Merging nanoparticles with different functions into a single microsphere can exhibit profound impact on various applications. However, retaining the unique properties of each component after integration has proven to be a significant challenge. Our previous research demonstrated a facile method to incorporate magnetic nanoparticles into porous silica microspheres. Here, we report the fabrication of porous silica microspheres embedded with magnetic and gold nanoparticles as magnetic recoverable catalysts. The as-prepared multifunctional composite microspheres exhibit excellent magnetic and catalytic properties and a well-defined structure such as uniform size, high surface area, and large pore volume. As a result, the very little composite microspheres show high performance in catalytic reduction of 4-nitrophenol, special convenient magnetic separability, long life, and good reusability. The unique nanostructure makes the microspheres a novel stable and highly efficient catalyst system for various catalytic industry processes. Owing to their higher catalytic activity, better selectivity, and longer stability than Pd and Pt catalysts, the catalysis of gold nanoparticles (Au NPs) in liquid-phase reactions has become the subject of increasing interest in recent years-15. It h3O4@SiO2 microspheres followed by a surfactant-assembly sol-gel process and synthesized multifunctional Fe3O4@SiO2-Au@mSiO2 microspheres with well-defined core-shell nanostructures, confined catalytic Au NPs, and accessible ordered mesopore channels[in situ load Au NPs on Fe3O4@SiO2 magnetic spheres[3O4@SiO2 magnetic nanoparticles were firstly prepared, AuCl4- was introduced to the surface and then reduced by Sn2+ species that were linked to the surface of the Fe3O4@SiO2 precursor. The synthesis step and the reaction cost were remarkably decreased. Despite of these researches, in situ fabrication is limited[Several strategies have been developed to immobilize Au NPs onto/into the magnetic composite supports-35. Genechannels. However spheres. After t limited,36-39, a2O3) are synthesized via the method in our previous report. Secondly, the surface of the porous magnetic polymer microspheres are modified by a quaternary amine via ring-opening reaction of epoxide groups of GMA with trimethylamine (TMA). Thirdly, the gold precursor (AuCl4-) is adsorbed onto TMA-treated magnetic polymer composite microspheres through the ion exchange between quaternary ammonium ions and AuCl4-. Then, the silica nanoparticles are deposited into the channel of magnetic P(GMA/EGDMA)-N+/AuCl4- composite microspheres through sol-gel reaction with the silica precursor tetraethylorthosilane (TEOS). Finally, uniform mesoporous silica microspheres embedded with magnetic and gold nanoparticles, designated as γ-Fe2O3/Au/mSiO2, are obtained after calcinations to remove the polymer template and organic agents. The designed multifunctional microspheres possess uniform particle size, large magnetization, hierarchical mesopores, and stably confined but exposed active metal nanoparticles. The multifunctional porous microspheres show excellent catalytic performance towards the reduction of 4-nitrophenol by excess sodium borohydride (NaBH4) in aqueous solution and could be very useful in various catalytic reductions. With an external magnetic field, the catalyst can be easily recycled. Long lifetime and high reusability are demonstrated with negligible decrease in the catalytic performance after use for more than ten times.To overcome the above problems, herein, on the basis our group's previous work of incorporating magnetic nanoparticles into porous silica microspheres, we report an efficient method for the synthesis of uniform multifunctional mesoporous silica microspheres incorporated with magnetic and gold nanoparticles by using porous polymer microspheres as template. The preparation strategy is shown in Figure 3 · 6H2O), sodium oleate, trimethylamine (TMA) hydrochloride, sodium hydroxide, ammonium hydroxide (28% aqueous solution), and ethanol were purchased from Shanghai Chemical Reagent Corp. . Hexanes, chloroform, sodium borohydride (NaBH4), 4-nitrophenol (4-NP), and 1-octadecene were purchased from Alfa Aesar. Anhydrous alcohol and chloroauric acid tetrahydrate (HAuCl4 · 4H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd.  