Exaiptasia pallida anemones colonized by physiologically different symbiont species, in comparison with symbiont-free (aposymbiotic) anemones. We compared one homologous species, Symbiodinium linucheae, that is natively associated with the clonal Exaiptasia strain (CC7) to another heterologous species, Durusdinium trenchii, a thermally tolerant species that colonizes numerous coral species. This approach allowed the discovery of a core set of host genes that are differentially regulated as a function of symbiosis regardless of symbiont species. The findings revealed that symbiont colonization at higher densities requires circumvention of the host cellular immunological response, enhancement of ammonium regulation, and suppression of phagocytosis after a host cell in colonized. Furthermore, the heterologous symbionts failed to duplicate the same level of homologous colonization within the host, evidenced by substantially lower symbiont densities. This reduced colonization of D. trenchii correlated with its inability to circumvent key host systems including autophagy-suppressing modulators, cytoskeletal alteration, and isomerase activity. The larger capability of host molecular circumvention by homologous symbionts could be the result of a longer evolutionary history of host/symbiont interactions, which translates into a more finely tuned symbiosis. These findings are of great importance within the context of the response of reef corals to climate change since it has been suggested that coral may acclimatize to ocean warming by changing their dominant symbiont species.Reef corals and sea anemones form symbioses with unicellular symbiotic dinoflagellates. The molecular circumventions that underlie the successful intracellular colonization of hosts by symbionts are still largely unknown. We conducted proteomic analyses to determine molecular differences of  Symbiodinium [Coral reefs, frequently referred as “the rainforest of the sea,” represent the most diverse marine ecosystem. Comprising about 0.1% of the earth surface, coral reefs provide habitat for 93,000 known species , includiiodinium ), which Durusdinium  to another heterologous species, Durusdinium trenchii, a thermally tolerant species that colonizes numerous coral species (The present study leverages the power of comparing different symbiont identities as well as presence and absence of symbiont (symbiotic state) in a clonal host background. Here, we use a proteomic approach to determine differences within the protein profiles of these anemones hosting physiologically distinct Symbiodiniaceae species in comparison with aposymbiotic anemones. We chose to compare one homologous species,  species . FurtherSymbiodinium linucheae (2.87 ± 0.60 × 103 cells μg–1 protein) was four times denser than that of the heterologous symbiont, Durusdinium trenchii (0.65 ± 0.32 × 103 cells μg–1 protein). The level of endosymbiotic dinoflagellate densities within Exaiptasia pallida anemones reported in this study and the colonization differences between the two species of Symbiodiniaceae are consistent with values documented by other studies for homologous vs. heterologous symbionts  were compared in reference to proteomes of aposymbiotic anemones . The comparisons were conducted in anemones sampled both during day (solar noon) and during night (solar midnight). From these four comparisons (two symbionts species × two sampling times), 97 differentially expressed unique host proteins were identified among approximately 1,600 protein features visibly detected on the 2D DIGE gels. The data reveal a core proteome of symbiosis; that is, both homologous S. linucheae- and heterologous D. trenchii-hosting anemones shared expression commonalities in comparison with aposymbiotic anemones, but also exhibited distinct symbiont-specific responses both during the day and night, as described below.The proteomic profiles of symbiotic anemones colonized by different Symbiodiniaceae species  shared between symbiotic anemones compared to the day-sampled anemones (n = 9/56)  . The oven = 9) and night (n = 22) groups shared an overlap of four proteins  downregulation  and 39% (29/75), respectively (n = 14) and at night (n = 18) indicating that these proteins represent a core set that are regulated independently of symbiont type and day/night group  of up-regulated proteins concordant between day and night  concordance between up-regulated proteins and 46% (18/39) in down-regulated  from New Zealand where the heterologous symbiont Durusdinium trenchii colonized in considerably lower densities than the homologous Breviolum minutum  relative to the same heterologous species (D. trenchii) . An overarching pattern of differential protein expression detected in these Symbiodiniaceae-specific comparisons was a dramatically larger number of proteins regulated as a function of symbiosis  in anemones colonized by the homologous symbiont compared to those colonized by the heterologous symbiont. This finding supports similar patterns discovered in a different population of renchii) . The loweristics  that proeristics .We also discovered a congruent pattern of the symbiosis-regulated proteins between day and night in those anemones colonized by the homologous symbionts. By contrast, the anemones colonized by the heterologous symbionts share fewer differentially expressed proteins between day and night, which indicates a more dynamic pattern of expression. This dynamism of expression shows the existence of a diel regulation pattern in the anemones colonized by the heterologous symbiont. While the cause of this remains to be resolved, it is possible the heterologous partners more drastically alter expression levels to maintain homeostasis relative to homologous symbionts that drive a more constant state of host gene regulation.Despite these differences, a minimum of essential molecular circumventions is expected to occur for any symbiotic dinoflagellates to take residence inside a cnidarian the host cell. We were able to identify common core modulations of protein expression in response to symbiosis when colonized with either homologous or heterologous symbionts. Protein expression comparison between day and night provided further compelling evidence of the role of these core proteins in symbiosis. Below we discuss these shared molecular alterations between anemones colonized by homologous and heterologous symbionts. We also argue that lack of some alterations in those anemones colonized by the heterologous symbionts might be linked to differences in symbiotic engagement.c from the mitochondria and activation of caspases 3, 8, and 9, signaling that otherwise would lead to apoptosis  during day and night , consistDrosophila cells, NPC2 binds cholesterol in the lumen of the endosome and lysosome and transfers it to NPC1, a transmembrane protein that exports the cholesterol to other intracellular locations. The Anemonia viridis NPC2 (AvNPC2-d) was found to be up-regulated in symbiotic relative to aposymbiotic sea anemones and its expression was indeed higher in the gastrodermis (symbiont-containing tissue) closely localized with symbiosomes  in the anemones colonized by the heterologous symbionts. These data suggest that D. trenchii failed to circumvent key host systems including autophagy-suppressing modulators, cytoskeletal alteration, and isomerase activity , which was up-regulated during both day and night in S. linucheae-hosting anemones, but down-regulated in anemones hosting the heterologous symbiont D. trenchii during the day . These anemones host either the homologous (native) Symbiodinium linucheae  Durusdinium trenchii, well-known for its ability to convey thermal resistant attributes to the coral holobiont  of inucheae , which aolobiont . An apos–2s–1) on a diurnal 12 h light:12 h dark cycle, and fed twice weekly with freshly hatched Artemia franciscana nauplii. Seawater was changed 6–12 h after feeding.Stock anemones were kept at 26°C room in glass bowls containing autoclaved artificial seawater (ASW)  at 32–34 PSU. Anemones were grown under white fluorescent light , with remnant symbionts removed via DCMU treatment. Following 1 week of feeding cessation, anemones were incubated in darkness at 4°C for 8 h and then returned to room temperature. Daily water changes with autoclaved ASW were performed during this process. The cold treatment was repeated three times over the course of a week, with 24–48 h between treatments to allow for host recovery. Following cold treatments, anemones were incubated in 50 μM of the herbicide and PSII inhibitor 3-1-1 dimethylurea (DCMU) in autoclaved ASW and exposed to 1,000 μmol m–2s–1 of light for 6 h. After DCMU exposure, ASW containing DCMU was exchanged for autoclaved ASW without DCMU. The DCMU and light exposure treatments were repeated twice weekly for 3 weeks. Loss of symbionts was confirmed using fluorescence microscopy and the absence of detectable PCR amplification of Symbiodiniaceae chloroplast 23S DNA compared with an amplified positive control (Aposymbiotic  control . FurtherDurusdinium trenchii (Strain CCMP2556) obtained from the National Center for Marine Algae and Microbiota (NCMA) and grown in silica-free Prov50 medium. Log phase D. trenchii were mixed with A. franciscana nauplii and fed to the anemones to achieve colonization. Colonization was confirmed by fluorescence microscopy, chloroplast 23S DNA PCR amplification, and Sanger sequencing. Stocks of aposymbiotic anemones and anemones infected with D. trenchii were increased through asexual reproduction via pedal laceration (Durusdinium trenchii), and homologous-hosting (Symbiodinium linucheae) anemones were maintained under similar culture conditions for the next 12 months; that is, without any additional DCMU/stress treatments or re-inoculation with symbionts, to ensure that the symbiotic states or lack thereof were stable. While DMCU could have an effect on downstream proteomic analyses, it is important to note that an entire year elapsed between the bleaching of anemones with photosynthetic inhibitor DMCU before proteome experiments were conducted. Therefore, we assumed that this is a very conservative length of time to allow recovery from the chemical treatment. Prior to proteomic experiments, symbiotic state was again confirmed by fluorescence microscopy, chloroplast 23S DNA PCR amplification, and Sanger sequencing.A portion of the aposymbiotic anemones were colonized with the non-native, heterologous ceration . AposymbD. trenchii hosting, and S. linucheae hosting) were transferred to six-well culture plates containing 10 mL of autoclaved ASW. Each plate contained five anemones (one per well) ranging in size from 5 to 10 mm that together represented a single biological replicate for its respective treatment. Six biological replicates (each consisting of one plate) were used for each treatment. All culture plates were maintained under the same environmental condition for 7 days before sampling for the comparative molecular study. The plates were partially immersed in a temperature controlled (26°C) water bath and received ∼80 μmol m–2s–1 of light for 12 h daily . Anemones were fed immediately after their initial transfer to new culture plates and their water was replaced with autoclaved ASW 12 h after feeding. Following this, anemones were not fed to avoid detection of A. franciscana nauplii proteins in the experimental analyses.Following 1 year of similar culture conditions, anemones of the three symbiotic states  for each treatment group sampled in the day and again at night. The tubes were then centrifuged for 1-min at 78 × g. Liquid was removed from the tubes and samples were frozen in liquid nitrogen.Every anemone treatment group was sampled at two different times: one in the middle of the day (12 p.m.), and the other in the middle of the night (12 a.m.). For each sampling event, five anemones from each treatment groups were pooled into a cryogenic tube serving as technical replicates, forming together one biological replicate. This was repeated in triplicate  and correct for multiple tests. Of the differentially expressed protein spots detected on the gels, 115 were selected for MALDI-TOF mass spectroscopy using a 5800 System mass spectrometer (AB Sciex). The selection of these proteins was based on highly significant  differential expression. An additional 19 spots were also selected for mass spectrometry identification based on their large expression ratios (>3x relative expression) or their differential expression  in four or more of the nine comparisons. Proteins were identified by a genomic database for E. pallida  using the peptide mass and fragmentation spectra along with the MASCOT search engine (Matrix Science). Significant candidates had either protein score C.I.% or ion C.I.% greater than 95.Total host protein was extracted and proteomic analyses were performed by Applied Biomics  using two-dimensional difference gel electrophoresis (2D DIGE) gels, which allowed for the detection of differential protein expression. The analyses included nine pairwise comparisons: amongst symbiont identities during the day; amongst symbiont identities during night; and between day and night within each symbiont identity . The 2D Protein sequences were searched against the non-redundant (nr) NCBI database using Blast2GO . Gene onMR-L conceived the study. MR-L, DM, and AB planned the experimental design. EM conducted the study with the assistance of DM and AB. EM and MR-L conducted the analyses and interpretations, with contributions from DM and AB. EM and MR-L wrote the manuscript. DM and AB edited and reviewed the manuscript.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.


