Prospective and case-control studies which reported adjusted relative risk estimates and 95% confidence intervals of endometrial cancer associated with a hypertension diagnosis were included. Summary relative risks were estimated using a random effects model. Nineteen case-control studies and 6 cohort studies were included. The summary RR was 1.61  for all studies, 1.73  for case-control studies and 1.32  for cohort studies. The association between hypertension and endometrial cancer was weaker, but still significant, among studies with adjustment for smoking, BMI, oral contraceptive use, and parity, compared to studies without such adjustment. This meta-analysis suggest an increased risk of endometrial cancer among patients with hypertension, however, further studies with more comprehensive adjustments for confounders are warranted to clarify the association.A history of hypertension has been associated with increased risk of endometrial cancer in several studies, but the results have not been consistent. We conducted a systematic review and meta-analysis of case-control and cohort studies to clarify the association between hypertension and endometrial cancer risk. PubMed and Embase databases were searched up to 27 Hypertension is a major cause of morbidity and mortality worldwide and is an established risk factor for coronary heart disease and stroke158910Endometrial cancer is the eighth most common type of cancer in women with approximately 320 000 cases recorded in 2012, accounting for about 4.8% of all cancers in women 1420212223242526272829303132333435363739404142432021242526283032333435363738394222232729313942451620212533352021242528293335th February 2016 for eligible studies. We used the following search terms in the PubMed search: (hypertension OR high blood pressure OR blood pressure OR risk factor) AND . We followed standard criteria for reporting meta-analysesWe searched the PubMed and Embase databases up to 27We included published retrospective case-control studies and cohort studies that investigated the association between hypertension and the risk of endometrial cancer. Adjusted estimates of the relative risk  had to be available with the 95% CIs in the publication. A list of excluded studies and exclusion reasons is provided in The following data were extracted from each study: The first author’s last name, publication year, country where the study was conducted, study period, sample size, number of cases/controls, exposure and subgroups of tumor characteristics  or cancer type (type 1 vs. type 2), relative risks and 95% confidence intervals for the association and variables adjusted for in the analysis. Data were extracted by one reviewer (DA) and checked for accuracy by a second reviewer (AS).We calculated summary relative risks of developing endometrial cancer by history of hypertension using the random-effects model by DerSimonian and Laird2 statistics2 is a measure of how much of the heterogeneity that is due to between study variation rather than chance. I2-values of 25%, 50% and 75% indicates low, moderate and high heterogeneity respectively. We conducted main analyses  and stratified by study design  because of the greater potential for recall and selection bias in retrospective case-control studies and to investigate sources of potential heterogeneity. We also conducted subgroup analyses by other study characteristics such as sample size, number of cases, geographic location, and by adjustment for confounding factors. We also conducted a stratified analysis by whether the articles explicitly stated that participants with prevalent hysterectomies at baseline were excluded, and/or whether participants with incident hysterectomies were censored during follow-up in cohort studies, or excluded from the control group in case-control studies.Heterogeneity between studies was evaluated using Q and IPublication bias was assessed using Egger’s testOut of a total 7879 records identified by the search we included 25 studies with 28385 cases and 300598 participants in the meta-analysis of hypertension and endometrial cancer risk, including six cohort studies394041424320212223242526272829303132333435362 = 86%), and it was 1.73  for case-control studies and 1.32  for cohort studies  for studies with such exclusions or censoring and 1.81  for studies without such exclusions or censoring.In a further sensitivity analysis we also conducted a subgroup analysis by whether the studies explicitly stated that they excluded participants with prevalent hysterectomies at baseline and/or stated that they censored participants at the time of incident hysterectomy (cohort studies) or excluded participants who had undergone hysterectomy from the control group (case-control studies). The summary RR was 1.51  for all studies and 1.33  for cohort studies. Further including another cohort study2 = 85%) for all studies and summary RR = 1.28  for cohort studies. Mean (median) study quality scores were 7.3 (7.0) for all studies combined, 7.3 (7.0) for case-control studies, and 7.3 (7.0) for cohort studies).In a sensitivity analysis we also included a pooled analysis which assessed the association between quintiles of systolic blood pressure and endometrial cancer riskTo our knowledge this is the first meta-analysis of published observational studies of hypertension and the risk of endometrial cancer and our results confirm that hypertension is a strong risk factor for endometrial cancer with a 61% increase in the relative risk, however, the association was weaker in cohort studies (RR = 1.32) than among case-control studies (RR = 1.73). These findings are consistent with a large cohort study of 290 000 women in Austria, Norway and Sweden which found an increased endometrial cancer risk with increasing levels of diastolic blood pressure and in particular, systolic blood pressureThe present meta-analysis has some limitations. As hypertension is a condition that is strongly related to lifestyle factors and some medical conditions including diet, BMI, physical activity, and diabetes we cannot entirely exclude the possibility that the observed association between hypertension and endometrial cancer risk at least partly could be due to confounding. We found that the association was weaker, but still statistically significant, among studies that adjusted for smoking, BMI, oral contraceptive use, hormone replacement use, parity and age at menopause (RR = 1.14–1.34 for studies with such adjustment vs. 1.74–2.10 for studies without such adjustment). However, because there was still a significant association in subgroups that adjusted for these factors it could indicate that there is an adverse effect of hypertension on endometrial cancer risk, but that it may be slightly weaker than what was suggested from the overall summary estimates. Because the original studies did not stratify for BMI or diabetes it was not possible for us to investigate whether the association was limited to specific weight classes or if it was modified by diabetes status.We also found that the positive association between hypertension and endometrial cancer persisted when the studies were stratified by whether participants with prevalent hysterectomies at baseline were excluded and/or whether participants with incident hysterectomies were censored, or whether prevalent hysterectomies were excluded from the control group. Hypertension may also be related to hysterectomies5657Case-control studies are more likely to be affected by certain biases, such as recall bias and selection bias. Because we included both case-control and cohort studies there is a possibility that recall or selection bias might have affected the results in the case-control studies and the overall summary estimate. Although the association appeared to be stronger in case-control studies than among cohort studies, there was still a significant association among cohort studies, which suggest that recall bias or selection biases does not entirely explain the observed association. In addition, there was some indication of publication bias with Egger’s test, but this appeared to be restricted to the analyses of case-control studies and all studies combined, and was not observed among the cohort studies.The biological mechanism(s) that may explain an adverse effect of hypertension on endometrial cancer risk are unclear at present. It has been suggested that long-term hypertension may lead to cellular senescence and inhibition of apoptosisStrengths of the present meta-analysis include the comprehensive search strategy, the detailed subgroup and sensitivity analyses, and the large sample size providing a more robust estimate of the association between hypertension and endometrial cancer risk. To date relatively few studies have investigated the association between hypertensive disorders of pregnancy and endometrial cancer risk with one study suggesting an increased risk with hypertensive disorders overall66In conclusion, the results from this systematic review and meta-analysis suggest that women with hypertension may have a 61% increase in the relative risk of developing endometrial cancer. Any further studies should clarify potential effect modification by age, BMI and diabetes status, and the causality of the observed association, as well as the potential underlying mechanism(s).How to cite this article: Aune, D. et al. Hypertension and the risk of endometrial cancer: a systematic review and meta-analysis of case-control and cohort studies. Sci. Rep.7, 44808; doi: 10.1038/srep44808 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Multiple environmental stresses affect growth and development of plants. Plants try to adapt under these unfavorable condition through various evolutionary mechanisms like physiological and biochemical alterations connecting various network of regulatory processes. Transcription factors (TFs) like APETALA2/ETHYLENE RESPONSE FACTORS (AP2/ERFs) are an integral component of these signaling cascades because they regulate expression of a wide variety of down stream target genes related to stress response and development through different mechanism. This downstream regulation of transcript does not always positively or beneficially affect the plant but also they display some developmental defects like senescence and reduced growth under normal condition or sensitivity to stress condition. Therefore, tight auto/cross regulation of these TFs at transcriptional, translational and domain level is crucial to understand. The present manuscript discuss the multiple regulation and advantage of plasticity and specificity of these family of TFs to a wide or single downstream target(s) respectively. We have also discussed the concern which comes with the unwanted associated traits, which could only be averted by further study and exploration of these AP2/ERFs. Plants are mostly affected by multiple stresses like salinity, toxicity, pathogen attack, and wounding. To survive and reproduce in adverse conditions, plants develop various adaptive traits governed through a complicated system. The interconnected and diverse networks operated during downstream signaling cascades are regulated by TFs which bind to the cis elements present in their promoters , Trichodesmium erythraeum (cyanobacterium), Enterobacteria phage RB49 (virus), and Bacteriophage Felix 01 (Virus) are reported to have an AP2 domain , DREB (subgroup A1–A6), ERF (subgroup B1-B6), and others  from Poppy which bind to both DRE as well as GCC box elements thus provide abiotic and biotic stress tolerance in tobacco , ICEr2 and CM2-Conserved DNA Motif 2  to which a bHLH PIF7 (Phytochrome Interacting Factor) interacts and suppresses expression of DREB1C. PIF7 interacts with TOC1 (Timing of CAB expression 1) and PhyB (phytochrome B) to transcriptionally repress the expression  and LHY (Late Elongated Hypocotyl) that bind to the EE (AAAATATCT) and CCA1 (AATCT) motifs of DREB1C/CBF2 promoters and positively regulate their expression . ORCA3 has a regulatory role in primary and secondary metabolism in Catharanthus roseus, therefore it needs to be properly regulated at the transcriptional level. Its promoter carries a 74-bp long autonomous Jasmonate Responsive Element (JRE) having a quantitative sequence responsible for enhanced expression and a qualitative sequence that acts as an on/off switch in response to MeJA. CrMYC2 binds to T/G-box (CACGTG–G box with one mismatch) present in the qualitative sequence of JRE and mediate expression of ORCA3 that in turn regulate early jasmonate-responsive genes  alters its binding affinity to DRE/CRT cis-element  which are then recognized and degraded by N recognins ATE1/2 (Arginyl tRNA Protein Transferases) & PRT6 (Proteolysis 6) as shown in Figure Ubiquitination is another post-translational mechanism through which AP2/ERFs are regulated. It may affect proteins in number of ways, either it process them for proteasomal-mediated degradation, or change cellular location, or alter their activity or enhance/prevent interaction of protein. Adding to the list of tight regulation of DREBs is the ubiquitination by DRIP1/2 (DREB2a-Interacting Protein1) under