The advent of optical coherence tomography angiography (OCT-A) provides a new opportunity to visualize the retinal vasculature in a non-invasive and dye-free manner which may help identify vascular abnormalities in glaucoma. While a reduction in retinal and optic nerve head vessel densities and blood flow indexes measured by OCT-A has been demonstrated in patients with glaucoma in many studies, it is unclear whether OCT-A provides additional information for the detection and monitoring of glaucoma compared with OCT measurements such as retinal nerve fiber layer thickness, neuroretinal rim width, and ganglion cell inner plexiform layer thickness. Longitudinal studies are needed to elucidate whether vascular abnormalities detected by OCT-A are a cause or a consequence of optic nerve damage in glaucoma. Some of these measurements reported in the literature include vessel density (commonly annotated as the percentage of detected vessel area over the imaged area), flow index , and blood flux index . It is worth noting that these indexes are surrogate measures and their validity for measurement of blood flow remains to be investigated.The recent introduction of optical coherence tomography angiography (OCT-A) has sparked interest in evaluating vascular alterations in the retina and optic nerve head (ONH) for diagnosis, staging, and monitoring in glaucoma. OCT-A is an extension of OCT which allows non-invasive visualization of the retinal vasculature by detecting motion contrast from perfused blood vessels without the use of exogenous dye. In principle, OCT-A compares sequential B-scans acquired at the same location to detect change. As stationary structures would appear static in sequential B-scans, changes detected by OCT-A are largely attributed to erythrocyte movement in the perfused vasculatures. A number of algorithms such as split-spectrum amplitude decorrelation angiography (SSADA), OCT-A ratio analysis, and optical microangiography (OMAG) have been devised to compute blood flow measurements from the sequential B-scans3. They showed that the optic disc flow index was reduced by 25% in glaucomatous eyes (0.161 ± 0.008) compared with healthy eyes (0.121 ± 0.026). Using a cut-off value of 0.1515, they showed that the sensitivity and specificity for the detection of glaucoma were both 100% . The flow index was highly associated with visual field pattern standard deviation (R2 = 0.752). The same group then evaluated the peripapillary flow index and the peripapillary vessel density for discrimination between glaucomatous and healthy eyes by using a spectral-domain OCT and reported the area under the receiver operating characteristic curve (AUC) to be 0.892 and 0.938, respectively4. It remains controversial whether OCT-A measurements have a higher diagnostic performance for glaucoma detection compared with conventional OCT measurements such as the retinal nerve fiber layer (RNFL) thickness, neuroretinal rim width, and macular ganglion cell and inner plexiform layer thickness. Chen and colleagues demonstrated that the peripapillary blood flux index measured between the internal limiting membrane (ILM) and RNFL using OMAG and circumpapillary RNFL thickness had comparable diagnostic performance for the detection of glaucoma suspect  and glaucoma 5. In a recent study, Rao and colleagues compared the diagnostic performance for glaucoma detection between OCT-A vessel density measurements using SSADA and OCT measurements 6. All vessel density measurements, including the radial peripapillary capillary (measured between the ILM and RNFL), the ONH segment vessel , and the macular superficial plexus (between the ILM to the inner plexiform layer), were found to have significantly smaller AUCs compared with OCT measurements. Discrepancies among the studies are likely attributed to the different definitions adopted and varying stages of glaucoma patients included in the analysis.Jia and colleagues provided the first account of vascular abnormalities at the ONH measured by a swept-source OCT in glaucoma7. Examining 98 primary open-angle glaucoma eyes with a localized RNFL defect, the authors demonstrated that the radial peripapillary capillary vascular abnormality detected by OCT-A using SSADA exactly coincided with the RNFL defect in both the location and the extent, suggesting that vascular change is a consequence of optic nerve damage in glaucoma. On the other hand, Chen and colleagues studied the microvasculature density  and blood flux index between the ILM and RNFL measured using OMAG at the peripapillary region in glaucomatous eyes with single-hemifield visual field defects and reported that the intact visual hemifield showed reduced blood flux index and microvasculature density in eyes with glaucoma compared with healthy eyes but that no significant difference in circumpapillary RNFL thickness between the groups was detected8. While significant correlations between blood flow index/circumpapillary RNFL thickness and visual field mean deviation were observed in the normal visual hemifield in eyes with glaucoma, there was no correlation between microvasculature density/blood flux index and visual field mean deviation/circumpapillary RNFL thickness in the abnormal visual hemifield. Yarmohammadi and colleagues showed that while radial peripapillary capillary vessel density, macular superficial vessel density, RNFL thickness, and GCC thickness were all reduced in both the affected and the intact visual hemifields in eyes with glaucoma, the strength of association with visual field sensitivity measures was stronger for vessel density measurements using SSADA compared with RNFL and GCC thicknesses9. Longitudinal studies investigating the temporal sequence of OCT-A and ONH/RNFL changes are needed to address whether the vascular changes detected by OCT-A are a cause or a consequence of optic nerve damage in glaucoma.Lee and colleagues hypothesized that if vascular abnormality were a consequence of optic nerve damage, it would be observed only at the area of RNFL defect10. Poor-quality OCT-A scans are more common than poor-quality OCT scans. In a study in which both OCT-A and OCT measurements were performed by the same commercially available instrument, 17% and 29% of the OCT-A scans using SSADA were considered to have poor quality at the optic disc and the macula regions, respectively11. By contrast, only 9% of OCT scans at the optic disc region and 3% of OCT scans at the macula were graded as poor quality. With the currently available OCT-A instruments, the scan time typically varies from 3 to 6 seconds for a 3×3 to 6×6 mm2 scan12, which is longer than imaging the ONH or the macula for RNFL and ganglion cell layer/inner plexiform layer analyses. The longer scan time in OCT-A can contribute to a higher incidence of motion artefact.Motion artefacts and projection artefacts are common in OCT-A. A considerable proportion of OCT-A images remain suboptimal in quality for interpretation. For example, in a study evaluating the intra-visit and inter-visit variability of vessel density measurement in primary open-angle glaucoma and ocular hypertension patients, 78.3% of the participants were excluded because of suboptimal SSADA-derived OCT-A image qualityVascular abnormalities detected by OCT-A have been consistently observed in glaucoma. However, it remains unclear whether OCT-A provides additional diagnostic information for the detection of glaucoma compared with conventional OCT measurements such as circumpapillary RNFL thickness, neuroretinal rim width, and ganglion cell inner plexiform form layer thickness. Findings from the literature comparing OCT-A and OCT measurements for the detection of glaucoma and evaluation of structure function association are divergent. The temporal sequence of vascular changes and optic nerve damage in glaucoma remains to be elucidated.