Water was purified by distillation followed by deionization using ion exchange resins. Other chemicals were of analytical grade and used without any further purification.The silica precursor tetraethylorthosilane (TEOS) was purchased from Alfa Aesar . The template polymer microspheres are a polymer of glycidyl methacrylate (GMA) cross-linked with ethylene glycol dimethacrylate (EGDMA) supplied by Nano-Micro Technology Company . Ferric chloride hexahydrate  were added into 50 ml of toluene. After ultrasonic treatment in water bath for 1 h, a homogeneous yellow solution was obtained. Another 100 ml toluene containing 2 g of P(GMA-EGDMA) microspheres was prepared. Under stirring, the magnetic nanoparticle solution was added into the polymer microsphere solution. After 2 h, magnetic nanoparticle-embedded porous polymer microspheres were filtrated and washed repeatedly with toluene and ethanol. The brown magnetic polymer composite microspheres were dried at 50°C under vacuum.Brown composite spheres (2 g) were dispersed in 250-ml mixture of ethanol and water (volume ratio = 2:1). Then, 2 g of trimethylamine hydrochloride and 1 g of sodium hydroxide were added to the mixture solution. After the resulting mixture was stirred in water bath at 50°C for 24 h, the resulting TMA-treated magnetic P(GMA-EGDMA) composite microspheres were filtrated and washed repeatedly with distilled water. The brown functionalized magnetic polymer composite microspheres were dried at 50°C under vacuum.4 · 4H2O (1.0 wt%) were subsequently added at room temperature with mechanical stirring. After 4 h, the reddish brown precipitate was recovered by a magnet and washed with water for five times. The precipitate for further characterization was dried at 60°C for 6 h.TMA-treated magnetic P(GMA-EGDMA) composite microspheres (1.0 g) were added to a 100-ml round-bottomed flask, and then 50 ml deionized water and 5 ml aqueous HAuCl+/AuCl4- composite microspheres and 2 ml of ammonia hydroxide were introduced over a period of 0.5 h. A 10% TEOS solution (in ethanol) of 30 ml was then added dropwise into the mixture in 1.5 h. The sol-gel transformation of TEOS to silica in the pore of the composite polymer microspheres was carried out at 30°C for 24 h. The brown γ-Fe2O3/polymer/gold/silica microspheres obtained were washed repeatedly with ethanol and distilled water before being dried at 50°C overnight. The dried microspheres were calcined at 600°C for 10 h (ramp rate of 10°C/min) under air. After calcination, yellow hierarchically porous silica microspheres embedded with γ-Fe2O3 and Au nanoparticles were obtained.In a 250-ml three-necked, round-bottomed flask equipped with a mechanical stirrer, 80 ml of ethanol and 20 g of water were placed. With vigorous stirring in the flask, 0.5 g of magnetic P(GMA-EGDMA)-N4 was chosen as a model reaction for investigating the catalytic performance of the porous SiO2/Au/γ-Fe2O3 composite microspheres. Typically, aqueous solution of 4-NP  was mixed with fresh aqueous solution of NaBH4 . Two milliliters of aqueous suspension of the SiO2/Au/γ-Fe2O3 composite microspheres (1.0 mg) was rapidly added. Subsequently, 2 ml aqueous suspension at a given interval was sampled and filtered through 0.45-μm membrane filters. The UV-visible absorption spectra of the filtrates were recorded at room temperature.The reduction of 4-NP by NaBH2/Au/γ-Fe2O3 composite microspheres were studied using a field emission scanning electron microscope  and a transmission electron microscope . The particle hydrodynamic size was measured by using a Beckman Coulter Counter laser size analyzer . The thermogravimetric analysis was conducted on a DuPont TGA 2050 , with a temperature ramp of 10°C/min. The magnetization curve was measured at room temperature under a varying magnetic field with a vibrating sample magnetometer . N2 adsorption and desorption isotherms were measured at 77 K on a Micromeritics TriStar II 3020 . The X-ray diffraction (XRD) pattern of the prepared powder sample was collected using a Rigaku D/Max-2200PC X-ray diffractometer with Cu