non-stressed condition. These are RING E3 ligases that interact with DREB2A in the nucleus and leads to its ubiquitination in the cytosol  while some are involved in development (OsERF3 and WOX11 interact with cytokinin-responsive gene RR2 to regulate crown root development in rice)  and AREB2/ABF4 proteins to regulate ABA response  adjacent to DBD that makes DREB2A unstable under normal condition. Deletion of NRD converts DREB2A into its constitutively active form whose overexpression displays reduced growth but improved drought and salt tolerance , GmERF6 (enhanced drought tolerance), and NtERF3 (increased resistance to TMV) positively regulates stress response while AtERF7  negatively regulates stress response  in the alternative respiratory pathway and is involved in seed germination and seedling growth  gene that encodes an isoform of the cytochrome c oxidase zinc binding subunit involved in respiratory chain complex  and Sub1C from rice are induced by ethylene but acts antagonistically to each other in response to flooding  and provide enhanced cold and salt stress tolerance  involved in BR biosynthesis. DWF1/4 leads to accumulation of DELLA protein such as SLR1 (Slender Rice 1) and SLRL1 (SLR1 like) which inhibits GA responsiveness  BR (brassinosteroid) also affect ERF mediated regulation. Hypoxia responsive HRE1 and HRE2 show ethylene response, but unlike RAP2.2 they lead to inhibition of ethylene signaling and provide GA response  gene was found to play an important role in regulating determinacy of spikelet meristem and floral organ identity. MFS1 is associated with unidentified function clade in the family of AP2/ERF which certainly regulates the expression pattern of Long Sterile Lemma (LSL) and the Indeterminate Spikelet1 (IDS1) like genes Supernumerary Bract and OsIDS1 , 10 subgroups, and two soloists  transgenic wheat showed reduced electrolyte leakage and H2O2 content, while increased soluble sugar and proline content , Chit1 (Chitinase 1), RAB18 (ras-related protein 18), LEA3 (Late embryogenesis abundant 3), TIP2 (Delta tonoplast intrinsic protein 2), POX2 (Peroxidase 2), and GST6 (Glutathione S-Transferase 6) while could not bind with DRE cis-element  and displayed better tolerance to bacterial pathogen Ralstonia solanacearum. Constitutive expression of SlRAV2 in tomato also led to enhanced tolerance to R. solanacearum bacterial wilt and increased expression of SlERF5 and PR5 genes  and the zinc finger oxidative stress 2 are predicted targets of this ERF  1long-chain acyl-CoA synthetase1, PAS2 (Pasticcino2), KCR1 (β-ketoacyl CoA reductase1), ECR , and WSD1 . Wax layer in turn protects plants from high irradiance, desiccation and UV radiation  genes  and post-translational level . Therefore, these aspects need to be exploited at a large level so that specific targets in the complex mechanism could be identified, which could be modulated for development of improved varieties.cis-elements present in the promoter of that gene. We believe that this specificity to identify a single or multiple cis-elements is regulated not only by the rare anomalies in the DBD but also by the motifs flanking it. These bindings are also influenced by protein-protein interactions through different domains. So these domains and motifs, which lead to specificity or plasticity in the interaction of AP2/ERFs with their downstream targets to achieve multiple responses, should be thoroughly studied.AP2/ERFs activate or repress expression of a particular gene by interacting specifically to the Also with recent findings, it is evident that retrograde signaling affects several AP2/ERF-mediated responses. Signaling from mitochondria or chloroplast to the nucleus and its feedback is not properly understood. Therefore, new insight is required in the field of retrograde regulation of AP2/ERFs.AP2/ERFs from different plant species.Recent molecular tools like CRISPR  and CRISPR-associated 9 (Cas9) genes could be applied to different non-model plants where functional mutants are not available. CRISPR/cas9 genome editing tool would have an edge over conventional tedious silencing approach (through pART vectors) or VIGS (through TRV vectors) in the coming era. Therefore, this technique could be exploited for functional characterization of UP and GJ has compiled and written the review. VT and RS has edited and finalized the draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MgF2 conversion layer was first produced by immersing Mg alloy substrate in hydrofluoric acid solution. The outer PCL coating was then prepared using dip coating technique. Morphology, elements profile, phase structure, roughness, mechanical properties, invitro corrosion, and biocompatibility of duplex MgF2/PCL coating were then characterized and compared to those of fluoride coated and uncoated Mg samples. The invivo degradation behavior and biocompatibility of duplex MgF2/PCL coating with respect to ZK60 Mg alloy were also studied using rabbit model for 2 weeks. SEM and TEM analysis showed that the duplex coating was uniform and comprised of porous PCL film (~3.3 μm) as upper layer with compact MgF2 (~2.2 μm) as inner layer. No significant change in microhardness was found on duplex coating compared with uncoated ZK60 Mg alloy. The duplex coating showed improved invitro corrosion resistance than single layered MgF2 or uncoated alloy samples. The duplex coating also resulted in better cell viability, cell adhesion, and cell proliferation compared to fluoride coated or uncoated alloy. Preliminary invivo studies indicated that duplex MgF2/PCL coating reduced the degradation rate of ZK60 Mg alloy and exhibited good biocompatibility. These results suggested that duplex MgF2/PCL coating on magnesium alloy might have great potential for orthopedic applications.The present work addresses the performance of polycaprolactone (PCL) coating on fluoride treated (MgF Such closely matched mechanical properties can minimize stress shielding effects caused by difference in elastic modulus between foreign implant and host hard tissue -2,5-diphenyltetrazolium bromide  solution (100 μl/well) was then added and incubated at 37°C for 4 hr. At the end, 100 μl of dimethyl sulfoxide  solution was added to each well. The sample plate was incubated for 1 hr and the absorbance value or the optical density (OD) of the resulting purple colored solution was measured by using an ELISA plate reader  at wavelength 595 nm.MC3T3-E1 preosteoblast cells  were employed in the 5 cells/well were seeded direct onto sterilized MgF2 and MgF2/PCL samples in 24 well plates. These samples were incubated at 37°C with 5% CO2 atmosphere for 1, 3, and 5 days. After a stipulated time, samples were washed thrice with PBS solution and proceed to fixing with paraformaldehyde  for 15 min at room temperature. The cells were later permeabilized with 0.5% Triton X-100  for 10 min. After subsequent blocking with 2.5% bovine serum albumin  solution for 60 min, cell membrane was immunostained with fluorescein isothiocyanate conjugated phalloidin  solution overnight at 4°C. Samples were later stained with 10 μg/ml Hoechst 33342  for 5 min for staining cell nuclei. The stained samples were finally washed with PBS solution thrice, mounted onto slides, and visualized under confocal fluorescent microscope  along with the accompanied FV10i-ASW2.0 viewer software.For cell proliferation studies, 2x105) seeded samples were incubated for 4 hr in humidified atmosphere with 5% CO2 at 37°C. Following the same procedure steps until BSA blocking as mentioned above for proliferation, the cells were further immunostained using vinculin antibody  and placed overnight at 4°C, followed by washing thrice with PBS. Afterwards, samples were incubated with Alexa Flour 594  conjugated secondary antibody for 1 hr at room temperature. At the end, cells were stained with FITC conjugated phalloidin and Hoechst 33342 at the specified interval, mounted and analyzed by confocal microscope.For cell adhesion studies, cell . Fur on the right knee of the rabbit was shaved, the exposed skin was thoroughly cleaned with 70% ethanol and disinfected with povidone iodine solution. A full thickness incision measuring 3–4 cm in length was made along the lateral aspect of the knee to access the underlying joint. The distal femoral head was accessed by creating another incision on the overlaying soft tissues. The exposed distal femoral head was then drilled with a trephine drill with a 6 mm diameter at a height of 5 mm with constant irrigation with sterile saline solution. The implant  was then carefully placed within the bone defect. Control defect was left without any implant material. Three rabbits were used for each sample. All incisions were then closed with 0–5 Vicryl suture and disinfected with povidone iodine solution. Tramadol and Baytril were administered based on recommended doses to manage pain and as prophylaxis respectively. After 2 weeks, the animals were sacrificed via overdose of ethyl ether anesthetic. Tissue samples containing the implants were extracted and fixed using 10% formaldehyde solution. Extracted samples were then scanned with Skyscan 1076 microCT scanner . Data scans were reconstructed and analyzed to determine the progression of implant biodegradation and tissue development. Using CTAnalyzer software , the implanted sample was selected using a series of region of interest to create a volumetric selection. The data set was then segmented through two ranges of gray intensities to separate the implant and the surrounding bone tissue. Volume of the remaining sample was calculated using the same program. Three-dimensional reconstruction was created using the same corresponding data set using CTVox software .To determine the effect of the duplex MgF2/PCL were then dehydrated using subsequent series of ethanol then acetone immersion for 1 day each. After dehydration, the samples were infiltrated and embedded in methyl methacrylate (MMA) resin using benzoyl peroxide as a hardener. MMA embedded blocks were transversally cut using a diamond abrasive cutter  and were polished to 10–45 μm thickness. The slides were washed with distilled water and finally stained with Villanueva Osteochrome Bone stain. Micrographs of the stained tissue sections taken using an optical microscope  equipped with a DP72 digital camera.The fixed tissues ZK60 Mg alloy, duplex MgF2, and duplex MgF2/PCL coatings are shown in 2/PCL coating was found to be uniform and crack-free  and zinc (Zn) elements were present in ZK60 Mg alloy with minute impurities of carbon (C) and oxygen (O) (2 film on the substrate. The oxygen content might be attributed to the formation of Mg(OH)2. Minute traces of carbon are also seen in the film  . Quantitthe film . Small cthe film . The pre surface .2/PCL coating was investigated by TEM micrograph along with selected area electron diffraction (SAD) patterns and mapping profile. Results are shown in 2, and Mg substrate. The thickness of compact MgF2 film was approximately 2.2 μm and that of outer PCL coating was around 3.3 μm . The amorphous nature of PCL coating contributed to the diffused ring observed in SAD pattern  peak with tetragonal structure was primarily found along with Mg. However, the intensity of MgF2 peak was quite low compared to that of Mg, suggesting that the fluoride coating layer was thin as depth of CuKα line for Mg is around 0.8 μm at grazing angle of 1°C [2 by displacement reaction and insoluble MgF2 can form a barrier coating on Mg [2 in MgF2/PCL coatings  coating was significantly increased to 66.3 nm. Minute pores generated on the surface due to acid immersion might be ascribed to the above increment. The surface morphology was described by the precipitation of intermetallic particles (MgZn) on the surface and at grain boundaries  studied that PCL film exhibited poor bonding strength with substrate which might be due to its molecular structure. PCL possess lower ratio of oxygen by its weight, which leads to weak electrostatic interaction with Mg substrate [2 coating. The relatively increased adhesion of MgF2/PCL coatings over PCL coatings was more likely due to the mechanical interlocking as PCL solution was infused into MgF2 film sealing the micro pores and thus forms a stronger bonding layer with MgF2 film. In addition, the higher interfacial adhesion strength of MgF2 film with the PCL might be due to the formation of hydrogen bonding between -OH group present in fluoride treated surface and = O group of PCL [2 film showed the existence of O along with F elements as evident from EDX  degrades to acids [2 could be possibly due to more rigorous destruction of MgF2 coating than that of duplex MgF2/PCL coating. Hence, Mg substrate with MgF2 coating was more degraded compared to that with MgF2/PCL coating at later stage. The reduction in long-term protective ability of MgF2 coating might be due to the existence of micro-cracks in the coating evident in SEM micrographs shown in 2/PCL coating could effectively prevent the penetration of immersion fluids  protection on the ZK60 Mg alloy which retards the corrosion rate. ZK60 Mg alloy exhibited the highest weight loss rate