Taenia solium is a cestode with humans acting as the definitive hosts and pigs as the intermediate hosts. The infection is endemic in areas where sanitation is poor, pigs scavenge for food and veterinary meat inspection is limited. In addition to the economic losses farmers face from selling infected pork, cysticercosis incurs a significant public health burden when humans ingest parasite eggs, which liberate larvae that may establish in the nervous system, a condition called neurocysticercosis (NCC). The past 10 years have seen two independent groups publish, for the first time, NCC-associated epilepsy Disability Adjusted Life Years (DALYs) estimates. The 2010 NCC-associated DALYs was estimated at 0.5 million  by the Global Burden of Disease [The availability of an increasing number of clinical and epidemiological studies on NCC and cysticercosis has resulted in the publication of several systematic reviews and meta-analyses in the past 10 years –6. TheseT. solium- specific copro-antigen ELISA, Real-time polymerase chain reaction (RT-PCR) on stool extracts and serological diagnosis using the recombinant rES33 antigen have also been developed. A point of care test for simultaneous taeniosis and cysticercosis diagnosis with rES33 and rT24 antigens is currently being field evaluated. In contrast to developments of human T. solium infections diagnostic tools, diagnosis of porcine cysticercosis has not progressed much although accurate diagnosis is needed for food safety, epidemiological studies and public health intervention monitoring. While current antibody detection methods overestimate the prevalence due to transient antibodies, antigen detection methods show poor specificity due to cross reactions with T. hydatigena. Currently, laborious and expensive full carcass detection remains the only reliable method calling for urgent development of pen-side tests [Several researchers have been developing alternatives to the still preferred, yet complex and expensive, enzyme linked immunoelectrotransfer blot (EITB) assay for the immunodiagnosis of cysticercosis . The mosde tests .There has been an increasing understanding of the immunological response to NCC in the past 10 years. Viable cysticerci has been shown to provoke a downregulation of inflammatory Th1 response and a switch to Th2 cytokines associated with asymptomatic disease, while a Th1 host response predominantly follows the degeneration of the cysts . CytokinT. solium taeniosis is generally lower, but this could be due to the two-step approach needed to confirm microscopy- or copro-antigen-positive samples with RT-PCR. The new rES33 test is likely to improve our understanding of the prevalence and factors associated with T. solium taeniosis. Several studies have identified older men as more often infected with cysticerci [A recent systematic review of the frequency of and risk factors associated with human cysticercosis and taeniosis has emphasized the clustered nature of this infection and very large between- and within- country variations in sero-prevalence estimates depending on which communities are selected for study. Human active cysticercosis prevalence has been reported to vary from 0.6% in Ha Tinh, Viet Nam to 21.6% in one Democratic Republic of Congo community [The past 10 years have also seen several research initiatives to control the infection through community-based interventions , 20. Unf


The purpose of this study was to analyze and compare the enamel surface roughness before bonding and after debonding, to find correlation between the adhesive remnant index and its effect on enamel surface roughness and to evaluate which clean-up method is most efficient to provide a smoother enamel surface.135 premolars were divided into 3 groups containing 45 premolars in each group. Group I was bonded by using moisture insensitive primer, Group II by using conventional orthodontic adhesive and Group III by using self-etching primer. Each group was divided into 3 sub-groups on the basis of type of clean-up method applied i,e scaling followed by polishing, tungsten carbide bur and Sof-Lex disc. Enamel surface roughness was measured and compared before bonding and after clean-up.Evaluation of pre bonding and post clean-up enamel surface roughness  with the t test showed that Post clean-up Ra values were greater than Pre bonding Ra values in all the groups except in teeth bonded with self-etching primer cleaned with Sof-Lex disc. Reliability of ARI score taken at different time interval tested with Kruskal Wallis test suggested that all the readings were reliable.No clean-up procedure was able to restore the enamel to its original smoothness. Self-etching primer and Sof-Lex disc clean-up method combination restored the enamel surface roughness  closest to its pre-treatment value. Key words:Enamel surface roughness, clean-up method, adhesive remnant index. At completion of ﬁxed appliance therapy, one of the orthodontist’s primary concerns is to return the enamel surface to its original state as far as possible. The ideal would be minimal enamel loss at each stage of the bonding, debonding, and enamel clean-up process and the production of an enamel surface with the same degree of roughness or smoothness as the original, untreated tooth . During A certain amount of enamel loss is almost inevitable because of the failure of micromechanical bond between the composite resin bonding agent and the acid-etched enamel -5. At prThe exploration of an efficient and safe method of adhesive resin removal following debonding has attracted the interest of many investigators, resulting in the introduction of a wide range of instruments and procedures. Residual adhesive on the enamel surface after debonding can be removed in various ways, including hand instruments , various burs, Sof-lex discs, ultrasonic devices, air abrasion units and lasers but studies have shown that some recommended modalities damage enamel surfaces ,7. It haAssessment of the effectiveness of rotary instruments was limited to inspecting the surface under scanning electron microscopy (SEM) to see the morphology of the enamel surface . HoweverThe hypothesis tested in this study is that the method of clean up may affect enamel roughness. Therefore, the purpose of this study was to analyze enamel surface roughness before bonding with various adhesives and after debonding followed by various clean-up methods, and to provide a quantitative data on which clean-up method is most efficient to provide a smoother enamel surface and to correlate between the adhesive remnant after debonding and its effect on surface roughness.-Study sample Before the start of the study ethical approvals were sought from the institutional review board for conducting the study. A total of 135 extracted premolars were selected, cleaned and stored in distilled water at room temperature. The inclusion criteria included: teeth extracted within 45 days, teeth with no caries or restorations on the buccal surface, teeth having no enamel defects, hypocalcifications or fluorosis on the buccal surface and teeth with no visible cracks on the buccal surface. The extracted premolars were mounted in acrylic custom made blocks which were colour coded and were divided into three different groups on the basis of the type of adhesive used  Group 2 = Yellow  Group 3 = Red (Self etching primer) -Estimation of surface roughnessAfter mounting, the buccal enamel surfaces were pumiced, washed thoroughly and dried with moisture free spray to remove the organic layer. On the buccal surface of all the teeth two layers of nail varnish were applied keeping a circular area of 3 mm in diameter on the middle part exposed to provide the same area of measurement in different treatment stages and to avoid contamination of the prospective bonding buccal surface. Before bonding the labial enamel surface of all the samples were subjected to Mitutoyo Surface Roughness Tester SJ-210)  for 30 seconds, rinsed and dried. A chalky appearance of the enamel surface verified successful etching. Then group 1 was bonded using moisture insensitive primer, group 2 by using conventional orthodontic adhesive and group 3 by using self-etching primer using a micro-brush and lightly dried with compressed air. In all the groups, the primer was cured for 10 seconds using mini LED black (Satelec Aceteon) curing light. Stainless steel Pre Adjusted Edgewise premolar brackets  with slot size of 0.022” x 0.028” were bonded on each specimen using light cure adhesive paste . The brackets were placed in the middle of the tooth in occlusal-gingival and mesial-distal directions, and seated with firm pressure. Excess adhesive resin was carefully removed with an explorer. The curing time was 40 seconds from occlusal, gingival, mesial and distal directions (10 seconds each) as per the manufacturer’s instruction.-Debonding and evaluation of remaining adhesive All the cured specimens were kept for 24 hrs before debonding. Debonding was carried out by debonding plier by applying a gentle squeezing force on the outer wings of the bracket. After debonding, Adhesive Remnant Index (ARI)  was evalScore 0,- 0% of adhesive remaining on the tooth;Score 1, - ≤ 50% of adhesive remaining on the tooth;Score 2, - > 50% of adhesive remaining on the tooth;Score 3, - 100% of adhesive remaining on the tooth.Each group was further subdivided into 3 sub groups on the basis of the type of clean-up method applied for the removal of remaining adhesive resin left after debonding, which are as follow.Group A: Ultra sonic piezo scaling followed by pumice application Group B: 12 fluted Tungsten carbide burGroup C: Sof-Lex disc (3M ESPE)The Sof-Lex discs (3M ESPE) were used in decreasing order of coarseness starting initially with coarse disc followed by medium, fine and finally super fine disc. Final polishing was carried out using rubber cup and zirconium silicate paste . After complete removal of the remaining composite resin from the enamel surface of all the teeth, which was veriﬁed by visual inspection of the teeth under a dental operating light under wet and dry condition, all the samples were once again subjected to Mitutoyo Surface Roughness Tester (SJ-210) for evaluation of surface roughness for each particular tooth.To assess the quality of the enamel surface after the debonding procedures and to find any correlation between the ARI score and post clean-up enamel surface roughness , the sub-groups under each group were combined and treated as one group.-Statistical Analysis p=0.05).The data obtained was subjected to statistical analysis which was performed using SPSS  version 16.0 for Windows. One-way Analysis of Variance (ANOVA), Kruskal Wallis test, Post hoc Tukey test Honestly Significant Difference (HSD) tests, t-test and Pearson‘s correlation test were used for statistical analysis to compare the enamel surface roughness of teeth before bonding and after debonding and clean-up with various adhesives, which clean-up method produced a smoother enamel, reliability of the ARI score and to correlate between the ARI & post clean-up enamel surface roughness. The level of statistical significance was set at 95% (p=0.000) except in teeth bonded with self-etching primer cleaned with Sof-Lex disc (p=0.098) (p=0.000) (p=0.030). This difference in other clean-up methods was inconclusive (Sub-groups A&B).Post clean-up enamel surface roughness  was greater than Pre bonding enamel surface roughness  in all the groups. This difference in Ra was found to be highly statistically significant (p=0.098) . Enamel p=0.000) . Enamel p>0.05) whereas teeth bonded with moisture insensitive primer or conventional orthodontic adhesive and cleaned by any type of clean-up method showed negative correlation which was also statistically non-significant (p>0.05).Clinical orthodontic treatment has been revolutionized during the past decades with the advent of direct bonding. Placement of orthodontic attachments on the surface of teeth can be accomplished by using bonding materials. However, unlike when used for restorative dentistry, these materials must be removed from the surface of enamel at the completion of orthodontic treatment. The concern over debonding-induced enamel surface alterations derives from the importance of the uppermost layer of enamel attributed to its hardness, higher mineral content and more fluoride relative to deeper zones .Extracted human teeth are very difficult to collect, store, and standardize. The teeth used in this study were premolar teeth previously extracted for orthodontic purposes. Since the enamel surface was the focus of this investigation, great care was taken to select the surfaces with the least amount of pre-existing damage and defect which was the basis for the inclusion criteria.Debonding and cleanup are operator-dependent procedures, so the results might vary between operators. In order to minimize this error, only one operator carried out all the clinical procedures in the present study. Results of the present study revealed that post clean-up enamel surface roughness  was more than the pre bonding enamel surface roughness  in all the groups. This means that no clean-up method was able to fully restore the enamel surface roughness to its original state.et al.  was non-significant i,e. Sof-Lex disc was able to restore the enamel surface roughness close to its original state. This finding is similar to the finding of some other study conducted by Ozer et al. . This maet al. . On the et al. . Howeveret al. .et al. (p>0.05). Surface roughness increased significantly after clean-up with the diamond bur and the Er:YAG laser (p<0.01)  .et al. , although the result was inconclusive for all the groups. This may be because the bond failure occurred at the bracket adhesive interface (adhesive failure) or between the adhesive itself (cohesive failure) . These hIt can be concluded from the study that-a) No clean-up procedure was able to restore the enamel to its original smoothness.b) Sof-Lex disc was the only clean-up method to be able to restore the enamel surface closest to its original state.c) Self-etching primer and Sof-Lex disc clean-up method combination restored the enamel surface closest to its pre-treatment value.d) Correlation between ARI score and post clean-up enamel surface roughness  shows non-significant result.