The recombinant ScFv was generated in Nicotiana benthamiana by transient expression, and was chemically conjugated (ScFv/RVG) to a 29 amino acid peptide, specific for nicotinic acetylcholine receptor (nAchR) binding in the CNS. This conjugated molecule was able to bind nAchR in vitro and enter neuronal cells more efficiently than ScFv. The ability of the ScFv/RVG to neutralise virus in vivo was assessed using a staggered administration where the molecule was inoculated either four hours before, two days after or four days after infection. The ScFv/RVG conjugate was evaluated in direct comparison with HRIG and a potential antiviral molecule, Favipiravir  to indicate whether there was greater bioavailability of the ScFv in the brains of treated mice. The study indicated that the approach taken with the ScFv/RVG conjugate may have utility in the design and implementation of novel tools targetting rabies virus infection in the brain.Rabies causes more than 60,000 human deaths annually in areas where the virus is endemic. Importantly, rabies is one of the few pathogens for which there is no treatment following the onset of clinical disease with the outcome of infection being death in almost 100% of cases. Whilst vaccination, and the combination of vaccine and rabies immunoglobulin treatment for post-exposure administration are available, no tools have been identified that can reduce or prevent rabies virus replication once clinical disease has initiated. The search for effective antiviral molecules to treat those that have already developed clinical disease associated with rabies virus infection is considered one of the most important goals in rabies research. The current study assesses a single chain antibody molecule (ScFv) based on a monoclonal antibody that potently neutralises rabies  RABV is a non-segmented negative strand RNA virus in the order RABV is almost always transmitted following a bite injury from an infected animal that is excreting virus in its saliva. The mechanism of virus infection once it has crossed the dermal barrier is poorly defined. Lyssaviruses are strongly neurotrophic, however, replication in the musculature, prior to entry into the peripheral nervous system occurs, and is likely to contribute to the variation in incubation times seen following infection Current options for rabies post exposure treatment include immunoprohylaxis with human or equine rabies immunoglobulin (H/ERIG) at the site of the infection and vaccination at a site distant from the exposure to ensure that the application of RIG does not interfere with the humoral immune response Rabies glycoprotein (G), present as a trimeric peplomer on the viral envelope, contains a short conserved motif which serves to bind cellular receptors in planta and expression of a single chain antibody fragment (ScFv) of a previously defined rabies neutralising monoclonal antibody in E. coliN. benthamianaN. benthamiana but chemically conjugated to synthetic 29 amino acid peptide (ScFv/RVG) for evaluation. The ScFv/RVG conjugate retained the ability to neutralise RABV. In comparison to ScFv alone, ScFv/RVG demonstrated enhanced ability to cross an in vivo 3D cell culture BBB model via a mechanism that involves the N-acetylcholine receptor. Finally, the ability of ScFv/RVG to act as a potential post-exposure tool was assessed in vivo. Direct in vivo comparisons with the action of HRIG demonstrated that ScFv/RVG may have future utility as a post-exposure alternative to HRIG for rabies virus post exposure treatment.Previous studies have described the application of monoclonal antibody preparations as an alternative to RIG 22.1Agrobacterium tumefaciens LBA4404 was separately transformed with the pEAQ-62-71-3 IgG N. benthamiana plants, by vacuum infiltration as described The pEAQ-62-71-3 IgG 2.2Crude protein extracts from plant leaves were prepared 5 days after agro-infiltration and denatured by boiling in NuPAGE® LDS Sample Buffer. Proteins were separated on 4–12% gradient polyacrylamide gels . Proteins were visualised by Coomasie staining, or electrophoretically transferred to a nitrocellulose membrane for immunoblotting. Nitro-cellulose membranes were blocked  before being probed with horseradish peroxidase (HRP) conjugated mouse anti-E-tag antiserum  diluted at 1:5000 in 1% non-fat dried milk in PBST. Bands were visualised following addition of ECL plus detection reagent .2.3Human embryonic kidney 293 cells expressing human α7-nicotinic acetylcholine receptor (HEKnAchR7) were reported previously 2.43) in PBS, pH 7.4. For the entry assay, samples were washed 3 times with permeabilization buffer  before the cells were incubated with 1:1000 mouse anti-E tag antiserum at 4 °C, overnight. The cells were then washed as before, before incubation with a goat anti-mouse IgG antiserum conjugated with cy5  at 37 °C for 1 h. After further washing, the cells were resuspended in staining buffer and analysed by flow cytometry, using FACS CellQuest software . For the competition assay, cells were pretreated on ice with either 2 × 107 PFU of UV inactivated Rabies virus (CVS) HEK 293 cells or Neuroscreen-1  cells were seeded on 6-well plates. After 24 h, cells were placed on ice and incubated with ScFv or ScFv/RVG for 5 min (binding assay) or 30 min (entry assay). The cells were washed with PBS, then harvested into FACS tubes and incubated in cell fixation solution  for 15 min. For the binding assay, samples were washed 3 times with 1% inactivated foetal calf serum . The restrictive paracellular permeability of hCMEC/D3 cells was assessed by their low permeability to the non-permeant fluorescent marker Lucifer Yellow (LY) 2) and samples were taken after 2 h and 18 h to assess the media in the bottom chamber for the presence of antibody by virus neutralisation.An immortalized human brain capillary endothelial cell line (hCMEC/D3) was kindly provided by Prof. Pierre-Olivier Couraud  and Prof. Pierre-Emmanuel Ceccaldi  2.6in vivo work was undertaken in BSL3/SAPO4 containment at the Animal and Plant Health Agency (APHA), following independent ethical review under strict Home Office guidelines (PPL70/7394). Molecules were administered to groups of mice by intraperitoneal inoculation. Intra-peritoneal administration (IP) of ScFv/RVG was compared to treatment with human rabies immunoglobulin (HRIG) as both a pre- and post-exposure treatment. Treatments with Favipiravir , and T-705 with ScFv/RVG were also included in the study.All Mice (n = 12/group) received ScFv/RVG (40 IU/kg), HRIG (40 IU/kg), T-705 (300 mg/kg) or ScFv/RVG (40 IU/kg) + T-705 (300 mg/kg) or were controls receiving PBS following the same treatment schedule. Mice were tagged and numbered before using a random number generator to distribute mice into groups. Each group of 12 mice was randomly split across two boxes of 6 mice each, to take account of interactions among mice sharing boxes and any other differences between boxes. Groups of mice were treated for 10 consecutive days. The treatments were initiated either four hours before virus inoculation (−4hr), two days (+2d) after virus infection or 4 days (+4d) after virus inoculation. Virus used for inoculation was a bat rabies strain originally isolated from a human fatality following infection from an insectivorous bat The data were analysed for treatment effects as a factorial design (5 treatments × 3 timings) by applying a multilevel mixed effects logistic regression to take account of potential correlation among mice in each box . Treatment effects were calculated as logits of mortality, where a logit is the logarithm of the odds ratio 33.1The purified ScFv and the ScFv/RVG conjugate were assessed by SDS-PAGE gel followed by Coomassie staining A or by i3.2The parent monoclonal antibody (62-71-3) and two versions of ScFv were tested to determine their capability to neutralize rabies virus (ERA strain) using a plaque-inhibition assay. The starting concentrations for all three antibodies was 0.5 mg/ml and the results suggest that the neutralizing activity of ScFv and ScFv/RVG conjugate was not significantly different to that of 62-71-3 mAb .Fig. 2Ra3.3The binding and penetration of ScFv and ScFv/RVG conjugate in HEK 293 cells overexpressing nAchR were tested by flow cytometry. A greater proportion of ScFv/RVG bound to the 293 cells as evidenced by the shift to the right of the dotted line compared to ScFv (solid line) A. After The specificity of binding between ScFv/RVG and HEK 293 cells via nAchR was tested by a competitive assay using irradiated rabies virus and α-bungarotoxin. The HEK 293 cell line was pre-incubated with each inhibitor, before incubation with ScFv or ScFv/RVG. No effect of either irradiated virus or α-bungarotoxin was observed in the case of ScFv E–H.Fig. 3.4in vitro BBB transport experiment was conducted on an hCMEC/D3 cell monolayer as described previously An 3.5in vivo. There was a clear trend showing greatest mortality in PBS treated groups, compared with those treated with HRIG (lowest) and ScFv/RVG  and the role of nAchR in this interaction was demonstrated by the ability of both rabies virus and alpha-bungarotoxin to competitively inhibit ScFv. Alpha-bungarotoxin is a neurotoxin that binds nAchR at the same site as rabies glycoprotein in vitro model was utilised to investigate the potential transport of different antibody based molecules across the blood brain barrier. This model was impermeable to the full length 62-71-3 IgG mAb as expected. Conjugating RVG to 62-71-3 IgG made no difference, indicating that that the size of IgG is a limiting factor. Although there was some apparent passage of ScFv across the BBB model, this was significantly enhanced in the case of ScFv/RVG. The increasing concentration of neutralising activity in the lower chamber of this assay with time, in comparison with the result using unconjugated ScFv alone, suggests that transport was mediated by an active mechanism.An in vivo assessment of ScFv/RVG was subsequently attempted using a murine model of rabies virus infection and different treatment schedules with either HRIG or the ScFv molecule. For this experiment, treatment schedules were designed on the hypothesis that at 4 h before inoculation and 2 days post inoculation, the infecting virus would still be in the periphery and that an established neuronal infection had not yet been initiated. The 4 day post inoculation treatment schedule was chosen because it was expected that an infection of the central nervous system would have established, so it should be possible to demonstrate protective efficacy from ScFv/RVG due to greater accessibility to the brain An in vivo, and demonstrates that the chemical conjugation process does not affect the viral neutralisation properties of the ScFv in vivo. A definitive pre-clinical study demonstrating protective efficacy in a robust model for central nervous system infection by rabies virus is now required.However, the results suggest that the virus took longer to reach the CNS than expected. HRIG was protective when delivered at all time points, even though it is well established that HRIG does not provide protection once rabies virus infection enters the CNS. So unfortunately, no conclusions can be drawn regarding potential ScFv mediated protection within the CNS. With no significant effect from the timing of treatments, ScFv/RVG halved mortality relative to the control treatment, but did not match the 90% protection observed for HRIG. Although the dosages administered were equivalent in terms of International Units/kg, ScFv/RVG performed less effectively than HRIG. This is likely to be due to different pharmacokinetics, as without Fc, ScFv/RVG would be expected to have a shorter serum half life In conclusion, the adaptation of ScFv through conjugation to a 29 amino acid RVG peptide has enabled greater bioavailability of the molecule. In particular, the approach adopted in this study overcomes the problem of low yield, and the scalable production of rabies ScFv molecule in plants is promising. RVG peptide synthesis and the conjugation process are readily available commercially and available under Good Manufacturing Practice when necessary. This leads to the possibility for rapid large scale production of the conjugated molecule and relatively quick translation to clinical trial. The development and clinical evaluation of new tools for post exposure control for rabies virus infection in endemic areas is a matter of some urgency.


