Recent studies have shown that RD3 interacts with guanylate cyclases GC1 and GC2 in retinal cell extracts and HEK293 cells co-expressing GC and RD3. This interaction inhibits GC catalytic activity and promotes the exit of GC1 and GC2 from the endoplasmic reticulum and their trafficking to photoreceptor outer segments. Adeno-associated viral vector delivery of the normal RD3 gene to photoreceptors of the rd3 mouse restores GC1 and GC2 expression and outer segment localization and leads to the long-term recovery of visual function and photoreceptor cell survival. This review focuses on the genetic and biochemical studies that have provided insight into the role of RD3 in photoreceptor function and survival.Retinal degeneration 3 (RD3) is an evolutionarily conserved 23 kDa protein expressed in rod and cone photoreceptor cells. Mutations in the gene encoding RD3 resulting in unstable non-functional C-terminal truncated proteins are responsible for early onset photoreceptor degeneration in Leber Congenital Amaurosis 12 patients, the  In the dark the basal catalytic activity of GC in photoreceptor outer segments (OS) is balanced by the basal activity of phosphodiesterase (PDE) to maintain cGMP at a level sufficient for maintaining a significant fraction of cGMP-gated channels in their open state. This allows the influx of Ca0–500 nM . At thisal level . Followixchanger . The redation GC . The incGUCY2D gene encoding human GC1 cause Leber Congenital Amaurosis (LCA) Type 1 (LCA1), a severe early onset retinal dystrophy, and cone-rod dystrophy  and structural organization . They coystrophy .rd3 mouse, rcd2 collie, and LCA12 patients has been shown to play a crucial role in the trafficking of GC1 and GC2 in photoreceptors (Photoreceptor cells are highly polarized neurons with the OS segregated from the rest of the cell by a thin cilium. OS proteins must be efficiently transported from the endoplasmic reticulum (ER) in the inner segment through the cilium since OS turnover every 10 days through the phagocytosis of aged OS membranes by retinal pigment epithelial cells and the addition of new membrane at the base of the OS . The moleceptors . In thisrd3 mouse was first identified as an uncharacterized transcript in an in silico search of retina-specific transcripts and called C1orf36 for Chromosome 1 open reading frame 36 (RD3 consists of 3 exons spanning the 5′ and 3′ untranslated region with the open reading frame comprised of part of exon 2 (amino acids 1–99) and exon 3 (100–195). RT-PCR and in situ hybridization confirmed the presence of RD3 in the retina with high expression in the photoreceptors.The gene responsible for retinal degeneration in the frame 36 . The genRD3 gene is highly conserved across vertebrates with the human protein consisting of 195 amino acids and sharing 95% sequence identity with other primates, 86% with mouse and rat, 83% with bovine, 67% with chicken, and 50–60% with lower vertebrates including Danio rerio (Zebrafish) and Xenopus tropicalis (Western clawed frog). Computer algorithms indicate that RD3 lacks any known homology domains or transmembrane segments. RD3 is predicted to have a high α-helix content (~43%), no β-sheets and considerable disordered regions . There are four conserved stretches of predicted α-helices (H1–H4) with the first helix consisting of 34 amino acids and the last helix having 39 amino acids. Additional conserved features include a putative coil–coil region between amino acids 22–54 and several predicted phosphorylation sites.The RD3 protein encoded by the RD3 has been isolated from retinal homogenates and HEK293 cells and bacteria expressing the recombinant protein . On SDS rd3 mouse was one of the first naturally occurring murine strains found to display early onset rod and cone degeneration  in a strain of collies  mice by western blotting and confocal microscopy 30RK/J strain 4Bnr rd3 mice were similar to age-matched WT mice. Up-regulation of endothelin 2 (EDn2), glial fibrillary acid protein (Gfap), and complement component factor 1 (Cf1) and down-regulation of phosducin (Pdc), gap junction protein α5 (Gja5) and retinal G protein – coupled receptor (Rgr) genes, however, were observed for the rd3 retina , GC1 and GC2 expressed at levels comparable to that of WT mice and localized normally to the photoreceptor OS layer 30RK/J strain of croscopy . GC1 wasOS layer . CollectThe effect of RD3 expression on the localization of GC was also examined in transfected culture cells . GC1 pri2+ sensitivity of GCAPs. It remains to be determined if RD3 plays a significant role in modulating the activity of GCs during phototransduction. It is possible that RD3 only plays an important role in inhibiting cyclase activity during the trafficking of GCs within the inner segments. At present the quantity of RD3 in photoreceptors remains to be determined, but in in vitro measurements, RD3 at nanomolar concentrations competitively inhibited the activation of GC by GCAP  expressed at levels comparable to WT RD3 and bound GC1. The F100ter mutant had no effect of the cyclase activity, whereas the missense mutations inhibited GC activity to varying degrees. The W6R/E23D and G35R mutations inhibited the cyclase activity at a similar concentration as WT RD3, whereas the R68W, K130M and G57V were less effective. It remains to be determined if these missense mutations in RD3 alter the ability of RD3 to function in GC trafficking in photoreceptors.The effect of the RD3 mutation (F100ter) associated with LCA12 and other possible disease-linked missense mutations on the interaction of RD3 with GC was studied . The F10Figure 2). RD3 may also be involved in the trafficking of GC within the cilium although this remains to be determined.Studies carried out to date suggest that RD3 serves two roles in photoreceptor cells. A primary function of RD3 is to facilitate the exit of GC from the ER and its trafficking to the photoreceptor cilium (rcd2 collie showed a 10 times higher level of cGMP than control retinas during a 2–8 week period with only 25% reduction in PDE activity (A second function of RD3 is to inhibit GC catalytic activity . The inhactivity .2+ levels can be toxic to cells (2+ through the cGMP-gated channels and the efflux through the Na/Ca-K exchanger. In the absence of GC in the OS, cGMP will not be produced resulting in permanent closure of cGMP-gated channels and a reduction in intracellular Ca2+ below a threshold required for photoreceptor survival. In this case the mechanism of photoreceptor degeneration in the rd3 mouse and LCA12 patients would be similar to photoreceptor degeneration in the GC1/GC2 knockout mouse and LCA1 patients. Direct comparison of the rate of photoreceptor degeneration in the rd3 mouse and GC1/GC2 knockout mouse is complicated by the large variation in the rate of degeneration observed for different strains of rd3 mice, although in general degeneration in the rd3 mouse appears to be more rapid. Comparison of LCA12 with LCA1 patients is complicated by the presence of functional GC2 in rod photoreceptors of LCA1 patients. However, limited clinical assessment of these patients suggests that LCA12 is more severe (The mechanism by which loss in RD3 causes photoreceptor degeneration remains to be determined. Here we discuss two possible mechanisms. The first mechanism is centered on the role of GC and cGMP in maintaining calcium homeostasis in photoreceptors. It is generally known that too high or too low Cato cells . Calciume severe .rd1 and rd10 mice linked to mutations in the β-subunit of PDE (A second mechanism involves the role of RD3 in the inhibition of GC activity . In the t of PDE , mice det of PDE , and trat of PDE . Both thThe 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.


Considered an epicenter of pandemic influenza virus generation, southern China has recently seen an increasing number of human H7N9 infections. However, it is not the only threat. On 30 November 2013, a human H10N8 infection case was first described in China. The origin and genetic diversity of this novel virus is similar to that of H7N9 virus. As H10N8 avian influenza virus (AIV) was first identified from a duck in Guangdong Province during 2012 and there is also evidence of H10N8 infected dogs in this region, we sought to examine archived sera from animal workers to see if there was evidence of subclinical human infections before the first human H10N8 cases.We studied archived serum samples  collected between May and September 2013 from 710 animal workers and 107 non-animal exposed volunteers living in five cities of Guangdong Province. Study participants’ sera were tested by horse red blood cells (RBCs) hemagglutination inhibition (HI) and microneutralization (MN) assays according to World Health Organization guidelines. The A/Jiangxi-Donghu/346-1/2013(H10N8) virus was used. Sera which have an HI assay ≥1:20 were further tested with the MN assay. Questionnaire data were examined for risk factor associations with positive serological assays. Risk factor analyses failed to identify specific factors associated with probable H10N8 infections.Among the 827 sera, only 21 animal workers had an HI titer ≥1:20 (18 had an HI titer of 1:20 and 3 had an HI titer of 1:40). None of these 21 subjects reported experiencing any influenza symptoms during the three months before enrollment. Among the three subjects with HI titers of 1:40, two had MN antibody titers of 1:40, and one had a MN antibody titer of 1:80 (probable H10N8 infections).Study data suggest that animal workers may have been infected with the H10N8 virus before the first recognized H10N8 human infection cases. It seems prudent to continue surveillance for H10N8 viruses among animal workers. Located in southern China, Guangdong Province is home to some of the world’s largest populations of humans, chickens, ducks and pigs and has been associated with human outbreaks of severe acute respiratory syndrome (SARS) and highly pathogenic H5N1 avian influenza infections. This region of China has been considered an epicenter of novel influenza virus generation . In receAs H10N8 AIV was first identified in a duck from Guangdong Province in 2012  and therThe animal worker sera were collected during the period June to August 2013 during a surveillance program for novel zoonotic influenza virus among animal workers living in five cities of Guangdong Province. Non-animal-exposed participants’ sera were similarly collected during the period May to August from middle school teachers and students in Guangzhou and Foshan cities who were healthy, reported no history of having received an influenza vaccine or having direct contact with swine or poultry, during the six months before enrollment. This study was approved by Guangdong Centers for Disease Control and Prevention and begun in early 2013. Study subjects were screened by telephone call and enrolled by informed consent. Participants completed a questionnaire which collected various relevant data including demographic, recent clinical signs and symptoms, and animal occupational exposure history.Sera were first screened by a horse red blood cells (RBCs) hemagglutination inhibition (HI) assay against influenza virus A/Jiangxi-Donghu/346-1/2013 (H10N8). Sera with HI titers ≥1:20 were further studied with microneutralization (MN) assays against the same virus. It is important to note that horse RBCs show a high proportion of sialic acid α2,3-Gal binding, which is preferential for AIV. It had been observed that the use of horse RBCs significantly increased the sensitivity of detection of HI antibodies in the sera of confirmed H7N9 cases compared with the use of turkey RBCs, thus the World Health Organization recommends that horse RBCs should be used to detect HI antibodies for H7N9 virus infection [50 of virus were added to the serially diluted serum at a 1:1 ratio (V/V) and incubated at 37°C for one hour. Finally, 0.2 ml of the virus-serum mixtures was transferred to 96-well monolayer plates and incubated in 5% CO2 at 37°C for 72 hours. Three wells were run for every dilution of each serum sample. We observed the pathogenic effects every day and tested the cell supernatants with a HA test to confirm the infection.Both assays were performed according to the World Health Organization guidelines . During Study sera which had titers <1:20 in endpoint serum dilution against H10N8 antigens used in this study were considered to be negative. As there was no established HI titer standard to detect a mild or asymptomatic human H10N8 infection, and the positive-control samples have shown HI antibody titers ranging from 1:40 to 1:1280, in this seroepidemiological study we considered sera with HI titers above 1:40 as having possible evidence of previous H10N8 virus infection but only those sera with both HI and MN titers ≥1:40 as having probable evidence of previous infection with influenza A(H10N8) virus.Archived sera and data from 827 poultry workers, poultry retailers in agricultural-trade markets, swine workers, veterinarians, slaughterhouse workers, zoo workers and non-animal-exposed volunteers were studied Table . The totHI titers of ≥1:20 were detected in 21 (2.54%) of 827 study subjects (18 with a HI titer of 1:20 and 3 with a HI titer of 1:40). None of these 21 participants  reported having influenza symptoms during the three months before enrollment. Three of these 21 subjects also had MN antibody titers ≥1:40 against H10N8 AIV  and more than 70 human cases with 50% mortality have since then been reported in mainland China . Since 2Our retrospective serologic study is the first to document serological evidence of human asymptomatic infection with H10N8 influenza virus among animal workers before 30 November 2013 (the time of the first index human H10N8 AIV case ) and mayThe results of the present study suggest that workers occupationally exposed to poultry may be at risk of H10N8 AIV infection. It is reassuring that more animal workers were not found to be seropositive and that these findings are consistent with other studies of H7N9 and H5N1 infections among China’s poultry workers. However, it seems appropriate that more extensive serological investigations regarding asymptomatic or subclinical H10N8 infections should be performed among groups expected to be at high risk .Study data suggest that animal workers may have been infected with the H10N8 virus before November 30, 2013, when the first human H10N8 case was recognized. It seems prudent to continue surveillance for H10N8 viruses among animal workers.This study protocol was reviewed and approved by the Institutional Review Board at the Guangdong Center for Disease Control and Prevention.