TLR)/CD14 genes and environmental microbes. We examined the associations between single-nucleotide polymorphisms (SNP) in the TLR/CD14 genes and asthma, and their interaction with proxies of microbial exposure . Ten SNPs in four genes  were genotyped for 1,116 participants from the Tasmanian Longitudinal Health Study (TAHS). Using prospectively collected information, asthma was classified as never, early- (before 13 years) or late-onset (after 13 years). Information on childhood farm exposure/childhood rural environment was collected at baseline. Those with early-onset asthma were more likely to be males, had a family history of allergy and a personal history of childhood atopy. We found significant interaction between TLR6 SNPs and childhood farm exposure. For those with childhood farm exposure, carriers of the TLR6-rs1039559 T-allele (p-interaction = 0.009) and TLR6-rs5743810 C-allele (p-interaction = 0.02) were associated with lower risk of early-onset asthma. We suggest the findings to be interpreted as hypothesis-generating as the interaction effect did not withstand correction for multiple testing. In this large, population-based longitudinal study, we found that the risk of early- and late-onset asthma is differently influenced by the interaction between childhood farming exposure and genetic variations.Asthma phenotypes based on age-of-onset may be differently influenced by the interaction between variation in toll-like receptor ( Farm and rural environment are associated with high load of microbial compounds from gram-negative and gram-positive bacteria and fungi4TLRs and CD14 genes have been associated with a change in amino acid sequence leading to a change in protein structureTLRs and CD14 genes and asthma risk have been inconclusiveThe TLRs and CD14 receptors are pattern recognition receptors (PRR) found on the surface of macrophages and dendritic cellsTLR/CD14 genes and the environment, studies that have looked at either factor individually have produced inconsistent results. We recently conducted a systematic review of gene-environmental interactions for the CD14 gene and markers of microbial exposure. We found that high endotoxin exposure in early-life was associated with a lower risk of atopy in children with the CC genotype of the CD14-rs2569190 SNP13Given the complex interaction between TLR2 , TLR4 , TLR6  and CD14  genes and exposure to childhood farm exposure or childhood rural environment on the risk of early-onset asthma or late-onset asthma. The selection of SNPs were based on previous candidate gene studies that had examined the relationship with either asthma, hay fever or atopic sensitisation A total of 1,116 participants had both genotyping and data collected asthma and early-life environmental exposures and were included in this analysis . GeneralWe did not find an association between childhood rural environment and early- nor late-onset asthma . There wThe characteristics and distributions of the genotypes for each SNP is shown in TLR and CD14 SNPs and risk of early- and late-onset asthma. There was strong evidence for interactions between the TLR6-rs1039559 and TLR6-rs5743810 SNPs and childhood farm exposure for early-onset asthma , but not for those without childhood farm exposure. Again in those with childhood farm exposure having the TLR6-rs5743810 C-allele was associated with a lower risk of early-onset asthma . No interactions were observed for those with late-onset asthma . Nor was there any evidence for an interaction between any of the SNPs and either childhood farm exposure or childhood rural environment in relation to the risk of early- or late- onset asthma . The p-values for the analyses are presented without adjustment and the results should be viewed as hypothesis-generating rather than proof of a causal association.TLR6 SNPs and childhood farm exposure for these early-onset asthma phenotypes and found comparable findings for both phenotypes. The findings for each groups were comparable to the combined early-onset asthma group  into adult-life , we identified an effect modification by farm exposure in childhood on the association between TLR6 is located on chromosome 4p14, a region that was consistently associated with atopy in three GWAS1617TLR6 SNPs and early-onset asthma but not late-onset asthma in our study. In our data, early-onset asthma were more likely to have a history of atopy in childhood than those with late-onset asthma. Only about 22% of those with early-onset asthma had adult current asthma whereas about 59% of late-onset asthma had adult current asthma by age 44 years. We have also shown previously that early- and late-onset asthma exhibit different clinical characteristics in our studyTLR6-rs5743810 T-allele and atopic asthmaThe TLR6 SNPs and proxies of microbial exposure and allergic disease outcomesTLR6-rs1039559 T-allele and early farm exposure, their study found higher IgE levels at age two years in individuals with the TLR6-rs1039559 CC-genotype and exposure to two or more older siblings. The inconsistency with our findings may be due to the difference between our proxy measures of microbial exposure  and that used by Reijmerink et al. (siblings). Reijmerink et al. finding with regard to exposure to siblings leading to higher IgE is in contrast to most existing literature.We are aware of only one previous study that examined the interaction between in vitro studies of a variable response to bacterial cell products with the TLR6-rs5743810 SNP. A polymorphism of C to T in the TLR6-rs5743810 resulted in a change in amino acid from proline to serine at position 249 in the extracellular domain of the receptor. Although the effect on the structure is still unknown, studies using peripheral blood cells shown that the TLR6-rs5743810 C-allele showed greater NF-κB signalling activity in response to stimulation by lipopeptide when compared to the TLR6-rs5743810 T-alleleTLR6 mRNA gene expression and higher IFN-γ levels, thereby promoting greater Th1-immunity with a matching reduction of Th2-immunityTLR6-rs5743810 C-allele who were exposed to a farm environment in our study.There is some evidence from CD14 SNPs and asthma risk. This is consistent with a case-control study of adults that found no association with the CD14-rs2569190 SNPs and asthma for those who lived in the country in childhoodCD14 gene showed consistent evidence of a decreased risk of allergic sensitisation in childhood when exposed to high levels of endotoxin in carriers of the CD14-rs2569190 CC-genotypeCD14 and microbial exposure and asthma risk.We did not find any evidence of modification by childhood farm exposure or childhood rural environment on the relationship between any of the TLR2 and TLR4 gene polymorphisms on asthma is still largely undetermined. A recent meta-analysis showed no evidence of association between TLR4-rs4986790 and asthmaTLR2, two studies found a reduced risk of asthma in children with A-alleles of the rs4696480 SNP19TLR2-rs4696480 SNPTLR2-rs4696480 in childrenTLR2-rs4696480 and asthma, and the association was not modified by childhood farm exposure or childhood rural environment. The inconsistency in the findings across studies may be due to variation in the definition of asthma across studies.At present, any effect of the Farm exposure is commonly used as a proxy for microbial exposureStrengths of our study include the large cohort of individuals and the prospective data collection enabling us to accurately classify participants into different asthma phenotypes based on age of onset of disease and also to define the proxies of microbial exposure prior to the baseline assessment of asthma. The use of self/parental-reported asthma is a limitation as it is likely to be less reliable than a physician’s diagnosis. However our definitions of self/parent-reported asthma has been validated against a definition that included bronchial hyper-responsivenessTLR6 gene and asthma that started in early life. Our finding may provide some explanation to the lack of consistent evidence in genetic and gene by environment interaction studies especially when asthma phenotypes based on age of onset were not clearly defined. Replication of our finding using longitudinal studies with repeated measurements of objective markers such as endotoxin and distinct phenotypes of asthma are needed to fully explore these relationships. Our work has provided some indication of a possible mechanism in which microbial products may interaction with TLR in a well-defined asthma group.In conclusion, we have shown for the first time that farming exposure in early-life modified the relationship between polymorphisms in the The design of the TAHS, as well as the prevalence of asthma at baseline and follow-up, has been reported elsewhereThe 2002 follow-up survey traced 7,380 (87.31%) of the original 1968 cohort to an addressThis study was conducted in accordance with the amended Declaration of Helsinki and approved by the Human Ethics Review Committees at The Universities of Melbourne , Tasmania (040375.1) and New South Wales (08094), the Alfred Hospital (1118/04), and Royal Brisbane & Women’s Hospital Health Service District (2006/037). Informed written consent was obtained from all study participants at each follow-up.Asthma was defined by an affirmative response to the question: “Has he/she at any time in his/her life suffered from attacks of asthma or wheezy breathing?” This question was asked in all follow-up surveys and in the 1968 and 1974 studies the parents answered on behalf of their children, but in 1992 and 2004 the participants answered for themselves. Participants who reported asthma in the 1968 or 1974 surveys were classified as having “Early-onset asthma” and asthma reported in the 1992 or 2004 surveys was classified as “Late-onset asthma”. The reference group was those who did not report asthma at any survey. The cut-off for early onset was set at the age of 13 years which has been consistently used in previous studiesTwo proxy measures of exposure to a farming or rural environment in our study:Childhood farm exposure” was defined from the father’s occupation obtained from the school medical record completed by the parents before the child commenced primary school when aged 5 years. Occupations were coded using the Australian Standard Classification of Occupations (ASCO) four-digit classification“Childhood rural environment” was measured by residential remoteness using the postcode of the school that the child attended in 1968 and was based on the Australian Standard Geographical Classification (ASGC)“‘Maternal asthma and/or hay fever’ and ‘paternal asthma and/or hay fever’ were defined by a parent having asthma and/or hay fever from the 1968 survey. ‘Parental smoking’ was defined as probands with either or both parents who reported a history of smoking in the 1968 survey. Childhood eczema and hay fever was reported by parents at the 1968 survey.TLR2 , TLR4 , TLR6  and CD14 . The selection of SNPs were based on previous candidate gene studies that had examined the relationship with either asthma, hay