among other samples. An increasing trend in rate of weight loss was noticed for all the samples with increase in exposure time. The % weight loss of coated MgF2 and MgF2/PCL samples were about 13.6% and 4.5%, respectively compared to ZK60 Mg alloy (21.2%) after 14 days of immersion. The continuous increment in weight loss of coated samples with prolonged exposure time indicated the delamination of the coating layers on Mg substrate. It signified that both MgF2 and MgF2/PCL coatings were not providing adequate protection for longer immersion time. In addition, it was found that the corrosion rate was more accelerated in the initial 3 days of exposure compared to prolonged immersion time (14 days) for ZK60 Mg alloy  2 into soluble MgCl2 (H2) (Eq ). Hence,gCl2 (Eq ). Hence,EM image . Also, t2, and MgF2/PCL coating after immersion in PBS solution for 14 days along with EDX are shown in 2, showed network-like cracks with small precipitates evenly distributed over the entire surface of the alloy. The thicker corroded layer might be ascribed to the presence of micro-cracks in the coating caused by the shrinking of surface corrosion product during drying. However, a few white precipitates were observed on duplex MgF2/PCL coated surface. EDX analysis of corroded surface compositions for different samples showed the presence of O, C, Na, K, Cl, and P elements. The existence of elements suggested the formation of MgCl2, Mg(OH)2 and other Na and K rich compounds in the corrosion product. Therefore, contents of these elements on the surface after immersion can be used to estimate the degree of corrosion occurred in each sample. It was observed that ZK60 Mg alloy had much more corrosion products on its surface than MgF2 or MgF2/PCL coated sample. Duplex coated surface showed significantly lower concentrations of P and O with the absence of K or Na compared to ZK60 Mg alloy. The lower concentration of Mg and higher concentration of C in MgF2/PCL coated sample indicated that PCL coating was still present after 14 days of immersion. However, in case of MgF2, traces of Na and P with relatively higher concentration of Mg depicted the destruction of fluoride coating on Mg substrate (2/PCL coated sample after 14 days of immersion. The cracks in MgF2 coating can provide a suitable path for penetration of PBS solution into the coating (2 is more stable than MgO and Mg(OH)2 and efficient in resisting corrosion attack [2/PCL coating systems, outer PCL layer retard the corrosion attack while the inner MgF2 layer prevent the ZK60 Mg alloy substrate from aggressive medium when it crosses from the outer PCL layer. These results suggested that duplex MgF2/PCL coated sample exhibited higher corrosion resistance compared to MgF2 coating or ZK60 Mg alloy.SEM images of surfaces of ZK60 Mg alloy, MgFubstrate . SEM ima coating . Hence,  coating . All corn attack . Hence, 2 and MgF2/PCL coatings after 4 hr of incubation is shown in 2/PCL coatings exhibited higher cell attachment and more spreading than MgF2 coating.The degree of proliferation and adhesion of host cells on the substrate specifies its biocompatibility . Cell ad2 and MgF2/PCL coatings using indirect cell assay with increasing extract concentrations after 1 day of incubation are shown in 2 and 95% to 87% for MgF2/PCL coatings. MgF2/PCL coatings showed higher cell viability than MgF2 coating at all extract concentrations.Results of cell viability studies of MgF2 and MgF2/PCL coating were examined by confocal imaging  shows gradual reduction of the inflammatory phase after 2 weeks , ZK60 Mg alloy (12.34±1.99 mm3) and duplex MgF2/PCL coating (13.56±0.47 mm3) within 2 weeks  and MgF2 film (~2.2 μm) onto ZK60 Mg alloy. Duplex MgF2/PCL coatings showed better adhesion strength than PCL coatings. No significant change was found in the microhardness of duplex MgF2/PCL coating when compared to ZK60 Mg alloy. Hydrogen evaluation, pH change, and degradation tests showed that the duplex protective layer of MgF2/PCL exhibited an enhancement in corrosion resistance compared to single layered MgF2 or ZK60 Mg alloy. The barrier properties of uniform PCL film along with compact MgF2 film contributed to lowest corrosion rate. Such duplex MgF2/PCL coating also supported cell viability, cell adhesion and proliferation better than MgF2 coated and uncoated alloy sample. Invivo studies indicated that duplex MgF2/PCL coating reduced the degradation rate of ZK60 Mg alloy and exhibited good biocompatibility for 2 weeks of implantation. These results indicated that outer PCL coating might be a promising approach after fluoride treatment. However, long-term invivo studies are required to further validate the application of duplex coated implant for orthopaedic applications.Duplex MgF


Although numerous studies explore the effects of emotion on decision-making, the existing research has mainly focused on the influence of intrapersonal emotions, leaving the influence of one person’s emotions on another’s decisions underestimated. To specify how interpersonal emotions shape decision-making and delineate the underlying neural dynamics involved, the present study examined brain responses to utilitarian feedback combined with angry or happy faces in competitive and cooperative contexts. Behavioral results showed that participants responded slower following losses than wins when competitors express happiness but responded faster following losses than wins when cooperators express anger. Importantly, angry faces in competitive context reversed the differentiation pattern of feedback-related negativity (FRN) between losses and wins and diminished the difference between losses and wins on both P300 and theta power, but only diminished the difference on FRN between losses and wins in cooperative context. However, when partner displays happiness, losses versus wins elicited larger FRN and theta power in competitive context but smaller P300 in both contexts. These results suggest that interpersonal emotions shape decisions during both automatic motivational salience valuation (FRN) and conscious cognitive appraisal (P300) stages of processing, in which different emotional expressions exert interpersonal influence through different routes. How would you react to a competitor or a cooperator who displayed happy expressions when you lost money in a poker game? Would you trust a salesperson who smiles at you, or give money to a grumpy beggar muttering angry phrases? While such decisions are routinely made, little is known about how emotional expressions shape interpersonal decisions. Although contemporary scholars show increasing interest in identifying the effects of emotion on decision making, current efforts mainly focus on how individuals’ judgments and decisions are influenced by their own mood state 134So far, some studies have investigated the interpersonal effect of emotional expressions. For instance, it has been shown that people are more likely to cooperate with smiling partners7111217Despite the fact that an interpersonal effect of emotional expressions has repeatedly been demonstrated, the mechanisms underlying this are not fully understood. One line of study implicates emotion contagion in the effect. Emotion contagion occurs when people automatically mimic each other’s facial expressions and bodily postures. Interoceptive feedback from these reactions then produces corresponding emotions19212345While previous research depicts the interpersonal effect of emotional expression in decision making and external moderators thereof26272829The quick detection of the motivational or valence significance of a feedback is associated with feedback-related negativity (FRN), a negative component peaking at fronto-central electrodes roughly 250–300 ms after presentation of relevant feedback information262632333435363827282930354041424329354327293541424748495048495051475348Although some studies have examined the neural dynamics underlying emotional feedback processing32732For these reasons, the current study aims to specify the neural dynamics of interpersonal effects of emotional expressions in decision making, after taking account of the interaction between utiliarian and emotional cues. We carried out an experiment with a modified version of Gehring and Willoughby’s gambling taskWe focused on the FRN, feedback-related P300, as well as theta synchronization to seek converging evidences, because of their strong link with feedback processing2635595961625963646166596869596061715932747576404142We hope to replicate the interpersonal effect of emotional expressions345305t(36) = −4.86, P < 0.001] of the shortened Chinese version of the Costa and McCrae NEO-FFIForty right-handed university students  ages 18–27 years were recruited to participate in this experiment. The mean age of the sample was 20.5 years old (SD = 2.14). They were randomly assigned to either competitive or cooperative games, with 20 participants  in each. All participants reported normal auditory and normal or corrected-to-normal visual acuity, and were free of neurological or psychiatric problems. They were emotionally stable, as evidenced by the significant below-threshold (0) scoring in neuroticism assessment , (see The participants selected more high-risk options after losses (0.55 ± 0.02) than after wins (0.43 ± 0.03), [39], see .outcome valence × context  for all these components. Critically, no significant main effect of emotion  and interactions involving emotion  were observed, suggesting that emotional expressions did not differ regardless of utilitarian cues and contexts.The analysis of VPP, N170, EPN and SPW amplitudes showed a significant main effect of This study examined behavioral and brain responses to utilitarian feedback combined with emotional expressions in both competitive and cooperative contexts to explore the neural dynamics underlying how emotional expressions shape interpersonal decisions. In line with previous studies 2628More importantly, angry faces in the competitive context reversed the differentiation pattern of FRN, and diminished the difference between losses and wins on both feedback P300 and theta band power, partly consistent with our prediction. FRN323334353647484950272935404142Even though the new interpretation of FRN can account for the current results, the bigger FRN for wins relative to losses is still a little bit elusive. We speculate the comparison between the ERPs associated with two emotional expressions following wins and losses separately may help to explain this. It has been proposed that the negative activity in the 200–300 ms time window, referred to as the FRN or N2 component, has consistently been observed to reflect valence difference697074757659When a competitor displayed happiness, losses were associated with larger FRN and theta power, but smaller P300. In this regard, happy expression in competitive context seems to have no profound influence on the valence of the utilitarian feedback. This might result from that the happy expressions following losses were taken as negative feedback  suggested that both happy and angry expressions share same level of attentional allocation and emotional arousal in the current experimental context, in line with the previous studies reporting no significant difference in early components between angry and happy expressions5959Despite the contributions of this study, some limitations should be noted. For the sake of brevity, we did not examine the potential effects of different magnitudes of monetary rewards. Though findings have not been completely consistent, some research suggests that magnitude of monetary reward may influence modulation of emotion expression on utilitarian feedback. For instance, magnitude of reward has been reported to affect FRNThe present study examined both behavioral and brain responses to utilitarian feedback combined with emotional expressions in both competitive and cooperative contexts to specify the neural dynamics underlying the interpersonal effects of emotional expressions. As predicted, interpersonal emotional expressions affect one’s response latency, FRN, P300, and theta power change associated with outcome evaluation, and such modulation varies depending on context. These findings indicate that emotional expressions shape interpersonal decisions during both automatic motivational salience valuation and the conscious cognitive appraisal stages of processing, in which happy expressions mainly elicit inferential processing while angry expressions elicit affective reactions in exerting interpersonal influence.How to cite this article: Chen, X. et al. The neural dynamics underlying the interpersonal effects of emotional expression on decision making. Sci. Rep.7, 46651; doi: 10.1038/srep46651 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


