AtBAG4 (Arabidopsis), Hsp70 (Citrus tristeza virus) and p35 (Baculovirus), significantly improves salinity tolerance in rice at the whole plant level. Physiological, biochemical and agronomical analyses of transgenic rice expressing each of the anti-apoptotic genes subjected to salinity treatment demonstrated traits associated with tolerant varieties including, improved photosynthesis, membrane integrity, ion and ROS maintenance systems, growth rate, and yield components. Moreover, FTIR analysis showed that the chemical composition of salinity-treated transgenic plants is reminiscent of non-treated, unstressed controls. In contrast, wild type and vector control plants displayed hallmark features of stress, including pectin degradation upon subjection to salinity treatment. Interestingly, despite their diverse origins, transgenic plants expressing the anti-apoptotic genes assessed in this study displayed similar physiological and biochemical characteristics during salinity treatment thus providing further evidence that cell death pathways are conserved across broad evolutionary kingdoms. Our results reveal that anti-apoptotic genes facilitate maintenance of metabolic activity at the whole plant level to create favorable conditions for cellular survival. It is these conditions that are crucial and conducive to the plants ability to tolerate/adapt to extreme environments.Environmental factors contribute to over 70% of crop yield losses worldwide. Of these drought and salinity are the most significant causes of crop yield reduction. Rice is an important staple crop that feeds more than half of the world’s population. However among the agronomically important cereals rice is the most sensitive to salinity. In the present study we show that exogenous expression of anti-apoptotic genes from diverse origins,  By 2050, the world population is expected to reach 9.6 billion people . To sustOryza sativa L.) is an important crop that feeds more than half of the world’s population and is the model system for monocotyledonous plants that include members of the agronomically important cereals. Approximately 90% of the world’s production and consumption of rice are in Asia  conventional breeding, (ii) marker assisted selection and (iii) genetic engineering. Of these, genetic engineering displays great potential and has become a powerful tool in plant breeding programs since it allows the introduction of select gene(s) without affecting the desirable characteristics of an elite genotype .Genetic Programmed cell death or simply “the decision of whether a given cell should live or die” is essential for all multicellular (Metazoan) organisms . Under sAtBAG4, Hsp70 and p35 are anti-apoptotic genes that have been reported to confer tolerance to salinity and drought stresses in transgenic tobacco. AtBAG4 is a Bcl-2- associated athanogene from Arabidopsis thaliana. The A. thaliana genome contains seven homologs of the BAG family, including four with a domain organization similar to animal BAGs  by constitutive-overexpression of AtBAG4 (NCBI Reference Sequence: NM_115037), Hsp70 (GenBank: EU857538) and p35 (GenBank: KF022001) and evaluation of their ability to confer salinity tolerance in rice using agronomical, biochemical and physiological assessments. By investigating the ability of anti-apoptotic genes (from different sources) to confer tolerance to salinity stress via maintenance of ROS, photosynthetic efficiency and growth rate thereby minimizing yield loss, a possible mechanism of anti-apoptotic genes to maintain metabolic activity in plants under salinity stress is also elucidated.Here we present for the first time the development of salinity tolerance in one of the most important staple crops – rice  promoter were cloned into the binary vector pCAMBIA1301. All constructs were sequenced prior to the transformation for confirmation of fidelity.O. sativa L. ssp. Japonica cv. Nipponbare, and transformed by Agrobacterium-mediated transformation as described in Agrobacterium tumefaciens (strain AGL1) harboring the respective genes of interest at 25–27°C in dark for 3 days. Following co-cultivation contaminating Agrobacteria were removed by washing in 2N6 liquid media and sterile water containing 200 mg/L-1 timentin before blotting dry on sterile Whatman® filter paper and cultivation on 2N6 selection media supplemented with 200 mg/L-1 timentin and 25 mg/L-1 hygromycin; calli were transferred onto fresh 2N6 selection media containing 50 mg/L-1 hygromycin and 200 mg/L-1 timentin every 14 days. Seven weeks post-co-cultivation, individual proliferating callus clumps (cream white in color) were transferred to regeneration media supplemented with 200 mg/L-1 timentin and 25mg/L-1 hygromycin and incubated at 27°C in the dark for the first 7 days and then exposed to day/night cycle of 16/8 h at 25°C for shoot formation. Shoots with at least three well-formed leaves were transferred to rooting media supplemented with 200 mg/L-1timentin and 25 mg/L-1 hygromycin. Culture vessels were incubated in tissue culture growth room at 25°C with a 16 h photoperiod and monitored for plant growth, elongation and root development.Embryogenic calli were initiated from mature seeds of Table S1). Briefly, genomic DNA was isolated from 100 mg of fresh leaf tissue using a DNeasy® Plant Mini Kit . PCR analysis was carried out in a 20 μL reaction mixture containing 2X GoTaq green , 5 pM each forward and reverse primers, 100 ng of genomic DNA and a volume of DNase, RNase free water up to 20 μL using standard PCR parameters. For RT-PCR, total RNA was isolated from 50 mg of leaf tissue using an RNeasy Plant Mini kit  following the manufacturer’s instructions and treated with RNase free DNase  RT-PCR was carried out using the SuperScript® III first strand synthesis system  using gene specific primers following the manufacturer’s instructions. Plants that were confirmed transgenic by PCR and having the gene of interest expressed (confirmed by RT-PCR) was considered as one transgenic line. This plant was multiplied in vitro until at least 30 clones (plants) were obtained.One plant from each petri dish was sampled for PCR and RT-PCR using gene specific primers and hygromycin specific primers  Enhanced Liquid Substrate System for Immuno-histochemistry solution, D3939  following the manufacturer’s instructions. Briefly, pieces of the youngest fully expanded leaf (~1 cm long) were excised from rice plants and immediately immerging into 0.5 mL of mixed D3939 solution for 60 min at room temperature. The DAB solution was decanted and replaced with 1 ml of Ethanol: Acetic acid (3:1 v/v) for destaining of chlorophyll.In Situ Cell Death Detection Kit, Fluorescein  following the manufacturer’s instructions. Briefly, root tips fragments (~1 cm) were taken from plants, washed three times with fresh phosphate buffer saline (PBS) and fixed in 4% paraformaldehyde solution at 4°C for 1 h. Once fixed, root tips were washed two times with fresh PBS before immersing in fresh permeability solution (0.1% triton X 100 and 0.1% sodium citrate) and microwaving at 700 W for 1 min. Fresh PBS were used to immediately cool the sample after microwaving, followed by two more PBS washes. The samples were then mixed with a 50 μL aliquot of TUNEL reaction mix in a 1.5 mL Eppendorf tube. As a negative control, a 50 μL aliquot of TUNEL label solution without enzyme was also included. Samples were incubated at 37°C for 1 h under high humidity, washed two times with fresh PBS and counterstained with 0.5 mg/mL propidium iodide  for 15 min in the dark at room temperature. Stained root tips were washed two times with fresh PBS and squash mounted onto slides and examined under a Nikon A1 confocal microscope.The Terminal deoxynucleotidyl transferased UTPNick End Labeling (TUNEL) assay was carried out using an Electrolyte leakage from rice leaves at the seedling and reproductive stages was measured using a CM 100-2 conductivity meter . Briefly, detached leaves were sectioned into 0.5 cm pieces, washed two times with deionised water, blotted dry with paper towel and loaded into wells of the CM 100-2 conductivity meter containing 1.25 mL of deionised water. Measurements were taken at 2 min intervals over a 60 min period. Dry weight (DW) was determined after the samples were dried overnight in an oven at 70°C. Electrolyte leakage was normalized by DW and was calculated as the slope of electrolyte leakage over time.AtBAG4, Hsp70 and p35 leaves exposed to 0 and 100 mM NaCl. For analysis, 2 cm leaf sections excised 5 cm from the base of the flag leaf, were lysophilised in 2 mL Eppendorf tubes overnight at -83°C using a Flexi-Dry MPTm Freeze Dryer  under a vacuum pressure of <500 Kpa. Once freeze dried, samples were ground to a fine powder using a Qiagen Tissue Lyser and analyzed by ATR/FTIR spectroscopy. IR spectra were collected in the range of 4000 to 525 cm-1 using a Nicolet 870 Nexus FTIR spectrometer equipped with a smart endurance single bounce diamond ATR accessory .Fourier transform infrared (FTIR) was used to analyze the chemical composition of wild type (WT), vector control (VC), Leaf relative water content (RWC) was calculated using the method described by Net photosynthesis (A) was measured using a LiCOR Infra-Red Gas Analyser LI-6400 XT . Measurement of net photosynthesis at seedling stage was performed on the third leaf and was recorded at 0, 3, 7, 10, and 13 days after salinity treatment; reproductive stage measurements were taken on the flag leaf.+ and K+ in the leaf tissue of salinity-stressed plants at both the seedling and reproductive stages were determined using an Atomic Absorption Spectrophotometer . Fifteen milligrams of dried leaf was cut into 0.5 cm long segments and immersed in 30 mL deionised water in a 50 mL Falcon tube as described in The amount of NaAll experiments in this study were conducted using a randomized block design and where applicable, data were analyzed using one-way ANOVA and Tukey’s HSD (honest significant difference) or Fisher’s LSD (least significant difference) tests (Minitab Version 16).Figure S1). To study the potential of the manipulation of PCD pathways for enhancing tolerance to salinity stress in rice, the coding regions of the AtBAG4, Hsp70 and p35 genes were placed under the control of the maize ubiquitin-1 promoter . The respective expression cassettes and a VC  were introduced to O. sativa L. ssp. japonica cv. Nipponbare by Agrobacterium-mediated transformation of Nipponbare embryogenic calli. Fifty-six PCR-confirmed transgenic lines were generated . The expression of AtBAG4, Hsp70 and p35 in the transgenic lines was examined using RT-PCR . The RT-PCR results show that 100% of PCR-positive transgenic lines of AtBAG4 and Hsp70 have the gene of interest expressing and approximately 90% of PCR-positive lines expressing p35 (Supplementary Table S1).The TUNEL assay confirmed that salinity induces PCD in WT Nipponbare rice .The WT, VC and the transgenic plants expressing anti-apoptotic genes were grown under normal growth conditions to reveal any differences in morphology and physiology between the plants at the seedling and reproductive stages. The results show that no significant difference was observed between WT, VC and the transgenic