fever or atopic sensitisation121619DNA samples were isolated from whole blood samples obtained from each participant. Genotyping was performed for the following genes: To further explore other phenotypes within early-onset asthma, we have categorised early-onset asthma into transient early-onset asthma and persistent early-onset asthma. Individuals with early-onset asthma who also reported having asthma at age 45 years was categorised into early-onset persistent asthma while individuals who did not have asthma at age 45 years was group into early-onset transient asthma. Asthma under 5 years of age may not be true asthma and hence we repeated our main analysis by excluding participants who had their first asthma attack before the age of 5 years.All analyses were performed using STATA ver13.1 . Deviation from Hardy-Weinberg equilibrium was tested using a Pearson chi-squared test in the reference group (never asthma).The association between proxies of microbial exposure or SNPs and early-onset/late-onset asthma was assessed using multinomial logistic regression. All analyses were adjusted for sex, maternal and paternal asthma and/or hay fever at baseline, and atopy at age 7 years.The association between the SNP and asthma outcomes was examined using all four types of genetic models . To determine the best fitting genetic model for the interaction analysis, we compared the Akaike’s information criteria (AIC) and Bayesian information criteria (BIC) for each genetic model for each SNP . The modHow to cite this article: Lau, M. Y. Z. et al. The interaction between farming/rural environment and TLR2, TLR4, TLR6 and CD14 genetic polymorphisms in relation to early- and late-onset asthma. Sci. Rep.7, 43681; doi: 10.1038/srep43681 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Plant Ribosome-inactivating proteins (RIPs) including the type I RIP Saporin have been used for the construction of Immunotoxins (ITxs) obtained via chemical conjugation of the toxic domain to whole antibodies or by generating genetic fusions to antibody fragments/targeting domains able to direct the chimeric toxin against a desired sub-population of cancer cells. The high enzymatic activity, stability and resistance to conjugation procedures and especially the possibility to express recombinant fusions in yeast, make Saporin a well-suited tool for anti-cancer therapy approaches. Previous clinical work on RIPs-based Immunotoxins (including Saporin) has shown that several critical issues must be taken into deeper consideration to fully exploit their therapeutic potential. This review focuses on possible combinatorial strategies  to augment Saporin-targeted toxin efficacy. Combinatorial approaches may facilitate RIP escape into the cytosolic compartment (where target ribosomes are), while genetic manipulations may minimize potential adverse effects such as vascular-leak syndrome or may identify T/B cell epitopes in order to decrease the immunogenicity following similar strategies as those used in the case of bacterial toxins such as Pseudomonas Exotoxin A or as for Type I RIP Bouganin. This review will further focus on strategies to improve recombinant production of Saporin-based chimeric toxins. Saponaria officinalis. It was firstly described by the group of Fiorenzo Stirpe [Since the idea of the “magic bullet” was conceived by Paul Erlich  over oneo Stirpe , and appSaponaria officinalis L., a member of the Caryophyllacea family that is classified as a Type I ribosome-inactivating protein (RIP). This plant produces several isoforms of Saporin, the major one being SO6 from seeds, also named Sap-SO6. Mixtures of closely related isoforms and several cDNA and genomic clones have been isolated from Saponaria officinalis L. Sap-SO6 represents the major HPLC peak (peak 6) of purified seed-extracted protein and constitutes up to the 10% of the total protein content Seed protein sequencing revealed heterogeneity at two positions, showing either an aspartic or a glutamic acid in position 48, and either lysine or arginine present in position 91, thus indicating that the SO6 peak contains a set of closely related Saporin isoforms. Single recombinant expression of these seed isoforms demonstrated they had same catalytic activity, except for a leaf-derived cDNA that corresponded to a previously identified SAP-5 isoform that showed a 10-times lower catalytic activity [Saporin is a toxic protein produced by activity . We and Saporin is comprised of a single catalytic chain that is devoid of any lectin-like binding domain. This is in contrast to ricin holotoxin that is comprised of two protein chains, the A chain possessing N-glycosidase catalytic activity and the B chain possessing lectin binding activity. The two chains are linked together by a single disulfide bond whose reduction is necessary to allow the free A chain ,5 damagiSaporin and all plant RIPs possess N-glycosidase activity (EC 3.2.2.22), which removes a specific adenine  located in a universally conserved GAGA-tetra loop also termed the α-sarcin/ricin loop. This specific depurination leads to a permanent inactivation of the large ribosomal subunit, blocking the recognition and binding of the elongation factor EF-1 and the subsequent formation of the EF-2/GTP-ribosome complex thereby preventing the translocation of the tRNA from the A site to the P site  of the rType I RIPs have high isoelectric points pI > 9.5), while on the contrary Type II catalytic A chains are either neutral (pI 7.09 for RTA) or acidic (pI 5.25 for Abrin A chain) . Despite.5, whileThe mature form of Sap-SO6 consists of 253 amino acids almost ten percent of which is represented by lysine residues, a structural feature that confers an extremely high pI of approximately 10 .The crystal structure of seed-extracted Sap-SO6 (PDB Code: 1QI7) has revealed that this protein contains two main domains: an N-terminal domain with a predominantly beta-sheet structure and a C-terminal domain with a prevalent alpha-helix structure A. The N-The active site  is conseThe key catalytic residues Glu176, Arg179 and Trp208 also take up the same position in other RIPs while Tyr72 assumes a different conformation, with this residue being responsible for the interaction with the target ribosomal adenine [The Sap-SO6 isoform is extremely resistant to high temperatures, to denaturation with urea or guanidine or to the attack by proteolytic enzymes . FurtherIn order for Saporin to show therapeutic utility, the toxin must be able to gain entry into the cytosol of the target cells; it must therefore overcome the fundamental barrier presented by the plasma and intracellular membranes that constitute the endo-lysosomal system. Type II RIPs, due to the presence of a lectin moiety that binds to sugar residues on the plasma membrane surface, have an efficient route of access to the cell interior via endocytic processes, that subsequently lead them to a retrograde transport pathway trafficking through the Golgi complex to the ER . On the With regard to Saporin, the observations that some cell types show a moderate resistance to its cytotoxicity and that other cell types are much more sensitive, led some researchers to identify specific surface receptors that may mediate the internalization of this plant toxin. A mechanism of endocytosis mediated by the α-2-macroglobulin receptor  was propSaporin has been attached to a variety of different murine monoclonal antibodies to generate first-generation “immunotoxins” directed against several different cancer types, particularly hematological malignancies .Pioneering work was done with chemically linked immunotoxins by the group of Stirpe in Bologna and Flavell in Southampton, directing Saporin to human leukemia and lymphoma cells expressing differentiation antigens such as CD2, CD7, CD19 and CD22 on their plasma membrane surface ,34,35,36Moreover, a clear cellular cytotoxicity (ADCC) depending on the presence of the Fc portion of the antibody  suggesteSaporin-based immunotoxins containing anti-CD22 bispecific antibodies were also tested in pilot clinical trials, with heterogeneous clinical effects, low signs of general toxicity, however, with some patients developing grade I VLS or myalgia, fever and development of anti-Sap-SO6 or anti-mAb antibodies ,38,39. 50 values in the nano to pico molar range displaying differences from hundreds to thousands-fold increases compared with cells exposed to Saporin toxin alone.A comprehensive list of Saporin-based immunotoxins reported in the literature has been recently published . All havThere is a considerable amount of data available on the in vivo performance of Saporin-based immunotoxins used individually or in combinations in preclinical xenograft models of human cancers in severe combined immunodeficient (SCID) mice. A selected number of these are presented in + Hodgkin’s lymphoma [There is some limited clinical experience with Saporin-based immunotoxins and other types of targeted toxin and these are listed in lymphoma . Only folymphoma ,42 treatIt has been reported that approximately 40% of patients with a variety of hematological tumors respond to immunotoxins, produced using both bacterial or plant toxins, with the production of anti-toxin antibodies ,48,49, bRecently Cizeau et al.  describePioneering work was undertaken by Mulot et al.  on the dLeung et al.  mapped tAnalysis of the primary structure  of Sap-SPhytolacca americana and Phytolacca dioica  share a peculiar YHIF motif in the same region.By means of structural alignment of the Type I RIPs  it can bIf the structural localization of the three residues is analyzed,  it can bThus, taking into account what is observed for Trichosanthin, it is reasonable to suppose that this conserved motif may be safely mutagenized in other plant RIPs including Saporin, resulting in a potential alteration of immunological epitope(s) without affecting the catalytic activity. This might therefore be considered as a good starting point for the de-immunization of plant RIP toxins in general.The precise prediction of immunogenic epitopes eliciting immune response in humans is a complex issue, because of the presence of allelic variants of MHC class II. However, if the analysis is restricted to the most diffuse allelic forms of MHC class II  it cIt should be further emphasized that, notwithstanding a difference in amino acid sequence between Sap-SO6 and Bouganin, the critical antigenic regions of Bouganin correspond (on the basis of the prediction made by the PropRed application) to peptides predicted to also be antigenic in Sap-SO6  some common features can additionally be observed. As shown in Among these, if we exclude those containing the key catalytic residues and those represented by a single amino acid, we can focus on clusters 2, 4 and 5. Looking at the position of peptides originating from the first amino acid in the cluster , for Sap-SO6 see  we can oThus, it could be inferred that residues present in the two loops exposed in clusters 2 and 4 should be investigated as potential candidates for de-immunization of this toxin.Vascular