target . The γ-Fe2O3 content in the silica microspheres was determined by atomic absorption spectroscopy  of an extract from the sample obtained with dilute HCl (1:1) and HF (1:1) at 80°C for 6 h. UV absorbance spectra were measured using a NanoDrop 2000 spectrophotometer .The morphology and structure of the porous SiO2O3 and gold nanoparticles. The composite microspheres are highly monodisperse with the diameter about 4.4 μm which are assembled by nanoparticles of about 30 nm. The surface morphology of the composite microspheres is a porous structure which is similar to that of the porous polymer template microspheres , Fe (Fe2O3), and Au is confirmed by STEM-energy-dispersive X-ray (EDX) analysis. To further verify the formation of Fe2O3 and gold nanoparticles, Figure θ = 30.3°, 35.6°, 43.2°, 53.5°, 57.2°, and 62.9°), related to their corresponding indices ((220), (311), (400), (422), (511), and (440)), are clearly observed in Figure 2O3 in the products. The four peaks positioned at 2θ values of 38.2°, 44.4°, 64.5°, and 77.4° could be attributed to the reflections of the (111), (200), (220), and (311) crystalline planes of cubic Au, respectively. In addition, we find that only a weak peak (2θ = 38.2°) clearly shows up in Figure 2O3 nanoparticles are incorporated into the hybrid materials as well  to characterize the sample. As shown in Figure 4 is chosen as a model reaction for investigating the catalytic performance of the porous γ-Fe2O3/Au/SiO2 microspheres. There is no appreciable by-product formation during this reaction. The extent of the reaction could be determined by measuring the change in UV-visible (UV-vis) absorbance at 400 nm. As shown in Figure 4, the adsorption maximum shifts to 400 nm immediately, due to the formation of 4-nitrophenolate. No change is observed after standing for a long time, indicating that the reduction does not proceed without a catalyst.4-nitrophenol (4-NP) is one of the most common organic pollutants in industrial and agricultural wastewater, while 4-aminophenol (4-AP) has important applications in many fields, such as medicine, photography, anticorrosion, etc. The conversion of 4-NP to 4-AP under the catalyst of noble metal NPs, which simultaneously realizes the degradation of 4-NP and the efficient production of 4-AP, has attracted the interest of researchers. Here, the reduction of 4-NP by NaBH2O3/Au/SiO2 microspheres is added and the adsorption peak at 400 nm significantly decreases as the reaction proceeds, revealing the reduction of 4-NP to form 4-AP. Figure 4 is completed within approximately 13 min, and the bright yellow solution gradually becomes colorless. Linear relationships between ln(Ct/C0) and reaction time are obtained in the reduction catalyzed by the γ-Fe2O3/Au/mSiO2 microspheres  of the γ-Fe Figure C, which 4- to the acceptor 4-NP after the adsorption of both onto the catalyst surface. The hydrogen atom, which is formed from the hydride, after electron transfer to the Au NPs attacks 4-NP molecules to reduce them. For comparison, the catalytic ability of the equal amount of γ-Fe2O3/mSiO2 is also studied. Without Au catalyst, the reduction reaction does not proceed, as evidenced by a nonvarying absorption spectrum.The reduction reaction occurs via relaying electrons from the donor BH2O3/Au/mSiO2 microspheres, we use a magnet to separate the catalysts from the solution and then rinse it with deionized water. Then, the microspheres are dispersed into deionized water for the next cycle of catalysis. As shown in Figure 2O3/Au/mSiO2 microspheres could be successfully recycled and reused for at least ten times within 10 min. The microstructures are well retained after the repeating catalytic processes.To investigate the reusability of the γ-Fe2O3/Au/mSiO2 microspheres with high surface area. Quaternary ammonium in the surface of the microspheres serves not only as a reducing agent but also as a protecting ligand, which makes the adsorption of gold nanoparticles simple and convenient. Gold nanoparticles are reduced in situ and incorporated into the matrix of porous microspheres. The resulting multicomponent microspheres have high magnetization and can be conveniently separated from the reaction