Changes in the expression and activity of PDK1 and several AGC kinases have been linked to human disease, including cancer.PIK3CA mutation status.We used immunohistochemical analysis to determine PDK1 expression in 241 tumors from patients with breast cancer in which we had previously analyzed PIK3CA mutation status and PDK1 overexpression.Moderate or high expression of PDK1 was observed in 213 of the 241 cases (88%). There was no correlation between Our findings indicate that PDK1 is independently activated in breast cancer and not only as part of the PIK3CA pathway, suggesting that PDK1 plays a specific and distinct role from the canonical PIK3/Akt pathway and promotes oncogenesis independently of AKT. Our data implicate PDK-1 and downstream components of the PDK-1 signaling pathway as promising therapeutic targets for the treatment of breast cancer. The protein kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a key role in signaling pathways activated by several growth factors and hormones. PDK1 functions downstream of phosphoinositide 3-kinase (PIK3) and activates members of the AGC family of protein kinases such as protein kinase B (Akt), protein kinase C (PKC), p70 ribosomal protein S6 kinases, and serum glucocorticoid-dependent kinase by phosphorylating serine/threonine residues in the activation loop. AGC kinases are known to play crucial roles in the regulation of various physiological processes relevant to metabolism, growth, proliferation, and survival.PTEN +/- mice and the resulting mice with deficient PDK1 levels had a reduced prevalence of tumor development. Subsequent studies demonstrated the role of PDK1 in a variety of different cancers; in particular PDK1 appears to play a decisive role in the development of breast cancer [The first evidence that PDK1 might be a viable target in cancer came in 2005 when Bayascas et al. [PIK3CA displayed a reduced dependence on Akt for tumorigenicity, and instead relied on PDK1-dependent activation of another AGC kinase, SGK-3. Breast cancers are known to be a group of diverse diseases, and cellular heterogeneity has been shown to affect disease-free survival in patients with breast cancer [Increased PDK1 expression has also been reported in 45% of patients with acute myeloid leukemia, and PDK1 seems to be a viable target in head and neck cancer, multiple myeloma, pancreatic cancer, and colorectal cancer -7. Vasudt cancer . For exat cancer . MoleculPIK3CA mutations, but also in the absence of these genetic alterations [There is accumulating evidence that PDK1 is overexpressed in particular cancer settings and activates cancer cell growth and survival independent of Akt signaling. These findings suggest that PDK1 is not just an Akt-activating player, but rather an important oncogenetic regulator and a potential therapeutic target in cancer. Recently, it has been shown that PDK1 regulates anchorage-independent growth, resistance to several anticancer drugs, and tumor formation in breast cancer cells–not only in tumors harboring erations .PIK3CA mutations?This study aimed to answer the following questions: (1) Is PDK1 overexpressed in breast cancer and to what extent? (2) Is there any correlation between PDK1 overexpression and PIK3CA mutations [To address these questions, we examined the phosphorylation status of PDK1 in a group of tumors in which we had previously analyzed utations . Four tiPIK3CA mutations were identified in 15.8% of cases in the same patient collective. There was no correlation between PIK3CA mutation status and PDK1 overexpression.Moderate or high expression of PDK1 was observed in 213 of the 241 cases (88%). The fact that some cases without PIK3CA mutations had a moderate or high expression of PDK1 suggests that PDK1 can be independently activated in breast cancer and not only as part of the PIK3CA pathway. Our results indicate that PDK1 plays a specific role distinct from the canonical PIK3/Akt pathway.PIK3CA mutation status and PDK1 expression in tissues from the same tumor group. The results of our study suggest that the PDK-1 signaling pathway might be a promising therapeutic target for the treatment of breast cancer and that PDK1 expression should be tested in addition to PIK3CA status in patients with breast cancer prior to beginning therapy.To the best of our knowledge this study is the first to compare PDK1: 3-phosphoinositide-dependent protein kinase-1; PIK3CA: phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha.The authors declare that they have no competing interest.


Toxoplasma gondii AMA1, we engineered a form of the protein where the mobile portion of the loop was replaced with a short Gly-Ser linker (TgAMA1ΔDIIloop). Isothermal titration calorimetry measurements with a panel of RON2 peptides revealed an influential role for the DII loop in governing selectivity. Most notably, an Eimeria tenella RON2 (EtRON2) peptide that showed only weak binding to TgAMA1 bound with high affinity to TgAMA1ΔDIIloop. To define the molecular basis for the differential binding, we determined the crystal structure of TgAMA1ΔDIIloop in complex with the EtRON2 peptide. When analyzed in the context of existing AMA1-RON2 structures, spatially distinct anchor points in the AMA1 groove were identified that, when engaged, appear to provide the necessary traction to outcompete the DII loop. Collectively, these data support a model where the AMA1 DII loop serves as a structural gatekeeper to selectively filter out ligands otherwise capable of binding with high affinity in the AMA1 apical groove. These data also highlight the importance of considering the functional implications of the DII loop in the ongoing development of therapeutic intervention strategies targeting the AMA1-RON2 invasion complex.Apicomplexan parasites are the causative agents of globally prevalent diseases including malaria and toxoplasmosis. These obligate intracellular pathogens have evolved a sophisticated host cell invasion strategy that relies on a parasite-host cell junction anchored by interactions between apical membrane antigens (AMAs) on the parasite surface and rhoptry neck 2 (RON2) proteins discharged from the parasite and embedded in the host cell membrane. Key to formation of the AMA1-RON2 complex is displacement of an extended surface loop on AMA1 called the DII loop. While conformational flexibility of the DII loop is required to expose the mature RON2 binding groove, a definitive role of this substructure has not been elucidated. To establish a role of the DII loop in  Plasmodium, Toxoplasma, and Eimeria genera, for example, are the etiological agents of malaria, toxoplasmosis, and coccidiosis, respectively. P. falciparum causes the most severe cases of human malaria . Sp. SpTgAMAl groove . TgRON2Dine loop . Importaine loop , and alscontacts . The difTgRON2D3 . Togethein silico docking experiments and corresponding proof of principle invasion inhibition studies [High affinity AMA-RON2 complexes anchor the junction between apicomplexan parasites and host cells and play an important role in invasion , 14, 36. studies , 41–44.  studies , 23, 24.T. gondii AMA1 where the flexible region of the DII loop was replaced with a short Gly-Ser linker (TgAMA1 selectivity. Notably, TgAMA1 bound both TgRON2D3 and EtRON2D3 but only the former with high affinity, while TgAMA1ΔDIIloop showed moderate binding to each tested RON2D3 and bound both TgRON2D3 and EtRON2D3 with high affinity (TgAMA1ΔDIIloop in complex with EtRON2D3. Notably, this is the first cross-genus AMA-RON2 pair to be structurally characterized (TgAMA1-TgRON2D3 and TgAMA1ΔDIIloop-EtRON2D3 structures enabled the identification of key anchor points in the AMA1 groove exploited to outcompete the DII loop and bind TgAMA1 with high affinity (PfAMA1 [T. gondii and P. falciparum.Initially, we engineered a form of r linker . Solutioaffinity . To estacterized . A compaaffinity . Intrigu (PfAMA1 , 45. ThePlasmodium, and it is even suggested that a complex between AMA1 and a host surface protein may support a pre-invasion step [TgAMA1 that the ΔDIIloop construct displays a more permissive binding profile. Furthermore, engineering parasites to express ΔDIIloop constructs of AMA1 will enable the investigation of potential DII loop dependant signalling events associated with high affinity AMA1-RON2 binary complex assembly and moving junction formation.The biological implications of truncating the DII loop have proven difficult to assess. However, since the presence of the AMA1 DII loop results in a more selective receptor, this substructure may act as a structural gatekeeper to restrict formation of unproductive AMA1 complexes. Thus, a more discriminating AMA1 apical groove would ensure the parasite-host cell junction is competent for invasion because only a cognate RON2 would bind with sufficient affinity to outcompete the DII loop. This gatekeeping function may ultimately serve as a molecular switch to signal the parasite that a functional junction has been formed and invasion can proceed. It follows that an apicomplexan parasite expressing the truncated DII loop form of AMA1 might experience more abortive invasion events resulting from unproductive, non-RON2 based AMA1 complexes that are not linked to junction integrity. The potential for AMA1 to form non-RON2 based complexes has been proposed –50, partion step . In thisTgAMA1 and TgAMA1ΔDIIloop reveal a previously underappreciated role for the DII loop in selectively filtering out ligands otherwise capable of binding in the AMA1 apical groove. Companion structural studies offer important insight into molecular recognition thresholds that likely need to be achieved by ligands able to outcompete the DII loop and form high affinity complexes capable of anchoring the moving junction during invasion of the host cell. While the biological implications of apicomplexan AMA1 DII loop conformational changes remain an intriguing topic for future studies, these results provide a molecular basis for understanding AMA1 selectivity, which can support the development of novel therapies targeting the important AMA1-RON2 invasion complexes.The solution binding studies of several RON2 peptides with


Wolfram syndrome 1 (WFS1) is a genetic disorder which has been associated both with impaired early brain development and neurodegeneration. Recent studies have suggested regulation of Ca2+ homeostasis by wolframin (Wfs1) and have demonstrated the involvement of endoplasmatic reticulum (ER) stress in Wfs1 deficiency. Despite the ER dysfunction WFS1 shows several characteristics of pathologies related to mitochondrial dynamics. Therefore our aim was to examine the hypothesis that Wfs1 deficiency could disturb mitochondrial dynamics contributing to impaired neuronal functioning. First we show that Wfs1 deficiency induces mild ER stress leading to Inositol 1,4,5-Trisphosphate Receptor (IP3R) dysfunction and disturbed cytosolic Ca2+ homeostasis, which, in turn alters mitochondrial trafficking, inhibits mitochondrial fusion and augments mitophagy. The overexpression of the active IP3R fragment restores IP3R-mediated Ca2+ release and corrects all perturbations in mitochondrial dynamics suggesting that these events are causally linked. We further demonstrate that suppressing the expression of two Parkinson disease-related proteins, Pink1 and Parkin, leads to reduced Wfs1 deficiency-induced mitophagy and also to the correction of the fusion-fission dynamics and mitochondrial motility. These data suggest that Wfs1 deficiency may over-activate Pink1 and Parkin pathways. Our most important discovery is that Wfs1 deficiency delays neuronal development and axonal growth in primary rat cortical neurons. According to our data, the link between Wfs1 deficiency and delayed neuronal development appears to be mediated by impaired mitochondrial dynamics because suppression of the Pink1-Parkin pathway corrected also the developmental delay. Our data shed light on the mechanisms of neuronal abnormalities in WFS1 and point out potential therapeutic targets. This work may have broader implications for understanding the role of mitochondrial dynamics in neuropsychiatric diseases.