PRDM13 (MCDR1) and a duplication including IRX1 (MCDR3) have been identified. However, the underlying disease-causing mechanism remains uncertain. Through a combination of sequencing studies on eighteen NCMD families, we report two novel overlapping duplications at the MCDR3 locus, in a gene desert downstream of IRX1 and upstream of ADAMTS16. One duplication of 43 kb was identified in nine families , and another one of 45 kb was found in a single family. Three families carry the previously reported V2 variant (MCDR1), while five remain unsolved. The MCDR3 locus is thus refined to a shared region of 39 kb that contains DNAse hypersensitive sites active at a restricted time window during retinal development. Publicly available data confirmed expression of IRX1 and ADAMTS16 in human fetal retina, with IRX1 preferentially expressed in fetal macula. These findings represent a major advance in our understanding of the molecular genetics of NCMD and provide insights into the genetic pathways involved in human macular development.Autosomal dominant North Carolina macular dystrophy (NCMD) is believed to represent a failure of macular development. The disorder has been linked to two loci, MCDR1 (chromosome 6q16) and MCDR3 (chromosome 5p15-p13). Recently, non-coding variants upstream of  Understanding the genetics of rare developmental macular conditions is key for unravelling the mechanism of development of this structure that is found only in higher primates within mammals1. NCMD shows fully penetrant inheritance and is considered a non-progressive disorder with a wide range of phenotypic manifestations, usually affecting both eyes symmetrically3. Phenotypic presentation varies from mild cases with drusen-like deposits covering the macular region but with little or no visual impairment, to severe cases with marked central chorioretinal atrophy and poor vision. Although generally non-progressive, complications associated with choroidal neovascularization can contribute to visual deterioration.North Carolina macular dystrophy (NCMD) is a rare autosomal dominant disorder in which there is abnormal development of the macula, a crucial structure of the central retina responsible for central vision and colour perception10. A similar phenotype has been assigned to a second locus at 5p15-p13 11. Interestingly, several studies reported evidence for ancestral haplotypes at the MCDR1 locus13. Early sequencing studies of the two disease intervals failed to identify exonic disease-causing variants14. More recently, three novel single nucleotide variants (SNVs) were identified in 11 families at the MCDR1 locus, within a DNase1 hypersensitivity site (DHS), in the non-coding interval between PRDM13 and the neighbouring overlapping genes CCNC/TSTD315. Two tandem duplications including the full coding region of PRDM13, with some additional upstream and downstream sequence included, were also identified16. One MCDR3-linked family of Danish origin3 was found to carry a 900 kb tandem duplication15 that includes the entire coding sequence of IRX1. However, duplications of IRX1 have been observed in several normal individuals from the Database of Genome Variants17 and the significance of this reported variant is uncertain. Thus, the causative mechanism at the 5p15-p13 NCMD locus remains unclear.The molecular genetics of NCMD has been extensively investigated with the disorder being mapped to chromosome 6q16  in multiple families of different ethnic origins since the early 1990sIn this report we present a combination of genomic investigations in a cohort of 18 NCMD families. The aim of this study was to identify any causative molecular changes and mechanism of disease in these families.14, family 2 was originally reported to be linked to the MCDR3 locus11, and families 12 and 13 were linked to MCDR19, with family 13 recently found to carry the SNV V2 upstream of PRDM1315. All families  showed autosomal dominant inheritance and had at least one individual with Grade 3 disease. DNA samples from a total of 56 affected and 33 unaffected family members were available for genetic analysis.Eighteen families with phenotypes consistent with a diagnosis of NCMD were included in the study Table . Four faFigure 18 to search for shared identical-by-descent (IBD) chromosomal segments among affected individuals within each family. This analysis was performed in those families for which Illumina single-nucleotide polymorphism (SNP) array data were available for more than one affected family member (families 1–5 and 12–13). The 6q16 MCDR1 locus was excluded in four families, including the two previously MCDR3-linked families 114 and 211 and unreported families 3 and 4  at the 6q16 locus, and not at the 5p15-p13 locus  methodPRDM1315. In addition to the previously reported V2 family 1315, two more NCMD families were found to harbour the variant V2 (family 11 and the previously described MCDR1-linked family 127).All families, except families 1–4 for which linkage at the 6q16 locus had been excluded via haplotype sharing analysis, were tested with Sanger Sequencing for the three previously reported SNVs (V1-V3) upstream of IRX1 and upstream of ADAMTS16  data (CNV browser)20.To investigate the MCDR3 locus for the presence of structural variants (SVs), an aCGH experiment using 10,000 probes spanning the region at chr5:11882–10140073 (GRCh37/hg19) was performed in three affected individuals from families 1–3 which did not show linkage at the 6q16 locus 1–3 Fig. . PrecisePolymerase chain reaction (PCR) primers were designed to amplify the novel sequence across the breakpoint between duplicated copies Table , Fig. 3 The remaining 8 unmapped families were tested with the established PCR assay for both duplications, and 3 of them (families 8–10) were also found to carry the 43 kb duplication Table . Thus, a15. Therefore, haplotype sharing analysis was performed using available Illumina SNP array data from 14 affected individuals in 3 families carrying the 6q16 V2 variant (families 11–13) and 14 affected individuals in 6 families carrying the 5p15 43 kb duplication (families 2–7). Using a cut-off of 2.0 cM and 2.5 cM respectively, the results confirmed that all the genotyped 6q16 individuals collectively shared a RCHH of approximately 2.5 Mb from GRCh37/hg19 coordinate chr6:98962591 (rs150396) to chr6:101468591 (rs1321204) at the MCDR1 locus, and all the genotyped 5p15 individuals collectively shared a RCHH of approximately 0.9 Mb from GRCh37/hg19 coordinate chr5:4327455 (rs155354) to chr5:5210050 (rs1560063) at the MCDR3 locus 26. Different sites were identified to be open/active within the 39 kb shared region at four out of five time points (~10–15 weeks of gestation) available during retinal development 16 was suggested. However, no causal relationship between the non-coding variants and PRDM13 expression has been identified.Eye development, like other organogenesis processes, requires the precise spatio-temporal and quantitative expression of genes, orchestrated by a complex network of regulatory mechanisms influencing critical transcription factors and other developmental genes. The lack of readily accessible animal or PRDM13 at the MCDR1 locus and the most likely candidate gene IRX1 at the MCDR3 locus exists warrants further investigation. iPSC technology and CRISPR manipulation in eye cups from normal and affected individuals may help elucidate the molecular mechanism33 and the potential molecular links between the two genes. Importantly, the involvement of ancestral variation at both the 6q16 and 5p15 loci  and family 139 . When possible, retinal imaging was undertaken using colour fundus photography, fundus autofluorescence and OCT imaging. Blood/saliva samples were collected for DNA extraction, genotyping and sequence analyses. The study protocol was approved by the local ethics committees  and conformed to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all participants, or their parents, before inclusion in the study.All families were ascertained at Moorfields Eye Hospital, London, United Kingdom, expect for family 1Genomic DNA was extracted from whole blood/saliva and genotyped using the Illumina HumanOmniExpress-24 v1.0 beadchip . Genotypes were determined using the Genotyping Module in the Illumina GenomeStudio v2011.1 software.18 was used for the analysis of those affected individuals that were genotyped with the Illumina array. The HH is a type of haplotype described by the homozygous SNPs only  and, therefore, can be uniquely determined on each chromosome. Since affected family members who inherited the same mutation from a common ancestor share a chromosomal segment IBD around the disease gene, they should not have discordant homozygous calls in the IBD region and thus they should share the same HH. The HH approach predicts IBD regions through the identification of RCHHs defined as those regions with a shared HH among affected individuals and a genetic length longer than a certain cut-off value .In order to search for chromosomal segments sharing the same haplotype across affected individuals (within the same family or across different families), the non-parametric HH method2 ratio of the Cy3/Cy5 intensities of a region encompassing at least four probes was > 0.3 or < −0.6, respectively (software default settings).aCGH was performed at Oxford Gene Technology (OGT)  using a custom design consisting of 10,000 probes spanning the MCDR3 locus at GRCh37/hg19 chr5:11882–10140073 , designed with Agilent e-Array software , in three individuals from families 1–3  and WGS data from 650 individuals with inherited retinal disease19.WGS was performed using the Illumina HiSeq X10 platform , generating minimum coverage of 30X. Reads were aligned to the hg19 human reference sequence (build GRCh37) with novoalign (version 3.02.08). The aligned reads were sorted by base pair position and duplicates were marked using novosort. Discordant reads were marked with samblaster (version 0.1.20) and sent to a separate file for manual inspection of breakpoints using the IGV (version 2.3.61). SVs were manually investigated using the IGV by identifying peaks of discordant reads which were interpreted as breakpoints. The identified duplicated regions were also screened for the presence of common copy number variants using data from the CNV browserSegregation analysis of the duplication events identified by WGS was performed using primers Table  designed26 was interrogated for fetal retina datasets of interest. Bed files from DNA-seq datasets  were downloaded and investigated at the shared duplicated region with R Studio. A second microarray expression dataset on human fetal retina (19–20 gestation week) was queried for the genes of interest24 using the platform GENEVESTIGATOR23.The Encyclopedia of DNA Elements (ENCODE)The datasets generated during the current study are not publicly available due to limitations imposed by the scope of participant consent, but are available from the corresponding authors on reasonable request.Supplementary Information


To study use of feature tracking technique in evaluation of regional and global diastolic strain rate and to define the best predictor of diastolic dysfunction by studying its correlation with echo grading of diastolic dysfunction.Echocardiographic examination is currently the gold standard for the non invasive assessment of diastolic dysfunction. Although strain is not load independent, studies have shown that systolic strain rates can assess myocardial contractility and are relatively load independent. It remains to be established if diastolic strain parameters correlate with the degree of diastolic dysfunction and if so which parameter correlates best with echo derived grade of diastology.We performed a retrospective, IRB approved, review of 46 patients who underwent cardiac MRI at our facility for evaluation of cardiac amyloidosis. All patients had an echocardiographic assessment of diastolic function . Conventional short axis, vertical long axis and 4-chamber cine SSFP (Single shot Free Precession) images from the cardiac MRI scans were used to generate 2D  and 3D  diastolic strain rate maps using myocardial feature tracking software. The diastolic strain rates were compared with echocardiographic grades of diastology.Patients had an average age of 61.5 years; males 61%, 17 patients had grade 0, 16 had grade 1 and 13 had grade 2/3 diastolic dysfunction. Base level parameters had the strongest correlations with diastolic dysfunction. The strongest correlations were found for base radial 2D  and longitudinal 3D diastolic  diastolic strain rates. Across all levels on average, radial 2D diastolic strain rate had the strongest correlation with diastolic dysfunction (0.46) followed by circumferential 3D (-0.36) and longitudinal 3D (-0.36) diastolic strain rates.Also, the base diastolic parameters separated best according to echo grade of diastolic dysfunction: The base longitudinal 3D differed the most between grade 0 vs. all grades of diastolic dysfunction . The radial 2D diastolic strain rate differed the most between grades 1 vs. Grades 2/3 .MRI feature tracking is a useful tool for detection of diastolic dysfunction. Our study demonstrated that global radial 2D strain rates and global longitudinal and circumferential 3D diastolic strain rates correlate best with the degree of diastolic dysfunction. For segmental analysis, measuring basal 2D and longitudinal 3D diastolic strain rates will provide the best measure of diastology.