Amputation in adults is associated with an extensive remapping of cortical topography in primary and secondary sensorimotor areas. Here, we used tactile residual limb stimulation and 3T functional magnetic resonance imaging in humans to investigate functional connectivity changes in the sensorimotor network of patients with long-term lower limb traumatic amputations with phantom sensation, but without pain. We found a pronounced reduction of inter-hemispheric functional connectivity between homologous sensorimotor cortical regions in amputees, including the primary (S1) and secondary (S2) somatosensory areas, and primary (M1) and secondary (M2) motor areas. We additionally observed an intra-hemispheric increased functional connectivity between primary and secondary somatosensory regions, and between the primary and premotor areas, contralateral to amputation. These functional connectivity changes in specialized small-scale sensory-motor networks improve our understanding of the functional impact of lower limb amputation in the brain. Our findings in a selective group of patients with phantom limb sensations, but without pain suggest that disinhibition of neural inputs following traumatic limb amputation disrupts sensorimotor topology, unbalancing functional brain network organization. These findings step up the description of brain plasticity related with phantom sensations by showing that pain is not critical for sensorimotor network changes after peripheral injury. About 90% of individuals suffering amputation report “phantom sensations”, the term used to define the perception that the missing part is still present9. Often these phantom sensations include pain. Studies have correlated phantom limb pain to a primary sensorimotor functional remapping after amputation, suggesting a maladaptive plasticity11. More recently, the widely-accepted hypothesis that maladaptive plasticity is related to phantom pain in amputees has been challenged. An increased activity in the deprived sensorimotor cortical region corresponding to the amputated hand was reported13. These apparent conflicting results suggest that a more complex and perhaps multifactorial explanation to the phantom phenomena may be at play. Accordingly, cortical plasticity has also been identified in amputees without phantom limb pain15, but non-painful phantom sensations were not consistently related to primary somatosensory cortex changes after amputation1.Amputation in adults is associated with the remapping of sensorimotor cortical representations. Changes in the primary sensorimotor cortical areas in amputees have been described employing different electrophysiological and imaging techniques15. Taken together, these functional and structural findings suggest that limb amputation lead to marked changes of sensorimotor network as a whole, potentially unbalancing its functional organization16.Structural changes were also demonstrated in amputees. Gray matter reduction within the hand representation in upper limb amputees as well as white matter changes in the corpus callosum in lower limb amputees were identified18 and to identify the contribution or influence of different brain regions to accomplish a specific task or behaviour, and the degree of coupling between components of distributed neural systems19. A resting-state approach – during which participants are not engaged with any external stimulus or explicit tasks – was recently employed to show a reduced interhemispheric functional connectivity between the two hand areas in hand amputees compared to controls12. Additionally, a reduction in functional connectivity between the missing hand motor representation and the sensorimotor network was observed11. However, because these studies investigated resting-state connectivity, it is unknown whether such changes would be present while participants are engaged in sensorimotor tasks. Additionally, these studies focused on the hand representation and its relations in particular and include participants with different pain ratings.Brain networks currently can be investigated in greater depth by functional magnetic resonance imaging functional (fMRI) connectivity methods during the performance of specific tasks. These methods can be used to analyse the functional relationships among different brain areas in detailIn the current study, we aimed to investigate the connectivity of a specialized network related to somatosensory stimulation in amputees without pain. To achieve this goal, we employed functional connectivity methods to analyse task-based fMRI time series data of lower limb amputees and controls.21. A priori ROIs were: primary motor cortex (M1); premotor cortex/supplementary motor area (M2); primary somatosensory cortex (S1); and secondary somatosensory cortex (S2).We report extensive functional connectivity changes in lower limb amputees without pain during two tactile stimulations: residual limb stimulation and remaining foot stimulation, as compared to controls. Sensorimotor brain connectivity was investigated using the region of interest (ROI-to-ROI) connectivity methodP < 0.05 for false discovery rate (FDR-corrected) , were also calculated. There was an increase in intra-hemispheric functional connectivity contralateral to amputation in the amputees. Ipsilateral connectivity from S1 to M1 , S1 to S2 , and S1 to M2  were increased in the “deafferented” hemisphere  , M1 , M2 , and S2   statistical comparisons of ROI-to-ROI functional connectivity during tactile stimulation of the residual limb from the amputees’ group (see Table ed) Fig. . Family-T = −4.82, PFDR = 0.008), S1 , and S2  , and deafferented M1 with the contralateral M2 and S1  . Functional connectivity during stimulation of the spared foot in amputees was also significantly different from foot stimulation in the control group Fig. . SpecifiIn the present study, we described functional connectivity changes in the sensorimotor network in lower limb amputees with phantom sensation, but without pain. Remarkably, during somatosensory stimulation, extensive sensorimotor functional connectivity changes were found. A marked reduction of inter-hemispheric functional connectivity between homologous sensorimotor areas was found along with an increased intra-hemispheric connectivity contralateral to the amputated limb.16. In accordance the sensorimotor system usually exhibits high levels of inter-hemispheric functional connectivity between homologous brain areas 22.Typically, normal inter-hemispheric functional connectivity of the brain is characterized by higher symmetrical brain network patterns between homologous areas, as compared to asymmetrical connectivity between heterotopic regions24. Forepaw denervation in animal models also seems to produce connectivity reduction25. Furthermore, a study has shown inter-regional connections asymmetry between sensorimotor regions in individuals with congenital upper limb absence26.During both residual limb and foot stimulation in the amputees Figs  and 2, h11. Nevertheless, the statistical effect observed by Makin and co-workers11 was lost when the phantom limb pain magnitude was accounted in the model. Although this result reinforces the idea that phantom limb pain can influence sensorimotor system organization, it also suggests a potential additive effect of brain changes related to limb amputation - as observed in the present investigation - and the ones related to the presence of pain per se.Weakened inter-hemispheric functional connectivity between hand representations during rest has already been described in upper limb amputees29. Additionally, this same circuitry has been recently described in humans through magnetic resonance imaging (MRI) and electroencephalography (EEG) approaches31. Considering the importance of transcallosal connection between sensorimotor areas of both hemispheres33, the functional connectivity decoupling found here could be related to fractional anisotropy reduction in the CC previously reported in the same group of patients15. Despite containing mostly excitatory fibres that contact the opposite hemisphere36, the net result of CC activity is importantly inhibitory, mainly intermediated by GABAergic interneurons40. Considering this inhibitory role, a reduction in the integrity of the corpus callosum in amputees should probably lead to a decrease of functional connectivity between the two hemispheres, as indeed was found in the present work. Other structural findings also suggest that inter-hemispheric relationships can be changed after amputation. For instance, S1 cortex thickness between the two hemispheres did not correlate in lower limb amputees as it does in controls41.In non-human primates, electrophysiological studies associated with anatomical tracer injections have demonstrated a predominantly homotopic connection between S1, S2 and M1 of both hemispheres via the corpus callosum (CC)15. Animal models support the existence of long-range inter-hemispheric cortical reorganization, probably mediated by the CC. In rodents, unilateral hind limb denervation leads to bilateral functional activation of the primary somatosensory cortex during sensory stimulation of the intact hind limb. This ipsilateral activation, however, no longer exists after ablation of the cortex contralateral to the healthy paw, highlighting the role of CC projections to this phenomenon42. In addition, microstructural cortical changes in callosal axons were also recently reported in a neonate rat model of amputation43. Is this study, the callosal axons originated from the deafferented S1 area expressed an expansion of their terminal arbors and an increase of the number of terminal boutons within the homotopic representation (S1) at the contralateral cerebral hemisphere.Functionally, a reduction in inter-hemispheric inhibition may also help explain the bilateral somatosensory cortex activation found during somatosensory stimulation of the residual limb and the contralateral foot previously reported in the same group of amputees15.During tactile residual limb stimulation, along with interhemispheric connectivity reduction, we have found an increase of functional intra-hemispheric connectivity between primary and secondary somatosensory regions, and also between primary and premotor/supplementary motor areas of the hemisphere contralateral to amputation. Conceivably, this increase in functional connectivity between areas within the same hemisphere may be related to the augmented cortical activation during stimulation of the residual limb and in the intact foot previously reported in amputees45 as well as in humans46. This expansion can parallel an increase of the intra-hemispheric functional connectivity and might be related to the unmasking of cortical representations, inhibited under normal conditions47.Functional expansion of the cortical representation of remaining body parts into those of deprived sensory input has already been demonstrated in different animal models50. By using voltage-sensitive dye imaging, Ferezou and his colleagues49, have shown that after a stimulus delivered onto a single mouse whisker, a response could be seen primarily at the corresponding S1 barrel cortex and then spread across S2 and M1 with small latency. In addition, if the S1 barrel cortex was inactivated by an ionotropic glutamate receptor antagonist, no response could be recorded in neither S1 nor any other cortical area49. These findings indicate a direct connection between S1 and different motor areas of the cortex.In fact, the