solution using external magnetic fields. They exhibit excellent catalytic performance and high reusability for the reduction of 4-NP in the presence of NaBH4. This functional microsphere holds great promise as a novel gold-based catalyst system for various catalytic applications. Additionally, the approach for the fabrication of γ-Fe2O3/Au/SiO2 microspheres can be extended to synthesize other multicomponent nanostructures for advanced applications in chemical/biosensor, environmental detection, and electromagnetic devices.In summary, by employing a functionalized magnetic polymer microsphere template, we have successfully synthesized monodisperse, hierarchically mesoporous γ-Fe4-AP: 4-aminophenol; 4-NP: 4-nitrophenol; Au NPs: gold nanoparticles; BET: Brunauer-Emmett-Teller; EDX: energy-dispersive X-ray spectroscopy; EGDMA: ethylene glycol dimethacrylate; GMA: glycidyl methacrylate; SEM: scanning electron microscope; TEM: transmission electron microscopy; TEOS: tetraethylorthosilane; TMA: trimethylamine; XRD: X-ray diffraction.The authors declare that they have no competing interests.LR carried out the synthesis and characterization of porous silica microspheres. CT participated in the morphology characterization. LZ drafted the manuscript. JH and You Wang participated in the UV and TGA analyses. XZ participated in the XRD characterization. Yong Wang and BJ conceived of the study and helped draft the manuscript. MH and JZ participated in the design of the study. All authors read and approved the final manuscript.2O3 nanoparticles.(A-B) SEM images of commercially available porous P(GMA/EGDMA) microspheres. (C-D) TEM images of synthesized magnetic γ-FeClick here for file


The aim of the study was to evaluate the effect of delay in treatment intensification  in conjunction with glycaemic burden on the risk of macrovascular events (CVE) in type 2 diabetes (T2DM) patients.A retrospective cohort study was carried out using United Kingdom Clinical Practice Research Datalink, including T2DM patients diagnosed from 1990 with follow-up data available until 2012.In the cohort of 105,477 patients mean HbA1c was 8.1% (65 mmol/mol) at diagnosis, 11% had a history of cardiovascular disease, and 7.1% experienced at least one CVE during 5.3 years of median follow-up. In patients with HbA1c consistently above 7/7.5%  during 2 years post diagnosis, 26/22% never received any IT. Compared to patients with HbA1c <7% (<53 mmol/mol), in patients with HbA1c ≥7% (≥53 mmol/mol), a 1 year delay in receiving IT was associated with significantly increased risk of MI, stroke, HF and composite CVE by 67% , 51% , 64%  and 62%  respectively. One year delay in IT in interaction with HbA1c above 7.5% (58 mmol/mol) was also associated with similar increased risk of CVE.Among patients with newly diagnosed T2DM, 22% remained under poor glycaemic control over 2 years, and 26% never received IT. Delay in IT by 1 year in conjunction with poor glycaemic control significantly increased the risk of MI, HF, stroke and composite CVE. Currently 8.3% of adults worldwide are estimated to have diabetes . The risGlycaemic management in patients with T2DM has become increasingly complex, and in some cases controversial, with a widening classes of pharmacological agents now available –12. Baseclinical inertia, has been discussed by some studies . The event rates were similar for those with HbA1c ≥7.5% (≥58 mmol/mol) during 1 year post diagnosis.Among all patients, compared to patients with HbA1c below 7% 53 mmol/mol) who received IT before 1 year of diagnosis, patients with HbA1c ≥7% (≥53 mmol/mol) not receiving IT within a year had significantly increased risk of MI, HF, stroke and composite CVE significantly by 67, 64, 51 and 62%, respectively, after adjusting for various confounding factors  consistently during 2 years post diagnosis , patients who did not receive intensified treatment had 82%  increased risk of composite CVE. The risk estimates were similar for patients with and without history of cardiovascular diseases. These estimates were obtained after adjusting for all factors mentioned in the method section, except any adjustment for HbA1c levels.Irrespective of glycaemic