EphA2 and EphA4 are expressed at the tips of the closing spinal neural folds prior and during neural tube closure. We investigated the possible role of murine EphA2 and EphA4 during the last step of primary neural tube closure, which is adhesion and fusion. The individual mouse knockouts of EphA2 and EphA4 per se do not exhibit neural tube defects (NTDs). The embryos generated by the crossing of double heterozygotes Epha2tm1Jrui/+Epha4rb-2J/+ displayed NTDs with a wide degree of severity including close exencephaly and close spina bifida (spina bifida occulta). Interestingly, mutants displaying NTDs had skin covering the underlying lesion. The tissue sections revealed the elevated neural folds had not adhered and fused. The phenotypes seen in Epha2tm1Jrui/+Epha4rb-2J/+ double heterozygous embryos suggest both genes play a compensatory role with each other in the adhesion and fusion of the neural tube. In this study, there exists a >50% penetrance of NTDs in the mouse mutants, which genetically have a single allele each of EphA2 and EphA4 absent.Members of the Eph receptor tyrosine kinase have previously been implicated in cranial neural tube development. Failure of neural tube closure leads to the devastating conditions known as anencephaly and spina bifida.  Genotyping of Epha2tm1Jrui/tm1Jrui and Epha4rb-2J/rb-2J mice was carried out according to the protocol provided by The Jackson Laboratory (stock # 006028 and stock # 003129 respectively) . Pregnant females were euthanized by cervical dislocation and an incision was made at the abdominal area. The uterine horns were incised and immediately transferred into cold Dulbecco's Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). The embryos were dissected out of the decidua and washed briefly with phosphate buffered saline (PBS) (Sigma) before overnight fixation in 4% paraformaldehyde (PFA) (Sigma). Subsequently, the embryos were washed and agitated in PBS for 10 min at 4°C. All steps from this point were agitated to ensure thorough washing. Then the embryos were dehydrated by ascending ethanol washes a concentration of 30, 50, and 70% for 20 min on each wash at 4°C. The embryos were kept in 70% ethanol at 4°C for downstream experiments. The embryos were analyzed in detail and documented under a high-resolution stereomicroscope (Leica MZ16).in situ hybridization, was performed using digoxygenin-labeled cRNA probes  incapable of having a close neural tube that adheres and fuses in the region whereby these genes are expressed  of the E11.5 mouse embryo in Figure Epha2tm1Jrui/+Epha4rb-2J/+) reveals bilateral elevated neural folds, which remain unfused in the dorsal midline at the point of closure of the neural tube highlighted by boxed region in the figure  embryo  that successfully completes closure 1  in more than 50% of the population of double heterozygotes (Epha2tm1Jrui/+Epha4rb-2J/+). The embryos have lipomyelomeningocele : 79% (Epha2tm1Jrui/+Epha4rb-2J/+): 2% (Epha2tm1Jrui/+Epha4rb-2J/rb-2J): 5% (Epha2tm1Jrui/tm1JruiEpha4rb-2J/+): unknown (Epha2tm1Jrui/tm1JruiEpha4rb-2J/rb-2J) (Table Epha2tm1Jrui/tm1JruiEpha4rb-2J/rb-2J) embryos were resorbed by E8.5. The parental double heterozygotes were considered robust without any gross abnormalities. Double heterozygous pups with abnormalities could not survive because the pups die within 2 h after birth  Table  apart frJ) Table . In cont Figures  due to mEphA2 and EphA4 gene expression was studied in the early E11.5 double heterozygotes  mutant embryos to observe for difference in expression pattern. Figure EphA2  among our crosses. However, we note that the numbers of animals given birth to in any of the crosses are much smaller in number than if the embryos were harvested during embryogenesis. There were between 10 and 12 embryos in each litter but if allowed to birth, the numbers dwindled to between 5 and 6 pups per litter. This gives rise to the possibility that if the embryos are unable to survive being a double heterozygote mutant, it gets resorbed; hence failure to complete embryogenesis successfully. Again, this system is similar to what has been observed in the EphB2; EphB3 double knockout (EphB2−/−EphB3−/−) that suffer embryonic lethality  have close spina bifida, which would translate clinically as spina bifida occulta. The implication of this finding is tremendous; that this is the first spina bifida occulta mouse model arising from failure of primary neurulation. Therefore, our mouse model can potentially explain the embryogenesis of lipomyelomeningocele as well as it is at odds with the current dogma of occulta-type spina bifida arising from failure of secondary neurulation. A previous study had reported Trpm6h (Walder et al., Trpm6h to understand the structure of the neural tube regulated by the Trpm6h protein and whether this occurs during primary neurulation (Walder et al., Our double heterozygotes (EphA2 and EphA4 the phenotype representation would be more severe. A further 7% have a more severe neural tube defect when either both the EphA2 allele or both EphA4 allele is completely deleted simultaneously with a single allele of either EphA2 or EphA4 i.e., Epha2tm1Jrui/+Epha4rb-2J/rb-2J or Epha2tm1Jrui/tm1JruiEpha4rb-2J/+ (Figure EphA2 and EphA4 during neurulation (Figure EphA2 and EphA4 are not expressed at the closure 1 site during neurulation but are expressed in the rhombomeres and the posterior neuropore. This phenomenon also further illustrates the specificity of the perturbation of neural tube development in this model and supports the haploinsufficiency theory. Gene dosage determines severity of the phenotype. The defect is selective enough not to phenocopy craniorachischisis, yet its caudal and anterior neuropores remain remarkably open in the areas where EphA2 and EphA4 would be expressed in the wildtype. It is striking that Closure 1 is achieved (Shum and Copp, EphA2 and EphA4 such as EphA1 and EphA5 which have a far broader expression domain than EphA2 and EphA4 (Abdul-Aziz et al., EphA2 and EphA4 in neural tube closure.These mutant embryos showed gene dosage pattern whereby with every loss of an allele of + Figure . Closure+ Figure . This isn Figure . EphA2 aEpha2tm1Jrui/+Epha4rb-2J/+ shows a defective phenotype encompassing spina bifida occulta, close exencephaly, gastrochisis, caudal dysgenesis and cyclopia. It is interesting to note that in the double heterozygotes, the expression of the “dot” which is visible at the point of adhesion and fusion is absent (Figures Epha2tm1Jrui/+Epha4rb-2J/+ are most likely attributed to the Epha4rb-2J/rb-2J mutant isoform that generates a protein size of 104 kDa (Mohd-Zin et al., Epha2tm1Jrui/tm1Jrui is a complete targeted knockout of the EphA2 protein (Ruiz and Robertson, Epha4rb-2J/rb-2J is not (Mohd-Zin et al., EphA4 gene were reviewed in Mohd-Zin et al. (Epha2tm1Jrui/+Epha4rb-2J/+. The background strain is a mix of C57BL/6J (Cook et al., More than 65% of  Figures  in refer Figures . The muln et al. . The fusn et al. . TherefoEphA2 in the notochord could explain a potential role between Sonic hedgehog and the EphA receptor family seeing that the double heterozygotes in our study display cyclopia (Cooper et al., EphA2 is expressed in the region surrounding the gut, which could explain the potential role of the EphAs in gastrochisis.Skin covering of exencephaly; poses the question whether malformations of the brain such as callosal dysgenesis with interhemispheric cyst is implicated in neural tube closure as they occur during the period of neural tube closure or early during post-closure of the central nervous system (Barkovich et al., Our report provides the genotypic and phenotypic embryonic evidence of the occulta-type NTDs arising from failure of primary neurulation.NA, AA-A, and NA-A: Conceived and designed the experiments; NA, SM-Z, and NA-A: Performed the experiments; All authors: Analyzed the data; AA-A and NA-A: Contributed reagents, materials, analysis tools; NA and NA-A: Wrote the manuscript. All authors read and approved the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Olig1, Olig2 and Prdm12 are excluded dorsally. PRDM13 does this via recruitment to chromatin by multiple neural bHLH factors to restrict gene expression in specific neuronal lineages. Together these findings highlight the function of PRDM13 in repressing the activity of bHLH transcriptional activators that together are required to achieve precise neuronal specification during mouse development.The mechanisms that activate some genes while silencing others are critical to ensure precision in lineage specification as multipotent progenitors become restricted in cell fate. During neurodevelopment, these mechanisms are required to generate the diversity of neuronal subtypes found in the nervous system. Here we report interactions between basic helix-loop-helix (bHLH) transcriptional activators and the transcriptional repressor PRDM13 that are critical for specifying dorsal spinal cord neurons. PRDM13 inhibits gene expression programs for excitatory neuronal lineages in the dorsal neural tube. Strikingly, PRDM13 also ensures a battery of ventral neural tube specification genes such as  The process of progenitors undergoing cell fate decisions to determine specific cellular identity and tissue patterning is fundamental in the field of developmental biology. Neural development is an excellent model system to study cell fate processes to understand how neural restricted progenitors differentiate into diverse types of neurons through complex interactions of transcriptional activators and repressors. Multiple regulatory design principles involving combinations of transcription factors have emerged through studies from vertebrate and invertebrate model systems that have significant repercussions in determining and maintaining cell identity . During The dorsal spinal cord functions as an essential center for integration of somatosensory information . ApproprPRDM13 is a transcriptional repressor present in progenitor domains of the dorsal but not the ventral neural tube from hindbrain regions to the spinal cord  Figure . It is aPrdm13 mutant mouse strains. In contrast to the previously reported Prdm13 mutant deleting exons 2 and 3  of PRDM13 ΔZF)  . This ZFdm13ΔZF) . The phend PTF1A . PRDM13-nd PTF1A  do not dnd PTF1A . A thirdm13∆115) . Surpris protein . Westerndm13∆115 . This muPrdm13 was identified as an important specification gene in the dorsal neural tube required for the balance of inhibitory  interneurons and excitatory  interneurons in the chick neural tube  and an increase in dI3/5 (TLX1/3+) in both the GFP/GFPPrdm13 and ΔZF/ΔZFPrdm13 neural tubes was seen at this stage. These phenotypes are identical to the Ptf1a null mouse  (for review see ). Prdm13ral tube . Overexping PAX2 . Supportulations . A complll mouse   in the Prdm13 mutants that is not seen with the loss of Ptf1a , a marker of excitatory neurons, is detected in the lateral region of the domain lacking Gad1  in dorsal neural tube cells in the presence and absence of PRDM13. RNA-seq was performed on GFPal tubes . 