unmasking of pre-existent projections is a potential mechanism for the increases observed in intra-hemispheric connectivity. Previous work with animal models has shown functional connectivity between primary somatosensory barrel cortex and other cortical areas in normal mice51. Indeed, experiments with anatomical tracers have shown a direct anatomical and reciprocal connection between S1 and M1, and between S1 and perirhinal cortex of the same hemisphere in normal mice33. The modulation of these connections may correspond, at least in part, to the underpinning mechanism of the increased intra-hemispheric sensorimotor functional connectivity seem in amputees in the present study.In animal models, anatomical connectivity can be studied by directly injecting tracers into specific brain regions51. Using tracers and also S1 and S2 focal cortical lesions in rats with forelimb amputation, Pearson and colleagues51 suggest a major role of subcortical sites in cortical reorganization. Accordingly, one could hypothesize a disinhibition of the anatomical connections between the residual limb area in somatosensory brainstem nuclei and the leg and foot areas in the thalamus, produced by amputation. This disinhibition could make the leg and foot representation areas in the thalamus become responsive to an input from the residual limb area, and then relay this input to the leg and foot areas in S1. Thus, when the residual limb is stimulated, this input would be delivered to the leg and foot representation areas in addition to the corresponding S1 area. Therefore, residual limb stimulation would activate more cortical areas than a stimulus made in an equivalent area in control subjects. In accordance to this proposal, thalamic mapping with microelectrodes in humans revealed an enlargement of the residual limb representation in amputees52. Aronoff and colleagues33 suggested that S1 has a functional and anatomical connection with motor areas; consequently, an increase of the number of cortical areas activated by a stimulus could lead to an increase of functional connectivity between S1 and S2, S1 and M1, and S1 and M2. All those findings are in agreement with our intra-hemispheric connectivity results.As an alternative explanation of our results, we could speculate about subcortical mechanisms that could account for it. Somatosensory cortical areas have extensive long-range connectivity with subcortical nuclei along the somatic pathwayIn this study, we selected patients with phantom limb sensation but who did not report phantom pain (five with permanent phantom sensations and four with intermittent phantom sensations). Whether there are functional connectivity differences between amputees without phantom limb pain who have permanent vs. intermittent phantom sensation is an important question that was beyond the scope of our study and remains unanswered. We believe that the perception of phantom limb sensation, whether intermittent or permanent, should play an important role in altering the balance of the sensorimotor cortical networks. The investigation of these possible differences can be the object of future studies. Furthermore, one of the main limitations of this study was the lack of a specific validated scale to quantify the phantom sensation reported by each of the amputees. Therefore, it was not possible to make any assumption about the relationship between the degree of phantom sensation and connectivity changes.To conclude, our results of functional connectivity in long term lower limb amputees with phantom sensations, but without pain, advance the current knowledge about brain correlates of phantom sensations by showing sensorimotor network changes pointing to interhemispheric decrease and intrahemispheric increase between the connected cortical areas.15 and are summarized here in Table 53.This study investigated functional connectivity in nine patients  with unilateral lower limb amputation (8 traumatic amputees and one amputated after an oncologic procedure) with phantom limb sensation and without pain, selected from the Amputee Unit Database of the Brazilian Beneficent Rehabilitation Association . Exclusion criteria for the study included: presence of phantom pain and/or amputation residual limb pain; amputation caused by peripheral obstructive vascular disease; diabetes mellitus with associated peripheral neuropathy; arterial hypertension; renal disfunction; history of neurological disease; neurotrauma; neurosurgical intervention or cerebrovascular disease; use of psychotropic, neuroleptic, anxiolytic or anti-depressant medication; and any contraindication to magnetic resonance imaging. A trained neurologist collected the clinical history of all subjects. The anamnesis included questions about the circumstances of amputation and a detailed assessment of phantom sensations, encompassing the time of onset, the pattern (intermittent/permanent), and the presence of pain, among others. The characteristics of the amputees were reported in a previous study3; field-of-view, 230 mm; 24 slices in the axial plane). Foam padding and straps over the forehead and under the chin were used to restrict head motion during acquisition. An fMRI task-based approach was performed using a block design model, with two experimental conditions (rest and somatosensory stimulation): for each condition, 10 dynamic volumes (20 seconds) were acquired 5 times, totalling 100 dynamic volumes per acquisition (3 minutes and 40 seconds of duration). The participants were instructed to stay relaxed and still during the experiment. The amputees received a somatosensory stimulation applied 2 cm above the residual limb end, and over the dorsal aspect of the remnant hallux and foot (“intact foot”). Matched controls received the stimulation on the same region corresponding to the level of amputation  and over the dorsal aspect of both hallux and feet (“control foot”). The somatosensory stimulation was a non-painful cutaneous tactile stimulation (5–6 Hz), employing a soft brush over a skin area of 10 × 2 cm applied manually by the experimenter54. To allow statistical comparisons, images from three right side amputees and their matched controls were left-right flipped before image processing steps were taken. This procedure has been previously used successfully by other investigators55. The hemisphere contralateral to amputation (“deafferented”) is therefore always displayed as the right hemisphere, while the hemisphere ipsilateral to amputation (“non-deafferented”) is displayed as the left hemisphere.Images were acquired on a 3T magnetic resonance scanner , equipped with a high-performance gradient system  and eight-channel sensitivity encoding head coil . fMRI images based on the blood oxygenation level-dependent (BOLD) signal were acquired with a T2*-weighted, single-shot, fast-field-echo, echo-planar imaging sequence . SPM Anatomy toolbox  was used to extract anatomical regions of interest. Connectivity Toolbox  was used for temporal processing and functional connectivity analysis. For each subject, the skull and other non-brain tissue were removed from the high-resolution T1 anatomical images using Brain Extraction Tool 57 and the extracted brain was used later on corregistration and normalization steps. The fMRI processing steps includes motion correction (6-parameter rigid body) and spatial smoothing (4 mm Gaussian filter kernel)58. For each subject, an averaged functional volume was created to corregister with high-resolution anatomical data. Then, a non-linear normalization between each subjects’ brain and the ICBM152 high resolution (1 mm isotropic voxels) anatomical brain template was performed . The non-linear anatomical normalization parameters were then applied to each acquired functional data. The resulting functional data in Montreal Neurological Institute (MNI) coordinates was utilized to perform functional connectivity group analysis. Segmentation was performed on the ICBM152 template59, obtaining probabilistic maps of gray matter (GM), white matter (WM), and cerebrospinal fluid areas (CSF). Next, the maps where eroded using a morphological erosion mathematical operator (2 mm3), in order to minimize potential partial volume effects between them, and binarized, resulting in three binary masks .The fMRI data was processed using Statistical Parametric Mapping , along with their first derivatives, as well the mean BOLD signals from WM and CSF voxels were selected as temporal confounding factors62. Also, task paradigm regression (TPR) procedure was used to remove the experimental cyclic task/condition effects from fMRI block-design acquisition. Thus, all the selected temporal confounding factors was then regressed out from the GM BOLD time series at each voxel, using the component-based noise correction method61. Finally, the residual BOLD signals were filtered with a 0.01 Hz high-pass filter, in order to remove excessive low-frequency drifts, which could introduce spurious correlations in the functional connectivity analysis.To avoid or minimize the impact of spurious correlations between voxels induced by different sources of physiological and non-physiological noises, which in turn could potentially increase the chance of false positives on functional connectivity results, several confounding variables were mitigated and extracted Two task-based fMRI stimulation data were used: (1) tactile stimulation of the residual limb from the amputees group compared with its homologous region on control group ; and (2) tactile stimulation of the remaining foot in the amputees compared to the corresponding foot stimulation in the control group (“foot” connectivity model). To identify possible differences in sensorimotor brain connectivity in lower limb amputees without pain, as compared to controls, we employed the ROI-to-ROI connectivity method.63, a priori anatomical ROIs were defined as follows: (1) primary motor cortex (M1), corresponding to the pre-central gyrus, including Brodmann areas BA4a and BA4p64; (2) premotor cortex/supplementary motor area (M2), corresponding to Brodmann area BA665; (3) primary somatosensory cortex (S1), including Brodmann areas BA3a, BA3b, BA1 and BA267; and (4) secondary somatosensory cortex (S2), corresponding to parietal operculum OP1, OP2, OP3 and OP4, including Brodmann area BA4068. Before functional connectivity analysis, all selected ROIs were segmented and masked by GM map.We addressed functional connectivity changes between primary and secondary sensorimotor cortices. Using SPM Anatomy toolboxr between all ROIs time courses were calculated, creating a correlation matrix for each subject. Afterwards, the correlation coefficients were converted to normally distributed scores using Fisher’s transform to allow for second-level General Linear Model (GLM) group analysis69. Thus, the resulting Fisher’s z-transformed correlation coefficients (z-scores) were used as a measure of “total” functional connectivity between ROIs at subject-level.After the average BOLD time series for each subjects’ ROIs were computed, linear measures of functional connectivity analysis were performed at first-level by calculating the bivariate correlation (Pearson correlation) of BOLD signals between each pair of ROIs (representing the effect size or strength of connection between two ROIs), ranging from −1 (maximum negative correlation) to 1 (maximum positive correlation). For each functional connectivity model , Pearson’s correlation coefficients 70. Between-subjects’ contrasts of interest were inferred by comparing functional connectivity patterns between the two groups of subjects at connection-level. The resulting connectivity matrix displays T values for the group comparisons. False positive control in ROI-to-ROI analysis was applied using false discovery rate71 (FDR) correction for multiple comparisons and significant connections were thresholded at P < 0.05. Furthermore, family-wise error (FWE) values were calculated between-groups and the FWE-corrected values (thresholded at P < 0.05) were presented at Tables 72.Following the calculation of ROI-to-ROI connectivity matrices for each subject, these measures were entered into a second-level random-effect GLM analysis to obtain group differences between amputees and controls