control, failure to intensify anti-hyperglycaemic treatments was associated with 42%  and 48%  significantly increased risk of CVE among patients with and without the history of cardiovascular diseases, respectively.Subgroup analyses with adjustments for available data on BMI, systolic blood pressure, LDL-cholesterol and total cholesterol at diagnosis revealed similar risks on cardiovascular outcomes, associated with clinical inertia.Among patients receiving IT before 12 months, those with HbA1c above 7% 53 mmol/mol) and 7.5% (58 mmol/mol) during 1 year post diagnosis had significantly increased risk of any CVE by 24%  and 33% , respectively compared to those with HbA1c below 7% (53 mmol/mol) and 7.5% (58 mmol/mol) , respectively.Our population level study, based on more than 100,000 newly diagnosed T2DM patients with median 5.3 years of follow-up, reveals that (1) 26% of patients with HbA1c above 7% (53 mmol/mol) during 2 years post diagnosis of diabetes did not receive any intensified treatment for hyperglycaemia during follow-up, (2) 32 and 46% of patients receiving early treatment intensification within 6 and 12 months of diagnosis continued to have poor glycaemic control over 2 years post diagnosis [HbA1c above 7.5% (58 mmol/mol)], (3) a 1 year delay in treatment intensification in conjunction with poor glycaemic control significantly increased the risks of HF, stroke and composite CVE in patients with and without history of CVD before diagnosis of diabetes, and (4) irrespective of early treatment intensification, the glycaemic burden was significantly associated with increased risk of MI, HF, stroke and composite CVE.In the study cohort 54% never received any intensified treatment, while their average HbA1c level remained above 6.5% (48 mmol/mol), but below 7% (53 mmol/mol), during 2 years post diagnosis  , high glThe ACCORD study in patients with 10 years of duration of T2DM could not establish any significant benefit of intensified glucose lowering treatment on macrovascular event during 3.5 years of median follow-up . HoweverAlthough early treatment intensification did not reduce the HbA1c below clinically acceptable limit in a significant proportion of patients, the residual benefit of intensified treatment on macrovascular risk is considerable. Compared to patients with HbA1c <7% <53 mmol/mol) during 1 year post diagnosis, those with HbA1c ≥7% (≥53 mmol/mol) and received IT within a year of diagnosis had 38% lower risk for any CVE (HR: 1.24) compared to those who had delayed treatment intensification  the time to treatment intensification was based on the dates of available prescriptions longitudinally, with the likelihood of missing data; (2) non-availability of adherence data on medications; (3) non-availability of prescription on diet and exercise; (4) failure to account for the socio-economic status of the patients, which may well be associated with poor glycaemic control and with an elevated risk of cardiovascular diseases; (5) non-availability of complete and reliable data on alcohol consumption; (6) non-availability of complete and reliable longitudinal data on doses for individual medications, (7) non-availability of hospitalisation data for MI and stroke, and (8) the potential for residual confounding, as common in any clinical epidemiological study. Due to lack of data on longitudinal doses of individual anti-diabetes medications, we could not consider the dose escalation for some anti-diabetes therapies as potential treatment intensification. However, these issues are unlikely to affect the robustness of the results of this study. The large analysis cohort was selected from the validated CPRD database should be considered as a representative sample, and as such, provides a good picture of the state of diabetes control in routine practice. Apart from complete data on demographics, we had complete data on HbA1c measured within 3-month window of diagnosis of diabetes. The 6-monthly follow-up measures of HbA1c were imputed for only 9% missing cases with random missing pattern. Finally, careful design of the study by defining sensible exposure time, time line for events, and appropriate adjustments for various aspects while