These data provide insight into the mechanism of nuclear penetration by 3E10 and demonstrate the potential for use of 3E10 in therapeutic approaches to diseases ranging from malignancy to ischemic conditions such as stroke.A nuclear-penetrating lupus anti-DNA autoantibody, 3E10, has been found to inhibit DNA repair and selectively kill certain cancer cells that are highly vulnerable to DNA damage. In addition, a 3E10 single chain variable fragment (scFv) has been developed for use as a delivery vehicle to carry therapeutic cargo proteins into cell nuclei. A greater understanding of the mechanism by which 3E10 penetrates cell nuclei is needed to help determine the scope of its potential therapeutic applications. Here we show that the presence of extracellular DNA significantly enhances the nuclear uptake of 3E10 scFv. In addition, we find that 3E10 scFv preferentially localizes into tumor cell nuclei  Antibody-based immunotherapy has improved treatment outcomes in diseases ranging from rheumatologic conditions to cancer, and efforts are underway to optimize these approaches to yield even greater clinical impact. Most immunoglobulins are unable to penetrate into living cells and therefore cannot be used to target key intracellular antigens or pathways, and this has been a critical factor that has limited the expansion of the use of antibodies in molecular therapy. However, a select group of lupus autoantibodies penetrate into living cellsin vitro and in vivo3456An unusual lupus anti-DNA antibody that penetrates cell nuclei without causing any apparent harm to normal cells or tissues, 3E10, has been found to inhibit DNA repair and thereby selectively kill cancer cells that are vulnerable to DNA damage due to BRCA2-deficiencySome cells take up immunoglobulins and immune complexes through Fc-receptor mediated endocytosis810H contains the sequence RGLLLDY, and mutation of the arginine to an aspartic acid residue takes away a positive charge and abrogates DNA binding by 3E10H by mutating an aspartic acid residue to asparagine greatly enhances the affinity of 3E10 for DNAThe ability of 3E10 to bind DNA is critically linked to its ability to penetrate cell nuclei. 3E10 is a cationic antibody, like most anti-DNA antibodies, and this contributes to its ability to bind to negatively charged DNA. For example, CDR3 of the 3E10 VOverall, the previous work strongly suggests that 3E10 scFv penetrates cell nuclei by binding to DNA or its components and then following it into cells through the ENT2 nucleoside salvage pathway. If this hypothesis is correct then 3E10 scFv should not be able to penetrate into cells in the absence of extracellular DNA. Moreover, addition of extracellular DNA would be expected to enhance nuclear uptake of 3E10 scFv. We therefore set out to test our hypothesized model of nuclear penetration by 3E10 scFv by evaluating the impact of the presence of extracellular DNA on efficiency of nuclear uptake of 3E10 scFv as described below.We sought to test the efficiency of nuclear penetration by 3E10 scFv into cells in the absence of extracellular DNA, and for these studies selected the GM02605 human fibroblast cell line because it maintains a high degree of viability (>99%) with minimal cell death even while maintained in culture for several days. With minimal cell death in culture the confounding effects of DNA released by dead cells are minimized. The GM02605 cells were washed with serum free media and then treated with 10 μM 3E10 scFv for one hour, after which cells were fixed and immunostained for presence of the fragment. Remarkably, 3E10 scFv did not penetrate into most cells. Instead, 3E10 scFv was found only in the nuclei of cells centered around what morphologically appeared to be rare dead cells, with a gradient effect observed with decreased amounts of intranuclear fragment detected with increasing distance from the central dead cell. A representative image demonstrating this effect is shown in To test the hypothesis that a factor released by dead cells enhances nuclear uptake of 3E10 scFv we compared the efficiency of nuclear penetration of the fragment into the GM02605 fibroblasts in the presence or absence of a cell lysate. As shown in We hypothesized that DNA is the critical factor in cell lysate that promotes nuclear uptake of 3E10 scFv. To test this, the GM02605 fibroblasts were treated with 10 μM 3E10 scFv in the presence of cell lysate that had been passed through a Centricon cellulose filter with a molecular weight cut off of 10,000 kDa to remove DNA content. In contrast to the complete cell lysate, the DNA-depleted lysate did not enhance nuclear uptake of 3E10 scFv , which sTo confirm that extracellular DNA enhances nuclear penetration by 3E10 scFv, we next treated the GM02605 fibroblasts with 10 μM 3E10 scFv in the presence of purified DNA (0.5 g/L). As shown in in vivo due to an expected higher concentration of extracellular DNA in the tumor vicinity released from dead cells in regions of tumor ischemia and necrosis. To test this, subcutaneous U87 human glioma xenografts were generated in immunodeficient mice, and once tumors grew to size of ~100 mm3 mice were treated with intraperitoneal injection of control buffer or 3E10 scFv. Mice were then sacrificed 4 or 24 hours after treatment, and tumors and select normal tissues were immunostained for the presence of 3E10 scFv. Four hours after treatment 3E10 scFv was not detected in the cell nuclei of normal tissues including heart, kidney, skeletal muscle, and liver. By contrast, cell nuclei in the tumor xenografts stained positive for presence of 3E10 scFv  grows to confluence in 96-well tissue culture plates with remarkably high viability . Cells were grown in MEM with 15% FCS and washed with MEM without serum before incubation with 10 μM 3E10 scFv for one hour. Nuclear penetration by 3E10 scFv was then examined by anti-Myc immunostaining as previously describedCOS-7 cell lysate was prepared by subjecting cells to multiple freeze-thaw cycles in liquid nitrogen. Cell debris was removed by centrifugation. DNA-depleted COS-7 cell lysate was prepared by passing the lysate through a Centricon cellulose filter with a molecular weight cut off of 10,000 kDa.Purified calf thymus DNA sheared to an average length of 2000 bp was purchased from Invitrogen .3 mice were treated with intraperitoneal injection of control PBS buffer or 0.8 mg 3E10 scFv in PBS. Mice were sacrificed 4 or 24 hours after treatment, and tumors and selected normal tissues were fixed in formalin and embedded in paraffin. Tissues were then surveyed for nuclear penetration by 3E10 scFv by immunohistochemistry (IHC). Tissue sections were deparaffinized, rehydrated, and incubated at 95–99 C for 30 minutes for epitope retrieval. Sections were washed, blocked with peroxidase, and probed with a 9E10 anti-Myc  primary antibody directed at the C-terminal Myc tag in 3E10 scFv followed by additional washes and then incubation with a labeled polymer-HRP secondary antibody . After additional washes color development was performed using DAB followed by counterstaining with hematoxylin. All in vivo studies were conducted in accordance with institutional guidelines. The protocol for the in vivo work was approved by Yale University’s Institutional Animal Care and Use Committee.U87 human glioma subcutaneous xenografts were generated in nude mice as previously describedHow to cite this article: Weisbart, R. H. et al. DNA-dependent targeting of cell nuclei by a lupus autoantibody. Sci. Rep.5, 12022; doi: 10.1038/srep12022 (2015).