A major goal of cancer genomics is to identify somatic mutations that play a role in tumor initiation or progression. Somatic mutations within transcription factors are of particular interest, as gene expression dysregulation is widespread in cancers. The substantial gene expression variation evident across tumors suggests that numerous regulatory factors are likely to be involved and that somatic mutations within them may not occur at high frequencies across patient cohorts, thereby complicating efforts to uncover which ones are cancer-relevant. Here we analyze somatic mutations within the largest family of human transcription factors, namely those that bind DNA via Cys2His2 zinc finger domains. Specifically, to hone in on important mutations within these genes, we aggregated somatic mutations across all of them by their positions within Cys2His2 zinc finger domains. Remarkably, we found that for three classes of cancers profiled by The Cancer Genome Atlas (TCGA)—Uterine Corpus Endometrial Carcinoma, Colon and Rectal Adenocarcinomas, and Skin Cutaneous Melanoma—two specific, functionally important positions within zinc finger domains are mutated significantly more often than expected by chance, with alterations in 18%, 10% and 43% of tumors, respectively. Numerous zinc finger genes are affected, with those containing Krüppel-associated box (KRAB) repressor domains preferentially targeted by these mutations. Further, the genes with these mutations also have high overall missense mutation rates, are expressed at levels comparable to those of known cancer genes, and together have biological process annotations that are consistent with roles in cancers. Altogether, we introduce evidence broadly implicating mutations within a diverse set of zinc finger proteins as relevant for cancer, and propose that they contribute to the widespread transcriptional dysregulation observed in cancer cells. Recent large-scale cancer genomics initiatives have revealed that tumor cells typically have numerous genetic alterations, only a small subset of which play a functional role in cancer. Thus, discovering cancer-relevant mutations from tumor sequencing data is a major goal of cancer genomics. Here, we aimed to uncover functional mutations occurring in transcription factors, as gene expression dysregulation is frequently observed in human cancers. In particular, we analyzed Cys2His2 zinc finger genes, as they comprise the largest family of transcription factors in human. We aggregated somatic mutations within these genes by their positions within zinc finger domains, and found that two specific positions within zinc finger domains are recurrently mutated in three cancer types—Uterine Corpus Endometrial Carcinoma, Colon and Rectal Adenocarcinomas, and Skin Cutaneous Melanoma. The two heavily altered positions are known to influence whether and how the domains bind DNA, and thus mutations within them can substantially alter gene function. Further, these mutations, observed across a wide range of transcription factors, converge on at least two processes—chromatin remodeling and dysregulation of retroelements—that are increasingly being linked to human cancers. More generally, we propose that these uncovered mutations contribute to the widespread transcriptional dysregulation commonly observed in cancer cells. Recent cancer genomics efforts have sequenced the tumors of hundreds of individuals across tens of different cancer types , 2. AnalPreviously, analysis of protein structures revealed that many well-known cancer genes are enriched in mutations that affect protein stability or participation in interactions with nucleic acids, small molecules and peptides –6. Thus,To expand our knowledge of DNA-binding proteins that may play a role in human cancers, we analyzed somatic mutations found in Cys2His2 zinc finger (ZF) domains. ZF domains are major determinants of human regulatory networks, as they are contained in nearly half of human transcription factors . DespiteThe structurally and functionally important positions within ZF domains are well known , and thiOur main finding is that two specific positions within ZF domains are mutated in three cancer types—Uterine Corpus Endometrial Carcinoma (UCEC), Colon and Rectal Adenocarcinomas (COAD/READ), and Skin Cutaneous Melanoma (SKCM)—significantly more often than expected. These two heavily altered positions are known via previous structural studies to influence DNA-binding activity , 23. Theα-helix that binds DNA) is mutated frequently in the UCEC and COAD/READ cohorts, largely in the form of an arginine to isoleucine mutation (R9I). Position 11 (p11) is mutated frequently in the SKCM cohort, almost exclusively as a histidine to tyrosine mutation (H11Y). These mutations affect a considerable fraction of tumors, with missense mutations at p9 found in 97 of 543 UCEC and 57 of 594 COAD/READ tumors, and missense mutations at p11 found in 204 of 470 SKCM tumors  are ultramutated , where Ψ is a large, hydrophobic amino acid and the final amino acid can be H or C.The HMMER function hmmsearch (versions 2.3.2 and 3.0) was run on Ensembl human protein sequences using 12 Pfam HMM profiles from the Cys2-His2 ZF clan: Mutations across individual ZF domains were aggregated according to the position in which they occurred within the domain. Mutations were only considered if the entire ZF domain exactly matched the protein sequence from RefSeq. These criteria only excluded a few mutations in each cancer type due to variations between Ensembl and RefSeq sequences.p-values were obtained by calculating, for each position, the fraction of permutations where the total number of mutations were at least as high as the actual number.For each cancer type, we considered all ZF genes, and computed for each position within the ZF domain the sum of all missense mutations observed across the samples at that position  than in the unpermuted data, as well as the number of permutations that had a higher fraction of p9  missense mutations occurring in KRAB-containing ZF genes.We performed a related but different permutation analysis to determine whether mutations are concentrated within particular subsets of ZF genes. In particular, we repeatedly performed context-sensitive randomizations of the locations of p9 mutations in UCEC and COAD/READ samples across all p9 codons in the ZF genes considered in our analysis, and likewise for p11 mutations in SKCM samples across all p11 codons. To obtain empirical poibin R package [We used the Poisson binomial distribution to determine whether the cumulative numbers of mutations affecting ZF p9 in COAD/READ and UCEC and p11 in SKCM were significantly higher than expected when taking into account per-patient context-dependent mutation rates. The parameters required to compute the Poisson binomial distribution are: (1) the number of trials, expressed as the total number of positions of interest  across all ZF genes matching the context/s of interest ; and (2) the per-individual mutation rates for the context/s of interest, computed across the whole exome. The  package  was usedFor each cancer type, we computed the per-gene missense mutation rate as the total number of missense mutations observed in a gene, divided by the number of nonsynonymous sites in the gene, and likewise with synonymous mutations and sites. The nonsynonymous/synonymous sites are proportional to the outcomes of the possible mutations per position, and together sum to the length of the coding sequence.q-value correction.Gene Ontology (GO) and KEGG functional enrichments on mutated gene sets were determined using the hypergeometric distribution, with the background set of all annotated genes, and with p-values using hypergeometric tests and then computed q-values [To test enrichment of annotation sets in the interaction partners of the mutated ZF genes, we used the undirected form of the NEAT method , which iq-values  across oS1 Text(PDF)Click here for additional data file.S1 Figp-values obtained from binomial tests using the larger set in the comparison as the expected fraction and the subset as the trial group.Four sets of ZF domains are compared: all ZF domains in the human genome, those with any missense mutation in any sample from the three cancer types, those that contain a position 11 histidine to tyrosine substitution at least once in any of the three cancer types, and those that contain at least one position 9 arginine to isoleucine substitution. The fraction of ZF domains in each set whose C-terminal boundary is at most 12 amino acids from a subsequent zinc finger domain is shown, with (PDF)Click here for additional data file.S2 FigBars give the actual missense mutation counts for each of 21 positions in a classic Cys2His2 zinc finger domain. Data over 5483 domains in 642 genes. Boxplots show the distributions of counts after observed mutations are shuffled 10,000 times across each gene in a trinucleotide context-preserving manner.(PDF)Click here for additional data file.S3 Fig(a) To check whether the mutation peaks were robust with respect to the choice of mutation caller, we repeated our domain analysis with the three other versions of each mutation dataset, as processed by the MuSE, SomaticSniper, and VarScan2 callers. (b) We repeated our domain analysis with a filtered version of mutation calls (see (c) Position 9 and 11 peaks remain when only mutations at genomic locations with the highest possible 36-mer mappability scores are considered. (d) Position 9 peaks remain when samples with excessive sequencing damage are removed. (e) Even when all mutations in ZF domains that had at least one R9I mutation in that cancer type are excluded, more missense mutations occur at position 9 than at other positions.alls see . The num(PDF)Click here for additional data file.S4 FigFor each cancer type, the tumor sample allele frequencies of R9I or H11Y mutations are compared with those of all other exome mutations involving the same base substitution, as well as those of all exome mutations involving other base substitutions.(PDF)Click here for additional data file.S5 Fig(a) Missense mutations in ZF domains are shown for the nine cancer types in which they occur most frequently, as well as for the remaining 23 cancer types in aggregate. (b) Missense mutation counts from panel A normalized by number of samples per cancer type . Colors correspond to the groups in panel A. (c) For each cancer type, normalized nonsynonymous to synonymous mutation ratios Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 DatasetFor each gene, the number of missense mutations at p9 or p11 in each cancer type is given. Also shown is whether the gene has a KRAB domain, and the number of ZF domains in the gene that were considered in this study.(TXT)Click here for additional data file.