determining the time-to-events are the primary strengths of the study.As with all observational studies, we were unable to provide definitive evidence for direct cause–effect relationships, and the risks of false positive or negative findings due to selection bias and residual confounding, even after the most stringent corrections, may not be trivial. However, although the results from large controlled clinical trials evaluating the effect of intensified glucose lowering treatment on macrovascular complications in patients with T2DM have been inconclusive, our longitudinal study with primary care level data demonstrates the beneficial effect of guideline recommended tight glycaemic targets on long-term cardiovascular risks.


Streptococcus pneumoniae. Dendritic cells (DCs) play a central role in orchestrating the adaptive and innate immune responses by communicating with other cell types via antigen presentation and secretion of cytokines. In this study, we set out to understand how pneumococci activate human monocyte-derived DCs to produce interleukin-12 (IL-12) p70, an important cytokine during pneumococcal infections. We show that IL-12p70 production requires uptake of bacteria as well as the presence of the adaptor molecule TRIF, which is known to transfer signals of Toll-like receptor 3 (TLR3) or TLR4 from the endosome into the cell. While TLR4 is redundant for IL-12p70 production in DCs, we found that TLR3 is required to induce full IL-12p70 secretion. Influenza A virus (IAV) infection of DCs did not induce IL-12p70 but markedly upregulated TLR3 expression that during coinfection with S. pneumoniae significantly enhanced IL-12p70 secretion. Finally, we show that pneumococcal RNA can act as a bacterial stimulus for TLR3 and that it is a key signal to induce IL-12p70 production during challenge of DCs with pneumococci.A functional immune response is crucial to prevent and limit infections with  Streptococcus pneumoniae, a common colonizer of the nose, is the causative agent of severe and deadly diseases. A well-orchestrated immune response is vital to prevent and limit these diseases. Dendritic cells (DCs) reside in the mucosal linings of the lungs and sample antigens. They are activated by pathogens to present antigens and secrete cytokines. While many studies focus on murine models, we focused our work on human monocyte-derived DCs. We found that pneumococcal RNA is an important stimulus in DCs to activate the endosomal receptor TLR3, a receptor previously not identified to sense pneumococci, and its adaptor molecule TRIF. This leads to secretion of the cytokine interleukin-12 (IL-12). Severe pneumococcal pneumonia occurs closely after influenza A virus (IAV) infection. We show that IAV infection upregulates TLR3 in DCs, which sensitizes the cells to endosomal pneumococcal RNA. This new insight contributes to unlock the interplay between pneumococci, IAV, and humans. Streptococcus pneumoniae is a common colonizer of the upper respiratory tract, with the potential to cause mild diseases, like otitis media and sinusitis, or life-threatening diseases, such as pneumonia, sepsis, or meningitis. It is estimated by the WHO that more than 800,000 children under the age of 5 years die annually as a consequence of pneumococcal infection , such as the membrane-bound Toll-like receptors (TLRs) and cytosolic NOD-like receptors (NLRs), play an important role in innate detection of pneumococci. Several pneumococcal components have been implicated in the activation of NLRs and TLRs . These responses are common in infections with intracellular pathogens, but they are also found in infections with the extracellular pathogen S. pneumoniae , an important proinflammatory cytokine which drives the differentiation of Teumoniae –23. IL-1umoniae – which caumoniae –, 24. It umoniae –, which uin vitro studies have shown that IAV infection of human monocyte-derived DCs triggers an enhanced secretion of IL-12p70, IL-6, tumor necrosis factor alpha, and IFN-γ in response to secondary pneumococcal infection  receptor TLR3. Furthermore, we show that pneumococcal RNA activates TLR3 and that it is a sufficient and required stimulus for IL-12p70 production in DCs.Cytokine