leak syndrome (VLS) may occur due to the nonspecific binding of the plant toxic domains to vascular endothelial cells ,70. VLS Pseudomonas aeruginosa, when linked to a monoclonal antibody , inducenti-CD22 . A very nti-CD22 . Vitettanti-CD22 ,76,78,79Peptides with altered or deleted “LDV” sequence in RTA were in contrast unable to cause damage to HUVEC cells. Further experimental work also identified other flanking structural amino acids as potentially contributing to the observed toxicity, while a mutagenesis strategy was been adopted to reduce the VLS properties of dgRTA by changing Arg48 and Asn97 that are in close proximity to the LDV sequence in the crystal 3D-structure of RTA . AspartiMomordica balsamina, see What can instead be clearly noted in If the crystal 3D-structures of other plant RIPs belonging to cluster 2 (including Trichosanthin and Momordin) and Sap-SO6 see B is examIris hollandica,  and subsequently also the ER when A431cells were treated with butyric acid by following a retrograde transport route  to be fiCellular toxicity of RIPs may be mainly attributed to their rRNA depurination activity, which in turn induces a “ribotoxic stress response” characterized by activation of several protein kinases. Generation of reactive oxygen species (ROS) in response may also cause oxidative stress to cells, which might be one of the key factors in inducing apoptosis through the mitochondrial pathway . It has Saporin cytotoxicity has been shown to act in a caspase-dependent manner since inhibition of caspase-3 also results in the inhibition of apoptosis. Furthermore, it has been shown that Saporin-SO6, unlike many other RIPs, did not necessarily depend only on N-glycosidase activity for cellular toxicity, it having been reported that internucleosomal fragmentation of DNA may also contribute to cellular damage . HoweverEscape of toxin across the endo-lysosomal membrane system represents a major bottleneck that prevents the efficient translocation of Type I RIPs from the lumen of intracellular vesicles to the cytosol. Several endosomal escape enhancers have been investigated with the goal of disrupting the intracellular vesicular membrane without increasing any non-specific off-target toxicity. Other such examples include the use of lypopolyamines or DMSO, both of which lead quantitatively to an increased access of Saporin to the cytosolic compartment with a commensurate increase in cytotoxicity. However, neither of these compounds enhances the cytotoxic activity of RTA, probably reflecting the different retrograde trafficking pathway this toxin takes through the TGN and ER ,94.Saponaria officinalis). Weng et al. [A very promising means to augment Saporin-based targeted toxins is the use of glycosylated triterpenoid saponins  against the EGFR-expressing TSA mammary carcinoma cell line TSA growing in syngeneic mice [Another saponin species, SO-1861 from the roots of eic mice . In thiseic mice .Gypsophila paniculata L.) was used as a co-treatment together with a chimeric Saporin protein, containing the PDZ domain from hCASK  binding extracellular CD98 as the targeting moiety. This antigen is used as a marker for several human tumors and is particularly considered as a negative prognostic marker in human glioblastoma. The recombinant fusion toxins with either one (h(CASK)-Saporin) or two (h(CASK)2-Saporin) PDZ domains were tested against several glioblastoma cell lines. Saponin SA1641 was able to increase the toxicity of Saporin and h(CASK)2-Saporin chimera in U87 Glioblastoma cell line (IC50 < 10−11 M and << 10−12 M respectively), and allowed hCASK-Saporin to reach an IC50 of 10−10 M [Saponin SA1641  [Saponinum album saponins (SA) augmented the PE40-based ITx by 170-fold while the Saporin-based ITx was augmented by more than 6 orders of magnitude following this co-treatment. This great difference in the saponin enhancer effects on PE40-ITx versus Saporin-based ITxs reflects the preferential interaction of some Type I RIPs with these triterpenoid glycosylated saponins . Holmes et al. [Saponinum album saponins on Saporin-based ITxs constructed with antibodies directed against CD7, CD19, CD22, CD38 and CD71 on three different human leukemia or lymphoma cell lines, but found considerable cell line and target molecule variation on the extent of augmentation observed, ranging from just 174-fold to greater that 31.5 million-fold for Daudi lymphoma cells targeted via CD19 antigen. These authors also showed that a proportion of the augmentative effect of SA on ITx cytotoxicity was off-target, a problem that was partially resolved by scheduling exposure to ITx first followed by SA second. Smith et al. [Lombardi and co-workers compared the performances of anti CD22 single chain variable fusion constructs/scFv to identify novel recombinant single chain antibodies showing nanomolar affinity for the CD22 antigen able to deliver, either Saporin or n below) ,101. Saps et al.  investigh et al.  have furh et al.  to hypotper se for the target cells; (ii) their co-adjuvant role requires their concomitant presence at the site of dislocation during the passage of the toxin “en route”—to its final destination (iii) these agents should not facilitate toxin cytosolic entry into off-target cells.As a general rule endosomal escape enhancers: (i) should not exert any appreciable toxicity These optimal conditions should be ideally tested in in vivo systems. Unfortunately, most of the data published using endosomal escape enhancers to date have been examined mostly in vitro. Further studies in in vivo model systems are therefore urgently required to validate the in vitro results necessary for pre-clinical development. Some authors have however, demonstrated efficacy of endosomal escape enhancers in animal models . For a cAnother innovative methods to enhance the delivery and uptake of therapeutic agents, as Saporin, is the encapsulation of drugs using specific exosomal membranes modified with different peptides including a cationic pH-sensitive fusogenic peptide known as the GALA peptide . These vMacropinocytosis is up regulated in several cancer cells and is a more efficient internalization pathway capable of bulk uptake of fluids from the external milieu. Macropinosome vesicles are on average of 200–300 nm diameter and additionally provide a further opportunity for nano-delivery approaches. Using an automated microscopy-based imaging platform to identify phage antibodies co-localizing with macropinocytotic markers, Ruan and co-authors screened their single chain antibody libraries enriched in internalizing antibodies by a laser capture micro-dissection selection, which bound preferentially to tumor cells . These wAntineoplastic treatments with ITx are often only partially effective or even completely ineffective when only one ITx targeting a single molecule on the tumor cell is administered. To enhance and optimize the anti-cancer potency, multiple ITx could be administered simultaneously. As a “proof of principle” of the possibility of targeting two overlapping but distinct subpopulations of cells by using a secondary immunotoxin treatment is the dual treatment used against Glioblastoma Multiforme (GMB) by combining the use of the “Mab-Zap” Saporin immunotoxin system developed by Kohl and co-workers . The “MaAn interesting example of a co-treatment with a Saporin-based immunotoxin and anti-CD20 antibody was described by Flavell and colleagues . These wRituximab has also been conjugated to seed extracted Saporin in combination with another anti-CD22 ITx containing Saporin. These two ITxs were created by chemical conjugation of the appropriate antibody to Saporin. Rituximab/Saporin and OM124/Saporin are respectively directed against CD20 and CD22, both antigens highly expressed on Non-Hodgkin’s lymphomas (NHLs) and found particularly expressed at high levels on normal mature B-cells and on a large population of B-lymphoma cells but absent in the normal tissues and hematopoietic stem cells. A way to increase the therapeutic efficacy of these two Saporin-based ITxs was obtained by the simultaneous administration of chemotherapeutic agents or cell inhibitors, such as the proteasome inhibitors PS-341, MG-132, or the purine analogue fludarabine .Finally, a protocol termed 3BIT consisting of a combination of three different saporin-based ITx directed against CD19, CD22 and CD38 was reported by Flavell’s group . The com+ lymphoma cell lines, Raji and D430B, the HMW-ITx was more cytotoxic than the LMW-ITx. Such a double ITx system (HMW and LMW-ITx) is suggested by the authors to be flexible in a potential clinical use, devoting the dimeric HMW use as endocytosis enhancer  or as an improved penetration system for solid tumors due to the reduced molecular size of the LMW ITx [An interesting issue is correlated to the chemically linked immunotoxins, where both the number of toxins/antibody or the total number of both the components may affect the activity of the conjugate. A Saporin-Rituximab immunotoxin was obtained by chemically conjugating via a disulfide bond rituximab to Saporin to yield two immunoconjugate species, a HMW-ITx (Dimeric) and LMW-ITx (Monomeric) each of which had similar activity in inhibiting protein synthesis in a cell-free system. However, in two CD20 LMW ITx . These aFlavell and collaborators  showed tThe classical approach to site-specific drug delivery of a target-specific immunotoxin against tumors cells requires receptor-mediated endocytic uptake of the drug and efficient release of the therapeutic molecule from intracellular endocytic compartments, into the cytosol. This process has several limitations among which the low rate of penetration through the membranes of endocytic vesicles and/or compartments and the degradation of therapeutic macromolecules trafficked to lysosomes by proteolytic enzymes .To at least partially circumvent this problem a light-based, an innovative technology has been developed termed photochemical internalization (PCI).PCI may help to release mostly endocytosed macromolecules into the cytosol being based on the use of specific photosensitizers located within endocytic vesicles that, upon activation by light, induce the drug release due to photochemical-mediated rupture of endocytic membranes .+ rat epithelial ovarian cancer cell line NuTu-19 cells approximately 1000-fold relative to EGF-Saporin treatment of cells without PCI [Weyergang and colleagues demonstrated that PCI significantly increases the toxicity of EGF–Saporin on the EGFRhout PCI .The evolution of PCI technology as an in vivo treatment has been developed through the use of laser-controlled endosomal escape in localized tumors. An example of such an application is the treatment with a CD133-targeting immunotoxin AC133–Saporin (PCIAC133–Saporin) that co-localizes with the PCI-photosensitizer TPCS2a in CD133 positive cancer stem cells. After light exposure, cytosolic release of AC133–Saporin was induced in target cells with a concomitant increase in cytotoxicity. The advantages of this novel technology is the ability of PCI-photosensitizer to preferentially accumulate within tumor tissues, allowing the possibility to trigger the PCI photosensitizer in vivo locally in tumor tissue combined with the tumor-selectivity of the immunotoxin .PCI is also valuable for treatment of CSCs expressing CD44, that characterize tumor cells highly resistant to ROS attack, rendering originally resistant cells to be more sensitive to chemo- and radiotherapy. CD44 has also been reported to be involved in multidrug resistance (MDR). Bostad and coworkers made an ITx using a biotinylated-CD44 mAb and an avidin-Saporin. After administration of the PCI-photosensitizer TPCS2a and ITx in seven different carcinoma and sarcoma cell lines an efficient and specific cytotoxicity in CD44-expressing but not in CD44-negative cancer cells was observed .