Spodoptera frugiperda with Bt corn expressing Cry1Fa has decreased, forcing them to use chemicals to reduce the damage caused by this insect pest. A colony of S. frugiperda was established from individuals collected in 2013 from Cry1Fa corn plants (SfBt) in Brazil and shown to have at least more than ten-fold higher resistance levels compared with a susceptible colony (Sflab). Laboratory assays on corn leaves showed that in contrast to SfLab population, the SfBt larvae were able to survive by feeding on Cry1Fa corn leaves. The SfBt population was maintained without selection for eight generations and shown to maintain high levels of resistance to Cry1Fa toxin. SfBt showed higher cross-resistance to Cry1Aa than to Cry1Ab or Cry1Ac toxins. As previously reported, Cry1A toxins competed the binding of Cry1Fa to brush border membrane vesicles (BBMV) from SfLab insects, explaining cross-resistance to Cry1A toxins. In contrast Cry2A toxins did not compete Cry1Fa binding to SfLab-BBMV and no cross-resistance to Cry2A was observed, although Cry2A toxins show low toxicity to S. frugiperda. Bioassays with Cry1AbMod and Cry1AcMod show that they are highly active against both the SfLab and the SfBt populations. The bioassay data reported here show that insects collected from Cry1Fa corn in the Cerrado region were resistant to Cry1Fa suggesting that resistance contributed to field failures of Cry1Fa corn to control S. frugiperda.Brazil ranked second only to the United States in hectares planted to genetically modified crops in 2013. Recently corn producers in the Cerrado region reported that the control of  Bacillus thuringiensis (Bt) have been used in the field since 1996 and are important tools to control insect pests reducing the use of chemical insecticides [Genetically modified (GM) plants expressing insecticidal proteins from the bacterium cticides  but thoscticides , 4.2, constituting one of the largest areas of agricultural activity in Brazil, as it represents almost 25% of the national territory. Annual cultivation of corn, soybean and cotton in the Cerrado begins in October and extends until June [In 2013, the global area planted with GM Bt plants was 75.9 million ha in different countries . In 2010til June . This arInsects can evolve resistance to Bt toxins in laboratory and field conditions , 9, 10. The area of refuge recommended for the cultivation of GM corn in Brazil, was equivalent to 10% of the total area planted with Bt corn and that it should be located at a distance less than 800 meters from the Bt corn field . FurtherS. frugiperda populations to Cry1Fa Bt corn was previously reported in Puerto Rico [S. frugiperda [S. frugiperda populations to Cry1Fa corn in different regions of Brazil including state of Goiás in the Cerrado region was confirmed [Resistance of rto Rico . From 20ugiperda , suggestonfirmed . The resonfirmed .Pectinophora gossypiella on Cry1Ac Bt cotton in India [Busseola fusca on Cry1Ab Bt corn in South Africa [Helicoverpa zea on Cry1Ac Bt cotton in USA [Diabrotica virgifera in Cry3Bb Bt corn in USA [Helicoverpa armigera on Cry1Ac Bt cotton in China [Field-evolved resistance in different insect pests in other regions such as in India , Busseolh Africa , Helicovn in USA  and Heliin China  were alsS. frugiperda population that has evolved resistance to Cry1Fa toxin resulting in the survival of this insect pest in Cry1Fa Bt corn. We analyzed the stability of the resistant phenotype after several generations without selection pressure, studied the cross-resistance of this population to other Cry1A and Cry2A toxins and determined whether Cry1AMod toxins that have been reported as effective for the control of various lepidopteran species resistant to Cry1A toxins [S. frugiperda larvae collected from Brazilian Bt corn fields.The objective of this study was to further analyze the A toxins , 20 coulInsects were collected during 2013 on a farm in the municipality of Cabeceiras in the state of Goiás, located in the Cerrado region of West-Central Brazil . The property is about 700 acres where Bt corn, conventional corn and herbicide tolerant soybeans and common beans are grown. Cry1Fa Bt corn had been cultivated for four years and Cry1A.105 corn, Cry2Ab corn and herbicide tolerance corn had been cultivated for the past two years. Insect collection activity and maintenance was authorized by the Ministry of Environment of Brazil (MMA), Instituto Chico Mendes de Biodiversidade—ICMBio, Sistema de Autorização e Informação em Biodiversidade-SISBIO Number: 39022–1.S. frugiperda larvae were collected in 2013 from Cry1Fa Bt corn variety 30F53H, taken to the insect breeding laboratory at Embrapa Genetic Resources and Biotechnology and maintained individually until pupating in 50 ml plastic cups with acrylic covers containing 10 ml artificial diet [Four hundred ial diet . The pupS. frugiperda originated from mass rearing in Embrapa, and named SfLab. This breeding was implemented in 1988 [The susceptible colony of  in 1988  and the cry1Aa, cry1Ab, cry1Ac, cry2Aa, cry2Ab genes, obtained from HD-1 strain were individually cloned into pHT315 vector [B. thuringiensis serovar. israelensis acrystalliferous strain 4Q7 (Bacillus Genetic Stock Center) [xg for 30 min at 4° C. The spore-crystal mixtures were frozen and lyophilized for 18 h in a Labconco Lyphlock model lyophilizer. Crystal protein concentration was measured by using the Bradford reagent from BioRad. Bioassays were carried out by spreading 35 μl of 10 serial dilutions containing different protein concentrations of Cry1Fa protein or mixture of freeze-dried spore crystals, on the surface of artificial diet previously distributed in 24-well plates. After the protein samples were dried in the diet, one second-instar larvae was placed in each well. We tested 24 larvae per toxin concentration in triplicate, and analyzed ten different concentrations of toxin, making a total of 720 larvae per LC50 value determination. As negative control one plate was left without toxin addition. Three biological replicates were performed. The first reading of mortality was made 48 hours after start of the test, at which time the larvae were transferred to 50 ml plastic cups containing diet without toxin. On the seventh day, the second and final reading was taken [50 was determined using generalized linear models (GLM) with probit link function [50 and the slope of the dose were calculated. This assay is accredited by ISO/IEC 17025. Bioassays were performed with larvae of the F1 generation of SfBt and SfLab during 2013. Bioassays were also performed with larvae from F2 and F8 generations of SfBt colony in 2013 and 2014 respectively, using a diagnostic dose corresponding to 10 times the LC50 dose on the SfLab strain . Bioassays for other Cry and Cry1AMod toxins were performed as described above with larvae from the F8 generation.Bioassays were performed with activated Cry1Fa toxin  and a mixture of spores and crystals from Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab, Cry1AbMod and Cry1AcMod produced in Bt transformant strains. The 5 vector  and expr Center) . The Cry Center) . The rec Center)  supplemeas taken , 26. Theas taken , where tanalysis). The conth generation) were used per treatment in three repetitions. Mortality was recorded after 7 days.Bioassays with corn leaves were also analyzed under laboratory conditions. Corn leaves from Cry1Fa corn variety 30F53H were collected during 2013 in the Cabeceiras Cerrado region of Brazil. Leaves from non transgenic plants variety 30F53 were also collected from the same region at the same time. Leaves were taken to the insect breeding laboratory at Embrapa Genetic Resources and Biotechnology and maintained at-20°C. Frozen leaves were defrost in February 2014 and placed in Petri dishes containing a moisture paper with water. A total of 20 larvae in the second instar from SfLab and SfBt  with 1 mM EDTA and 0.1 mM PMSF. The crystals were separated by sucrose gradient centrifugation [2CO3, 0.2% β-mercaptoethanol, pH 10.5). The protoxins were activated with trypsin (1:50 w/w)  for 2h at 37° C; the reaction was stopped by addition of PMSF . Activated toxins were purified as previously described [The mixture of spores/crystals produced by the recombinant Bt strains was washed three times with PBS buffer , aliquoted and stored at-80° C. Enrichment of 4–6 fold of ALP enzymatic activity compared to the initial gut homogenate was observed in BBMV preparations.Brush border membrane vesicles (BBMV) from the larval gut were purified according to Wolfersberger et al. , from miThe trypsin activated proteins Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab and Cry1Fa were biotinylated using the ECL Protein Biotinylation System  kit according to the manufacturer's recommendations. After biotinylation, detection was performed with peroxidase-conjugated streptavidin to confirm the biotin labeling of proteins as previously reported  using stS. frugiperda BBMV were incubated with10 nM of each biotinylated toxin for 30 min at 25°C in 100 μl total volume of binding buffer . Unbound toxins were removed by centrifugation . The BBMV containing bound Cry toxins were washed three times in binding buffer and suspended in 15 μl of 1X PBS and 5 μl of 4X Laemmli sample buffer . The samples were boiled for 3 min, resolved by SDS-PAGE at 9% and transferred to nitrocellulose membrane as described above. Labeled proteins bound to BBMV were visualized with streptavidin-peroxidase conjugate followed by incubation with SuperSignal chemiluminescence substrate as described above. The optical density of the bands was scanned with ImageJ program and the percentage of each band on the blot was calculated.Binding analyses were performed in February 2014 using BBMV from SfBt and SfLab populations as previously reported . BrieflyS. frugiperda larvae (LC50) from the different populations was determined. 