837 gengulation .Tlx3 , and used only those sites identified in at least two experiments. We identified 220 PRDM13 binding sites largely located at distal sites relative to the transcription start sites of genes. These targets included a known target of PRDM13, Tlx3 . The gen of Tlx3 . This reenhancer .De novo motif analysis failed to identify a novel binding motif unique to PRDM13, but PRDM13 bound sites were enriched for motifs for known neural TFs such as E-box (bHLH) (40%), Rfx (17%), Pdx (HD) (25%) and Sox (30%) motifs . Indeed,GFPPrdm13 neural tubes  whereas only six are decreased   .Prdm13 mutants. For example, PRDM13 binds the Tlx3 enhancer and restricts its expression at E10.5/E11.5 (eTlx3). Consistently, Tlx3 is elevated 17-fold, while Pax2 is reduced 6.3-fold in the absence of Prdm13  ( control . To dete control . Becausecription  these dacription  where th (Ptf1a) .Ptf1a transcription is regulated through a 2.3 kb autoregulatory enhancer (e2.3Ptf1a)    . PTF1A b.3Ptf1a) . One posr region .Ptf1a autoregulatory enhancer using the chick electroporation assay and a GFP reporter under the control of the 2.3 kb Ptf1a autoregulatory enhancer (e2.3kb-Ptf1a::GFP) , and HEB (Tcf12) (Ptf1a transcription by interrupting PTF1A auto-regulation through binding with PTF1A to the Ptf1a autoregulatory enhancer (We next tested the ability of PRDM13 to regulate transcription through the 1a::GFP) . As was activity . In contmpletely . Finallympletely . Becausempletely   . We propenhancer .Prdm13 and Ptf1a loss of function phenotypes in the second wave of neurogenesis in the dorsal neural tube ). The latter finding is striking because these ventral specification factors are restricted to the ventral neural tube. Three factors with the highest fold increase in the Prdm13 mutant are Olig1, Olig2 and Prdm12 , as well as a number of neural related factors misregulated upon loss of PRDM13 . Notablyd Prdm12 . We usedOlig1 and Olig2 show striking ectopic dorsal expression within GFP/GFPPrdm13 neural tubes  in the ventral spinal cord at these early stages of neurogenesis, and it is required for specification of motor neurons  Figure . Upon loal tubes . This ab mutants . Co-labe mutants . Notably mutants , factorsOlig1 and Olig2, we identified three genomic regions surrounding the Olig genes bound by PRDM13 from ChIP-seq  and is required for generation of V1 interneurons  survived to adulthood and exhibited a retinal phenotype  for function  activity dependent on the PR domain of the protein . Those PDecades of work have gone into defining progenitor domains and resulting neuronal populations in the developing spinal cord mainly by combinatorial TF expression of HD and bHLH factors . RepressPrdm13 mutants they are aberrantly present dorsally, altering the identity of these neurons. Taken together, PRDM13 is a transcriptional repressor essential in the dorsal spinal cord development to modulate the transcriptional activity of neural bHLH factors to generate precise neuronal identities.Here we provide evidence for an additional variation of these models whereby the transcriptional repressor is broadly expressed and it limits inappropriate gene expression driven by progenitor-specific activators, the bHLH factors. PRDM13 is identified as a key transcriptional repressor of a whole battery of genes, not just those in domains adjacent to the domain giving rise to the dorsal inhibitory neurons, but also factors normally restricted to the ventral neural tube and required for neuronal identity in that region  Figure . PRDM13 Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Jane E. Johnson (jane.johnson@utsouthwestern.edu).GFPPrdm13 mutant and the fusionGFPPrdm13 mouse model were developed using zinc-finger nuclease technology (Prdm13 gene (CACCAGCGTGAACGCTGActgctGCATCCCGGCTGGCT) were purchased from Sigma-Aldrich  and delivered by pronuclear injection into fertilized mouse eggs along with a donor plasmid. For GFPPrdm13 mutant mice, the donor plasmid encoded GFP followed by a stop codon, and contained two homology regions each 750 bp in length to allow for homologous recombination of the insert into the Prdm13 locus (fusionGFPPrdm13 mouse that was used for ChIP-seq. Homozygous fusionGFPPrdm13 mice live to adulthood and show no changes in PAX2 or TLX1/3 expression at E10.5 (data not shown). Mice were genotyped by PCR with 5’-GCTGCTCCTGGTTCTGTCA-3’, 5’-CCTTTTCTCTGCTGCTCGTC-3’ and 5’-GCTGGAGTACAACTACAACAGCCA-3’ .The chnology . mRNA en13 locus . Out of Δ115Prdm13 mutant line is a 115 bp deletion generated within the first exon of Prdm13 upon injection of the ZFN mRNA . Homozygous Δ115/ Δ115Prdm13 mice live to adulthood, and show no changes in PAX2 or TLX1/3 expression at E10.5. PCR genotyping used primers 5’-GCTGCTCCTGGTTCTGTCA-3’ and 5’-CCTTTTCTCTGCTGCTCGTC-3’  (The  198 bp) .ΔZFPrdm13 mutant mouse line was generated by pronuclear injection of CAS9 mRNA and two sgRNA targeting the 3’ end of Prdm13 . The CREPtf1a mouse line replaces the coding sequence for Ptf1a with that for Cre recombinase  and cryosectioned at 20 µm (E10.5-E11.5) and 30 µm (E12.5-E16.5) focusing on sections from the upper limb level.20/1X loading dye and injected into the lumen of the closed neural tube at stages HH13-15 along with either a pMiWIII-Myc epitope tagged vector serving as an electroporation control or a TF containing expression vector. The injected embryos were then electroporated with 5 pulses of 25 mV each for 50 msec with intervals of 100 msec. Embryos were harvested 48 hr later at stages HH22-23, fixed with 4% paraformaldehyde for 45 min, and processed for cryosectioning and immunofluorescence.For in ovo chick electroporation assays, fertilized White Leghorn eggs were obtained from the Texas A and M Poultry Department  and incubated for 48 hr at 37⁰C. The supercoiled reporter plasmids were diluted to 1.5 mg/ml in HImmunofluorescence was performed using the following antibodies: guinea pig anti-PRDM13 , mouse Prdm13 and Ptf1a (Prdm12 (Gad1 and Vglut2 (Slc17a6) . The chick Prdm12 probe was generated starting from a pBSK+ cPrdm12 cDNA (EST BU233582). Probes for Ccbe1 and Mfng were generated by PCR from mouse neural tube cDNA. Imaging was performed with a Hamamatsu Nanozoomer 2.0HT digital slide scanner.In situ hybridization was performed as previously described . Probes nd Ptf1a , mouse P  , mouse Od Phox2b , mouse Ob , e2Prdm12 , e1Olig , e2Olig , e3Olig . E1Olig-EboxMut, e2Olig-PTF1Mut and e2Olig-allEboxMut were subcloned using gBlocks with E-box mutations . KpnI/SpeI restriction sites were introduced on each end for cloning adjacent to the β-globin minimal promoter in the reporter cassette. The Ptf1a 2.3 kb enhancer reporters are as described   with pMiWIII  expressi in pCIG , and toto acids) . The HEKGFPPrdm13 embryos either heterozygous or homozygous for GFP were individually placed into cold DMEM/F12 and dissociated in 0.25% trypsin for 15 min at 37 degrees C. Trypsin activity was quenched with 2% fetal bovine serum, and GFP positive cells were purified from the resulting single cell suspension by fluorescence activated cell sorting (FACS). Total RNA from FACS isolated cells was extracted and purified with Mini RNA Isolation Kit . Genotypes were determined and RNA from 9 homozygous and 7 heterozygous embryos were combined, mRNA was purified, reverse transcribed and amplified for sequencing with Illumina’s mRNA-Seq kit  . Gene exfusionGFPPrdm13 embryos were used for GFP ChIP. ChIP was performed as previously described (PTF1A and ASCL1 ChIP-Seq were previously published  (GSE5584escribed . Notablyescribed . LibrariSequence reads for each sample were mapped to the mm10 genome assembly by using Bowtie2 (v.2.2.6) . Mapped enh::GFP expression was measured by ImageJ on the electroporated side of the neural tube with the background levels measured from the non-electroporated side subtracted. Significant differences between control and experimental samples in each case were calculated using a two-tailed two-sample unequal variance (homoscedastic) Student’s t-test in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ns = not significant. Error bars indicate SEM. The enrichment of PRDM13 ChIP targets in the list of genes that showed significant expression change in mutant samples relative to controls was calculated using the hypergeometric test.In sections from mouse embryos with cells labeled by immunofluorescence  and 3, qRNA-Seq and ChIP-Seq data generated here are available on the GEO database (GSE90938). In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Didier Stainier as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Diogo S Castro (Reviewer #1); John LR Rubenstein (Reviewer #2).Thank you for submitting your article "Repression by PRDM13 is