induction was investigated after infection of human DCs with the T4R strain. The nonencapsulated strain T4R was used as an alternative to opsonization of the encapsulated wild-type T4 strain, since it was shown previously that both induce comparable amounts of uptake and cytokine induction in DCs . Hence, TRIF is an important adaptor molecule for signaling from the endosomal compartment, since it mediates the signals from TLR3 and TLR4 into the cell. We studied the impact of TRIF on IL-12p70 production by silencing TRIF in DCs via small interfering RNA (siRNA). Upon stimulation with T4R, we found that IL-12p70 production was significantly reduced by the TRIF knockdown . The TRISince TRIF mediates the signals from both TLR3 and TLR4 into the cell and pneumolysin has been suggested to activate TLR4, we first set out to investigate whether TLR4 is activated by T4R in our infection model. We silenced TLR4 with siRNA in DCs and found that IL-12p70 production in response to T4R was independent of TLR4 . SimilarSince IL-12p70 production was independent of TLR4 but required TRIF, we next tested whether pneumococci are recognized by TLR3, an endosomal receptor shown to recognize dsRNA but that has not previously been identified as a receptor for pneumococci. Upon silencing of TLR3 with siRNA, a significant reduction in IL-12p70 production was observed in DCs challenged with T4R . In accoWe previously described a type I IFN-dependent cytokine boost in IAV-pneumococcus coinfections. IAV triggers type I IFN production in infected DCs, which in turn primes the surrounding DCs to secrete increased amounts of IL-12p70 . We set To understand whether pneumococcal RNA alone is sufficient to induce IL-12p70 production, total pneumococcal RNA from T4R or RNA digested with a cocktail of the endonucleases RNase A and RNase T1 was transfected into DCs. In combination, RNases A and T1 cleave RNA behind the C, G, and U residues, which results in nearly complete degradation of RNA. Total pneumococcal RNA was indeed a sufficient stimulus to induce IL-12p70 production in DCs, and RNA digested with the cocktail of RNases A and T1 lost its ability to activate DCs, indicating that there were no other components in our RNA preparation that activate the cells . To deteOur results showed that pneumococcal RNA can induce production of IL-12p70 by DCs and that pneumococcal RNA can act as a stimulus for TLR3.To investigate the role of RNA during the infection of DCs with whole pneumococci, we treated T4R with UV radiation or heat. We found heat treatment, in contrast to UV treatment, abolished bacterial induction of IL-12p70 . TreatmeTo study whether depletion of RNA from whole bacteria could decrease IL-12p70 production, we next treated UV-killed T4R with the cocktail of RNase A and RNase T1 prior to DC challenge. We used UV-killed bacteria for the RNA digestion, since the enzymatic treatment induced death in live bacteria (data not shown), which also could result in reduced cytokine production by DCs. The RNase treatment of UV-killed T4R led to a significantly reduced cytokine secretion by DCs in a dose-dependent manner, and the effect was specific for degradation of RNA, since pretreatment of LPS with the endonucleases did not affect cytokine secretion . In contWithin recent years, bacterial RNA has emerged as an important trigger of host immune responses to sense live bacteria , in both36–43–The role of pneumolysin as a TLR4 agonist is still being debated in the literature. Depending on the model system and cell type, it has been reported that pneumolysin activates TLR4 and induces the production of cytokines –16, wher–46–in vivo has also been found in mice in relation to pneumococcal infections for the receptors TLR1, TLR2, TLR4, and TLR6 .In murine-derived DCs infected with T4R, we previously found a much smaller effect on IL-12p70 secretion than in T4R-infected human DCs . Additioand TLR6 , 49, 50.and TLR6 . Monocytand TLR6 , and thein vitro. The presence of bacterial RNA was required and sufficient to induce IL-12 responses, and we found that completely digested pneumococcal RNA lost its capacity to induce IL-12p70 secretion. Moreover, we demonstrated that pneumococcal