50 value was at least ten times greater in the SfBt population compared with the control population SfLab. The exact LC50 values could not be calculated for the SfBt colony since the highest dose of Cry1Fa that we could assayed was 3500 ng/cm2 and at this concentration the mortality was not enough to determine LC50 values.The Cry1Fa lethal concentration required to kill 50% of 2) 10 times higher than the LC50 obtained with Cry1Fa in larvae of the SfLab colony (The colony SfBt was maintained in the laboratory in the absence of selection pressure for eight generations. Each of these generations was used to determine their susceptibility to a Cry1Fa diagnostic concentration (3,500 ng/cmb colony . At thisIn order to determine whether the level of resistance of the SfBt population to Cry1Fa affects the efficacy of Cry1Fa corn plants, the number of survival larvae of the susceptible SfLab and the Cry1Fa-resistant population SfBt on leaves of Bt corn plants was determined. The larvae of the population SfLab did not survive on leaves from Cry1Fa corn while the F8 generation of SfBt population showed 100% survival. The SfLab and SfBt populations had similar 100% survival on leaves from non-transgenic corn.S. frugiperda populations to other Cry proteins as described in Materials and Methods. The F8 generation of SfBt colony showed LC50 values 30 times higher for Cry1Aa toxin than those of the susceptible SfLab colony (50 values of Cry1Ab and Cry1Ac were 4 and 2.5-fold higher respectively when compared to the values obtained in the SfLab colony. The fiducial limits values for these bioassays do not overlap, indicating that these differences are significant. In contrast, analysis of susceptibility to Cry2Aa and Cry2Ab showed that there are not significant differences in susceptibility to these toxins which showed low or no-toxicity to both SfLab and SfBt insects (S. frugiperda larvae in the Cerrado region of Brazil due to their low toxicity. It is worth to mention that these assays were performed with spore/crystal suspensions. In all cases the same acrystalliferous Bt 4Q7 strain was transformed with the pHT315 vector containing the different cry genes as reported in Material and Methods section. There is evidence in the literature that Bt spores produce virulence factors that could be important for toxicity [Subsequently, we compared the susceptibility of the two b colony . The LC5 insects . These dtoxicity . However50 value of a native toxin divided by the LC50 of the corresponding modified toxin, is very high in the resistant strain since Cry1AbMod show a potency of 8.5 fold higher than the native Cry1Ab toxin and Cry1AcMod of 5.7 fold higher than Cry1Ac against the SfBt population (50 value of a Cry1Fa toxin and of the modified toxins against SfBt (Toxicity assays of Cry1AbMod and Cry1AcMod toxins showed that these toxins are highly active against both the SfLab and the SfBt populations. The Cry1AMod proteins showed an important insecticidal activity against the SfBt population. In the case of the SfLab population Cry1AbMod and Cry1AcMod showed insecticidal effect comparable to that of Cry1Aa . Howeverpulation . It woulnst SfBt  indicate50 of a native toxin divided by the LC50 of a modified toxin (based on data from LCS. frugiperda BBMV is specific [S. frugiperda populations. Previously it was shown that binding of Cry1A and Cry1Fa toxins to specific . We analS. frugiperda BBMV showed that Cry1Fa share binding sites with Cry1A toxins but not with Cry2A [Homologous and heterologous binding competition analysis of Cry1Fa toxin to th Cry2A . We perfS. frugiperda colony with significant resistance to Cry1Fa toxin from individuals collected in a region where Bt corn expressing the Cry1Fa toxin was grown for four years. Recently, a parallel study also reported high resistance levels of S. frugiperda populations to Cry1Fa corn in the same Brazilian region [In this work we report the establishment of a n region . Our datS. frugiperda larvae. It remains to be determined if the SfBt population is capable of surviving in other crops expressing Cry1Ab, Cry1Ac or Cry2Ab toxins. Our data indicated no cross-resistance to Cry2A toxin and no susceptibility to Cry2Ab. Due to the low susceptibility of S. frugiperda to Cry2Aa and Cry2Ab toxins it is expected that GM plants expressing Cry2Ab are not likely to be effective in controlling Cry1Fa resistant insects. Resistance of S. frugiperda to Cry1Fa corn was previously documented in Puerto Rico [S. frugiperda resistant population from Puerto Rico showed a similar cross-resistance pattern to Cry toxins to the SfBt reported here, with low cross-resistance to Cry1Ab and Cry1Ac and no cross-resistance to Cry2Ab toxin [Heliothis virescens [Plutella xylostella [B. thuringiensis var. kurstaki strain that expresses the three Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa toxins while XenTari express Cry1C and Cry1D that are toxic to S. frugiperda [S. frugiperda populations. Here we show that Cry1Aa is highly toxic to the susceptible strain of SfLab (S. frugiperda populations from different Latin American regions showed different susceptibility to selected Bt toxins [The resistance mechanism in SfBt associated with reduced binding of Cry1Fa to BBMV, suggesting that the resistant allele may be affecting expression of a Cry1Fa binding molecule. However, these data remain to be confirmed by further studies for Cry1Fa protein receptors identification. The Cry1Fa resistant population SfBt showed high cross-resistance to Cry1Aa, and low cross-resistance to Cry1Ab and Cry1Ac toxins. The latter proteins are expressed in other transgenic plants used in Brazil, including cotton and soybean, which are also host plants for rto Rico . The S. Ab toxin . These direscens , and NO-lostella . It was lostella . DiPel iugiperda . The higof SfLab . It was t toxins  suggestiH. virescens [Helicoverpa armigera, Helicoverpa zea, Spodoptera exigua [Ostrinia nubilalis and also in S. frugiperda [Competition binding assays have shown that Cry1As and Cry1Fa share binding sites in different Lepidopteran insects such as irescens , Helicova exigua  Ostriniaugiperda , 40. CroS. frugiperda BBMV, but we showed here that these toxins showed low toxicity to SfLab or SfBt and did not compete the binding of Cry1Fa to SfLab BBMV. These data indicate that these toxins would not be an alternative to control Cry1Fa resistant populations. Other examples showing lack of correlation between Cry toxin binding to BBMV and insect susceptibility were previously reported [As previously reported , we founreported , 42. Alsreported –46.50 value of Cry1AMod toxins to SfBt is comparable to the LC50 value of Cry1Fa to SfLab population. The increase in potency ratio is higher than the resistance ratio of both Cry1AMod toxins indicating that even though that SfBt show cross resistance to Cry1AMod, these Cry1AMod toxins are effective in countering resistance of S. frugiperda to Cry1Fa.The SfBt population showed a significant cross resistant ratio of 3.1 fold to Cry1AbMod and 5.6 fold to Cry1AcMod toxins . It is iS. frugiperda larvae (S. frugiperda molecule involved in Cy1F toxin binding and oligomerization.In the model of the mechanism of action of Cry1A toxins, it was proposed that binding to a membrane bound cadherin receptor facilitates removal of the N-terminal region of the toxin including helix alpha-1 and part of alpha-2 resulting in oligomerization of the toxin . Cry1AMoa larvae . In thisS. frugiperda from other regions of Brazil should be determined in the future. The data presented here showed the rapid evolution of insect resistance to Bt crops in Brazil and is a warning that in the future a proper use/management of Cry toxins would be needed. Also, we showed that the use of Cry1AMod toxins could provide alternative choices to target S. frugiperda and possibly manage resistance to Cry1F.The analysis of resistance mechanisms and the effectiveness of Cry1AMod toxins in other populations of


S2 FigInternal fragments of the p48 , type II restriction endonuclease  and xer1  genes were amplified from M. bovis strain PG45 with appropriate primers and inserted between the NotI and PstI sites of the IRR based oriC plasmid. To promote homologous recombination, the recA gene was amplified from M. gallisepticum strain S6 and cloned between the PstI and SalI cleavage sites of the construct.(PPT)Click here for additional data file.


CRB1 lead to a spectrum of autosomal recessive retinal dystrophies with variable phenotypes suggesting the influence of modifying factors. To establish the contribution of the genetic background to phenotypic variability associated with the rd8/rd8Crb1 mutation, we compared the retinal pathology of rd8/rd8/JCrb1 inbred mice with that of two rd8/rd8Crb1 lines backcrossed with C57BL/6JOlaHsd mice. Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three rd8/rd8Crb1 lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines. Optical coherence tomography, semithin, ultrastructural morphology and assessment of inflammatory and vascular marker by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction revealed that the lesions were associated with photoreceptor death, Müller and microglia activation and telangiectasia-like vascular remodelling—features that were stable in the inbred, variable in the second, but virtually absent in the third rd8/rd8Crb1 line, even at 12 months of age. This suggests that the rd8/rd8Crb1 mutation is necessary, but not sufficient for the development of these degenerative features. By whole-genome SNP analysis of the genotype–phenotype correlation, a candidate region on chromosome 15 was identified. This may carry one or more genetic modifiers for the manifestation of the retinal pathology associated with mutations in Crb1. This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers.Understanding phenotype–genotype correlations in retinal degeneration is a major challenge. Mutations in  Understanding the genotype–phenotype correlation in retinal degenerations remains a major challenge even for monogenetic diseases. Although the causality of many primary mutations has been convincingly established, the clinical manifestation of even identical mutations can be highly variable. This may be due to additional genetic polymorphisms in the same gene  or in additional genes (genetic modifiers) as well as environmental factors. The combination of all these factors in a patient may influence age of onset, progression and severity of disease as well as the development of particular phenotypic features associated with a primary mutation ,2.CRB1 gene (OMIM #604210). They typically cause a spectrum of autosomal recessive (ar) rod-cone dystrophies that ranges from retinitis pigmentosa (RP12)    to Leber OMIM #60210. Theya (RP12) . Both arlopathy) –7. So faCA cases –7. This  disease ,6,8.CRB1 is a member of the highly conserved CRB protein family that in mammals comprises two additional members, CRB2 and CRB3 ,10. WhilIn the retina, CRB1 is part of the CRB/Crb complex, which is localized at the outer limiting membrane (OLM) where it sits just apically to the adhesion junctions (AJ) that connect photoreceptors with each other and with Müller glia cells . The CRBCrb1 alleles exist  that causes a frame shift and premature stop codon resulting in a predicted truncated protein that only consists of the N-terminal extracellular domain  and (2)]. Autofluorescent lesions in C57BL/6 Crd8/rd8rb1 (1) mice were small, weak, reduced in number and located more inferiorly  mice, but with a high degree of variability  mice only small inferior lesions were visible , AF-SLO, optical coherence tomography (OCT) and semithin histology revealed that this pathology was mainly confined to the inferior retina of ice Fig. . White/oice Fig. A–C. Corrble Fig. D that weble Fig. D since tble Fig. E.FigureCrb1rd8 allele, possibly by altered expression of the predicted truncated CRB1 protein . Crb1 transcripts were significantly reduced to ∼20% of wild type in all rd8/rd8Crb1 mice, independent of the severity of their phenotypes . Consistent with these data, immunohistochemistry using a C-terminal antibody for Crb1 revealed a weak specific signal across the whole superior and inferior OLM in wild-type retina  mice is macroscopically similar to that of wild-type mice  mice  lines. Also the expansion of the glial component at the OLM seems similar at the inferior and superior OLM within each line, but different between them  per 10 µm in wild-type mice to 12 ± 3 per 10 µm (range: 7–14) in the superior and inferior retina of –18 per 1rd8/rd8Crb1 mice lead us to further investigate an association between photoreceptor death and Müller and microglia activation  and TUNEL+ photoreceptor nuclei in sections  mice. Without exception, these areas were closely associated with localized activation of microglia  and Müller glia cell . However, no significant differences in expression of chemokine  or microglia activation marker  were detected in retinas of