MLH1 is usually due to epigenetic silencing with associated promoter methylation; coding somatic mutations rarely occur. Here we use the presence of a colorectal cancer (CRC) risk variant (rs1800734) within the MLH1 promoter to investigate the poorly understood mechanisms of MLH1 promoter methylation and loss of expression. We confirm the association of rs1800734 with MSI+ but not MSS cancer risk in our own data and by meta-analysis. Using sensitive allele-specific detection methods, we demonstrate that MLH1 is the target gene for rs1800734 mediated cancer risk. In normal colon tissue, small allele-specific differences exist only in MLH1 promoter methylation, but not gene expression. In contrast, allele-specific differences in both MLH1 methylation and expression are present in MSI+ cancers. We show that MLH1 transcriptional repression is dependent on DNA methylation and can be reversed by a methylation inhibitor. The rs1800734 allele influences the rate of methylation loss and amount of re-expression. The transcription factor TFAP4 binds to the rs1800734 region but with much weaker binding to the risk than the protective allele. TFAP4 binding is absent on both alleles when promoter methylation is present. Thus we propose that TFAP4 binding shields the protective rs1800734 allele of the MLH1 promoter from BRAF induced DNA methylation more effectively than the risk allele.Expression of the mismatch repair gene MutL homolog 1 (MLH1) is silenced in a clinically important subgroup of sporadic colorectal cancers. These cancers exhibit hypermutability with microsatellite instability (MSI) and differ from microsatellite-stable (MSS) colorectal cancers in both prognosis and response to therapies. Loss of  MLH1). This loss of MLH1 is rarely caused by mutations. Instead, epigenetic silencing occurs, with high levels of DNA methylation present in the MLH1 promoter. Understanding how MSI+ CRCs develop is clinically important. Stage II/III MSI+ CRCs have a relatively favourable prognosis, whereas stage IV MSI+ tumours have a poor prognosis. MSI+ CRCs respond poorly to commonly used chemotherapy, such as 5-fluorouracil but are targetable by immune checkpoint inhibitors due to their high number of neoantigens2.Colorectal cancer (CRC) is a major cause of morbidity and mortality, with a lifetime risk of ~6% in the UK. About 15% of sporadic CRCs are deficient in DNA mismatch repair (MMR), a process that normally acts to correct spontaneous DNA replication errors. MMR-deficient cancers exhibit a high rate of mutation genome-wide, but this is most evident at short repeat sequences, causing these cancers to be termed “microsatellite-unstable” (MSI+). The majority of sporadic MSI+ cancers have reduced protein and mRNA expression of the MMR gene, MutL homolog 1  resulting in hypermethylation at MLH1 and other CIMP marker genes, via the transcriptional repressor MAFG4.The importance of MLH1 promoter methylation has come from the study of a single nucleotide polymorphism (SNP), rs1800734, that lies in the 5′ untranslated region of MLH1. An association between rs1800734 and CRC risk has been shown in multiple candidate studies8. However, this strong association is limited to MSI+ cancers, and is weak or absent in un-stratified data sets. Several groups have investigated possible mechanisms by which this SNP may confer increased CRC risk, although the results are not straightforward. There is an association between the rs1800734 risk (A) allele and (i) DNA methylation at the MLH1 promoter in cancers10, and (ii) CpG island shore methylation in normal tissue12. There is also evidence that binding of the transcription factor TFAP4 (AP-4) is modified by rs180073414in vitro and in vivo. However, Liu et al.13 detected no difference in MLH1 allele-specific expression as a result of TFAP4 allelic bias. Instead, they showed an effect on the expression of the gene encoding the protein kinase DCLK3 and long-range chromatin interactions between rs1800734 and the DCLK3 promoter.Some further understanding of the acquisition of MLH1 as the target gene, and investigate the relationship between the rs1800734 allele, MLH1 promoter methylation and, importantly MLH1 mRNA expression in normal tissue and the pathway to colon cancer. To add to this correlative data, we demonstrate the causal role of methylation by dynamically altering MLH1 promoter methylation levels, and showing that methylation loss and gain is modified by the rs1800734 allele with downstream effects on mRNA expression and TFAP4 binding.Here we confirm the association of rs1800734 with CRC risk in our own MSI+ data set, confirm its absence in MSS cancers and perform a meta-analysis with other publically available MSI+ datasets. We then describe a comprehensive study which confirms MLH1 promoter SNP rs1800734 has been assessed as a candidate for CRC susceptibility in a number of MSI+ colorectal cancer data sets. We confirmed these results in our own MSI+ data set from the VICTOR and QUASAR2 CRC clinical trials . A meta-analysis of these data and 5 other data sets showed a highly significant association with CRC risk  strongly suggesting that the SNP plays a mechanistic role in the silencing of MLH1 during MSI+ cancer development.The MLH1 promoter methylation and MLH1 mRNA expression in normal bowel  of amplicons of bisulphite-treated DNA. As expected, there was little or no methylation close to rs1800734 or in Deng region C  in any of the samples. There were, however, increasing levels of methylation towards the upstream CpG island shore and interestingly these were higher in low-risk GG homozygotes than in AG heterozygotes , suggesting a causal relationship.Similar analyses were carried out in tumours from MSI+ cancer patients  with loss of MLH1 protein expression Fig. . Methyland larger dataset with a broader demographic. We therefore carried out a similar analysis on colorectal cancer with matched normal data from The Cancer Genome Atlas . We found that methylation in region C and at rs1800734 was significantly higher in tumours with AA and AG genotypes . Early lesions in the pathway to cancer could represent intermediate stages in the process of MLH1 silencing. Sessile serrated adenomas (SSAs) are known precursors of BRAF-mutant MSI+ CRCs16. SSAs that develop dysplasia progress rapidly to cancer and approximately 75% of these have methylated MLH1 promoters and silencing17. Fennel et al.18 have shown that the rs1800734 risk (A) allele is associated with a dosage dependent increase in methylation in SSAs with dysplasia. The AA genotype was also associated with protein loss as measured by immunohistochemistry. We hypothesised that SSAs would show allele-specific expression at the mRNA level, so we investigated allele-specific MLH1 mRNA expression and methylation in a small set of fresh-frozen rs1800734-heterozygous SSAs with BRAFV600E mutations , possibly indicating that hypermethylation is spreading from the CpG island shore across region C towards rs1800734. The intermediate levels of methylation present in our SSAs are already sufficient to cause a difference in allelic mRNA expression levels  for 48 hours, we saw a reduction in DNA methylation at all CpGs , which gradually increased again over 11 days , BRAFV600E) has very high levels of methylation at all CpGs analysed in the PCR Fig. . Hypermeays Fig. . Intereset al.13 demonstrated in a cell-free system that the transcription factor TFAP4 binds in vitro to the protective (G) allele of rs1800734 with higher affinity than to the risk (A) allele. We hypothesized that preferential TFAP4 binding on the G allele may give some protective effect from de novo DNA methylation machinery. We therefore confirmed that in the heterozygous MSS cell line COLO320, TFAP4 binds strongly and in a localized fashion to the region containing rs1800734  rs1800734 allele has no measurable repressive effect on MLH1 in normal bowel tissues, even using highly sensitive techniques, and in fact associates with reduced DNA methylation at the upstream CpG island shore, in line with previous observations12. However, significant allele specific effects are seen on both methylation and mRNA expression in MSI+ cancers in our own and TCGA data sets. As expected, the risk (A) allele leads to significantly higher methylation levels and this strongly correlates with lower mRNA expression. The data of Fennell et al.18 and our small set of SSAs indicate that the allele methylation and expression bias probably arise very close together, during the serrated pathway.We have confirmed that the SNP rs1800734 in the promoter of MLH1 transcriptional silencing by treating with AzaC, removing methylation and demonstrating re-expression of MLH1 in MSI+ cells. This result implies that methylation is indeed the primary cause of MLH1 silencing in sporadic MSI+ cancers. Interestingly, even in this engineered situation the risk (A) allele is more prone to acquire methylation and its mRNA is re-expressed at lower levels. Thus it is likely that the mechanism by which rs1800734 mediates cancer risk is via methylation acquisition. The reason the A allele is more readily methylated could be due to the lack of binding of the TFAP4 transcription factor as shown by Liu et al.13. We confirm their result that there is indeed a strong bias in TFAP4 binding in unmethylated CRC cells and that this binding is dependent on promoter demethylation post AzaC treatment in MSI+ cells.We have also clearly demonstrated that DNA methylation is necessary for 20. This suggests that it could be a major factor in determining protein binding and potentially chromatin landscape across the region. The fact that TFAP4 also binds allele specifically to another disease associated SNP  suggests it might play a more generalised role in a subset of SNP trait associations, acting to recruit activating factors in an allele specific manner. Thus, as our data suggest, the effect of TFAP4 on de novo methylation of the MLH1 promoter is unlikely to be direct and therefore may only offer partial protection such that the rs1800734 protective (G) allele can still acquire methylation but at a slower rate or at lower levels than the risk (A) allele.It is unlikely that TFAP4 is the only transcription factor binding across rs1800734 and publically available genome wide ChIP-seq experiments show multiple proteins binding in the region . Indeed, TFAP4 has been shown to belong to a class of enhancer binding factors that are important for co-factor recruitment and activationet al.13 suggest that allele specific TFAP4 binding at rs1800734 may exert an effect on the cancer pathway via long range interactions with the promoter of the DCLK3 gene, causing enhanced expression of genes related to epithelial-to-mesenchymal transition. However we were unable to corroborate this finding. We failed to detect significant DCLK3 expression in either our MSS or MSI+ cell lines using sensitive Q-PCR based techniques. We have clearly demonstrated that rs1800734 is only associated with an increased risk of MSI+ cancers, ie those with a dysfunctional MMR pathway, and only modifies methylation in these and not in MSS cancers. Thus, it seems unlikely that a gene with no known role in MMR plays the primary or causative role in rs1800734 associated cancer risk.Liu et al.4. Figure Our data, taken together with other studies described above, support the prevailing hypothesis that MLH1 repression is the main mechanism by which rs1800734 confers cancer risk. Since the majority of our MSI+ cancers (23/35) and all our SSA samples also carry a BRAFV600E mutation we suggest it is likely that rs1800734 influences the acquisition of methylation via the BRAF/MAFG pathway described by Feng 22, and gastric23) cancers, as well as lung24, bladder25, and some haematological malignancies26. Drugs such as Azacitidine that inhibit DNA methylation are already approved for the treatment of some cancers. However with the advent of CRISPR technology, more precise demethylation is now possible27 and could be harnessed in the design of future therapies.While the functional role of rs1800734 in the pathways of CRC development is becoming clearer, it is also interesting to note how readily the accumulation of MLH1 promoter methylation could be reversed resulting in re-expression of MLH1. The importance of MLH1 promoter methylation in other cancer types is less well understood however it is frequently found in endometrialet al., Campbell et al., Raptis et al., Whiffin et al.28 plus our own dataset using clinical trial samples (QUASAR229 and VICTOR31). All patients were genotyped on Illumina tagSNP or custom arrays and quality control was performed as previously described.A summary of all patient sample sets used in this study is given in Supplementary Fig. 32, were used for allele specific analysis of methylation and MLH1 mRNA expression. Formalin Fixed Paraffin Embedded (FFPE) CRC MSI+ tumours for analysis of methylation, expression and rs1800734 were collected at the Department of Pathology of ASST dei Sette Laghi- University of Insubria. 