plants under normal growth conditions at both developmental stages . Morphological data such as leaf damage and survival rate were captured and analyzed . The results showed that more than 70% of the transgenic lines tested exhibited higher survival levels and less leaf damage in comparison to the WT and VC plants .The best transgenic lines expressing each of the above genes were selected for further study.An initial screening for salinity stress tolerance was conducted on 10 transgenic rice lines (10 plants/line) containing each of AtBAG4, Hsp70 and p35 after 36 h exposure to 100 mM NaCl; WT and VC plants were also included as controls. The data show that there was no or very little cell death in transgenic rice plants expressing anti-apoptotic genes while noticeable cell death was observed in WT and VC plants . As expected, no cell death observed in the non-stressed WT, VC or transgenic controls (data not shown).To examine whether expression of anti-apoptotic genes prevents salinity-induced PCD, TUNEL assays were conducted on root tips of rice plants expressing in situ H2O2 production by DAB staining 30 h post exposure to 100 mM NaCl. As shown in Figure 2 more H2O2 production was observed in WT and VC leaves while lower levels of H2O2 were detected in the leaves of transgenic plants expressing the anti-apoptotic genes. This result indicates that the expression of anti-apoptotic genes is associated with reduced H2O2 production during salinity stress.Programmed cell death has shown to be triggered by signals including ROS that originate from different organelles such as the chloroplast and mitochondria . ROS lev-1), and pectin accumulation  rather than the major band (1653 cm-1) have been associated with salinity tolerance and protein stability in the ice plant  due to more ordered hydrogen bonding between the peptide bonds  upon comparison of the 0–100 mM treated samples. In contrast, transgenic lines expressing AtBAG4 and Hsp70 maintained a higher proportion of the minor band and gave low major/minor band ratios (<1) indicated stable protein structures. The p35 expressing transgenic line displayed an intermediate major band ratio thus correlating with the tolerance levels observed in the salinity assays.Fourier transform infrared spectroscopy, FTIR, is an established and powerful technique for the analysis of structural and composition changes in both animal and plant cells . To invede bonds . Therefo-1. Studies by Arabidopsis plants which are sensitive to salinity treatment do not accumulate cell wall precursors. In contrast, tolerant ice plants continue to generate pectin and other cell wall precursors. As shown in Figure 3, cell wall synthesis in WT and transgenic controls plants was much more sensitive to salinity treatment compared to the respective transgenic lines which maintained cell wall synthesis rates. Taken together, the FTIR data clearly show that the transgenic lines contain proteins with a higher level of ordered H-bonds and continue to synthesize cell wall precursors during salinity stress than their WT and transgenic control counterparts. These results correlate with those observed in other salinity tolerant plant species such as the ice plant and suggest that the transgenic lines are able to maintain higher-ordered forms of proteins in leaves during salinity stress.Another key indicator of stress is the rate of cell wall synthesis and pectin accumulation which can be observed by analysis the ester at the absorption band at 1745 cmAtBAG4, Hsp70 and p35 as well as VC and WT plants. Under salinity stress, shoot growth and dry weight of WT and VC plants were significantly reduced compared to that of the transgenics . This result indicates that at the seedling stage, transgenic rice expressing AtBAG4, Hsp70 and p35 are more tolerant to salinity stress than the WT and VC plants. Salinity tolerance of the respective plants at the reproductive stage was also determined based on yield components including the number of tillers per plant, panicles length and the number of spikelet per panicle. As shown in Figure 5, salinity stress resulted in significantly reduced yields of VC and WT plants whereas transgenic plants expressing AtBAG4, Hsp70 and p35 genes exhibited significantly higher yield components.Previous reports have indicated that rice is relatively tolerant to salinity stress during germination, tillering and maturity but sensitive during seedling and reproductive stages . Salinit+, K+ concentrations and the ratio between Na+ and K+ are key parameters that differentiate sensitive and tolerant rice cultivars during salinity stress . Electrolyte leakage was also increased in transgenic rice plants expressing anti-apoptotic genes but at lower levels when compared to WT and VC plants. These results combined with TUNEL and ROS assays indicate that transgenic rice expressing anti-apoptotic genes unlike their WT and VC counterparts can maintain cell membrane integrity during salinity stress.Membrane integrity, Nay stress . Transge+ in the leaf and shoot than their sensitive counterparts . In contrast, the transgenic plants expressing AtBAG4, Hsp70 and p35 maintained lower Na+ levels and a lower Na+/K+ ratio following NaCl exposure at the seedling stage . As expected, no significant differences in leaf Na+ content or leaf Na+/K+ ratio was observed between the transgenic plants, WT and VC plants under non-stressed conditions .Under salinity stress, tolerant rice cultivars accumulate less Naterparts . In thisAtBAG4, Hsp70 and p35 were examined and compared with WT and vector-control plants at both the seedling and reproductive stages. As shown in Figures 7A,B, 100 mM NaCl stress treatment resulted in differential responses between the transgenic anti-apoptotic and control plants. Although the trend of net photosynthesis was different between the plants tested in the first 7 days of salinity exposure, this difference was not statistically significant. Net photosynthesis of all transgenic and control plants decreased at day 10 in both unstressed  and salinity stressed growth conditions . This decrease was possibly due to developmental effects such as senescence (photosynthesis was measured using the third leaf at seedling stage). However, net photosynthesis in WT and VC plants without anti-apoptotic genes subjected to salinity dropped significantly more rapidly than the transgenics. After 13 days exposure to 100 mM NaCl, net photosynthesis of WT and VC plants was significantly lower than that of the transgenic plants; and the net photosynthesis of AtBAG4, Hsp70 and p35 transgenic plants was not significantly different from that of their non-stressed counterparts.Photosynthesis is a fundamental physiological process that provides a vital energy source for plant growth as well as arsenal to facilitate plant adaptation to environmental and biotic stresses. During salinity stress, net photosynthesis and RWC were reported to be maintained higher in tolerant than sensitive rice cultivars . To inveFigures 7C,D under 100 mM NaCl treatment at the seedling stage, the transgenic plants maintained significantly higher leaf RWC compared to WT and VC plants. Leaf RWC of AtBAG4 and p35 transgenic rice plants following salinity treatment at the reproductive stage was significantly higher than that of the WT and VC plants . However, no significant differences were observed between Hsp70 transgenic plants, WT and VC plants when subjected to 100 mM NaCl at the reproductive stage .The previous data showed that the anti-apoptotic transgenic rice plants can maintain cell membrane integrity during salinity stress. To determine whether this correlated with higher water retention, leaf RWCs of the transgenic, WT and VC plants in unstressed and following NaCl treatment were assessed at both the seedling and reproductive stages. As shown in AtBAG4, Hsp70 and p35 for enhanced salinity tolerance using a variety of agronomical and physiological characteristics.Climate change forecasts indicate that vast areas of the globe will become significantly more prone to aberrant environmental conditions. This potential crisis is exacerbated by predicted trends in population growth that suggest global food production needs to increase significantly to reach sustainable levels. The next frontier of agronomic crops should be both high yielding and tolerant to a multitude of stresses, thus enabling them to survive and yield in future environments. In this study we generated and assessed transgenic rice plants expressing the anti-apoptotic genes AtBAG4, Hsp70 and p35 were found to possess many characteristics that have been reported in salt tolerant rice cultivars subjected to salinity stress. This includes maintenance of ROS, growth and yield levels, minimisation of cellular membrane electrolyte leakage, high photosynthetic efficiency and low Na+ accumulation. In contrast, the WT and the vector-control rice plants, remained salt sensitive with serious damage at the whole plant level such as stunted growth, cell membrane damage, low photosynthetic efficiency, high sodium ion content and finally death or yield losses.Transgenic rice expressing AtBAG4, Hsp70 and p35 increased during salinity stress. Importantly, however, despite showing increased levels, the amount of ROS observed in the transgenics was significantly lower than in the WT and VC controls. Although speculative, the genes used in this study may regulate ROS levels either directly (p35) or indirectly (Hsp70 and AtBAG4).Previous studies have shown that PCD pathways are triggered by increased ROS levels, among other signals, that originate from a variety of organelles including the chloroplast and mitochondria . Moreovep35 has been reported to inhibit H2O2-induced PCD in insect cells by directly sequestering ROS. This antioxidant function of p35 has been attributed to the presence of metal-binding sites in the proteins that could enhance its antioxidant property and/or its three-dimensional structure contains some amino acids that confer electro-dynamically stable configuration conducive to ROS-trapping. The antioxidant role of p35 is also supported by the chemical radio-protectors formed by six cysteine residues in its sequence which can react with certain ROS in a constant rate  constitutive expression of Hsp70 and AtBAG4 genes might reduce ROS levels indirectly by facilitating the function of cellular processes which if not working efficiently promote the generation of ROS and (ii) maintenance of ion homeostasis.The exact mechanistic details of how Hsp70 and AtBAG4 may assist in the folding of proteins and prevention of protein denaturation in high ROS environments, thus maintaining efficiency of cellular processes and mitigating the production of ROS and plant damage. The suppression of ROS in transgenic plants expressing anti-apoptotic genes is perhaps one of the most important steps to protect cells from oxidative damage and enable the plants to maintain photosynthetic efficiency which in turn provides energy for plants to grow and survive under salinity stress conditions.If left unchecked, copious ROS production can denature proteins. To mitigate the denaturation of proteins, cells employ a complex network of molecular chaperones and foldases which promote efficient and correct folding of cellular proteins . MembersAtBAG4, Hsp70 and p35 is less damaged than in WT and VC plants during salinity stress. Cell membrane integrity probably enables the plants to maintain ion homeostasis under salinity stress. During salinity stress, increased extracellular Na+ concentrations create a large electrochemical gradient that favors the passive transport of Na+ into the cell through K+ transporters result in high cytosolic Na+ concentration , protein oxidation (PO), enzyme inhibition and DNA, RNA damage . The celntration . To main+-ATPase . As the e plants .