different rd8/rd8Crb1 lines relative to wild type supporting a localized response of microglia cells to these degenerative events . In all these assessments, rd8/rd8C57BL/6 Crb1 (1) animals appeared similar to wild-type mice with the exception of the rare appearance of subtle aneurysm-like changes at the OPL . Three-dimensional (3D) reconstruction of large inferior lesions revealed how activated Müller and microglia cells surround several photoreceptor columns at the OPL  lines showed again the prominent irregular lesions in the inferior retina  line still only revealed very few small or even no inferior lesions   mice at 2 and 12 months of age did not show prominent vascular remodelling or microglia activation apart from a rare manifestation of a few small aneurysms at the OPL  . As earlbed Fig. . In contrd8/rd8/JCrb1 line, and the common ancestry of the rd8/rd8C57BL/6 Crb1 lines (1) and (2), there is a significant variation in the observed phenotype. We reasoned therefore that a modifying gene or genes may have been segregated between the lines. To test this, we first determined whether the different mouse lines carry any known mutant alleles commonly found in inbred mouse strains (rd1Pde6b (c.1041C > A) allele  (Cpfl3Gnat2 (c.598G>A) ], that has been previously shown to be a genetic modifier for retinal degeneration  allele , two mut.518A>G)  and Gnat.598G>A) , and theneration . None ofrd8/rd8/JCrb1 line for which we observed a higher proportion of C3H  SNPs to be present than in the two other rd8/rd8C57BL/6 Crb1 lines (data not shown) suggesting that there are distinct genetic differences between the inbred rd8/rd8/JCrb1 line and the two rd8/rd8C57BL/6 Crb1 lines (1) and (2) that are of common ancestry. Based on this common ancestry, and the distinct phenotypic differences shown by rd8/rd8C57BL/6 Crb1 line (1) and rd8/rd8C57BL/6 Crb1 line (2), we decided to generate preliminary data to help identify regions in the genome that may contain modifying factors. We therefore performed an ‘analysis of extremes’ using DNA SNP screen data from animals that are from either of the two related rd8/rd8C57BL/6 Crb1 lines (1) and (2), but show the most divergent (extremes) of the AF-lesion phenotype. Statistical analysis of this dataset identified a significant association between the genotype on a region of chromosome 15 and the phenotype , whereas mice with a high AF-lesion number are either heterozygous or homozygous for alternative SNPs. These data suggest that this region on chromosome 15 may carry one or several genetic modifiers that determine the manifestation of the phenotype associated with the rd8/rd8Crb1 mutation.To provide further insight into a potential genetic basis for the phenotypic variability, we decided to perform DNA SNP screen analysis for animals from the different lines. The SNP data confirmed the inbred status of the  (1) and  that arerd8/rd8Crb1 mutation and present initial genetic data from SNP screen analyses that suggest the existence of genetic modifiers on chromosome 15 that modulate the manifestation of the rd8/rd8Crb1 phenotype in mice. These findings indicate that this mutation alone is necessary, but not sufficient for the manifestation of the inferior retinal degeneration and specific associated features, which were only observed in two of the three evaluated homozygous rd8/rd8Crb1 lines. This study also highlights how pronounced the influence of small genetic background differences can be on the manifestation of this retinal degeneration and presents initial data for a candidate region that may carry the underlying genetic modifier(s). This is an important issue since this mutation has been found in many mouse strains across the world  occurs. NMD is a mechanism that results in the degradation of mRNA transcripts that carry premature stop codons and serves as a quality control to prevent the expression of deleterious mutant proteins  over multiple generations. Furthermore, our observation that the two closely related rd8/rd8C57BL/6 Crb1 lines (lines 1 and 2), still differ significantly in their phenotype suggests that a few genetic factors or even a single factor may be responsible for the modifying effects in the different lines. By using an ‘analysis of extremes’ on the SNP data obtained from animals with the most divergent phenotypes from the two related rd8/rd8C57BL/6 Crb1 lines (1) and (2), we identified a large region on chromosome 15 that is significantly associated with the phenotype and thus may carry such genetic modifiers.Because of the inferior nature of the degeneration, light was considered to be a good candidate for such an additional initiating factor for this type of retinal degeneration. Yet, this hypothesis was recently excluded by the observation that both, nditions ,20. Sincckground . Our stuC57BL/6 allele, animals with a many lesions are either heterozygous or homozygous for an alternative allele. Therefore, the modifying factor(s) on chromosome 15 may act either as recessive suppressor(s) or as dominant enhancer(s) for the manifestation of the rd8Crb1/rd8 phenotype. However, our data do not allow us to conclude with certainty about the origin of the modifying factor(s) from either of the genetic backgrounds since it could be the result of a spontaneous mutation in any of the alleles. To answer these questions and gain mechanistic insight into the function of these additional genetic factors, a more extensive mapping or QTL project would be required to confirm our initial results and narrow down the genomic region on chromosome 15 which may then result in the identification of the genetic modifying factor(s) for the rd8/rd8Crb1 phenotype.While animals with few retinal lesions are homozygous for one allele, consistent with a Crb1 pathway, or alter processes that define the threshold of Müller glia or microglia responses. A general innate immune activation during disease progression was indicated by the increased accumulation of subretinal macrophages in all rd8/rd8Crb1 mice during later stages. Additional modulating factors that further alter such inflammatory or gliotic responses may lead to an even more permissive inflammatory and pro-angiogenic environment in the retina and may result in vascular remodelling and additional degenerative features. However, the question of why the inferior retina is preferentially affected by these processes remains unresolved. Nevertheless, it is of particular interest that the localized aneurysms- and telangiectasia-like vascular lesions in rd8/rd8Crb1 mice were clinically similar to retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations and that the manifestation of these lesions in humans also seems to be dependent on genetic modifying factors  was compared with two genetically related homozygous rd8/rd8Crb1 lines obtained from a backcross experiment with C57BL/6J Ola Hsd mice, referred to as rd8/rd8C57BL/6J Crb1 (1) and rd8/rd8C57BL/6J Crb1 (2), respectively  amplification with flanking primers and subsequent sequencing  and ketamine (60 mg/kg body weight). Pupils were dilated with 1% tropicamide. Animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the UK Home Office licence (PPL 70/1279).To minimize environmental differences, all lines were housed under the same 12 h/12 h dark/light cycling. During light, animals were exposed to an illuminance of 38 ± 28 lux , when lids, food and water bottles were in place. Outside the cages 200 ± 136 lux (range 50–500 lux) were measured .As described previously, autofluorescent fundus images and OCT images were obtained using the HRA2 scanning laser ophthalmoscope with a 55° angle lens and the Spectralis™ HRA+OCT with a 30° angle lens, respectively  . The optThe severity of the phenotype  and the number of subretinal macrophages  were determined by counting respective lesions on images focussed on the superficial retina for pathology or on the outer retina for subretinal macrophages.m sodium cacodylate–HCl (pH 7.4) and processed for embedding in araldite resin . Semithin (0.7 µm) and ultrathin sections (70 nm) were stained accordingly and imaged by bright field microscopy  or transmission electron microscopy (TEM) with a JEOL 1010 connected to a Gatan Orius CCD camera. Calibrated images were imported into Image J for quantification . After counterstaining with Hoechst 33342  and mounting in fluorescent mounting medium (Dako), images were taken by confocal microscopy . High-resolution Z-stack images were used for 3D reconstruction with Imaris .Tissue was fixed and dissected in 4% PFA, cryoprotected with 20% sucrose and embedded in OCT . Eighteen micrometres of retinal sections or flat mounts were blocked with 1% BSA /5% goat serum  including 3% (flat mounts) or 0.3% (sections) Triton X-100 for permeabilization followed by overnight incubation at 4°C with respective primary and secondary antibodies  per animal were analysed on an SNP array containing 1449 allele-specific probes from across the genome distinguishing m of each primer and 100 nm of the corresponding FAM-labelled probe  followed by cDNA synthesis with the QuantiTect Reverse Transcription kit (Qiagen). For Real Time PCR analysis, 20 μl reactions were prepared in triplicates containing the FastStart TaqMan Probe Master Mix  with 5 µl of template cDNA, 200 nStatistical analyses were performed using GraphPad Prism 5 .HMG onlineSupplementary Material is available at .Department of Health's National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfields Eye Hospital and Alcon Research Institute. J.W.B.B. is supported by an NIHR Research professorship, C.J.C. is a joint MRC and Fight for Sight Clinical Research Training Fellow (G1100383), P.H. is supported by the German Research Foundation (DFG-He 6175/1-1). S.-M.k.H. is partly supported by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 281234, the Medical Research Council (core support at LMCB) and the Batten Disease Family Association, UK. Funding to pay the Open Access publication charges for this article was provided by RCUK.R.R.A. is partly funded by the


Skeletal muscle constitutes approximately 40% of body mass, and age-induced decrease of muscle strength impinge on daily activities and on normal social life in the elderly. Loss of muscle strength has been recognised as a debilitating and life threatening condition also in cachexia in cancer patients and in clinical conditions associated with prolonged bed rest. Skeletal muscle dihydropyridine receptors (Cav1.1) act as Ca2+ channels and voltage sensors to initiate muscle contraction by activating ryanodine receptors, the Ca2+ release channels of the sarcoplasmic reticulum. Cav1.1 activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Cav1.1 and the sarcoplasmic reticulum Ca2+ storage protein calsequestrin (CASQ1).We hypothesized that JP45 and CASQ1 form a signalling pathway which modulates Cav1.1 channel activity.We isolated flexor digitorum brevis (FDB) muscle fibres from JP45 and CASQ1 double knock-out mice (DKO) and tested whether there were differences in Ca2+ homeostasis between the different mouse lines.in vitro and in vivo.Our results show that Ca2+ transients evoked by tetanic stimulation in DKO fibres, result from massive Ca2+ influx due to enhanced Cav1.1 channel activity. This enhanced activity causes an increase of muscle strength both We conclude that skeletal muscle contraction is strengthened through the modulation of Cav1.1 channel activity by JP45 and CASQ1.