critical for generating precision in neuronal identity" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submissionThe described study utilizes newly generated null mutations in Prdm13 to investigate the function of this transcriptional regulator in the developing dorsal spinal cord. Resulting data supports previous findings that Prdm13 suppresses the fate of excitatory neurons in inhibitory lineages. By a combination of developmental and genomic assays, the authors demonstrate that the recruitment of PRDM13 to bHLH bound enhancers inhibits the expression of ventral transcription factors in the dorsal neural tube. While consistent with current models of neural tube patterning this study provides significant mechanistic insight into the Prdm13-mediated gene regulatory network that regulates neurogenesis in the dorsal spinal cord.While a carefully performed study, interpretation of the data would be further strengthened by additional analysis which must be adequately addressed in a revised manuscript.1) Prdm13 ChIP-seq peaks do not show enrichment for a specific motif but instead for several sequences recognized by various neural transcription factors, suggesting Prdm13 is indirectly recruited to DNA. Thus, it become even more important to check the specificity of the Prdm13 antibody. This should be done by validating a series of peaks (associated with the various DNA motifis) in chromatin from wild type and Prdm13 null embryos.2) In 3) Although the authors conclude that "these findings highlight the function of PRDM13 in repressing bHLH transcriptional activators" which implicitly includes Ascl1, the manuscript does not provide an example of a validated Ascl1/Prdm13 target. The authors should rewrite the conclusion accordingly, so that it is not misleading. While a carefully performed study, interpretation of the data would be further strengthened by additional analysis which must be adequately addressed in a revised manuscript.We thank the reviewers for their time and suggestions for improving this study. As you will see below, in performing additional analysis in response to the reviewer’s comments we have significantly strengthened our findings and thus, support for our conclusions.1) Prdm13 ChIP-seq peaks do not show enrichment for a specific motif but instead for several sequences recognized by various neural transcription factors, suggesting Prdm13 is indirectly recruited to DNA. Thus, it become even more important to check the specificity of the Prdm13 antibody. This should be done by validating a series of peaks (associated with the various DNA motifis) in chromatin from wild type and Prdm13 null embryos.Prdm13 mutant neural tubes. Instead we offer a different set of experiments that we believe serves a similar purpose and provides a high confident set of PRDM13 target genes. We now identify PRDM13 target genes based on 3 PRDM13 ChIP-seq data sets that were generated with 3 independent strategies. These include: 1) ChIP-seq data from the original submission that is from E11.5 neural tube tissue using a lab produced antibody generated against the terminal ZnF domain of mouse PRDM13. 2) ChIP-seq data from E11.5 neural tube tissue using an independently produced antibody generated against full-length mouse PRDM13. Here, telencephalon, a PRDM13 negative tissue, was used to control for non-specific binding of the antibody. 3) We generated a mutant mouse strain with a GFP fused in-frame N-terminal to the PR domain of PRDM13. Mice homozygous for this GFP-PRDM13 fusion are indistinguishable from wildtype. ChIP-seq data from E11.5 neural tube tissue from these animals using an antibody directed against GFP was generated.We agree it is important to strengthen the data identifying PRDM13 bound sites in the genome and provide some validation for these sites. While we would have liked to provide data from the suggested experiment, we have been unable to obtain efficient enough ChIP enrichment from the limited source material provided by Ptf1a, Olig2, and Prdm12 were all confirmed as PRDM13 targets providing the validation requested by the Reviewers. We show the genome browser tracks for all three ChIP-seq experiments in supplementary data associated with This exercise had a substantial positive impact on the support for the conclusions reported. We restricted our analysis to PRDM13 bound sites that were called as peaks in at least 2 of the 3 ChIP-seq datasets. This limited the number of PRDM13 target genes identified. The genes highlighted in the study including Prdm13 mutants relative to controls were identified as PRDM13 targets , whereas only 6 of 356 genes that decreased in the Prdm13 mutants were identified as targets . These findings support the role of PRDM13 as a repressor  In Olig1/2. We did this for e1Olig and e2Olig rather than e3Olig for reasons described below, and because e3Olig was not responsive to activation by PTF1A or ASCL1. These experiments demonstrate that the E-boxes are required for e1Olig and e2Olig enhancer activity. In the process of performing these experiments we corrected an error in the data reported in the original manuscript. The e1Olig sequence originally tested was not the correct sequence (consistent with its lack of activity in the reporter assay). In the current revised manuscript, we corrected this error. None of the 3 Olig enhancers direct reporter expression restricted in the endogenous Olig domains. Nevertheless, e1Olig and e2Olig require the Ebox motifs within the enhancers for their activity  Although the authors conclude that "these findings highlight the function of PRDM13 in repressing bHLH transcriptional activators" which implicitly includes Ascl1, the manuscript does not provide an example of a validated Ascl1/Prdm13 target. The authors should rewrite the conclusion accordingly, so that it is not misleading.Tlx3 previously in Chang et al., 2013 and this is described and referenced where relevant. In the current study, we provide additional support for those conclusions showing the increase in Tlx3 in the Prdm13 mutant mice. In the revised manuscript, we now point out that although this current study does not show an example of a validated ASCL1/PRDM13 target, this was shown in Chang et al., 2013 for regulation of TLX3 at the site highlighted in We disagree that this statement in the abstract is misleading. In this manuscript, we explicitly provide evidence for PRDM13 repressing multiple bHLH activators including PTF1A, NEUROG1, and NEUROG2. We don’t explicitly show a validated ASCL1/PRDM13 target here, but we have demonstrated this for


Live cell response to specific HER2 agonists (NRG1b and EGF) and antagonist (pertuzumab) was measured. Of the HER2− breast tumor cell samples tested, 7 of 34 patients  had HER2 signaling activity that was characterized as abnormally high. Amongst the tumor samples there was no correlation between HER2 protein status (by cell cytometry) and HER2 signaling activity  . One conclusion is that measurement of HER2 signaling activity can identify a subset of breast cancers with normal HER2 receptor levels with abnormally high levels of HER2 signaling. This result constitutes a new subtype of breast cancer that should be considered for treatment with HER2 pathway inhibitors.The results of clinical trials evaluating the efficacy of HER2 inhibitors in patients with breast cancer indicate that the correlation between HER2 receptor levels and patient outcomes is as low as 50%. The relatively weak correlation between HER2 status and response to HER2-targeting drugs suggests that measurement of HER2 signaling activity, rather than absolute HER2 levels, may more accurately diagnose HER2-driven breast cancer. A new diagnostic test, the CELx HER2 Signaling Profile (CELx HSP) test, is demonstrated to measure real-time HER2 signaling function in live primary cells. In the present study, epithelial cells extracted fresh from breast cancer patient tumors classified as HER2 negative (HER2 Breast cancer classification is largely based upon a patient's expression levels of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2).ERBB2) amplification and/or HER2 protein overexpression is detected in approximately 15%–20% of breast cancers and associated with more aggressive disease progression, metastasis, and a poorer prognosis  was used, where the values associated with the EGF and NRG1b stimulus and inhibition were combined to arrive at a total amount of HER2 signaling for a particular cell sample.− sample as having abnormal signaling, a test cut-off value of 250 was established that was 90% of the upper range of the healthy responses and coincidental with the median test value for the sample set of HER2+ cell lines previously tested [− patients have abnormal pathway signaling, assuming that 15% of the population would be abnormal, a sample size of 33 was required.To classify a HER2y tested . To conf+ cell lines generate different HER2 signaling levels using the CELx HSP test, it was first assumed that normal breast cells have a median CELx HSP test value of 100 with a standard deviation of 100. To detect a 150 absolute difference  in test value between the two types of cells, with a two-sided α at 0.05 and 90% power, the required number of patients in the normal population and HER2+ population arms is 10 each. Since only nine HER2+ positive cell lines were available, the sample size of the normal specimens was increased to 16 to offset the effect of the smaller HER2+ sample size.To confirm cells from normal patients and those from the HER2P < 0.05 was considered statistically significant.Pearson correlation analysis was performed with GraphPad Prism 6 to evaluate the relationships among the variables of interest. All dose-response curves were obtained using nonlinear regression curve fitting with GraphPad Prism 6. Box-and-whisker plots were constructed to analyze the different groups' CELx HSP test results.