+/K+ ratio improves photosynthesis and overall plant growth  and yield components (number of panicles per plant and number of spikelets per panicle) during salinity stress. This is probably also a result of the maintenance of high cytosolic K+ and homeostasis in transgenic rice plants expressing AtBAG4, Hsp70 and p35. In response to osmotic stress caused by salinity, shoot growth rate decreases immediately (+ in transgenic plants expressing anti-apoptotic genes allow plants to adjust to osmotic stress and maintain high growth rates (AtBAG4, Hsp70 and p35 in comparison to WT and VC which had very low cytosolic K+ under salinity stress condition. Another factor that causes reduced growth rates in high salt environments is inadequate photosynthesis due to limited carbon dioxide uptake as a consequence of stomatal closure (AtBAG4, Hsp70 and p35 transgenic rice plants maintained high net photosynthesis which provided ample energy for their growth and development.Transgenic rice expressing ediately . High cyth rates . The mai closure . AtBAG4,+ homeostasis. Low ROS levels reduce the risk of cellular damage caused by LPO and PO; two established hallmarks of oxidative damage. The maintenance of membrane integrity and Na+ homeostasis promoted sustained photosynthetic efficiency which in turn provided energy for growth. Maintenance of ROS levels also facilitates photosynthesis and growth by minimizing LPO in chloroplasts . Well-mIn conclusion this research focused on the improvement of salinity tolerance in rice by manipulating PCD pathways. The exogenous expression of three anti-apoptotic genes from different sources in this study displayed similar physiological and biochemical characteristics during salinity treatment thus providing further evidence that cell death pathways are conserved across broad evolutionary kingdoms. This study also provided mechanistic evidence for the biochemical and physiological basis of salinity tolerance in transgenic rice expressing anti-apoptotic genes and proposes that anti-apoptotic genes improve stress tolerance by “creating” an optimal cellular environment that minimizes pro-death signals including accumulation of ROS. Once created, this environment facilitates cellular metabolism even during stressful conditions to suppress flicking of the “cellular kill switch” that is apoptotic-like cell death.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Maintenance of redox balance is essential for normal cellular functions. Any perturbation in this balance due to increased reactive oxygen species (ROS) leads to oxidative stress and may lead to cell dysfunction/damage/death. Mitochondria are responsible for the majority of cellular ROS production secondary to electron leakage as a consequence of respiration. Furthermore, electron leakage by the cytochrome P450 enzymes may render steroidogenic tissues acutely vulnerable to redox imbalance. The adrenal cortex, in particular, is well supplied with both enzymatic (glutathione peroxidases and peroxiredoxins) and non-enzymatic  antioxidants to cope with this increased production of ROS due to steroidogenesis. Nonetheless oxidative stress is implicated in several potentially lethal adrenal disorders including X-linked adrenoleukodystrophy, triple A syndrome and most recently familial glucocorticoid deficiency. The finding of mutations in antioxidant defence genes in the latter two conditions highlights how disturbances in redox homeostasis may have an effect on adrenal steroidogenesis. At low concentrations, ROS modulate many cellular processes through redox-dependent signalling, including proliferation, differentiation, apoptosis, immune regulation and cellular adaptation to stress (Reactive oxygen species (ROS) are derived from Oo stress . A critic reductase), a small percentage of the molecular oxygen is converted to superoxide anions (O2·−) (2O2). H2O2 can also contribute to oxidative stress by reacting with free metals to form OH. In addition, in comparison to O2·−, which tends to remain at the site of production, H2O2 readily traverses membranes into other subcellular compartments where it may affect signalling pathways.Mitochondria are responsible for the majority of cellular ROS production secondary to electron leakage as a consequence of respiration. During respiration, electrons are transferred across to molecular oxygen to generate water via the four complexes of the electron transport chain. The electron–proton gradient established during this process is used to generate energy in the form of ATP. As a result of electron leakage at complex I (NADH dehydrogenase) and complex III (cytochrome (O2·−) . These s2O2  cysteine (Cys) residues in target proteins, and is detoxified by members of the glutathione peroxidase (GPX) and peroxiredoxin (PRDX) families, discussed later in this review. The relative contribution of each of these enzymes may be tissue- and compartment-specific and determined by the source of H2O2 and the expression levels of each individual enzyme.Several compartment-specific antioxidant mechanisms are in place to target superoxide production. Cytoplasmic copper/zinc-dependent SOD1 (CuZnSOD), mutations of which are associated with the neurodegenerative disorder, amyotrophic lateral sclerosis, and mitochondrial matrix manganese-dependent SOD2 (MnSOD) catalyse the conversion of superoxide anions into H2O2 . H2O2 haIn addition to electron leakage during respiration, other sources of superoxide production within the mitochondria include reactions catalysed by xanthine/xanthine oxidase, uncoupled nitric oxide synthases, NADPH-dependent oxidases (NOXs) and, most pertinent to the adrenal cortex, cytochrome P450 isoforms. The P450 enzymes are involved in the biosynthesis of cholesterol-derived steroidal compounds . In sterNADPH→ferrodoxin reductase→ferrodoxin→P450In a tightly coupled system, all electrons from NADPH are used in substrate hydroxylation; however, electron transfer within the P450 system can be relatively ‘uncoupled’ or ‘leaky’, with the rate of electron leakage varying depending on P450 subtype . Up to 4The adrenal cortex is particularly equipped to handle the increased risk of oxidative stress. Several antioxidants, both enzymatic and non-enzymatic, are highly expressed in comparison to other tissues. Ascorbic acid (vitamin C), which recycles α-tocopherol (vitamin A) radicals, is present at the highest levels in the adrenal cortex , indeed 2·− radicals are converted by SODs to H2O2. Mitochondrial H2O2 is further reduced by two major thiol antioxidant systems dependent on reduced glutathione (GSH) and the small protein thioredoxin 2 (TXN2), each system has several associated proteins :oxidised glutathione (glutathione disulphide (GSSG)) ratio is maintained. GSH, synthesised in the cytosol, is transported into the mitochondria by dicarboxylate and 2-oxoglutarate carriers . Within ficiency . Indeed,2O2 and lipid peroxides, albeit less efficiently than the GPXs. They comprise a family of six members, most of which (PRDX1–5) use TXN as an electron donor. PRDX3 and PRDX5 are present within the mitochondria; PRDX3 is mitochondria-specific and is seen in abundance particularly within the adrenal cortex, with a significant role in H2O2 detoxification within the mitochondrial matrix. It is estimated that PRDX3 is the catalyst for up to 90% of H2O2 generated within the matrix , inactivating its peroxidase function  can undergo hyperoxidation to Cys sulfinic acid , involving both adrenal and gonadal dysfunction to milder atypical or non-classic forms of LCAH which can resemble FGD  form of STAR, in response to ROS  and most recently FGD. Despite ubiquitous expression of the proteins involved, in all three conditions defects in antioxidant defence specifically affect the adrenal. Mechanisms involving mitochondrial and peroxisomal dysfunction in addition to defective nucleocytoplasmic transport of DNA repair proteins and antioxidants demonstrate how disturbances in redox homeostasis in several subcellular compartments can have an effect on steroidogenesis.MC2R), and its accessory protein MRAP (melanocortin receptor accessory protein) (MCM4), a DNA replicase and NNT, encoding NNT, a proton pump located in the inner mitochondrial membrane  and a deficiency in both antioxidants results in death within 1–2 days  is a rare autosomal recessive disease characterised by the triad of alacrima, achalasia of the oesophageal cardia and primary adrenal failure (syndrome AAAS gene product is the 60 kDa nuclear pore complex (NPC) protein ALADIN . AAAS mRNA and the ALADIN protein are ubiquitously expressed with predominance in the adrenal and CNS structures in the human and the rat , implying that correct NPC targeting is vital for its function , a member of the peroxisomal ATP-binding cassette (ABC) transporters. The disease is clinically heterogeneous and the clinical phenotype can be variable even within families. The three most common phenotypes reported are isolated adrenal insufficiency; a late onset slowly progressive disease adrenomyeloneuropathy in which two-thirds of patients also have adrenal insufficiency; and a cerebral inflammatory demyelinating form which can be fatal in early childhood  and Macaca mulatta (rhesus monkeys) have been used to study age-related changes in endocrine function (Non-human primate models such as function . These hfunction . Howeverfunction b, 2013.ROS play an important physiological role in steroidogenesis; however, in excess they can have deleterious effects on adrenal function. Steroidogenesis itself increases the generation of ROS, rendering the adrenal gland susceptible to oxidative damage. Inherited defects in the components of several different subcellular compartments have been described in association with increased susceptibility to oxidative stress and adrenal insufficiency. The use and further development of techniques pertaining to the detection of ROS in a site-specific manner will provide further information on the specific cellular targets involved. Further functional interrogation together with careful phenotyping of the respective patient cohorts will allow better understanding of the role of these proteins in human biology. Given the ubiquitous expression of the causative genes identified so far, this knowledge could be invaluable in directing patient surveillance and could also potentially provide targets for therapeutic intervention. Antioxidant defence within the adrenal certainly warrants further investigation and other components within this pathway may prove to be causative in the aetiology of as yet unaccounted for cases of primary adrenal insufficiency.