35 consecutive sporadic CRCs showing MSI and MLH1/PMS2 immunohistochemical loss were selected33. All CRCs were histologically reviewed according to the World Health Organization (WHO) classification of tumors of the digestive system34 and the TNM staging system35 These patients were 20 females and 15 males with a mean age at CRC diagnosis of 77 years (range: 55–88 years). Thirty-two neoplasms arose in right colon, one in descending colon and the remaining two cases in sigmoid colon. Sessile serrated adenomas were collected fresh and frozen following endoscopic resection.Normal colorectal biopsies from 317 individuals of white UK origin undergoing colonoscopy in Oxford as previously reportedCollection of blood and tissue samples and clinico-pathological information from patients and controls was undertaken with informed consent and ethical review board approval in accordance with the tenets of the Declaration of Helsinki.Victor and Ethical approval for the QUASAR study was obtained from the West Midlands Research Ethics Committee . The VICTOR trial was reviewed by the Cancer Research Campaign, the Multicenter Research Ethics Committee, and research ethics committees at participating centers. Ethical approval for the normal patient samples was obtained from the Oxfordshire Research Ethics Committee A (REC 10/H0604/72). The formalin fixed paraffin embedded (FFPE) CRC MSI+ tumours sample study was approved by the Ethics Committee of Ospedale di Circolo di Varese (No. 0037028). The sessile serrated adenomas was approved by Oxford Radcliffe Biobank ethics committee .TM technology according to the manufacturer’s instructions (LGC). Primers are listed in Supplementary Table Patient samples and cell lines were genotyped for rs1800734 using KASP36. Up to 96 samples were sequenced using 250 bp paired end sequencing on a MiSeq (Illumina) according to the manufacturer’s instructions. MiSeq output was demultiplexed and FASTQ files generated . The sequences were quality assessed, and trimmed  then aligned and the methylation called .DNA was extracted from fresh cells or tissue using the DNeasy kit (QIAGEN) or from FFPE tissue using the High Pure FFPET DNA Isolation Kit (Roche). Bisulphite conversion of DNA was carried out using the EZ DNA methylation kit (Zymo Research) according to the manufacturer’s instructions. Converted DNA was amplified with Pyromark PCR kit (Qiagen) using CpG free primers with Illumina specific sequence tags to ensure unbiased amplification of methylated and unmethylated template  . MS-MLPA was performed according to the manufacturer’s instructions and data analysis was carried out with Coffalyser software v.8 (MRC-Holland).37.RNA was extracted from fresh cells or tissue using the RNeasy kit (QIAGEN) or from FFPE tissue using the High Pure FFPET RNA Isolation Kit (Roche) and cDNA was generated  according to the manufacturer’s instructions. Gene expression was quantified and normalized using Taqman gene expression ready mixed assays . Allele specific MLH1 expression was assessed by amplification of cDNA using Illumina tagged primers  at 37 °C in 5% CO8 cells were crosslinked for 10 mins with 1% formaldehyde, neutralized with 125 mM glycine, washed with ice-cold PBS and scraped. After 2 further PBS washes, cells were resuspended in lysis buffer,  sonicated using a Bioruptor (Diagenode) for 7–15 × 15 s cycles, centrifuged at max speed for 10 min at 4 °C and diluted 1:10 in IP dilution buffer . Immunoprecipitation with approximately 5 ug of antibody  was carried out overnight at 4 °C and then incubated for 4 hours with 50 ul of protein G Dynabeads (Invitrogen). Bead/antibody complexes were washed with TSEI , TSEII , LiCl buffer  and TE according to standard protocols and eluted with 1% SDS, 0.1 M NaHCO3. 1 ul of DNA was analyzed in duplicate or triplicate by SYBR green qPCR using PowerUp SYBR™ Green Master Mix (Thermofisher) and primers covering the MLH1 promoter region  for 48 hours (with replenishment of AzaC after 24 hours). AzaC was removed, cells washed with PBS, and then cultured in standard medium for 0, 4, 7 and 11 days. RNA and DNA were extracted simultaneously using the AllPrep kit (Qiagen) and MLH1 mRNA expression and promoter methylation assessed as described above. ChIP was carried out post AzaC treatment as described above.SNP association studies were carried out using PLINK. All other statistical analysis was carried out using R unless otherwise stated. Graphs were drawn using core R functions, ggplot2 or excel.Supplementary InformationSupplementary tables


Aquatic ecosystems worldwide continue to experience unprecedented warming and ecological change. Warming increases metabolic rates of animals, plants, and microbes, accelerating their use of energy and materials, their population growth, and interaction rates. At a much larger biological scale, warming accelerates ecosystem-level processes, elevating fluxes of carbon and oxygen between biota and the atmosphere. Although these general effects of temperature at finer and broader biological scales are widely observed, they can lead to contradictory predictions for how warming affects the structure and function of ecological communities at the intermediate scale of biological organization. We experimentally tested the hypothesis that the presence of predators and their associated species interactions modify the temperature dependence of net ecosystem oxygen production and respiration. We tracked a series of independent freshwater ecosystems (370 L) over 9 weeks, and we found that at higher temperatures, cascading effects of predators on zooplankton prey and algae were stronger than at lower temperatures. When grazing was weak or absent, standing phytoplankton biomass declined by 85%–95% (<1-fold) over the temperature gradient (19–30 °C), and by 3-fold when grazers were present and lacked predators. These temperature-dependent species interactions and consequent community biomass shifts occurred without signs of species loss or community collapse, and only modestly affected the temperature dependence of net ecosystem oxygen fluxes. The exponential increases in net ecosystem oxygen production and consumption were relatively insensitive to differences in trophic interactions among ecosystems. Furthermore, monotonic declines in phytoplankton standing stock suggested no threshold effects of warming across systems. We conclude that local changes in community structure, including temperature-dependent trophic cascades, may be compatible with prevailing and predictable effects of temperature on ecosystem functions related to fundamental effects of temperature on metabolism. Temperature affects metabolic rates of all organisms, thereby affecting ecological patterns and processes across scales of organization—from individuals to ecosystems. Increasing temperature accelerates major metabolic processes that drive net ecosystem production (NEP) and ecosystem respiration (ER) in aquatic and terrestrial ecosystems –4. HighlReconciling the high explanatory power of general temperature-dependent metabolic scaling models at macroecological scales with the well-documented contingencies of how temperature affects community-level outcomes of population dynamics and species interactions at intermediate scales has been challenging –17. WhetOne way to reconcile the apparent context dependence of empirical results under controlled conditions with the generality of temperature dependence of ecosystem function at broader scales is to consider how the direct and indirect effects of temperature on population dynamics interact. Direct effects of temperature on per capita metabolic rates cause organismal photosynthesis and respiration rates to increase exponentially when temperatures increase, as long as resources are not limiting in algae and animals, up to an optimal temperature. This relationship between temperature and fundamental metabolic rates (photosynthesis and respiration) is referred to as general metabolic scaling . For anyThe temperature dependence of consumer-resource interactions—mediated by dynamics of two or more populations—has received substantial attention in this context, because these trophic interactions can influence many aspects of community structure and ecosystem function, including biomass, abundance, species composition, and stability ,31,32,34Here, we aimed to resolve the paradox between apparent direct effects of temperature on ecosystem functions  that emerge when comparing communities across larger gradients and the potentially more complex effects of temperature at the population and community scales. We experimentally tested the hypothesis that temperature-dependent trophic interactions in a trophic cascade alter the effect of temperature on community properties such as biomass, abundance, and body size, but have little or no effect on the variation in ecosystem functions  over a temperature gradient. In freshwater plankton communities, we compared the effects of temperature on community properties typically measured in warming experiments  with the effects of temperature typically measured in macroecological studies . We controlled variation in biotic and abiotic conditions other than temperature and trophic structure (presence of grazers and predators) . We quanbi, gO2/hour) and related biological functions for organism i to body size  and body temperature  water is the O2 concentration of water, [O2]sat is the concentration the water would have if it were at equilibrium with the atmosphere (390 μatm), and T is temperature of the observation (°C) E) (We estimated NEP and ER by converting changes in observed O ([O2]E)  due to cbi—in our case, oxygen production via photosynthesis or consumption via respiration—in this model is a special case of a more complex equation that allows each species to follow a thermal performance curve (TPC), often described by a modified Sharpe-Schoolfield equation [The expression of temperature effects on a per capita metabolic rate equation ,20,65, iequation ,23. We dB (Zj) in the intercept term  and normalization constants (Eb). In the absence of additional information about their functional forms, we used general Arrhenius functions, but we note that other functions could be used if appropriate. Consequently, the ratio of MB with and without predators may vary with temperature according to the relative temperature dependences of thermal traits and size distributions:We then simplified and added temperature dependence of mass  as a function of mean weekly ecosystem temperature Twj ranging from 19.7 (±3.15) °C to 26.1 (±3.59) °C  °C . The reg) °C ER,  of respo ER, ER,  fitted oa concentration, with a random intercept for individual ecosystems. We used a within-subject mean centering approach to distinguish temperature effects into those associated with an ecosystem’s average temperature (Tj) over the entire experimental period (a “between-ecosystem” effect) from effects variation in temperature over time (Twj) (a within-ecosystem temperature” effect) [j in week w was modeled as a continuous response to variation in inverted ecosystem temperature (1/kTwj) and trophic treatment (Zj):β0.j(i) represents an intercept allowed to vary randomly among ecosystems. The terms in the full model (β2), estimated as the slope of ln(Ywj) on the mean temperature over all weeks for ecosystem j, expressed as inverse temperature ; the within-ecosystem (β1) effect of temperature variation over time, estimated as the slope of ln(Ywj) versus centered weekly temperature ; interaction (β3) between within-ecosystem temporal variation in temperature and the experimental temperature treatment; trophic species interactions (β4); and interactions between species interactions and overall mean (β5) and weekly temperature (β6).We used a mixed effects model (lme function in the nlme package of R) to examine the main and interactive effects of temperature (a continuous fixed factor) and trophic structure  on net ecosystem oxygen production (NEP), net ecosystem oxygen consumption (ER), and chlorophyll  effect) . The resll model  are the ER, β4) and interactions between Zj and temperature . We also tested models without temperature terms for within-system variation (β4). In total, the model set included nine models  on the grand mean of all temperatures observations To test our hypothesis that species interactions modify the temperature dependence ER,  of respoe models . Responsj was modeled for each week w and for the temperature of the tanks, with random effects uj assigned for each power treatment (p).To test the effect of temperature on trophic cascade strength, we used the following statistical model:C). When two or more models were considered comparable or equivalent (δAICC < 2), we reported all models meeting this criterion and report averaged coefficients. We estimated the realized temperature dependence of our response variables (slopes) and intercepts for among-ecosystem responses to temperatures by first rearranging We ranked models using Akaike’s Information Criterion weights (using the MuMin package in R), adjusted for small sample sizes (AICβ2 + β5 * Zj) include the temperature dependence of ecosystem responses plus any variation with trophic structure, and give the slope of lines plotted in Figs βw)0.j.S1 TableThere was a significant temperature * week interaction when we included all the data, from weeks 2−9 (Table S1A). When we used a smaller data set including observations from only weeks 2 to 7, we found evidence for a slight increase in chlorophyll concentration over time (Table S1B). Together, these results suggest that the negative trend in chlorophyll concentration is driven by the drop in week 8 across all treatments. This is concurrent with a cooling event and a large storm.(DOCX)Click here for additional data file.S2 Tablewj), taxon (copepod or Daphnia), trophic treatment , and their interactions. NA indicates that the term was not included in the model. (B) Estimated lengths of Daphnia and copepods in treatments with and without predators, from model m2g (Table S2A). AG, algae and grazers; AGP, algae, grazers, and predators; NA, not available.(A) Zooplankton average body size model selection results. We measured sizes of 641 individual zooplankton. We modeled log(length) of zooplankton in terms of ecosystem weekly temperature (T(DOCX)Click here for additional data file.S3 Tablewww.algaebase.org and www.diatom.org. A water sample 100–300 mL in volume from each mesocosm was filtered onto a 0.2-μm GF/F filter; the water volume varied with the chlorophyll a content. Chlorophyll a was extracted from the filters in 90% acetone. Chlorophyll a concentration, measured in μg/L, was determined fluorometrically using a Trilogy fluorometer (Turner Designs) following Wetzel and Liken (2000).Weekly, we sampled algal assemblages in 100-mL water samples collected from approximately 40 cm below the surface. We used 10-mL subsamples for the identification and counting from each mesocosm. Subsamples were placed in settling chambers and allowed to settle for 24 hours. We counted and identified cells to taxon level using an inverted microscope and the Utermöhl sedimentation method . Phytopl(DOCX)Click here for additional data file.S1 FigNonmetric multidimensional scaling plot (NMDS) of temporal phytoplankton taxonomic composition for all temperature treatments and trophic levels (Taxa listed in (JPG)Click here for additional data file.S2 FigDifferences among ecosystems were maintained by heaters of different power (watts). Red colors indicate warmer ecosystems at higher wattage and blue colors indicate cooler ecosystems.(JPG)Click here for additional data file.S3 FigNonmetric multidimensional scaling plot (NMDS) of temporal phytoplankton taxonomic composition for all temperature treatments and trophic levels (taxa listed in (JPG)Click here for additional data file.S4 FigLines connect observations from the same ecosystem. Trophic treatments are separated: A, algae only; AG, algae + grazers; and AGP, algae + grazers + predators.(JPG)Click here for additional data file.


