines . Thus inBy the use of an efficient CTL based epitope delivery technology; the predicted epitopes could eventually become vaccines in their own or fused as polytopes. The design of the HCV vaccine using conserved epitopes can avoid viral mutation and thus provides more efficient results. The study shows that the predicted epitopes were highly conserved in HCV genotype 3 and also but less conserved in genotype 1 and 2 both for MHC I and MHC II. Moreover, to ensure the viral detection at all stages of its intracellular evolution we have used all the viral proteins. Therefore, the total number of predicted epitopes were also maximized in correspond to the number of covered proteins used for the analysis.HCV: hepatitis C virus; HLA: human leukocyte antigen; MHC: major histocompatability complex; CTL: cytotoxic T lymphocytes.The authors declare that they have no competing interests.AS and SH designed the study. AS performed the immunoinformatics analysis and drafted the manuscript. MI critically reviewed the manuscript. All authors have read and approved the final manuscript.