APETALA1 during the divergence of Brassicales clade.Transcriptional networks are central to any biological process and changes affecting transcription factors or their binding sites in the genome are a key factor driving evolution. As more organisms are being sequenced, tools are needed to easily predict transcription factor binding sites (TFBS) presence and affinity from mere inspection of genomic sequences. Although many TFBS discovery algorithms exist, tools for using the DNA binding models they generate are relatively scarce and their use is limited among the biologist community by the lack of flexible and user-friendly tools. We have developed a suite of web tools  based on the proven Position Weight Matrices (PWM) formalism that can be used without any programing skills and incorporates some unique features such as the presence of dependencies between nucleotides positions or the possibility to compute the predicted occupancy of a large regulatory region using a biophysical model. To illustrate the possibilities and simplicity of Morpheus tools in functional and evolutionary analysis, we have analysed the regulatory link between LEAFY, a key plant transcription factor involved in flower development, and its direct target gene  Being able to detect TF binding sites (TFBS) by inspecting genome sequences helps understanding how organisms work and how they evolved. Methods based on Chromatin Immunoprecipitation (ChIP) such as ChIP-Chip , which can optionally be indicated if available. Since this value is rarely available, as default, Morpheus uses [X] = e (b/a) corresponding to a TF concentration at which the best possible binding site  is bound with a probability of 0.5.Occupancy calculation is based on the relation between predicted score and relative dissociation constant , 24, scoA. thaliana genome a set of sequences that do not overlap with the positive set and with the same size distribution (Positive bound sequences was taken from  S3 TextS3 Text. ribution .AtAP1 (At1g69120), AtCAL (At1g26310) and AtFUL (At5g60910). Hits without transcripts information or with incomplete gene prediction were discarded. A region of 2 kb upstream the ATG were used for relative binding score calculation. All sequences used in this study can be found in Genomic sequences were obtained from Phytozome database  by BlastS1 Text(TXT)Click here for additional data file.S2 Text(TXT)Click here for additional data file.S3 Text(TXT)Click here for additional data file.S4 Text(TXT)Click here for additional data file.S5 Text(TXT)Click here for additional data file.


Streptococcus pneumoniae (Sp) from the respiratory tracts of children with cystic fibrosis (CF). Whether these highly mucoid Sp contribute to, or are associated with, respiratory failure among patients with CF remains unknown. Other mucoid bacteria, predominately Pseudomonas aeruginosa, are associated with CF respiratory decline. We used a mouse model of CF to study pneumococcal pneumonia with highly mucoid serotype 3 and non-mucoid serotype 19A Sp isolates. We investigated susceptibility to infection, survival, and bacterial counts from bronchoaviolar lavage samples and lung homogenates, as well as associated inflammatory cytokines at the site of infection, and lung pathology. Congenic CFTR–/– mice and wild-type (WT)-mice were infected intranasally with CHB756, CHB1126, and WU2 , or CHB1058 (non-mucoid serotype 19A). BAL, lung homogenates, and blood were collected from mice 5 days post-infection. Higher CFU recovery and shorter survival were observed following infection of CFTR–/– mice with CHB756 compared to infection with CHB1126, WU2, or CHB1058 (P≤0.001). Additionally, CFTR–/– mice infected with CHB756 and CHB1126 were more susceptible to infection than WT-mice (P≤0.05). Between CFTR–/– mice and WT-mice, no significant differences in TNF-α, CXCL1/KC concentrations, or lung histopathology were observed. Our results indicate that highly mucoid type 3 Sp causes more severe lung disease than non-mucoid Sp, and does so more readily in the lungs of CFTR–/– than WT-mice.Recent studies have reported the isolation of highly mucoid serotype 3  Streptococcus pneumoniae (Sp) is a leading cause of morbidity and mortality worldwide, especially in children, the elderly, and those with underlying diseases. Sp generally colonizes the upper respiratory tract asymptomatically, but can also cause serious invasive disease including complicated pneumonia, bacteremia, and meningitis . 19A isolates, which always have a relatively non-mucoid phenotype on blood agar, were common among sputum samples from patients with and without CF. In sharp contrast, serotype 3 isolates with their mucoid phenotype on blood agar, were disproportionately represented in CF sputum, compared to their low frequency in respiratory specimens from patients without CF [Coats et al. in preparation]. In contrast to Pa, the clinical significance of Sp with different amounts of mucoid capsule has not been well defined in CF lung disease.Acquisition of mucoid patients  and withpatients , 14–20. Sp of capsular serotypes 3 and 19A in mice with and without a CFTR defect. We compared the effects of capsule type and amount of capsule on mouse survival, pro-inflammatory cytokines TNF- α and CXCL1/KC production, lung histology, and on numbers of recovered bacteria from BAL fluid, lung homogenates and blood. The CFTR–/– mice used in this study were unique in that they had a secondary mutation, insertion of human CFTR (hCFTR) in intestinal epithelial cells, thus permitting the mice to be nutritionally healthy enough for studies of lung infection naphthothiazolium bromide, 3,3′-Diethyl-9-methyl-4,5,4′,5′-dibenzothiacarbocyanine  as previously described . Capsule1UNC−/− (FABP-hCFTR) mice, (referred to as CFTR–/–) and CFTRm1UNC+/+ (FABP-hCFTR) [referred to as wild type (WT)] were obtained from the Gregory Fleming James Cystic Fibrosis Research Center at UAB. The intestinal abnormalities, commonly seen in mice lacking systemic CFTR-dependent chloride transport were corrected by insertion of human CFTR (hCFTR) expressed from a rat intestinal fatty acid-binding protein (FABP) promoter in the intestinal epithelial cells, allowing long-term survival of CFTR–/– mice without a specialized diet [1UNC+/+ (FABP-hCFTR) mice were crossed with CFTRm1UNC+/- (FABP-hCFTR) mice in order to get CFTR–/– mice, and CFTRm1UNC+/+ (FABP-hCFTR) mice were crossed with CFTRm1UNC+/+ (FABP-hCFTR) mice to get WT-mice.Male and female CFTRmzed diet . CFTRm1U–/– and WT-mice were anesthetized lightly with isoflurane. Following anesthesia, suspensions of approximately 1x105 (low dose) or 7x105 (high dose) CFU bacteria in 40μL of lactated Ringer's solution were introduced into the nares of the mice to induce aspiration pneumonia. Five days later, mice were bled retro-orbitally under isoflurane anesthesia and then euthanized using CO2 narcosis. Following sacrifice, the tracheas were intubated and the lungs were lavaged once with 1 mL of lactated Ringer's to collect BAL fluid. Next, lungs were harvested and homogenized in 1 mL of lactated Ringer's. The BAL fluids, lung homogenates, and blood were plated in serial threefold dilutions onto blood agar supplemented with gentamicin (4μg/mL) and incubated overnight at 37°C with 5% CO2 before colonies were counted. Colony counts were expressed as CFU per lung, CFU per total BAL fluid, and CFU per ml of blood [CFTRof blood .–/– and WT-mice were intra-nasally infected with suspensions of 1x105 CFU bacteria in 40μL lactated Ringer's solution and sacrificed 24 hours post-infection. Lung tissue and BAL were collected as previously described. Bacteria from each tissue were quantified by plating onto blood agar, and levels of TNF-α  and CXCL1/KC  were determined using commercially available ELISA kits according to the manufacturer’s instructions.To examine the cytokine response early in infection, CFTRAfter 24 hour infection with CHB756 or WU2, lungs were collected, fixed in 4% paraformaldehyde–phosphate-buffered saline, sectioned, and stained with hematoxylin and eosin using standard techniques as described previously . The degp-values comparing the CFTR–/– and WT-mice challenged with each bacterial strain were calculated using non-parametric rank order statistics (Mann-Whitney (two-tailed) rank test). Quantified CFU were transformed to log10 CFU for statistical analysis. In some groups mice died prior to day 5 when CFUs were determined. Thus, the time to moribund data were included along with the CFU data from the euthanized mice such that a higher rank consistently represented shortened survival and/or worse disease as follows: mice dying on day 5 post-infection were assigned a rank order higher than that of any mice sacrificed on day 5. Mice dying on day 4 were given a higher rank than those dying on day 5, and so on. Mice dying 1 day post-infection were assigned the highest rank of all mice in the experiment. Using these ranks for the WT and CFTR–/– mice infected with each strain, the statistical comparisons were calculated using the Mann-Whitney (two-tailed) rank test. When statistical comparisons were made between infections with three challenge strains, 1-way ANOVA with Tukey's Multiple Comparison Post-test was used. Cytokine data were analyzed with the unpaired two-tailed t-test.All statistical evaluations were conducted using Graph Pad Prism software version 5.03 December, 10 2009 for Windows. In Figs This study was carried out in strict accordance with the policies and approval of the UAB Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham (UAB) (APN: 090708819). Bacterial isolates from CF patients were obtained under a protocol approved by the UAB Institutional Review Board. None of the authors have any intellectual property or non-grant compensation related to the results of these studies.Sp isolate CHB756 produced 428% and CHB1126 produced 122% more polysaccharide capsule relative to WU2 (100%)  all were designated as mucoid based on their highly glossy colony appearance on blood agar plates and their difficulty to manipulate with cotton swabs. The type 19A strain (CHB1058), which had a type 19A capsule, was referred to as non-mucoid and provided a comparison between a relatively non-mucoid capsular type 19A strain that was originally isolated from CF sputum and the 3 mucoid type 3 strains. Among Sp capsular serotypes, group 19 strains invariably exhibit very low virulence in mice when given parenterally [Serotype 3 2 (100%) . CHB756 nterally .