SCN5A gene encodes the alpha subunit of the main cardiac sodium channel Nav1.5. This channel predominates inward sodium current (INa) and plays a critical role in regulation of cardiac electrophysiological function. Since 1995, SCN5A variants have been found to be causatively associated with Brugada syndrome, long QT syndrome, cardiac conduction system dysfunction, dilated cardiomyopathy, etc. Previous genetic, electrophysiological, and molecular studies have identified the arrhythmic and cardiac structural characteristics induced by SCN5A variants. However, due to the variation of disease manifestations and genetic background, impact of environmental factors, as well as the presence of mixed phenotypes, the detailed and individualized physiological mechanisms in various SCN5A-related syndromes are not fully elucidated. This review summarizes the current knowledge of SCN5A genetic variations in different SCN5A-related cardiac disorders and the newly developed therapy strategies potentially useful to prevent and treat these disorders in clinical setting.The  SCN5A gene encodes the alpha subunit of the main cardiac sodium channel Nav1.5, which is known to be responsible for maintaining the normal function of inward sodium current (INa). INa current is the main component in fast depolarization phase after which the excitation–contraction coupling cascade and proper conduction of the electrical impulse is subsequently initiated within the heart , long QT syndrome (LQT3), and cardiac conduction system dysfunction. Meanwhile, SCN5A variants are also found to be correlated with myocardial contractile dysfunction, dilated cardiomyopathy, and heart failure. According to one of the most complete open database of on-line disease-related genetic variations “ClinVar”  with four internally homologous domains (DI-DIV), each containing six transmembrane spanning segments (S1-S6)  tract, and cancer tissues  Figure . PreviouSCN5A isoforms: “adult” isoform  and “neonatal”  isoform . The “adult” SCN5A isoform is abundantly expressed in adult human heart while the “neonatal” isoform is abundantly expressed in neonatal heart. The “adult” isoform differs from the “neonatal” isoform in exon 6 within a voltage-sensor domain (D1/S3-S4)  with one of SCN5A variants I230T. Interestingly, prolonged culture time increased the expression of adult sodium channel isoform, which was paralleled by a decrease in INa, representing a severe clinical phenotype in patients with recessive cardiac conduction disease  is also expressed in human heart and should be mentioned in some detail. SCN5A-014 includes an additional glutamine at position 1077 (1077Q) at the exon 17–18 splice boundary, resulting in smaller INa current and exhibiting functional differences with the previous standard isoforms  and a delay in inactivation, while the latter one is manifested by a decrease in sodium influx and an acceleration of inactivation. Different variants-related phenotypes correspond to different state.Sodium current participates in phase 0 and 2 of action potential (AP), which is an important determinant on the production of heart rhythms, normal conduction, and maintenance of excitement. Aberrant sodium channels resulting from SCN5A and 128 SCN5A variants were found to be causative of hereditary SSS by the complete sequencing of the human genome  is characterized by dysfunction of the sinus node. SSS patients exhibit inappropriate sinus bradycardia, sinus arrest, and reduced chronotropic response  is caused by atrial electrical and mechanical impairment, manifested by disappeared P wave, bradycardia, or borderline escape rhythm . The decreased expression of Cx40 jointly impaired the electrical coupling between atrial cells. It was thus speculated that genetic variants could synergistically enhance the functional effect of each individual genetic change, consequently leading to a complete loss of electrical and mechanical activity.Atrial arrhythmias associated with SCN5A have been shown to be associated with AF  is the most common sustained arrhythmia characterized by rapid and irregular beating of the atria  type 3; Brugada syndrome (BrS) and idiopathic ventricular fibrillation (IVF), which greatly increase the risk of sudden death in young individuals with a structurally normal heart. Decades of researches have raised enormous insights into the role of SCN5A variants in ventricular arrhythmogenesis.Variants of SCN5A accompanied with an increasing risk to sudden death during rest or sleep and electrocardiographically characterized by prolonged QT/QTc interval, accentuated QT dispersion, late onset of T wave and frequent prominent U wave. The first description of LQT3 variant was presented in 1995 with a deletion of amino acids 1,505–1,507 (ΔKPQ) in a patient with prolonged QT interval , thereby easily induced Torsades de pointes (TdP) and ventricular fibrillation (VF)  and LQT3, but being influenced by both elevated temperature and cytosolic calcium  is an inherited arrhythmogenic disease characterized by prolonged QT intervals. Among the 15 current forms of LQTS, long QT syndrome type 3 (LQT3) is caused by inherited variants of SCN5A in 1998  is a familial arrhythmia syndrome characterized by ventricular arrhythmias and SCD, in which the signature feature of type-I BrS ECG pattern is ST-elevations in the right precordial leads  as well as variable region location  is characterized by spontaneous ventricular fibrillation and exclusion of any specific structural or functional cardiac diseases including structural cardiac diseases  and primary arrhythmia syndromes  , also called Lenegre syndrome or Lev disease, is one of the most common conduction disorders. It is characterized by progressive changes in the His-Purkinje system with either right or left bundle branch block and QRS complex broadening, eventually leading to complete atrioventricular block, syncope or sudden death. Variants in SCN5A  and truncation variants (2550-2551insTG) were successively discovered in DCM patients  is a myocardial disease characterized by left ventricular systolic dysfunction with dilatation of left ventricle or double ventricles. It is the most common type of primary cardiomyopathy and the most common reason for cardiac transplantation in adults and children Sanbe, . It was SCN5A variants were traditionally recognized as the cause of arrythmias in DCM patients, but did not directly affect the cardiac systolic function. A primary disruption of an ion channel through gene variants leading to DCM was explained that in one way, the ion channel disturbance caused dysfunction of cytoskeletal protein binding partners and resulted in a DCM phenotype. In another way, the electrical dysfunction caused by variants defects led to mechanical instability, and ultimately led to myocardial dilation . These results suggested that A1180V was a risk factor for familiar DCM with preceding atrioventricular block  denominates cases of infant death, which mostly occurs during sleep without any preceding symptoms. It is the leading cause of post neonatal mortality  expression induced an abnormal distribution of connexin43 (Cx43) and Nav1.5. As a result, the sodium current decreased and conduction velocity slowed down, which impaired the mechanical and electrical coupling, these factors also contributed to the pathogenesis of ARVC  is an inherited cardiomyopathy characterized by myocardial atrophy with fibro-fatty replacement, eventually resulting in wall thinning and cardiac dilatation. The inflow tract, outflow tract and apex of the right ventricle constitute the so-called “triangle of dysplasia”  stabilizing protein ELAVL1/HuR and to verify whether it was beneficial to control arrhythmia. Researchers found injection of viral particles carrying HuR increased SCN5A expression and improved AP upstroke and conduction velocity, which reduced reentrant arrhythmia. Increasing mRNA stability to rescue decreased SCN5A expression may represent another new paradigm in antiarrhythmic therapy (Zhou et al., In addition to pharmacological treatments and ICD implantation, some other new approaches are gradually emerging. Non-sense variant is one type of SCN5A variant are highly diverse. Besides, environmental and epigenetic alterations also determine variable disease severity. Continuous exploration and novel insight of this issue will contribute to clarify detailed mechanisms and ultimately enable improved diagnosis, risk stratification, and development of more effective treatment strategies.Cardiac sodium channel plays a central role in cardiomyocyte excitability and proper conduction of cardiac electrical impulses. The function and mechanisms of sodium channel are complicate and remain a lot of controversies. Even within families, the observed phenotypes carrying the same All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. In particular, WL, LY, and CS: drafting of the manuscript; KH, JG, and AS: revising the manuscript for important intellectual content critically and for final approval of the manuscript.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.