+ Zr-75-1 cells prior to light exposure  that increase the cytotoxic effect because “light first” procedure also reduces the trastuzumab-induced HER2 endocytosis of ITx [Another antibody whose cytotoxic performance is greatly improved by PCI is the commercially humanized HER2 mAb, trastuzumab. This FDA-approved antibody was used in chemotherapeutic treatment of HER-2 positive breast cancer. Unfortunately, the acquired resistance to trastuzumab treatment is a common outcome difficult to solve. A novel strategy to overcome this problem is by the use of PCI. Using biotinylated-Trastuzumab and avidin-Saporin a novel ITx was generated for PCI, and co-administered with PCI-photosensitizer TPCS2a to trastuzumab-resistant HER2s of ITx .Vikdal and colleagues demonstrated that PCI can also increase the accumulation of free Saporin and subsequent enhanced PDT induced cytotoxicity in the HUVEC cell line with respect to HT1080 cell line (fibrosarcoma). This strategy can be used as an antivascular treatment in the anticancer therapy .An alternative approach to overcome the potential barriers that can reduce the cellular uptake and intracellular release of Saporin is the use of generation four polyamidoamine (PAMAM) dendrimers carrier molecules. These molecular constructs are specifically capable of increasing the endocytic uptake, the passive tumor targeting, and could be utilized in the context of photochemical internalization (PCI) technology, facilitating the Saporin cellular uptake and cytosolic release .Recently a PCI-based drug delivery system was applied to EpCAM positive cancer cells. A novel ITx was generated using an EpCAM-targeting mAb 3–17I chemically coupled to Saporin and when ITx was tested in vitro showed a potent and selective reduction of cellular viability, proliferative capacity, and colony forming ability in breast carcinoma, pancreatic adenocarcinoma, and colon adenocarcinoma cell lines .As further innovative procedure under investigation consist of plasmid co-transfection using two toxins DNA sequences in an in vitro model. This technique, termed originally “Toxic gene therapy” (suicide gene therapy) has been successfully tested on various cancer cell lines  by using two gene toxins coding for the mature polypeptide inserted into two distinct mammalian gWIZ plasmids: pGEL (gWIZgelonin) and pSAP (gWIZ-Saporin). The co-transfection treatment in mammal tumor cell lines was achieved by using cationic polyethyleneimine (PEI) and exerted a potent and specific cytotoxicity .Ricinus communis protects itself from self-intoxication is to produce a long inactive precursor where the nascent polypeptide is efficiently co-translationally inserted inside the lumen of ER to avoid ricin mislocalization in the cytosol and inactivation of target ribosomes [E. coli were also found problematic, because, upon induction of RIP expression the bacterial growth rate was significantly impaired, as reported earlier in the case of Mirabilis antiviral protein [Another further relevant aspect for the development of Saporin chimeras (and other plant RIPs chimeric toxins) as pharmaceutical agents is their efficient expression and proper folding of the nascent fusion polypeptide chain in a host cell. To express plant protein toxins and especially recombinant fusion chimaeras, based on Saporin or Type I RIPs it would be preferable to use a eukaryotic expression system, firstly because Type I RIPs such as Saporin, are secretory proteins and secondly because correct folding by the cellular quality control system is most readily achievable in the microenvironment of the endoplasmic reticulum (ER). However, exogenous protein toxin expression may trigger autointoxication issues. The principal way in which ibosomes . When oribosomes . The Sapibosomes , thanks ibosomes . Other Tibosomes . It is,  protein , PAP [12 protein , dianthi protein  well as  protein . More re protein .E. coli strain BL21 (λDE3) pLysS to give satisfactory yields, issues such as endotoxin A contamination and processing of the insoluble bacterial pellets remained problematic [P. pastoris under an alcohol-oxygenase (AOX1) tight regulated promoter by inserting the prepro-alpha factor signal peptide at the NH2-terminus of Saporin L3, as we also previously reported to obtain high level of expression of Saporin seed isoform in P. pastoris [P. pastoris to grow at very high cell-densities (with a rapid growth rate) and an almost protein-free extracellular medium facilitating the purification procedures; high levels of foreign proteins expressed under the methanol-induced Promoter AOX1; post-translational modifications like N-glycosylation , methylation, acylation, proteolytic maturation and proper subcellular targeting of the heterologous polypeptide [Although toxin expression could be more tightly regulated by employing the blematic . Strikinpastoris . The choypeptide .P. pastoris by modifying transcription factors regulating AOX1 promoter and developing an efficient glucose-glycerol-shift induction bioprocess for foreign protein expression control. The newly constructed strain could efficiently replace the traditional glycerol-methanol induction in the wild-type strains using an insulin model polypeptide expression, exhibiting a more economic, safe and environment-friendly impact that has a great potential for the biopharmaceutical industry [Furthermore, this yeast expression system is under intensive investigation to eliminate the disadvantages of methanol-oxygen consumption in batch-fed-large scale fermenters, Wang and collaborators engineered a new methanol-free industry . Finally, a crucial parameter that must be considered to achieve high levels of protein expression in yeast expression systems is the use of optimized yeast codon-usage.P. pastoris [For many years it has been empirically assumed that was a relationship between ribosome translation speed and the levels of an exogenous protein synthesized. Codon-usage choice evolved as means to optimize single mRNA translation, essentially by changing the rare codons present in the heterologous gene into those preferred by the expression system (codons found in most highly expressed endogenous proteins). This is a common approach that was also employed for optimal expression of seed Saporin and its derived fusion chimeras in pastoris . Proteinpastoris . Chu andpastoris .Pichia pastoris expression Saporin reached just 10 times lower levels of secreted active polypeptides as compared with those of Saporin-KQ, an inactive catalytic mutant [Working on codon-usage optimization for c mutant . P. pastoris when a codon-usage optimized ATF domain (human) was used or an optimized single chain variable domain to obtain scFv fusions, such as the anti CD22 scFv termed 4KB that was obtained from the subcloning of VH and VL regions of a parental 4KB128 monoclonal antibody then used to construct Saporin-based ITxs. Target CD22+ human Daudi lymphoma cells could internalize this recombinant scFv with absolute immunospecificity as demonstrated by competitive inhibition by the parental monoclonal CD22 antibody [Intrinsic host cell toxicity was also consistently decreased, employing this optimization strategy, as also demonstrated for the expression of ATF-Saporin or other chimeric fusions better expressed in antibody .E. coli or P. pastoris expression systems [E. coli or in Pichia pastoris and the resulting fusion proteins affinity-purified. Protein synthesis inhibition assays showed that the selected recombinant ITxs were active, having comparable IC50 (inhibitory concentration by 50%) in the nanomolar range. Overall our results confirmed that E. coli is the system of choice for the expression of recombinant fusion toxins of bacterial origin (that otherwise underwent proteolytic degradation in the yeast cells). We demonstrated, in contrast, that Saporin-based ITxs are best expressed and recovered from P. pastoris cultures following yeast codon-usage optimization. Codon-optimization of the scFv domain would appear important in order to increase the potential number of secreting clones that were capable of producing at least 1–2 mg/L of fusion protein. In addition, among alternate design options, the best performing ones were those having a yeast codon-optimized scFv where a 18-amino hydrophilic 18-flexible linker GSTSGSGKPGSGEGSTKG, (amino acid one letter code) that showed enhanced resistance to proteolysis together with reduced aggregation of scFvs [Interestingly, we compared the expression of ITxs made by using two different toxic domains both capable of irreversibly blocking protein translation: PE40 of bacterial origin (endowed with eEF-2 ADP-ribosylation activity) or Saporin. We evaluated optimal microbial expression conditions of various scFv fusion formats made with the two toxin domains to then further evaluate the immunospecific cytotoxic performance of each different fusion construct, together with their achieved yields in  systems . A numbeof scFvs ,133 was 4S)3 linker between the VH and VL or other formats tested were either not producing viable clones or gave rise to high yields of mutated Saporin fusions that were found inactive. The scFv versions having either a  or to directly select scFv from humanized libraries, together with the possibility to express recombinant RIPs and to eventually engineering their sequences, may allow again for future attractive clinical applications of ITxs. Furthermore, the possibility to have new recombinant antibodies available as humanized molecules in a relatively short time and cost effective (several companies offer this service, see for example