–/– mice to clear lung infections of each challenge strain was directly compared to WT-mice . In order to focus on the disease process in the lung and to minimize the risk of mice becoming moribund before the end of the 5 day infection cycle, a separate cohort of CFTR–/– and WT-mice were infected with mucoid serotype 3 strain CHB 1126, which produces 122% more capsule than WU2  .The ability of CFTR WT-mice . Statistogenates  and BAL ogenates  than thethan WU2 , but we  0.0034)  and slig 0.0034)  of CFTR––/– and WT-mice to the high dose of Sp infection with CHB756, WU2, and CHB1058. Infections with the three strains in CFTR–/– mice produced very different results with in both BAL and lung. The highly mucoid serotype 3 CHB756 was significantly more virulent, as measured by mouse survival time and CFU recovered, than the laboratory serotype 3 strain WU2, which was significantly more virulent than the type 19A strain CHB1058–/– mice. Even so, CHB756 was statistically more virulent in WT-mice than CHB1058 when the combination of the survival data and CFU in either the BAL fluid or lung homogenates was examined. Although WU2 again appeared to be intermediate in virulence in WT-mice and its virulence was not statistically different from either CHB756 or CHB1058 in BAL.Infections with these bacteria in WT-mice did not show such marked differences between isolates of different serotypes or levels of mucoidy, in part, because none of the strains caused as much disease in WT-mice as in the CFTR–/– mice to highly mucoid CHB 756, we examined CFUs recovered from BAL and lungs as well cytokine production and tissue inflammation. We used the lower infectious dose  (–/– mice infected with CHB1126 (data not shown). However, in the case of the lung homogenates, although there were more median CFUs in the CFTR–/– than WT homogenates, the difference was not statistically significant for infections with CHB756  , which wh CHB756  or for CCFTR on early recruitment of inflammatory mediators in the respiratory tract. These two cytokines both increase robustly in the first 24 hours in response to Sp infection. There were no significant differences in production of TNF-α or CXCL1/KC in the lung homogenates  at these earlier time points. Furthermore cytokine levels were very low at these early time points.TNF-α and CXCL1/KC levels were quantified from lungs and BAL of the same mice examined above to assess the impact of ogenates  or the Bogenates  between –/– and 2 WT), 4 infected with WU2 (2 CFTR–/– and 2 WT), and 4 uninfected control mice (2 CFTR–/– and 2 WT) after 24 hours to evaluate lung histopathology , are associated with progression of CF lung disease [In CF disease, chronic infection is associated with deterioration of lung function and is the major cause of morbidity and mortality. Although it is clear that the fundamental cause of CF is the lack of functional CFTR protein, the mechanisms leading to chronic lung disease associated with infection need further study. Gut-corrected ry tract , and wer disease , 29–31.–/– mice compared to WT-mice infected with a highly mucoid type 3 Sp strain isolated from a CF patient (CHB756) and an intermediately mucoid type 3 Sp strain isolated from a CF patient (CHB1126). Other pneumococcal strains , were relatively avirulent in both CF and WT-mice. It should be noted that in less resistant strains of mice WU2 can cause disease even by the intravenous route [Sp could exploit the CF defect of the CFTR–/– mice. Lack of CFTR in transgenic CF mice has similarly been shown to lead to increased levels of Pa in the lung which are associated with hypersusceptibility of CF mice to mucoid Pa infection [Sp with poor prognosis in CFTR–/– mice, it is not clear why mice with this mutation are less able than WT-mice to clear mucoid Sp from their lungs. The recruitment of neutrophils to sites of infection was demonstrated histologically to be similar in the WT and CFTR–/– mice and there was no difference in pro-inflammatory cytokine secretion in lung homogenates and BAL.We have demonstrated more severe pneumococcal lung disease in an aspiration pneumonia disease model in CFTRus route . These rnfection , 33. AltPa. The impaired ability of CF airway cells to mobilize secretions due to increased mucous production and reduced ciliary movement may favor the adherence of certain bacterial species and drive shifts to mucoid phenotypes. Mucoid Pa in the CF lung has been specifically associated with compromised bacterial clearance [Pa, indicating that mucoidy may be an adaptive mechanism of the bacteria to permit prolonged survival especially within the CF host. Our observations that mucoid type 3 Sp isolates appear more frequently in the lungs of CF patients than in normal individuals, and that CFTR–/– mice are more susceptible to highly mucoid Sp than WT-mice, both support the notion that mucoid pneumococci may have an advantage in the environment of the CF lung which may lead to chronic infection.High susceptibility to infection in CF lung disease has been attributed in part to bacterial adaptation within the CF airways, specifically to the transition of bacteria to mucoid phenotypes such as learance –36 increlearance , 19–20 alearance –14 compaCFTR has been shown to regulate the functions of circulating and resident cells involved in immune surveillance, and dysfunction of CFTR directly alters innate immune responses [–/– mice actually had higher levels of baseline inflammation compared to uninfected WT-mice (CFTR deficient mouse model.Heightened bacterial susceptibility in CF lungs has also been credited to changes in the host that result in an impaired ability to regulate immune responses , 37–38. esponses , 37. In  WT-mice , thus a –/– mice and WT-mice after 24 hours of infection with highly mucoid Sp strain CHB756. TNF- α was selected for measurement because this cytokine is produced into the alveolar space within a few hours following lung infection of mice with many bacterial species including Pa [Staphylococcus aureus [Sp [Sp infection, TNF- α initiates a wave of cytokine production that affects the adaptive immune response [–/– mice to CHB756 when compared to congenic WT littermates, the production of TNF-α and CXCL1/KC were only slightly, but not significantly higher in CFTR–/– mice than in WT-mice. These results suggest that the CF genetic background in mice may not significantly affect cytokine production and neutrophil recruitment during the first 24 hours of infection with highly mucoid Sp. Differences in susceptibility between CFTR–/– and WT-mice do not appear to be dependent on these cytokines or necessarily effect production of these cytokines. Future studies should include analysis of biofilm formation by type 3 pneumococci under in vitro and in vivo conditions as a potential mechanism of increased virulence and persistence by highly mucoid strains.In this study, differences in TNF- α, CXCL1/KC, and lung histology were compared between CFTRuding Pa , Staphyls aureus , and Sp reus [Sp , and is response . The mouresponse , 45. DesSp in an animal model of CF lung disease. Because mucoid phenotype may protect organisms from clearance from the CF lung, it may exacerbate the cycle of chronic infection and exaggerated inflammation. Advancing our knowledge of the microbiology of CF lung disease, pathogenesis, and subsequent immune responses should improve the prospect for better therapies aimed to reduce inflammatory deterioration, regulate immune responses, and to effectively reduce the disease burden cause by highly mucoid bacteria.We have studied infections with mucoid and non- mucoid


The oral mucosal pellicle is a layer of absorbed salivary proteins, including secretory IgA (SIgA), bound onto the surface of oral epithelial cells and is a useful model for all mucosal surfaces. The mechanism by which SIgA concentrates on mucosal surfaces is examined here using a tissue culture model with real saliva. Salivary mucins may initiate the formation of the mucosal pellicle through interactions with membrane-bound mucins on cells. Further protein interactions with mucins may then trigger binding of other pellicle proteins. HT29 colon cell lines, which when treated with methotrexate (HT29-MTX) produce a gel-forming mucin, were used to determine the importance of these mucin-mucin interactions. Binding of SIgA to cells was then compared using whole mouth saliva, parotid (mucin-free) saliva and a source of purified SIgA. Greatest SIgA binding occurred when WMS was incubated with HT29-MTX expressing mucus. Since salivary MUC5B was only able to bind to cells which produced mucus and purified SIgA showed little binding to the same cells we conclude that most SIgA binding to mucosal cells occurs because SIgA forms complexes with salivary mucins which then bind to cells expressing membrane-bound mucins. This work highlights the importance of mucin interactions in the development of the mucosal pellicle. The inability to replicate the mucosal layer stems from two key factors. Firstly, purification of proteins leads to the loss of their tertiary conformation, even if mucin preparations are made taking extra care to preserve its gel properties. Secondly, the substrates for in-vitro measurements are usually inorganic (or plastic) materials that are significantly dissimilar from the native surface of the cell or connective tissue of the linings. Thus, it has been shown that MUC5B and MUC7 are strongly retained on the buccal cell surfaces, with minimal retention of other salivary proteins [The mucus layer is essential for protection, molecular transport and lubrication on soft tissues and linings of most of the essential organs. Typically in airways and gastrointestinal tract the mucosal film is formed primarily by mucins, while in other linings like that in the oral cavity the mucosal film  also contains globular proteins and proline-rich proteins. Among these globular proteins secretory IgA (SIgA) plays an important role in topical immune response of the adsorbed proteinaceous film. While mucins spontaneously assemble on mucosal surfaces proteins . This isproteins , 3.in vitro. Firstly we utilised saliva as a mucin source, since saliva is the only mucosal fluid that has ability to self-assemble onto the surface from the bulk solution. Physiologically, saliva is synthesised away from the epithelium and assembles only upon its excretion from the ducts, where the pellicle forms within minutes of exposure to the oral cavity [In this work we adopted an approach that that tackles both issues associated with studying mucus deposition l cavity . This apl cavity . SecondlPrevious studies of mucin binding to synthetic surfaces suggested hydrophobic interactions are a dominant force that drives mucin adsorption , 6–8, wiIt is possible that mucin adherence and retention may be aided by attachment to MUC1, a membrane bound mucin present on the buccal cell surface, through receptor like actions , 14. OthMUC7 may further aid in the pellicle formation by forming complexes with SIgA and lactoferrin, which may improve their incorporation into the pellicle layer , 18. IgAThe aim of this study was to better understand the development of the mucosal pellicle and specifically the role of mucins, both bound and free, in the