ADAM10 inhibition or knockdown enhanced trastuzumab response in naïve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p≤0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p≤0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p≤0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients.Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells  and  The HER  family of receptor tyrosine kinases includes four receptors: EGFR, HER2, HER3, and HER4. Several ligands bind to these receptors including heregulin and betacellulin . Ligand Protein shedding via various ADAMs  is important for cell fate determination, migration, and proliferation . ADAM10 in vivo and in HER2 positive breast cancer patients.We have previously shown that ADAM17 mediates HER receptor activation during trastuzumab treatment . Since ATo assess the effect of trastuzumab treatment on ADAM10 expression, BT474 and SKBR3 cells were treated with two doses of trastuzumab for 24 hours. ADAM10 mRNA levels were increased in a dose-dependent manner: a 3.6–fold increase in BT474 cells and a 2.5-fold in SKBR3 after 40μg/ml trastuzumab treatment, compared to untreated control  (figure in vitro and in vivo.To assess the relevance of this observation in an  vehicle  were sta) figure . Therefototal=8, p≤0.001) (figure We previously established that the PKB inhibition by trastuzumab  increase) figure . We show) figure . CollectThe upregulation of ADAM10 by trastuzumab treatment at 24h in both cell lines occurred with an increase of betacellulin (ligand for EGFR and HER4) in the media figure , correlaWe therefore proceeded to assess the effect of an ADAM10 inhibitor (ADAM10i) in HER2 positive breast cancer cells. Treatment of SKBR3 and BT474 cells with INCB8765 (specific ADAM10 inhibitor) for 24h led to a decrease in the level of basal figures  and trasIn addition, ADAM10i decreased the basal and trastuzumab induced activation of EGFR, HER4, and HER2 in both cell lines figures , S1C. AlTo further prove the role of ADAM10, we silenced ADAM10 using two different siRNAs (and a combination). The knockdown was first optimized and ADAM17 levels were not affected . ADAM10 In view of the upregulation of ADAM10 during trastuzumab treatment in naïve cells, we hypothesized that ADAM10 might be implicated in acquired trastuzumab resistance. ADAM10 mRNA and protein levels were increased in the resistant cells compared to parental cells figure . In addiWe have shown the role of ADAM10 inhibition and knockdown in naïve and resistant HER2 positive breast cancer cells. Previously, we established that ADAM17 inhibition also had an additive effect with trastuzumab treatment . TherefoWe showed that the upregulation of ADAM10 levels occurred during trastuzumab treatment and upon acquired resistance in HER2 positive breast cancer cells. We hypothesized that ADAM10 expression might be relevant as a biomarker to predict prognosis and trastuzumab response in HER2 positive breast cancer patients. Therefore we stained tumors from HER2 positive breast cancer patients and examples are shown in We assessed ADAM10 levels in patients who underwent a window study as outlined in figure We also compared basal ADAM10 levels with those of the tumors obtained at definitive surgery after neoadjuvant chemotherapy and trastuzumab treatment (8 paired samples). There also was an overall trend for increased ADAM10 expression in the post-neoadjuvant treatment samples , which has a stringent quality control in cell authenticity and has incorporated short-tandem repeat (STR) profiling for cell line validation. Cell culturing and the generation of trastuzumab resistant cells were described previously .ADAM10 expression level was scored semi-quantitatively using immunohistochemistry on tumor samples based on staining intensity and distribution using the immunoreactive score of Remmele and Stegner (IRS) . The scoTissue microarrays (TMA) were provided by Oxford Radcliffe Biobank in compliance with Human Tissue Act 2004 (UK). The window study of HER2 positive breast cancer patients was carried out as depicted in figure Prism 6 software (GraphPad software) was used for statistical analysis. The differences between two means were assessed by t-Test. When investigating one factor, one-way ANOVA was used with Bonferroni's multiple comparison. For two factors, two-way ANOVA with Tukey's multiple comparison  was usedFurther details on materials and methods can be found in the


Increased Intra-abdominal pressure is common in critically ill patients  and it mTo assess the role of measuring intra-abdominal pressure in predicting success of spontaneous breathing trial, and whether intra-abdominal pressure can predicit risk of re-intubation in the first 24 hours after extubation.Study included 123 patients who were ventilated due to respiratory failure and were ready for weaning.To be enrolled in the study, patients had to have an improvement or resolution of the underlying cause of acute respiratory failure; adequate gas exchange as indicated by a PaO2 above 60 mm Hg while breathing with an Fi O2 of 0.40 or less with a positive end-expiratory pressure (PEEP) of 5 cm H2O or less; awake patients with a Glasgow Coma Scale score above 13; a temperature below 38° C; hemoglobin level above 10 g/dl; and no further need for vasoactive or sedative agents. In addition, the responsible physician had to agree that the patient was in stable condition and ready to be weaned from the ventilator. To measure IAP 50 ml of sterilized normal saline was inserted into the bladder using the Foley catheter and pressure was measured using the Krons technique. Baseline IAP was measured before SBT (IAP1)and was measured at 15 minutes interval for 60 minutes(IAP2-IAP3-IAP4-IAP5)Patients with BMI more than 30 ,abdominal pathology and baseline intrabdominal hypertension were excluded.Out of123 patients,94 were successfully extubated after 60 minutes versus 29 who failed the SBT. Mean IAP at all time intervals was 7.4cmH20 in those who were successfully extubated versus 8.4 cmh20 in those who failed the SBT (p = 0.013). IAP was lower at all time intervals in patients who were extubated than those who failed  Out of the 94 patients who were successfully extubated, 31 patients were reintubated in the first 24 hours. The mean IAP in patients who were reintubated was 7.4 cmH20 versus 7.3 cmH20 in those who were not re-intubated.Intrabdominal pressure is higher in patients who fail the Spontaneous Breathing trial druing weaning from Mechanical Ventilation and can be used to predicit failure. However, Intra-abdominal pressure was not significantly different in patients who were re-intubated in the first 24 hours after extubation.


Kir7.1 is present in epithelial tissues where it colocalizes with the Na+/K+-pump probably serving to recycle K+ taken up by the pump. Human mutations affecting Kir7.1 are associated with retinal degeneration diseases. We generated a mouse lacking Kir7.1 by ablation of the Kcnj13 gene. Homozygous mutant null mice die hours after birth and show cleft palate and moderate retardation in lung development. Kir7.1 is expressed in the epithelium covering the palatal processes at the time at which palate sealing takes place and our results suggest it might play an essential role in late palatogenesis. Our work also reveals a second unexpected role in the development and the physiology of the respiratory system, where Kir7.1 is expressed in epithelial cells all along the respiratory tree.Kir7.1 is an inwardly rectifying K Kcnj13 gene is a K+ channel belonging to a group of membrane proteins described as K+-transport type channels of the inwardly-rectifying Kir family. Kir7.1 was first described in 1998 and it presents marked differences in sequence as well as functional properties when compared with those of other members of the Kir channel superfamily [+ concentration and more akin to leak-type K+ channels, which is unusual for a Kir family channel whose other members present strong inward rectification properties, that is to say they allow mainly currents associated with K+ influx into cells [Kir7.1 encoded by the to cells .+/K+-pump. Kir7.1 is present at the basolateral membrane of intestinal epithelial cells, thyroid follicular cells and epithelial cells of proximal and distal convoluted tubule [+/K+-pump at the same location, which is the most usual in polarised cell layers. Interestingly Kir7.1 is expressed at the apical membrane of retinal pigmented epithelium (RPE) and in the choroid plexus, epithelia where exceptionally the pump is also expressed apically [+/K+-pump suggests that Kir7.1 could serve the role of K+ recycling needed to keep up with high rates of epithelial to ion transport [Kir7.1 is present in epithelial tissues and shows a remarkable colocalization with the Nad tubule –7. Theseapically ,8–10. Thransport . An addiransport . Also inransport .KCNJ13 gene are associated with retinal diseases snowflake vitreoretinal degeneration (SVD) [Kcnj13 expression in the RPE and shown that those photoreceptors apposing RPE cells lacking, but not those expressing Kir7.1, degenerate. This suggests a role of Kir7.1 in the ionic regulation of the confined space between RPE and photoreceptors possibly in relation to lactate transport, perhaps in the way it has been proposed to explain a similar phenotype in mice deficient in the ClC-2 Cl- channel [Mutations in human on (SVD)  and Lebeon (SVD) ,16. The on (SVD) . Recentlon (SVD)  have gen channel .Kcnj13 gene. Homozygous null mutant mice die hours after birth and show cleft palate and moderate retardation in lung development. Kir7.1 is expressed in the epithelium covering the palatal processes at the time at which palate sealing takes place and our results suggest this channel has an essential role in late palatogenesis. Our work also reveals in addition a second unexpected role in the development and physiology of the respiratory system.In order to study the role of Kir7.1 in epithelial transport we generated a Kir 7.1 deficient mouse by ablating the Kcnj13 gene were generated using the Velocigene method [Kcnj13 gene removes the full coding sequence. Mice with gene-targeted disruption of the Kcnj13 gene encoding Kir7.1 K+ channel , wild-type  and heterozygous (Kcjn13+/-) littermates were used for all comparisons. Heterozygous mice were mated to produce Kcjn13-/-, WT and heterozygous progeny.Mice deficient in the e method  in the CAges of mice analyzed are given as embryonic day (E) or days post conception (dpc), where the presence of vaginal plugs was considered as embryonic day E0.5 or 0.5 dpc. Timed pregnant females (E15.5 to E18.5) were sacrificed by cervical dislocation. Embryos were dissected from the uteri and placed in PBS. Body weight of embryos or newborn pups (P0) was measured in an analytical balance after drying off excess fluid.Kcjn13-/- genotype, and were euthanized first. Mice were individually labelled and decapitated for further study. To obtain new Kcjn13+/- mice, only pups presumed to be of Kcjn13-/- genotype were removed and euthanized. The genotype was confirmed to check for a Mendelian distribution of genotypes.Cages containing pregnant mice were surveyed from 7:00 AM on the computed day of birth (E19.5) and new born pups checked for activity and signs of dehydration. After 2 h the whole litter was removed for study. Pups presenting smaller size, signs of dehydration, failure to suckle or breathing difficult were presumed to be of Kcnj13 exon 2 (5’ATCTTCCCGCTAACCTAT3’), SD rev located in the 3’ untranslated region of the kcnj13 gene (5’ CAGCTTTCTACCCAGGGAGC 3’) and Neo for, located in the selection cassette (5’ TCATTCTCAGTATTGTTTTGCC 3’). Mice were maintained on standard laboratory chow  and water ad libitum, under regulated temperature (22–26°C) and light (12:12-h light-dark cycle) conditions. Housing ad breeding was at the SPF animal facility of the Centro de Estudios Científicos (CECs), Valdivia, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Animal procedures were approved by the CECs Animal Care and Use Committee.Tail snip DNA was used for genotyping by PCR using the following 3 primers: MP08 for, located in 5’-ATTCTTCATCTTCCCGCTAACCTA-3’) and RevEx3Kir7.1 (5’-GATTTCCCCAGTGCCTTCTTG-3’). Cyclophilin A is used as housekeeping gene.For the analysis of Kir7.1 expression, total RNA was isolated from dissected lung and brain from WT, heterozygous and null mutant mice in TRIzol following the manufacturer’s instructions. The RNA was quantified and tested for quality using spectrophotometry and 1% agarose gel. Five μg of total RNA was reverse transcribed at 42°C for 90 min using random primers and ImProm II kir (Promega) to synthesize single-stranded cDNA. The amplicon of Kir7.1 was obtained by PCR using specific primers: For1Ex3Kir7.1  or days post conception (dpc), where the presence of vaginal plugs was considered as embryonic day E0.5 or 0.5 dpc. Birth took place at E19 (P0). Timed pregnant females (E15.5 to E18.5) were