KODAMA, a novel learning algorithm for unsupervised feature extraction, is specifically designed for analysing noisy and high-dimensional datasets. Here we present an R package of the algorithm with additional functions that allow improved interpretation of high-dimensional data. The package requires no additional software and runs on all major platforms.http://cran.r-project.org). The software is distributed under the GNU General Public License (version 3 or later).KODAMA is freely available from the R archive CRAN (Bioinformatics online. KODAMA package developed for use in the R programming environment.Knowledge Discovery and Data Mining is an interdisciplinary area focusing upon methodologies for extracting useful knowledge from complex data. With the explosive growth of high-throughput experimental data, data-based solutions are increasingly crucial. We recently published KODAMA, a novel learning algorithm for unsupervised feature extraction, specifically designed for analysing noisy and high-dimensional datasets (a priori information is needed). The cross-validated accuracy can be calculated by using any supervized classifier. In the current version of KODAMA, two classifiers are implemented: k-Nearest Neighbors (kNN) and Partial Least Squares (PLS)—Discriminant Analysis (DA). The iterative procedure used in KODAMA leads to suboptimal solutions and must be repeated (100 as default) to average the effects owing to randomness. External class information can be integrated in KODAMA before performing the iterative procedure thereby supporting a semi-supervized approach for highlighting otherwise hidden features of interest. After each run of the procedure, a classification vector with high cross-validated accuracy is obtained. KODAMA subsequently collects and processes these results by constructing a dissimilarity matrix to provide a holistic view of the data while maintaining their intrinsic structure.The core of the algorithm consists of two main parts. The first step involves random assignment of each sample to a different class. In the second step, the cross-validated accuracy is maximized by an iterative procedure by swapping the class labels  and PLS-DA (pls.kodama) classifiers and the iterative procedure of KODAMA. The pls.double.cv and knn.double.cv functions perform double cross-validation procedures using PLS-DA or kNN as classifiers, respectively (k) is optimized by maximising the cross-validated coefficient of determination (Q2y) obtained by an inner cross-validation on the training sets.The package is computationally efficient with the workhorse functions written in C ++ using Rcpp , RcppArmloads function can be used to extract the variable ranking. After each maximization of the cross-validated accuracy the final label set is used to calculate the loadings of PLS-DA or the logarithm of the P-value from the Kruskal-Wallis Rank Sums test. The output of LOADS function is the average of these values for each variable of the dataset. The highest values indicate the most important variables.The k.test function performs a statistical test to assess association between the KODAMA output and any additional related parameters such as clinical metadata. The coefficient of determination (R2) is used to assess the proportion of the variance in the dependent variable (KODAMA output) that is predictable from the independent variable  and can thus be used as a measure of the goodness of fit , a collection of 873 nuclear magnetic resonance spectra of urine samples from a cohort of 22 healthy donors . loads function. Further analysis using the k.test function shows a statistically significant association between the KODAMA output with clinical metadata including donor (P < 0.0001) and gender (P < 0.0001).To demonstrate briefly the performance of the KODAMA represents a valuable tool for performing feature extraction on noisy and high-dimensional datasets. Addition functions facilitate the identification of key features associated with the generated output and are easily interpretable for the user. The K-test permits the identification of significant associations between the KODAMA output and related information.Supplementary DataClick here for additional data file.


MTHFR, SATB2, PVRL1. The present study showed that collecting saliva samples is a non-invasive procedure and is well accepted by CLP patients. MiRNAs can be easily isolated and identified. The differences in regulation of miR-141, miR-223 and miR-324-3p between the two groups of salivary samples suggest that these molecules are valid prognostic biomarkers and therapy dynamic response indicators, also for the accuracy and non-invasive sampling and dosing system.MicroRNAs (miRNAs) are endogenous non-coding RNAs of about twenty-two nucleotides that regulate gene expression through post-transcriptional control. The purpose of the present study was to identify and describe the salivary miRNAs in cleft lip and palate (CLP) patients comparing them with a control healthy group. Twelve patients  formed the study group. The control group was created selecting twelve healthy subjects matched for age and sex with study group. We recorded differences in miRNA expression profile between the saliva of CLP patients and the control group. Specifically, miR-141, miR-223, and miR-324-3p were mostly deregulated between the study and control groups. Interestingly, these three miRNAs are the regulators of the following genes correlated to cleft palate and lip development:  Orofacial clefts  occur with a frequency as high as 1 in 700 live births and are the most prevalent birth defects affecting humans . PatientCL/P and CPO are distinct developmental defects with different underlying causes. Genetic and environmental factors contribute to the complex aetiology of CLP. Thirteen loci (gene positions) in different chromosomal regions and 20 genes associated with CLP have been reported from linkage and association analysis. Few studies, however, have addressed the potential involvement of epigenetic factors, such as expression and function of microRNAs (miRNAs) in facial development, and most of these have focused on the developing secondary palate –10.Caenorhabditis elegans [et al. showed in 2012 that in saliva, microRNAs are primarily present inside exosomes rather than associated to other proteins or freely circulating [Although multiple molecular mechanisms of epigenetic regulation exist, one of the most actively investigated to date is the action of miRNAs. MiRNAs are short noncoding RNA molecules –13 of 19 elegans . Recent  elegans –17. MiRN elegans –20. The  elegans . Gallo eculating . MiRNAs culating . TogetheIn the past few years, several research papers have been published reporting the presence of miRNAs in saliva and their potential as non-invasive biomarkers in oral cancer detection.Profiling arrays typically require several micrograms or more of input RNA, causing problems with sensitivity and specificity. There are also three different forms of miRNA ; thus, the profiling methods have to detect and distinguish between each type.The identification and characterization of miRNAs in patients with CLP may be useful in obtaining important diagnostic and prognostic information of these craniofacial abnormalities and can be useful to plan the most appropriate therapeutic approach.In the present study, we have standardized a protocol of sampling and analysis of saliva identifying miRNAs expressed in salivary samples. Moreover, we have compared miRNAs signatures expressed in CLP and healthy patients correlating these miRNAs to genes involved in CLP occurrence.-(ΔΔCT)) of –3.45; the most upregulated miRNA (miR-324-3p) with Log2 (2-(ΔΔCT)) of 3.12 and a miRNA with not very significantly changed expression (miR-223). We have selected three miR-141 and miR-324-3p miRNAs, which with have high values the of fold changes calculated relatively to the mean value of normal samples and miR-223 as it was previously reported to be involved in embryogenesis [p < 0.05). Thereafter, we analysed the upregulated miR-324-3p and miR-223. We analysed miR-223 in the saliva of 12 patients and we found that it was upregulated in all samples except in patient number 5 and, therefore, we again validated the results obtained with microarray. . Finally, we analysed miR-324-3p in 6 patients and we have found a significantly up regulation of this miRNA in all samples as compared to microarray analysis.  that contain the miR-binding site(s) in the UTR. TargetScan analysis predicted the binding of miR-324-3p e miR-223 to the 3′-UTR of methylenetetrahydrofolate reductase (MTHFR)  an amount of 131 miRNAs in the saliva of CLP and control samples. In Table RNA miR-3-3p with RNA miR-34-3p withRNA miR-34-3p withEvery year a large number of children in the world are born with structural birth defects. Despite intellectual and technological strides in the biomedical sciences, including sequencing of the human genome and advances in prenatal care/diagnostics, the underlying causes of nearly 70% of all birth defects and developmental disabilities remain unknown. Isolated oral-facial clefts are among the most common birth defects , 2. FailWe calculated the sample size using the following statistical formula: . Indeed, the importance of miRNAs for craniofacial development in general, is supported by the severe craniofacial malformations seen in mouse embryos with a conditional deletion of Dicer in Wnt-1 expressing neural crest cells [Dicer expression in Pax2-expressing cells also leads to craniofacial defects, including cleft palate and midfacial hypoplasia [Interestingly, sampling saliva from patients has been well accepted by the subjects enrolled in our study. In according to the age of subjects, parent’s authorization has been requested and in all cases they proved willing to collaborate. Sampling saliva was not perceived as invasive or sticky by either parents or patients themselves even if it must be collected in the morning without breakfast and brunch teeth. Indeed, saliva represents a non-invasive biological fluid sampling, well accepted by patients and easy to apply. The determination of genetic and epigenetic causes of palate alterations is still undefined although exploration of the epigenome—the epigenetic variability in cells (epimutations) — holds the hope for precise diagnosis of disease and congenital anomalies such as cleft lip. A small number of studies up to date have investigated the involvement of epigenetic factors in the determination of this kind of diseases. MiRNAs are among the most actively investigated epigenetic factors to date. Their role in embryonic development is well documented and now widely accepted , 26 but st cells . Loss ofpoplasia . The clipoplasia –34. Prevpoplasia . It is ppoplasia . In addiIn conclusion, the present study is the first one investigating on the role of salivary miRNA expression in CLP. CLP patients between 9 and 15 years are subjected to many treatments as bone grafting jaw, orthodontics, additional cosmetic corrections including jawbone surgery, speech therapy, pharyngoplasty. Some of these are very long, complex, difficult and expensive associated to different and critical prognostic evaluations. All therapeutic approaches are guided only by the patient’s clinical analysis and diagnosis without evaluating the genetic and epigenetic variability of these subjects. Our future objective is identifying the different ways to respond to therapy and consequently modulate all therapeutic approaches through not only clinical evaluation but with the epigenetic characterization achieved with a simple saliva collection.Our data suggest miR-141, miR-324-3p and miR-223 as possible salivary biomarkers of the malformation. A complete understanding of the regulation