absorption of SIgA to the mucosa. HT29 cells were selected as a model epithelial layer as they can be induced (by methotrexate) to secrete and form a layer of bound mucus containing a gel forming mucin MUC5AC . SIgA bi2 flask split when approximately 90% confluent. Cells where grown to 3 time points, day 4 (100% confluence), day 8 and day 16 for experimental procedures. At day 8 HT29-MTX cells have started to produce mucus containing MUC5AC [HT29 and HT29-MTX cell lines  were cultured in Dulbecco’s modified eagles medium (DMEM) containing 4500 mg/L glucose with 10% fetal bovine serum and 1% penicillin-streptomycin . Cells were seeded into 6-well plates at 1:10 concentration in each well from 75 cmg MUC5AC , which cConfocal microscopy on live cells was used to confirm the presence of mucus on cell layers. Images were taken using Leica Confocal imaging software, using FITC and TRITC fluorescence at emissions spectra 488 and 568 respectively. After a control image was taken, 10 μl of melamine formaldehyde particles (0.5 μm diameter) were added which are useful in showing the top layer of mucus by reflection and 10 μl of TRITC 4 (100 mg/ml) was added to visualise cell membranes.Unstimulated whole mouth saliva (UWMS) was collected from one female volunteer, on the morning of each experiment, at least 1 hour after consumption of any food or drink. Parotid saliva (PS) was collected using sweet stimulation whilst using a Lashley cup attached onto the inside of the cheek, covering Stenson’s duct from the parotid gland. Informed written consent was obtained for the volunteer to donate saliva following ethical review of the project by the Brent NRES NHS committee (11/LO/1121). SIgA purified from human colostrum  was prepared at a concentration of 50 μg/ml in serum free media. SIgA was later prepared at 25 μg/ml and 150 μg/ml corresponding to IgA levels in saliva. 1 ml UWMS, PS and SIgA was then added to cells in triplicate for 20 minute or 1 hour incubation at 37°C, after washing cells in PBS. Saliva or media was then removed, and kept and frozen at −20°C, cells were washed in 0.5 ml PBS and homogenised in 0.5 ml of RIPA buffer  according to manufacturer’s instructions.BCA assay kit (Thermo Scientific) was used to measure total protein in all cell homogenates according to manufacturer’s instructions. This ensured equal loading of all cell samples for SDS-PAGE, western blotting and IgA enzyme-linked immunosorbent assay (ELISA).2O2 and 500 μl of 3,3’, 5, 5’ tetramethylbenzidine (3 mg/ml in dimethyl sulfoxide). The reaction was stopped with 2 M sulphuric acid and absorbance was read at 450 nm using a microplate reader .IgA binding on all cell and saliva samples were measured using an ELISA, as previously described . Rabbit SDS-PAGE was completed on cell homogenates. Equal protein amounts of samples were added to LDS sample buffer (1:4)  and DTT reducing buffer (1:10) (Invitrogen), with water to make up volumes and heated at 100°C for 3 minutes. 15 μl of each sample was applied to each lane on a 4–12% Bis-Tris gel (Invitrogen). Electrophoresis was carried out in MES-SDS running buffer (Invitrogen) according to manufacturer’s instructions. Lanes of buccal cell homogenates and TR146  were run as control comparisons to proteins expected.After completion of protein separation within the gel, western blotting was completed according to manufacturer’s instructions and used to transfer proteins onto a nitrocellulose membrane. Immunoblotting was used to examine specific proteins of interest including: statherin (1:2000), as described previously ), cystatThe presence of the mucus layer was further confirmed by gel electrophoresis and western blotting . CoomassFollowing a 1 hour incubation of UWMS with both cell lines the amount of IgA bound to the cells was much greater in the presence of the MUC5AC on the HT29-MTX cells at day 8 and day 16, as seen in IgA binding from PS also appeared to show an increase in IgA binding onto the HT29-MTX cells ; howeverWhen a similar concentration of purified SIgA in media was incubated with the HT29 and HT29-MTX cells for 20 minute and compared to UWMS there was significantly less binding compared to the purified SIgA alone . At day When the same experiment was completed with PS, although there appeared to be increased binding from PS to HT29-MTX cells at day 16, this result was not statistically significant.The results from this work strongly show the importance of a mucin layer in the development and build-up of a mucosal pellicle. Previously  MUC5B waIgA deposition onto cell surfaces from different salivas as well as control IgA solutions also indicated a requirement for mucin-mucin interactions. Time dependent and concentration dependent trends were observed that characterise mucin-assisted IgA binding to the HT29-MTX cells. In In the case of whole mouth saliva, there is a clear concentration dependence (as evident from the time course trend) which allows the deposition of soluble mucins to be attributed to the level of mucus production by the cells. Finally we see that other proteins such as cystatin S that also forms heterotypic complexes with mucins show a similar trend. At the same time statherin was not typically prominent in the pellicle in contrast to those formed on hydroxyapatite surface and air-saliva interfaces .One surprising result was the lack of MUC7 binding, in particular to the HT29-MTX cell or when strong IgA adherence was observed. As described earlier in the paper, MUC7 is known to form complexes with IgA  and is aThese new insights into mucin binding to mucosal surfaces provide an important step in understanding mucosal pellicle formation as well as help to guide future work to understand pathological conditions such as xerostomia.S1 DataExcel spread sheets containing raw data for the paper entitled ‘SIgA binding to mucosal surfaces is mediated by mucin-mucin interactions’. Data includes the BCA total protein data for cell homogenates and ELISA results for IgA bound to cells.(XLSX)Click here for additional data file.


Plants adapt to environmental stress by modulating their growth and development along with the various physiological and biochemical changes. This phenotypic plasticity is driven by the activation of specific genes encoding signal transduction, transcriptional regulation, ion transporters and metabolic pathways. Rice is an important staple food crop of nearly half of the world population and is well known to be a salt sensitive crop. The completion and enhanced annotations of rice genome sequence has provided the opportunity to study functional genomics of rice. Functional genomics aids in understanding the molecular and physiological basis to improve the salinity tolerance for sustainable rice production. Salt tolerant transgenic rice plants have been produced by incorporating various genes into rice. In this review we present the findings and investigations in the field of rice functional genomics that includes supporting genes and networks (ABA dependent and independent), osmoprotectants , signaling molecules (CaThe online version of this article (doi:10.1186/1939-8433-6-27) contains supplementary material, which is available to authorized users. World agriculture faces a challenging task to produce 70% more food for an additional 2.3 billion people by 2050 FAO . The low+ and Cl- accumulation, enhanced lipid peroxidation and increased production of reactive oxygen species like superoxide radicals, hydrogen peroxide and hydroxyl radicals. Area under salt stress is increasing due to many factors including climate change, rise in sea levels, excessive irrigation without proper drainage in inlands and underlying rocks rich in harmful salts etc. It is estimated that if current scenario of salinity stress would persist, there may be loss of 50% of present cultivated land for agriculture by 2050  is the world’s most important food crop and a primary source of food for more than half of the population. More than 90 per cent of the world’s rice is grown and consumed in Asia, where 60 percent of the earth’s people live. Salinity is the most common abiotic stress encountered by rice plants and classified as a salt sensitive crop in their early seedling stages  are induced one hour after salt stress and their expression is well correlated to the level of ABA in rice roots , transcription factors (TFs), micro RNAs and reactive oxygen species (ROS) involved in the salt stress tolerance through ABA-dependent pathway  has been reviewed in rice  show higher tolerance to salt and drought stresses than control plants and the knock-out mutants sik1 as well as RNA interference (RNAi) plants  have important roles in plant growth, development and stress responses. Salt, drought, H2+-dependent protein kinases (CDPKs), OsCPK21, have been cloned and the OsCPK21-ox transgenic rice exhibited higher salt stress tolerance than wild-type plant with enhanced expression of the ABA and salt-stress inducible genes such as OsNAC6 and Rab21  gene, which negatively regulates stomatal closure and directs modulation of genes related to H2O2 homeostasis. DST mutant increases stomatal closure and reduces stomatal density, thus resulting in enhanced salt and drought tolerance in rice. Another salt responsive zinc finger protein gene ZFP179-ox leads to increased salt stress tolerance with increased level of free proline and soluble sugars in transgenic rice  gene from rice. GS2-ox rice showed increased salt stress tolerance retaining more than 90% activity of photosystem II in comparison to complete loss of photosystem II in control plant. Overexpression of a salt stress inducible JAZ protein gene, OsTIFY11a, resulted in increased salt and dehydration stress tolerance in rice, yet the function of the protein is not clear  expression is induced by various abiotic stresses and phytohormone treatments. Overexpression of OsSKIPa, also exhibited significantly improved growth performance in the salt and drought resistance with increased transcript levels of many stress-related genes, such as SNAC1 and rice homologs of CBF2, PP2C and RD22  play a key role in the regulation of gene expression at the post-transcriptional level. Under salt-stressed condition, expression of two microRNAs, HVA1 encodes a late embryogenesis abundant (LEA) protein, which is thought as a molecular chaperon. Expression of HVA1 gene led to significantly increased salt and drought tolerance in transgenic rice  of the catalase activity. Alternative oxidase (AOX) is an inner mitochondrial membrane protein that functions as terminal oxidase in the alternative (cyanide resistant) pathway, and AOX serves as oxidation stress reliever from environmental stresses, particularly salt and dehydration stress  regulate stress signaling pathways for maintaining an osmotic adjustment or homeostasis and regulating plant growth and development. Plant hormonal