Members of the calcium-activated chloride channel (CLCA) gene family have been suggested to possess a variety of functions including cell adhesion and tumor suppression. Expression of CLCA family members has mostly been analyzed in non-neural tissues. Here we describe the expression of mouse and human CLCA genes in the nervous system.We show that from the six mouse CLCA family members only Clca1, Clca2 and Clca4 mRNAs are expressed in the adult brain, predominantly in olfactory ensheathing cells. During mouse nervous system development Clca1/2 is more widely expressed, particularly in cranial nerves, the diencephalon and in the cerebral cortex. While human CLCA2 and CLCA4 genes are widely expressed in brain, and at particularly high levels in the optic nerve, human CLCA3, the closest homologue of mouse Clca1, Clca2 and Clca4, is not expressed in the brain. Furthermore, we characterize the expression pattern of mouse Clca1/2 genes during embryonic development by in situ hybridization.The data published in this article indicate that within the nervous system mouse Clca1/2 genes are highly expressed in the cells ensheathing cranial nerves. Human CLCA2 and CLCA4 mRNAs are expressed at high level in optic nerve. High level expression of CLCA family members in mouse and human glial cells ensheathing nerves suggests a specific role for CLCA proteins in the development and homeostasis of these cells. Calcium activated chloride currents have been characterized in a number of cell types including smooth muscle, skeletal muscle and epithelium. Physiologically, it has been shown that activation of calcium-activated chloride current plays a prominent role in among others the maintenance of smooth muscle tone, epithelial secretion and vertebrate olfactory transduction. The precise molecular identities of the currents are still hotly debated. Proteins belonging to CLC, CLCA, bestrophin and tweety gene families have been proposed to function as calcium activated chloride channels [reviewed in ].The CLCA gene family includes 4 genes in humans, 5 genes in rat and 6 genes in mouse. The nomenclature of the CLCA genes in different organisms is somewhat confusing since the numbering of different genes does not reflect the actual homologies between the genes in different organisms but rather the time of characterization [reviewed in ].Although a lot of evidence shows the involvement of CLCA proteins in mediating chloride conductance, it is still unclear whether CLCA proteins are channels themselves. It has been shown that there are differences in endogenous chloride current characteristics in normal versus CLCA over-expressed cells . Also, aIn addition to their functions as chloride channels or channel modulators, some CLCA family members function as cell adhesion molecules ,8 and tuExpression analysis by RT-PCR of mouse Clca family members, has revealed that mClca1 is expressed at high levels in spleen and bone marrow and mClca2 in mammary gland. Moderate or low expression levels of both genes were found in most tissues with only mClca1 expressed in brain tissue . ExpressTo date, expression analysis has shown that only mClca1 is expressed in the mouse brain. However, in case of mClca3 and 4, brain tissue was not included in the expression analysis. In this study we describe the spatio-temporal expression of mClca1, 2 and 4 genes in the nervous system. We show that these genes are expressed in the olfactory ensheathing cells. In addition we also describe the expression pattern of human CLCA2 and 4 genes in the nervous system. Finally, we describe the expression pattern of mClca1/2 during embryonic development by in situ hybridization.Total RNA from NMRI mouse tissues and total RNA from postmortem adult human brain regions was extracted using RNAWiz (Ambion) according to the manufacturers instructions. Total RNA from human non-neural tissues was obtained from Clontech. First-strand cDNAs were synthesized with Superscript III (Invitrogen) reverse transcriptase using 5 μg RNA as recommended by the manufacturer.PCR reactions were performed in a volume of 25 μl, using 1/50 of the first-strand cDNA reaction. Annealing temperature for different sets of primers ranged from 55–60°C. The number of cycles used varied from 25–35 for different primer sets. Number of cycles for different primer sets was determined empirically and we always analysed the PCR product in the exponential phase of amplification. PCR with primers specific for housekeeping gene HPRT and GAPDH were used as a control to determine the variation of the amount of cDNA in different PCR reactions.Real-time quantitative (Q) RT-PCR analysis of CLCA mRNA levels in adult mouse and human brain regions and during mouse brain development was performed in triplicates using qPCR Core Kit for SYBR Green I (Eurogentec) with Lightcycler 2.0 (Roche) according to manufacturers instructions. Data was normalized with housekeeping gene HPRT and analyzed with Lightcycler 4.05 software (Roche). Data was not normalized with HPRT in case of mClca1, mClca2 and mClca4 PCRs using equal amounts of cDNAs from different mouse brain developmental stages, since the level of HPRT mRNA is increased during development .Primers used in the experiments are the following:hclca1 sense ACGAACAAGGACACCAGCAAAhclca1 antisense AAGAGATCAGGTATGGGAGCAThclca2 sense TGCATGTCAATCACTCTCCCAhclca2 antisense GAGTTCCTATCCATTGCTCGThclca3 sense GAAGGAGCTCAAACAGACGAChclca3 antisense ACTTTCTACTGAACCAGGCTChclca4 sense GCCACAGTTCATGAGGATAAGhclca4 antisense CACAGACAATACCAGCGTAGmclca1sense CACCAGGATCACTGGCACCAATmclca1 antisense GCATCGATAAGGCTGTTTAGGTCmclca2 sense CGCCAGCATCACAGGCAAGAAGmclca2 antisense GCGTCGATAAGGCTGCTTACATGmclca4 sense TTCAGCAGGACAGCATCTGGmclca4 antisense TGCCACTTGTGCGATGTTGgapdh sense TTCCTACCCCCAATGTGTCCGTCgapdh antisense ACCCTGTTGCTGTAGCCGTATTCAhprt sense GATGATGAACCAGGTTATGAChprt antisense GTCCTTTTCACCAGCAAGCTTGhclca2real sense AGCACCTGGAGAAGACTTTGAhclca2real asense CTTGCTGAGGATTTCGCTTTGAhclca4real sense AGACCTTGATGCCACAGTTCAThclca4real asense TGGTGACAGATCAGTAGTATTTAmclca1 realS CACTGATAACTTGCGTATCTACmclca1 realAS CACAGTTGTGAACCACATTGGmclca2 realS TCACTGATAACTTGCGTATCTATmclca2 realAS ACACTCGTGGACCACCTTCTmclca4 realS AATGACAGCTCCTACCTAGCmclca4 realAS GGCTCCACTGTGTTTGACCT35S]UTP  for labeling. The hybridization specificity was confirmed using [α-35S]-labeled sense riboprobes synthesized from the same templates. All sense probes resulted in the hybridization signal equivalent to the background. This shows that cRNA labeling of different CLCAs was specific. Primers used to generate probes were the following:DNA fragments for riboprobe generation were subcloned into pCRII-TOPO vector (Invitrogen), sense and antisense cRNA probes were synthesized with the MAXIScript In Vitro Transcription Kit (Ambion) T7 or SP6 RNA polymerase, using [α-CLCA12 sense ATAGTATCTCTGCACTGGTGCLCA12 antisense GAATGGATATCTAATTTCCATAGCLCA4 sense CCTCCTGGTCTGGGTACCAACLCA4 antisense ATAGACGCAAATAGGAAATTTACSerial saggital and coronal sections (14 μm) from fresh-frozen NMRI mouse brain were analyzed by in situ hybridization analysis following the previously described protocol . EmulsioPrevious analyses have shown that out of six mouse Clca genes only mClca 1 is expressed in the brain and is expressed at relatively low levels compared to other tissues where the gene is expressed [Quantitative real-time PCR analysis of mClca1, 2 and 4 expression during mouse brain development showed that expression of mClca1 was increasing during postnatal brain development and reached maximum levels in the adult mouse brain. mClca2 expression did not change during mouse brain development and mClca4 expression was low during embryonic development, highest around birth of the animal and the level of respective mRNA was gradually decreasing during postnatal development Fig .In order to analyze the spatial distribution of mClca1, 2 and 4 mRNA expression in the adult brain we performed real-time PCR anaysis using cDNAs from various regions of mouse brain. Strikingly, all the mClca genes expressed in the nervous system, were highly enriched in olfactory bulb Fig . ExpressTo further analyze the cellular distribution of mClca1,2 and 4 expression in brain, we performed in-situ hybridization on adult brain sections. Since mClca1 and 2 genes share 95% identity, we were unable to design probes that distinguish between these genes. Therefore, we consider the expression pattern of mClca1 and 2 together (marked mClca1/2). It should be noted however, that mClca2 was expressed at very low levels in the adult mouse brain Fig  and therWe performed in situ hybridization analysis in order to characterize mClca1/2 expression during mouse development. Our analysis showed that at embryonic day 13 (E13) mClca1/2 is expressed at high levels in the developing urethra, midgut, aorta and heart Fig . High maIn situ hybridization analysis on E17 mouse embryos showed that high levels of mClca1/2 mRNA expression were retained in the developing urethra Fig . ExpressSince three mouse Clca gene family members were expressed in brain, we were interested if any of the four human CLCA genes are expressed in the nervous system. As the numbering of CLCA family members in human and rodents is different, we performed bioinformatic analysis to reveal which rodent Clca genes have closest homology to which human family members. Schematic depiction of human, mouse and rat CLCA loci is shown in Fig Our bioinformatic analysis showed that human CLCA3 is the closest homologue of mouse Clca1, 2 and 4 genes Fig . It sharhCLCA2 and 4 were differentially expressed in the adult brain as revealed by quantitative RT-PCR analysis Fig . The higIn this study we show novel expression sites for mouse Clca1, Clca2 and Clca4 genes. All six mouse Clca genes are located in chromosome 3 and are clustered in the same locus. Our RT-PCR and in situ hybridization analyses reveal that in the murine nervous system, mClca1, 2 and 4 genes are preferentially expressed in the olfactory ensheathing cells. In contrast to our findings, it has been previously shown that mClca2 is not expressed in the mouse brain . Our anamClca 1, 2 and 4 genes share highest similarity with each other within the gene family. At amino acid level mClca1 and 2 share 95% identity and mClca4 shares 81% identity with mClca2 and 80% identity with mClca1. Moreover, these three genes lie next to each other and form a 3' gene cluster in the Clca gene locus. Given their similar expression pattern in the nervous system, it could be argued that either their olfactory ensheathing cell specific expression is driven by a common regulatory element or each of these mouse genes has retained an olfactory ensheathing cell specific promoter element following gene duplication. Other reports have shown that the rat Clca genes most homologous to mouse Clca1,2 and 4, i.e. rbClca and rbClca2 are expressed in rat brain ,21. TheiOur analysis of mClca1/2 expression in the nervous system at early postnatal development revealed that it is expressed also in the layer II-III of the cerebral cortex. Interestingly the expression was seen only in the frontal part of the developing cortex. At E17 mClca1/2 expression was more widespread in the brain, with prominent expression in diencephalon. During embryonic development mClca1/2 expression was seen in the developing nerves of the peripheral nervous system. We could detect expression at E13 in the developing olfactory nerve and spinal nerves and at E17 in the optic nerve, trigeminal nerve and olfactory nerve. It is possible that mClca1/2 expression marks the glial cells ensheathing peripheral nerves.In this study we have also analyzed the expression of human CLCA genes in the nervous system. Human CLCA locus contains 4 genes. The most 3' of the genes is hCLCA3, which is also most closely related to mClca1, 2 and 4. RT-PCR analysis showed that unlike its mouse homologues, hCLCA3 is not expressed in the nervous system. In contrast, our results show that two other members of the family, hCLCA 2 and 4 are expressed in various parts of human brain. It has previously been shown, using RNA dot-blot analysis, that hCLCA4 is expressed rather uniformly in the brain with striking absence in the cerebellum . HoweverIn this study we have shown that mClca1, mClca2 and mClca4 are expressed in the olfactory ensheathing cells of the adult mouse CNS. During mouse development mClca1/2 widely expressed in the CNS but at particularly high levels in cranial nerves. In addition, we found that mClca1/2 is expressed in layer II of the developing cerebral cortex at P9. Our analysis also reveals that hCLCA2 and hCLCA4 are expressed in the CNS of adult humans.TT and DM contributed to the design of the experiments and to the preparation of the manuscript. MP performed the experiments and contributed to the design of the experiments and to the preparation of the manuscript.


It is now recognised that epithelial-stromal interactions are important in a wide range of disease processes including neoplasia and inflammation. Metalloproteinases are central to matrix degradation and remodelling, which are key events in tumour invasion and metastasis and may also be involved in tissue changes occurring in chronic inflammation. Immunohistochemistry was performed on sections from 50 patients with pancreatic cancer (n = 27), ampullary cancer (n = 12), low bile duct cancer (n = 3), neuroendocrine tumours (n = 3) and chronic pancreatitis (n = 5), using antibodies raised against collagenase (MMP2), stromelysin (MMP3) and tissue inhibitor of metalloproteinase (TIMP1) and developed using the avidin-biotin complex method. Abundance of MMP2, MMP3 and TIMP1 was greater in pancreatic and ampullary cancer than any other pathology and immunoreactivity in the malignant epithelial cells in pancreatic and ampullary cancer was greater than in the stromal tissues . There were strong correlations between the immunoreactivity of the two antibodies for MMP2 (P < 0.0001), between MMP2 and TIMP1 (P < 0.0001) and between MMP3 and TIMP1 (P < 0.0001). The immunoreactivity for TIMP1 in pancreatic and ampullary cancers with lymph node metastases was significantly less compared with those cases without lymph node metastases (P < 0.02) and there was an association between increased immunoreactivity for MMP2 and the degree of tumour differentiation (P < 0.01). The results implicate MMP2, MMP3 and TIMP1 in the invasive phenotype of pancreatic and ampullary cancer.