of expression of all genes involved in schisis development will require the integration of miRNA expression profiles. These include the transcriptome, proteome, methylome, and histone modifications. How these diverse regulatory mechanisms lead to the coordinated morphogenetic processes of CLP development is a prerequisite for developing strategies for the treatment or prevention of these craniofacial abnormalities.Appropriate ethical approval was secured by the Health Research Ethics Board of the University of Campania “L. Vanvitelli” (No.1032).All CLP patients were consecutively enrolled at the Orthodontics Clinic of the University of Campania “L. Vanvitelli” from January 2015 to January 2016. Patients with syndromes including cleft lip and/or palate were excluded from the study. From an initial sample of 25 patients we have selected 17 patients. Five patients refused to participate in the study so the sample became of 12 final patients.Anamnesis is of all patients was obtained. For all subjects included in the study intra-oral and extra-oral photographs, craniox-rays in latero-lateral and posterior-anterior view and dental casts were requested and examined. Twelve patients without CLP matching for age and sex formed the control group. The same records and anamnesis were required in these subjects. Parents of all children involved in the study signed an informed consent before the beginning of the study.A protocol for collecting the saliva samples was developed according with the most recent literature in evidence. Following the collection, saliva was processed as soon as possible, to avoid cell damage and subsequent miRNA release from exosomes in saliva. For the isolation of salivary exosomes two centrifuge steps were performed.miRNA extraction was performed using MIRVANA PARIS KIT, , it was adapted to our purpose for its higher sensitivity.® MicroRNA Reverse Transcription Kit (Thermofisher Scientific) and the Megaplex™ RT Primers, (Thermofisher Scientific) single-stranded cDNA was synthesized from total RNA samples. Then, our cDNA targets were preamplified to increase the quantity of desired cDNA for gene expression analysis using 384-well TaqMan® MicroRNA Arrays. The preamplified target cDNA was amplified by DNA polymerase from the TaqMan® Universal PCRMaster Mixusing sequence-specific primers and probes on the TaqMan® MicroRNA Array. About 100 μL of the PCR reaction mix were dispensed into each port of the TaqMan MiRNA Array. Then, the array was loaded and run on an Applied Biosystems Viia7 instrument by using the default thermal-cycling conditions. The presence of the target was detected in real time through cleavage of TaqMan probe by the polymerase 5′-3′ exonuclease activity. To validate the results of the array cards, the expression of miRNAs found to be differentially expressed in the screening phase of the study was independently determined by real-time PCR . cDNAs were synthesized as detailed above and the expression of individual miRNAs was determined using pre-designed probe-primer sets from Life Technologies. The expression of each miRNA tested was normalized by the ΔΔCt method [®MicroRNA Assays. They are designed to detect and accurately quantify mature microRNAs (miRNAs) using Applied Biosystems real-time PCR instruments . The TaqMan MicroRNA Assays use looped-primer RT-PCR, a new real-time quantification method, to accurately detect mature miRNAs. Single-stranded cDNA were synthesized from total RNA samples using the TaqMan® MicroRNA Reverse Transcription Kit.Subsequently, using the TaqMant method . To perf® Gold DNA polymerase amplifies target cDNA synthesized from the RNA sample, using sequence-specific primers from the TaqMan Assay Plates.During the target amplification step, the AmpliTaqUsing comparative CT method, we used endogenous controls to normalize the expression levels of target genes by correcting differences in the amount of cDNA loaded into PCR reactions.To normalize human total RNA samples, an appropriate constitutively expressed endogenous control was selected.CT method has been extensively used as a relative quantification strategy for quantitative real-time polymerase chain reaction (qPCR) data analysis. This method is a convenient way to calculate relative gene expression levels between different samples in that it directly uses the threshold cycles (CTs) generated by the qPCR system for calculation [The 2-ΔΔculation . RelativA database system using the Statistical Package for Social Sciences (SPSS) version 18 was made. The descriptive statistical analysis was performed on proportions calculations, measures of central tendency and variability for socio-demographic and clinical aspects.http://www.targetscan.org/, http://pictar.mdc-berlin.de/,http://www.microrna.org/microrna/home.do).Genes that contain the miR-binding site(s) in the UTR were obtained using the TargetScan program (


Adherence to a medication is generally defined as the extent to which patients take medications as prescribed by their health care providers. Poor adherence to study medication is not uncommon posing a major challenge to treatment trails. However, poor adherence may not be randomly distributed but rather be associated with demographic or illness factors. The aim of the present study was to identify factors associated with adherence to study medication in young people at ultrahigh risk of psychosis who participated in the NEURAPRO study.Secondary analysis of data collected in a multi-centre, double-blind, placebo-controlled, randomized clinical trial to prevent or delay the onset of psychosis in participants at ultrahigh risk of psychosis testing omega-3 polyunsaturated fatty acids (omega-3 PUFAs) vs. placebo, in combination with cognitive behavioural case management (NEURAPRO) were included in this analysis. Measures included the Brief Psychiatric Rating Scale (BPRS), the Scale for the Assessment of Negative Symptoms (SANS), the Montgomery Asberg Depression Rating Scale (MADRS), the Young Mania Rating Scale (YMRS), the Social and Occupational Functioning Assessment Scale (SOFAS), and the Global Functioning: Social and Role scales. Adherence to the study medication was assessed monthly for each participant based on capsule count. The mean adherence rating over the 6-month intervention period was then computed and categorized as either adherent (≤25% of capsules returned) or non-adherent (>25% of capsules returned). Transition to psychosis was defined on the basis of operationalized criteria and assessed with the Comprehensive Assessment of the At-Risk Mental State. Levels of ω-3 PUFAs in fish oil, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (amongst other fatty acids) were measured as percentage of total fatty acids in erythrocytes at baseline and at month 6 (end-of-intervention).Of 304 randomised participants, 57.9% (N = 176) were non-adherent (>25% of capsules returned) and 128 (42.1%) were adherent (≤25% capsules returned) to the study medication.No sex differences were observed for adherence rates. At baseline the omega-3 index (EPA+DHA) was significantly lower in the non-adherent group (P = 0.018). The non-adherent group had significant lower scores on the SOFAS (P = 0.001) and the Global Functioning: Social and Role Scale at baseline assessment  compared to the adherent group. No statistically significant differences were observed on symptom measures at baseline . The cumulative transition to psychosis rate at month 12 was significantly higher in the non-adherent group compared to the adherent group .Adherence to study medication was relatively low in NEURAPRO. Poor functioning and lower levels of ω-3 PUFAs at baseline were associated with non-adherence. Young people who were non-adherent had a significantly higher risk of progressing to first episode psychosis. Knowledge about factors associated with adherence could help to improve the delivery of interventions in young people at risk of psychosis.


In this review paper, we aim to provide an overview of the key findings related to D1/D5 receptor-dependent persistence of synaptic plasticity and memory in HPC, especially focusing on the emerging evidence for a role of the locus coeruleus (LC) in DA-dependent memory consolidation. We then refer to candidate brain areas and circuits that might be responsible for detection and transmission of the environmental novelty signal and molecular and anatomical evidence for the LC-DA system. We also discuss molecular mechanisms that might mediate the environmental novelty-associated memory enhancement, including plasticity-related proteins that are involved in initial memory consolidation processes in HPC.Most everyday memories including many episodic-like memories that we may form automatically in the hippocampus (HPC) are forgotten, while some of them are retained for a long time by a memory stabilization process, called initial memory consolidation. Specifically, the retention of everyday memory is enhanced, in humans and animals, when something novel happens shortly before or after the time of encoding. Converging evidence has indicated that dopamine (DA) signaling via D Many people have vivid memories of the first dinner date with their partner, including details like the name of the restaurant and the food they had. In contrast, it is very difficult to remember what you had for dinner a few weeks ago. Most everyday memories, including episodic-like memories that we may form automatically in the hippocampus (HPC) –3, are f1/D5 receptors and protein synthesis inhibitors in HPC. Pharmacological studies of hippocampal synaptic plasticity have supported the notion that D1/D5 receptors act as a gating mechanism for long-term persistence of plastic changes , 23+ axofrom VTA   an an+ neurication)  153] al al+ neurication) . SpecifiIf candidate PRPs would be identified, the next logical step is to assess whether the candidate PRPs are preferentially targeted to activated spines using two-photon glutamate uncaging with time-lapse imaging . Subsequ+ neurons to HPC can drive the postencoding environmental novelty-associated enhancement of memory retention through noncanonical release of DA in HPC. These studies also raise an intriguing possibility that the impact of environmental novelty may differ qualitatively from that of reward-associated novelty and projections from VTA-TH+ neurons to HPC might mediate reward-associated novelty which modulates the memory retention with a narrow time window. Initial consolidation triggered by two distinct dopaminergic novelty systems could help make encoded memory traces last long enough for the effective function of the more extended process of system consolidation by which hippocampus-dependent memories guide the eventual stabilization of neocortical memory networks.Most everyday memories may form automatically in HPC. The key role of this memory system is to filter our unnecessary information but keep the important memories by a mechanism that involves novelty-associated DA release in HPC. Recent optogenetic studies have revealed that projections from noradrenergic LC-TH