regulations and Ca2+ dependent modification of enzymatic activities are coordinately or independently integrated into the stress signaling pathways. In this section, we reviewed the recent advances in molecular mechanisms of how these signaling molecules are concerted to cytosolic and nuclear events for maintaining ionic homeostasis and salt stress tolerance in plants.Under salinity stress conditions, diverse signaling molecules such as phospholipids, hormones and calcium ions  DAG (Diacylglycerol) and PA (phosphatidic acid) seem to play an important structural roles during stress responses in inducing cytosolic Caance Zhu . Under sacco Zhu . Furtheracco Zhu . The forants Zhu . This im2+ spiking. This event spontaneously initiates the stress signaling pathways for stress tolerance via stimulating various Ca2+ binding proteins including CBL-CIPKs, CDPKs and calmodulins  mutants which are oversensitive to salt stress  and functioned in sensing the cytosolic Ca2+ concentration by direct binding to Ca2+. Indeed, the loss-of-function sos3-1 mutation reduced its Ca2+ binding capacity, indicating that Ca2+ sensing by SOS3 is an essential mechanism for salt tolerance in plant  proteins. As a SOS3 interacting CIPK, SOS2  was identified, and the activation of kinase activity of SOS2 by SOS3 was in a Ca2+ dependent manner  cells reduced total Na+ content in the cell. Other SOS2 and SOS3 homologs in rice were also identified as OsCIPK24 and OsCBL4, respectively  and OsCam1-1 genes led to accumulation of ABA and tolerance to salt stress in rice. OsDSM2 and OsCam1-1 genes encode an ABA biosynthetic β-carotene hydrolase and a Ca2+-binding calmodulin, respectively  and its free-acid form, JA, is an important signaling molecule for diverse developmental processes and defense responses  in regulating salt stress  gene led to decreasing stomata density and increasing drought stress tolerance  is a common metabolic adaptation found in diverse plant species. The osmoprotectants have been definitely proven to be among the most important factors to protect plant cells from dehydration and salinity  occurs most abundantly in response to dehydration stress  from Arthrobacter globiformis aids in improving the salinity tolerance in rice , mustard (Brassica spp.), Arabidopsis  and tobacco (Nicotiana tabacum) naturally do not produce glycine betaine under stress or non-stress conditions , deficiency of glycine betaine has been attributed to the absence of the two enzymes, choline monooxygenase and betaine aldehyde, in the biosynthetic pathway  bond. In plants, the synthesis of this sugar occurs normally by the formation of the trehalose-6-phosphate (T6P) from the UDP-glucose and glucose-6-phosphate, a reaction catalyzed by the trehalose 6-phosphate synthase (TPS). Afterwards the T6P is dephosphorylated by the trehalose-6-phosphate phosphatase (TPP) resulting in the formation of free trehalose Wingler . It has +/Na+ ions in the leaves of maize seedlings  - that possesses a remarkable tolerance against drought, high salinity, and cold stress inositol is methylated to D-ononitol and subsequently epimerized to D-pinitol. This plant accumulates a large amount of these inositol derivatives during the stress  from the wild halophytic rice relative Porteresia coarctata and transformation in tobacco has been reported  regulates sodium efflux in roots and the long-distance transport of sodium from roots to shoots  and one endosomal Na+/H+ antiporter (OsNHX5) have been reported  is a pump which helps intracellular Ca2+ ion homeostasis  and OsCAX1c, a pseudogene of CAX1, were retrived. Expression of all rice CAX genes except OsCAX2 showed Ca2+ tolerance in yeast . In Arabidopsis, mutant lacking vacuolar V-H+-ATPase subunits showed a reduced tonoplast V-ATPase activity, but did not show sensitivity to high salinity  genes. Those channel proteins are present in vacuole, Golgi body and chloroplast. A salt stress inducible OsCLC1 was identified from rice and the OsCLC1 was shown to operate as anion channels in one system, but H+/Cl- antiporter in another  mediates the influx of K+ on the plasma membrane and it selectively accumulates K+ over Na+ upon the plasma membrane hyperpolarization  have been functionally characterized. Salt stress inhibited OsAKT1 gene expression and the inward K+ influx was significantly decreased in root protoplasts by salt stress, suggesting that OsAKT1 is a dominant salt-sensitive K+ uptake channel  are proposed as an entry gate of NaAvailability of high quality rice genome sequence fast tracked the progress made in functional genomics of salinity tolerance in rice. This aids in the discovery of several genes that could be deployed for use in breeding for rice salt tolerance. The complex mechanism underlying salt tolerance as well as the complex nature of salt stress itself and the wide range of plant responses make the trait unexplainable. Even so, several evidences showed that members of protein families involved in signal transduction, osmoregulation, ion transportation and protection from oxidative damage are critical in governing high salt tolerance Figure . VariousAdditional file 1: Table S1: List of salt stress supportive genes described in this paper. (PDF 134 KB)Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Below are the links to the authors’ original submitted files for images.


CACNA1C gene is reliably associated with both bipolar disorder and schizophrenia. We investigated whether this risk variant affects reward responsiveness because reward processing is one of the central cognitive-motivational domains implicated in both disorders. In a sample of 164 young, healthy individuals, we show a dose-dependent response, where the rs1006737 risk genotype was associated with blunted reward responsiveness, whereas discriminability did not significantly differ between genotype groups. This finding suggests that the CACNA1C risk locus may have a role in neural pathways that facilitate value representation for rewarding stimuli. Impaired reward processing may be a transdiagnostic phenotype of variation in CACNA1C that could contribute to anhedonia and other clinical features common to both affective and psychotic disorders.The variant at rs1006737 in the L-type voltage-gated calcium channel   The risk variant  is significantly associated with bipolar disorder4 and schizophrenia,8 supporting the hypothesis that CACNA1C contributes to the genetic overlap between these psychiatric disorders.Although the heritability of psychiatric illness is high, the genetic mechanisms that confer susceptibility remain relatively unknown.12 and working memory,13 emotional regulation,16 and verbal fluency.18 Numerous studies also suggest the rs1006737 variant may affect components of cognition, such as logical/working memory20 and clinical symptomology.22 Furthermore, emerging evidence supports a role for rs1006737 in the neural processing of reward23 and learning.24 Considering that the CACNA1C variant appears to transcend diagnostic boundaries, we hypothesize that the risk variant may affect intermediate phenotypes that are associated with several psychiatric illnesses.The rs1006737 risk variant has also been associated with several intermediate phenotypes such as neural activity during episodic30 One promising candidate intermediate phenotype for several neuropsychiatric disorders is response bias, as measured by the probabilistic reward-learning task.31 The paradigm measures the development of response bias on the basis of a differential reinforcement schedule and indicates an individual's propensity to respond to reward. Reward responsiveness initially predicted an anhedonic phenotype,31 but has since been shown to be diminished in patients with bipolar disorder32 and more general depressive phenotypes.35A growing body of research suggests that the neural circuitry supporting reward processing may be a suitable platform of study, as impaired reward function is repeatedly observed as a component of neuropsychiatric disorders.37 and interactions between stress and genes that influence the stress-axis.40 Additional evidence supports a role for gene variants that influence dopaminergic regulation.42 These associations are supported by findings that reward responsiveness during the task is moderately heritable.43 However, no studies have looked at the potential association between GWAS-identified risk variants and reward responsiveness. We consider CACNA1C rs1006737 as a candidate variant to probe for effects in reward responsiveness considering previous literature suggests (a) CACNA1C is associated putative effects on reward/learning;24 (b) CACNA1C is associated with mood disorders at genome-wide level7 and (c) reward responsiveness is diminished as a component of these psychiatric illnesses.35 As the A allele at rs1006737 is overrepresented in neuropsychiatric illness, we anticipate that the genotype group with the A risk allele will show blunted reward responsiveness compared with individuals who do not carry a copy of the risk allele.Reward responsiveness may also be modulated by dopaminergic innervationCACNA1C variant (rs1006737). Participants in this panel were recruited by advertisement, from among the University community  on the basis of the following self-reported criteria: western European descent; no experience of psychiatric/neurological symptoms or diagnoses in either themselves or first-degree relatives; no illegal  substance use/dependence (excluding nicotine) and no alcohol abuse/dependence. Cardiff site: 34 Caucasian volunteers were genotyped for the CACNA1C variant (rs1006737). No participants reported any current mental illness44 or use of psychotropic medication. The study was approved by the ethics committee of each University and written, informed consent was given by all participants before gDNA extraction and participation. A total of 164 healthy individuals were included in the study . A power calculation45 suggested that we had 78% power to detect an effect of this size for rs1006737 on response bias .Bangor site: 131 subjects were recruited from Bangor University and genotyped for the http://support.illumina.com/array/array_software/assay_design_tool.ilmn). SNP sequence: 5′-ACTTGGCTCTATCAAAGTCTTGCTATCAATTACATAAGTTCCATTCCATCTCAGCCCGAA[A/G]TGTTTTCAGAGCCGGAGACCTCACAGTGTCTCTCAGGACAGTACCTTTCAGGTTTGAATG-3′. Nucleic acid concentration was evaluated using PicoGreen assay . Golden Gate genotyping was performed according to manufacturer's protocols. Genotype calling and annotation were performed using GenomeStudio (Illumina). CACNA1C rs1006737 was not available for five individuals from the Bangor site.Bangor site: genomic DNA was obtained from saliva using Oragene OG-500 saliva kits for 136 participants. Genotyping of rs1006737 was performed using the Illumina Golden Gate assay  using the BeadXpress platform, which allows