sacrificed by cervical dislocation. Embryos were dissected from the uteri and placed in PBS. Body weight of embryos or newborn pups was measured in an analytical balance after drying off excess fluid.Whole embryos (E14.5 to E18.5), P0 dissected tissues or adult tissues were fixed in 4% paraformaldehyde in PBS for 90 min at room temperature. Then, tissues were dehydrated in 30% (vol/vol) sucrose solution for a week, and cryoprotected with Optimal Cutting Temperature Compound . Cryosections (10 μm) were mounted on Superfrost/Plus  slides, and stained with hematoxylin and eosin (H&E) for lung morphometry or reserved for detection of β-galactosidase activity. For immunohistochemistry assays, newborn lungs (P0) or adult mice (2 months old) were fixed in 4% paraformaldehyde in PBS (pH 7.4) overnight at 4°C, washed in PBS, and embedded in paraffin.3Fe(CN)6, 4 mM K4Fe(CN)6, 1.2 mM MgCl2, 0.01% sodium deoxycholate and 0.02% Igepal) overnight at 37°C. Sections were washed with PBS and then counterstained with eosin. Digital images were collected using an Olympus CX31 microscope and recorded with a digital microscope camera (Mshot MD90).The expression of Kir7.1 channel was observed indirectly through the detection of β-galactosidase activity. OCT preserved tissues were cut into 10 μm sections and fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed with PBS and incubated in X-Gal staining solution  were treated with 10% hydrogen peroxide, blocked with 2.5% normal horse serum, and incubated with anti-Kir7.1 [[Ten random representative fields per H&E stained sample were acquired with the Mshot MD90 camera and Olympus CX31 microscope. All images at a magnification of 400X were taken under the same exposure settings. The percentage of terminal sac spaces is the proportion of white surface area of each field relative to the total image area.2O and then 1% KOH for 24 h. Clearing was carried out by consecutive changes of glycerol  in 1% KOH every 24 h and the embryos were stored in 80% glycerol/1% KOH. Littermates were used and the results were obtained from at least three mice per genotype.Newborn mice were sacrificed by intraperitoneal injection of anesthetic and the skin and internal organs were carefully removed. The remaining carcass was fixed in 100% ethanol for 24 hours. Fat was removed with acetone overnight. Staining was carried out with Alcian Blue (0.3% Alcian Blue 8GS) and 0.1% Alizarin Red for 3–4 days at 37°C on a rocker. The tissues were washed with deionized H+ channel we generated Kir7.1 deficient mice by ablation of its codifying Kcnj13 gene by the Velocigene method [Kcnj13-/- mutant mice as expected. Heterozygous Kcnj13+/- mice were indistinguishable from their wild type Kcnj13+/+ litter mates in growth and development. Homozygous mutant mice however failed to suckle, were often cannibalised by their mothers and did not survive beyond P0. Because of extensive cannibalism we were initially under the impression most of the null mice died in utero, but cesarean delivery and genotyping revealed a Mendelian distribution of the three genotypes in embryos generated after crossing heterozygous mice . Analysis of embryonic tissue  reporter that is driven by the activity  in the ec tissue  revealedc tissue .Kcjn13-/- tissue are smaller than in the WT or heterozygous tissue. This impression is borne out by the quantification of terminal sac spaces shown in Kcjn13-/- mice survived for up to 12 h after birth and did not appear cyanotic, suggesting an absence of respiratory distress. This is consistent with similar levels of expression of proSp-C (surfactant protein C) in lungs of Kcnj13+/+ and Kcnj13-/- P0 mice (not shown). Lungs dissected from Kcjn13+/+, Kcjn13+/- and Kcjn13-/- P0 mice floated when placed in saline solution suggesting that normal respiration had taken place  to then progressing horizontally towards the midline above the tongue (~14.5 dpc). Fusion of the palatal processes has already occurred by dpc 15.5 [Kcjn13+/+, Kcjn13+/- and Kcjn13-/- mice. Palatal processes that are evident at 13.5 and 14.5 dpc in WT and heterozygous embryos have disappeared at 15.5 dpc, at which time complete sealing of the palate is evident. This was not the case for Kcjn13-/- embryos that show a delayed horizontal growth from E14.5 and lack of fusion at 15.5 and 16.5 dpc. Kcjn13+/+, Kcjn13+/- and Kcjn13-/- newborn mice, with the latter presenting evident cleft palate. Also shown are preparations to reveal bone and cartilage (blue), where it can be seen that palatine (pp) and maxillary processes (mp) are extended to the midline in Kcjn13+/+ and Kcjn13+/- tissues. In the Kcjn13-/- mouse, these processes were absent thus exposing the vomer (v) and presphenoid (ps) bones to result in a complete cleft secondary palate.Another cause of early postnatal lethality in null mouse models is a developmental craniofacial alteration leading to cleft palate  and a reelopment . Kcjn13dpc 15.5 . Fig 5a Kcjn13+/+ and Kcjn13-/- embryos. Kcjn13+/+ and Kcjn13-/-, and already fused at E15.5 in the WT but not in the null mutant mouse. The sites of Ki7.1 expression were identified by β-galactosidase staining of Kcjn13-/- mice. Kir7.1 expression is absent at 14.5 dpc, a stage at which robust expression is already present in the choroid plexus (see inset). Kir 7.1 expression is evident at E15.5 in the respiratory epithelium as well as that covering the palatal processes but here only in the nasal side. The same is seen at E18.5, where the higher magnification picture corroborates that Kir7.1 expression stops towards the tip of the palatal process. Kcjn13+/- tissue. Expression is also present in the respiratory and olphactory epithelia after birth as seen in the Kcjn13-/- section.We undertook a histological examination of the developing palate in Kcjn13 null mutant mice suffer from early postnatal mortality with pups not surviving further than P0. Our initial impression that Kcjn13-/- mice might not experience normal in utero development were dispelled by examining the distribution of phenotypes of 11.5–18.5 dpc foetuses of pregnant Kcjn13+/- females that had been crossed with heterozygous Kcjn13+/- males. The distribution of genotypes was not significantly different from the expected Mendelian distribution of 25% Kcjn13+/-/50% Kcjn13+/+/25% Kcjn13-/-. The process of generation of Kcjn13-/- mouse results in the insertion of a DNA coding a β-galactosidase whose activity is now under the control of the Kcjn13 promoter and therefore reports the channel tissue distribution. Sites of expression of Kir7.1 thus identified included the brain, meninges and choroid plexus; in the eye the retinal pigment epithelium; the gall bladder, and the cricoid cartilage; the small intestine and the thyroid gland. Most of these locations have been identified before as sites of expression of Kir7.1 [Kcnj13 null mutants and heterozygous and WT mice, with the terminal sac spaces in tissue from the Kcnj13-/- mice significantly smaller from E18.5 through to P0. We do not think, however, that this difference can lead to a lethal phenotype as the surviving P0 mice were able of breathing and were not cyanotic, and lungs had inflated as attested by flotation tests.Spurred by the frequent occurrence of respiratory distress as a cause of perinatal lethality, we looked for Kir7.1 expression in the respiratory system. There is extensive expression of the channel in the respiratory tree, including the epithelia of the trachea, bronchioles and alveoli, where Kir7.1 appears to be associated with type II pneumocytes. Expression of Kir7.1 in the respiratory system arises at E16.5. There was some differences in the developing lung between + channel ENaC whilst secretion relies on Cl- channels such as CFTR or Ca2+-activated TMEM-16 Cl- channels. In both cases the activity of basolateral Na+/K+ pump is also essential, as is the presence of K+ channels required for K+ recycling and to maintain a negative membrane potential compatible with continued transport of Na+ or Cl- [+ channels have also been identified in the alveolar and airway epithelia [What might be the reason for this retardation at the saccular development phase or indeed the function of the Kir7.1 channels in adult respiratory epithelium? It is well known that the transport of ions and fluid across all parts of the respiratory system is vital for gas exchange function. Mucociliary clearance is a key element in normal lung innate immune system function. Clearing the airways of particles including pathogenic organisms occurs through ciliary beat and ion transport and its associated water flow into and from the fluid layer covering all portions of the respiratory tract. The electrolyte transport systems that control the airway fluid layer include absorptive as well as secretory functions. Absorption requires the presence of apically located epithelial Na+ or Cl- ,24. Kir7pithelia  suggestiKcnj13-/- mice we can only conjecture that this might be due to a requirement of the Kir7.1 channel in some aspect of the process of expansion of the intra-sac spaces. Fluid secretion is thought to play a role in lumen expansion in vertebrate organogenesis driving expansion of nascent lumens to form a single tube or a sac with CFTR and Na+/K+-pump being often central drivers of the process [Kcnj13-/- mice.As to the retardation in saccular expansion noticed in  process . There i process . It is t+ channels have been proposed to fulfil diverse functions in lung and airway physiology and unravelling the precise role of Kir7.1 will be a daunting task. In a recent review that summarises the evidence for the presence of more than 40 transcripts for K+ channels in airway and alveolar epithelium, Bardou et al. {Bardou, 2009 7315 /id} use the following understatement: “The physiological and functional significance of this high molecular diversity of lung epithelial K+ channels is intriguing”. This diversity includes members of all major classes of K+ channels: voltage-dependent Kv channels, leak two-pore domain K2P and inwardly rectifying Kir channels, and functions as diverse as gas exchange and alveolar stability, inflammatory responses, epithelial repair after injury, control of transepithelial fluid and ion transport, Cl- secretion and Na+ absorption. Kir7.1 might contribute to one or more of these functions, perhaps in conjunction with other K+ channels. Discovering its contribution to lung and airway physiology will call for more tissue/cell-directed animal modification to allow the study of Kir7.1 avoiding the lethality of the total gene inactivation presented here.KKcnj13-/- mice. Detailed observation revealed that these mice suffered from complete secondary cleft palate, which is the most probable cause of their early postnatal death [Kcnj13 null mutant embryos. Despite the fact that processes do make midline contact at 14.5 dpc in Kcnj13-/- embryos, fusion has not occurred at 15.5 dpc as it does in WT embryos.Examination of head morphology suggested some sort of subtle craneo-facial malformation in al death . PalatogCleft palate is one of the most common birth defects in humans, it includes syndromic and nonsyndromic forms and is influenced by genetic and environmental factors –30. A grDrosophila as a model, these authors show that the fly homologue of Kir2.1 affects development perhaps by interfering with Drosophila decapentaplegic (Dpp) signalling. As Dpp is considered analogous mammalian BMP, they propose that an effect of Kir2.1 activity on the BMP pathway could be responsible for the morphological defects in the Kir2.1 deficient mouse.Mice deficient in the inwardly rectifying Kir2.1 channel die postnatally as a consequence of a complete cleft of the secondary palate . Interes+ fluxes in the palatal shelf epithelium, might be a triggering element in palate sealing. It will be interesting to explore further this possibility perhaps in rescue experiments in vitro. Effects of K+ channels not involving their permeation properties have also been seen to play a role in cancer cell migration [+ channels have often been invoked as central participants in cell migration, proliferation and differentiation processes [The expression of Kir7.1 coincides with the moment of palatal process fusion, i.e. between dpc 14.5 and 15.5. Interestingly it appears that the channel is expressed by epithelial cells covering the dorsal aspect only of the palatal shelves and it might be absent from the point of contact between opposite between processes itself. This contrasts with the localization of TGF-β 3 that is present in the MEE edge and the tip epithelium of the nasal septum . It is cigration  and thisrocesses , and anyAlthough we lack mechanistic explanations for the effect of Kir7.1 inactivation on palatal formation and on lung development, we provide firm evidence of its involvement in these processes. We hope that this evidence will spur new work that might contribute to our understanding of these important biological processes.