high-throughput multiplex genotyping of SNPs. Assays were designed for the experiment using Illumina's Assay Design Tool (CACNA1C rs1006737 was genotyped using custom SNP genotyping arrays from Illumina (Illumina). Individuals were excluded for ambiguous sex, cryptic relatedness up to third-degree relatives by identity of descent, genotyping completeness <97%, and non-European ethnicity admixture detected as outliers in iterative EIGENSTRAT analyses of an LD-pruned data set.46 Thirty four of the 37 individuals included in the sample had genotype data available for rs1006737.Cardiff site: genomic DNA was obtained from saliva using Oragene OG-500 saliva kits for 37 participants. et al.31 and Heerey et al.47 Asymmetric reinforcement, in which correct responses to one stimulus receive more frequent rewards than correct responses to another, leads to the development of response bias by increasing participants' likelihood of reporting the more frequently reinforced stimulus.48 It is hypothesized that individuals who develop greater levels of response bias are more responsive to rewards.31 Trials began with a fixation cross (500 ms), followed by the presentation of a cartoon face with no mouth. After 500 ms, either a short (22 mm) or long (24 mm) mouth appeared on the face. It was visible for 100 ms before disappearing. The face remained on screen until the participant responded with a button press indicating the presence of either the short or long mouth. Following the response, participants saw a screen that either displayed feedback (‘correct +5 pence') or remained blank  for 1750 ms. Participants completed three blocks of 100 trials. Both versions of the mouth appeared equally often in pseudo-random order such that there were no more than four successive trials of the same mouth. Participants received reward feedback on 40 correct responses per block. To induce a reward-related response bias in the task, we distributed the rewards asymmetrically across the mouths. The more frequently reinforced mouth received 30 rewards per block and the remaining 10 rewards occurred after responses to the other mouth. We used a pseudo-random reward schedule such that no more than three correct trials in a row received reinforcements. Participants never received feedback on incorrect trials. When positive feedback was scheduled for a trial and the participant answered incorrectly, the reinforcement was postponed until a later unreinforced, correct trial (of the same mouth length) occurred. The length (short or long) of the more frequently reinforced mouth was counterbalanced across participants. All trials where reaction times faster than 200 ms and slower than 3000 ms were removed from the analysis as previously described.47 We measured the frequency of each participant's reward feedback schedule on the basis of the number of positive rewards (‘correct +5 pence') they received. The maximum bonus was £6 and individuals who earned <£5 were excluded from the analysis (n=1). The reward feedback schedule received did not significantly differ between gender ; rs1006737 genotype group ; across sample site  and did not correlate with age .To measure reward responsiveness, we used a line discrimination task with asymmetric reinforcement, closely modelled after that described in Pizzagalli d', a measure of discrimination accuracy [d=z(H)−z(F)] and ‘criterion,' the degree to which participants showed a bias towards the more frequently reinforced mouth (c=−1/2[z(H)=z(F)]48). Please note that we reversed the values for criterion in the analysis , for ease of interpretation.A post-task debriefing interview confirmed that no participants were aware of the reinforcement asymmetry. We used a standard signal detection analysis to calculate 1,163=1.999, P=0.159) or in the degree to which they developed response biases . Therefore, data from the two sites were combined. Moreover, data from men and women were analysed together, as there were no significant differences between gender groups in either discriminability (d') or criterion across the whole group  or within CACNA1C rs1006737 genotype groups . There was a significant positive association between age and discriminability at block 1 . Therefore age was entered as a covariate into a mixed-model analysis of covariance to assess the potential effects of CACNA1C rs1006737 on discriminability (d'). There were no associations between age and criterion within genotype groups . One additional individual's discrimination ability was above the expected frequency (GG rs1006737 genotype); however, removing this individual did not significantly affect any demographic or genetic analysis. There were no significant outliers for the criterion measure.49 We therefore used mixed-model analyses of variance to compare criterion across task blocks (1–3) and genotype groups . We also ran the same mixed-model analyses of variance for criterion using a dominant genetic model  as previously described.24There were no site specific differences in participants' ability to discriminate between the mouths , suggesting that genotype differences in reward responsiveness were larger toward the end of the task . Post hoc independent-sample t-tests also revealed a dose-dependent response, in which the largest differences in mean response bias occurred between the AA and GG groups . Independent samples t-test using the dominant genetic model suggested that A risk allele carriers (AA/AG) also had pronounced deficits in reward responsiveness compared with the nonrisk (GG) genotype group . On the basis of our smallest effect size (Cohen's d=0.37), we estimate we had 76% power to detect a significant main effect of rs1006737 on mean criterion .Participants performed the line discrimination task equally well, regardless of genotype group . We additionally performed post hoc one-sample t-tests to test whether the rs1006737 genotype groups (AA/AG/GG) response bias significantly differed from 0 at each block (1–3). The AA homozygous risk group did not begin with or develop any response bias at any block . The AG heterozygous risk group showed a trend towards developing response bias , but these results were not significant after correction for multiple comparisons. However, the GG homozygous group showed significant response bias during all three blocks . This analysis indicates that the GG genotype showed response bias early in the task, the AG genotype group started to show a Δresponse bias in later trials, but in comparison, the AA risk group showed no evidence of developing response bias at any point throughout the paradigm . A one-way analysis of variance suggested that rs1006737 genotype related to an individual's propensity to develop response bias  modulates an individual's propensity to respond to reward, without disrupting general task discrimination ability. It is an attractive feature of the reward-responsiveness paradigm that it allows for the investigation of cognitive biases in situations of normal discriminability, which makes it a particularly sensitive trait marker for healthy at-risk populations.Previous research suggests that the CACNA1C variant may affect the neural mechanisms underlying reward processing and learning.51 Blunted reward responsiveness was seen across the whole task for AA genotype, the rs1006736 AG genotype group showed response bias towards the end of the task, whereas the GG nonrisk group showed reward responsiveness throughout the task. We suggest that rs1006737 ‘A' risk allele dose may contribute to psychopathology by affecting the rate of reinforcement–based learning, although this would need to be formally tested in extended versions of the paradigm. This finding is comparable to previous reports where impairment in learning was most pronounced in rs1006737 A carriers at the beginning of the task.24 These results offer novel insight into how CACNA1C may confer susceptibility to the neuropsychiatric illness characterized by reduced reward responsiveness and impairments in reward-based learning. The association between CACNA1C rs1006737 A allele and blunted response bias may point to a genetic basis for anhedonia, which is a symptom common to affective and psychotic disorders.54 Although evidence suggests that anhedonia is a residual trait underlying euthymic bipolar disorder,54 it is less straightforward to align our findings with bipolar phenotypes associated with the hyperthymic state, as it is speculated that manic states could be associated with a potentiated rather than blunted response bias.32This finding adds to a growing body of literature suggesting that the 55 We suggest that the effect of the rs1006737 (and any other common variant) on reward responsiveness are likely to be small51 and thus, our sample may have been underpowered. We therefore recommend that our results should be treated with caution until replicated in larger, independent samples.58 Additional studies could further help to verify how much variance in response bias associated with rs1006737 genotype. Furthermore, we cannot be sure whether rs1006737 is responsible for these effects or rather SNPs that are in linkage disequilibrium with the variant. Nevertheless, recent GWAS implicate CACNA1C as a suitable candidate for probing intermediate traits associated with multiple neuropsychiatric illness.59 It is possible that additive variations within CACNA1C12 or interactions with other genes60 could further modulate reward responsiveness and explain larger proportions of variance.Although the genetic architecture of reward-related deficits and related clinical symptoms (such as anhedonia) remains unknown, reward responsiveness may be a promising neurobiological process that links novel risk loci (such as SNPs identified via genome-wide association studies) with core clinical symptoms. It could also emerge as a promising surrogate marker for treatment effects. A limitation of the present study was that we did not screen for nicotine use, which may have an interactive effect on reward responsiveness.CACNA1C (rs1006737) may affect an individual's ability to respond to reward. An attractive feature of the rs1006737 ‘A' risk allele is its association with increased CACNA1C mRNA expression in cortical tissue,16 which offers putative mechanistic insight into how the variant may affect neural circuitry. A growing body of knowledge implicates the CACNA1C gene product in animal models of reward processing,63 therefore translational animal models homologous to reward responsiveness64 may also give further insight into the molecular mechanisms underlying the association between CACNA1C and reward responsiveness. Future studies could also explore the neural dynamics that support reward responsiveness to understand how CACNA1C may exert these effects.In conclusion, our results suggest that the genome-wide identified psychiatric risk locus on