RNA activates TLR3.Here, we also identified a stimulus for IL-12p70 production by human DCs S. pneumoniae . Dilutions were made to obtain the desired concentration of bacteria, and viable counts were performed to retrospectively confirm the bacterial numbers. The X31 strain of IAV   of S. pntant T4R . Bacterin of IAV  was orign of IAV .6 to 1.5 × 106 cells/ml in R10  supplemented with granulocyte-macrophage colony-stimulating factor  and IL-4 (40 ng/ml) (both from PeproTech) for 6 days. Cells were given fresh media and cytokines at a ratio of 1:1 on day 4 and cultured until day 6. The human DC phenotype was assessed by examination of CD11c and CD1a expression via staining with allophycocyanin (APC)-conjugated mouse anti-human CD11c and fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD1a (BD Pharmingen). DCs used in these experiments were above 90% CD1a+/CD11c+.Monocytes were purified from buffy coats of healthy donors  by using a RosetteSep monocyte purification kit (Stem Cell Technologies) and Ficoll-Hypaque Plus (Amersham Biosciences) gradient centrifugation. Human DCs were seeded at 0.8 × 10LPS, PolyI:C, R848, cytochalasin D, and wortmannin were purchased from Sigma, and the TLR3/dsRNA complex inhibitor was purchased from Roche. Human type I IFN-α was purchased from PBL Assay Science. Cytochalasin D (0.5 mM), wortmannin (0.1 mM), and the TLR3/dsRNA complex inhibitor (270 µM) were applied 20 min prior to infection. LPS (100 ng/ml), PolyI:C (10 µg/ml), R848 (10 µg/ml), and IFN-α (500 U/ml) were used for stimulation of TLR4, TLR3, TLR7/8, and IFNAR, respectively.6) were electroporated with 5 µM siRNA against TRIF , TLR4 , TLR3 (s235 and s236), or random control siRNA (4390843 and 4390846)  on day 4 of DC differentiation. The cells were electroporated with the Bio-Rad Gene Pulser , immediately resuspended in fresh culture medium containing IL-4 and GM-CSF, and incubated for a further 2 days.DCs (6 × 105 per well) and exposed to pneumococci. If not otherwise stated, an MOI of 1 was used, and extracellular bacteria were killed with 200 µg/ml gentamicin after 2 h of infection and maintained in culture until the experiment was ended after 18 h. For opsonization, bacteria were incubated in the presence of pneumococcal antiserum for serotype 4 from Statens Serum Institut for the duration of the uptake period. For coinfection experiments, DCs were exposed to IAV at a MOI of 1 under serum-free conditions for 1 h and in the presence of serum for 3 h. Cells were pelleted, medium was removed, and T4R was added in fresh R10 medium. After a 2-h incubation, extracellular bacteria were killed with 200 µg/ml gentamicin and maintained in the cell culture until the experiment was ended after 18 h.DCs were seeded in 96-well plates  and the fixable viability dye (FVD) eFluor780 (eBioscience). Cells were infected as previously described, and 18 h after infection the numbers of apoptotic and necrotic cells were determined by staining cells with annexin V-FITC and the FVD eFluor780 in Annexin V buffer and fixation with 2% paraformaldehyde. Cells were assessed by flow cytometry in a Gallios flow cytometer.TC method. Each PCR run included no-template controls.Total cellular RNA was extracted from infected cells using the RNeasy kit (Qiagen). The concentration and purity of isolated RNA were determined spectrophotometrically with the NanoDrop ND 1000 apparatus. cDNA was synthesized from the isolated RNA using the High Capacity cDNA reverse transcription kit (Applied Biosystems). RT-PCR was performed using the iTaq Universal SYBR green supermix (BioRad). Predesigned primer mixes containing forward and reverse primers for the specific RT-PCR target were purchased from Qiagen (QuantiTect primer assay). The following primers were used: TRIF, Hs_TICAM1_1_SG; TLR4, Hs_TLR4_2_SG; TLR3, Hs_TLR3_1_SG; IL-12p40, Hs_IL12B_1_SG; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hs_GAPDH_1_SG. Each primer pair was validated for specificity by performing melt curve analysis of the PCR product to ensure the absence of primer dimers and unspecific products. For each sample, the mRNA expression level was normalized to the level of GAPDH, and relative expression was determined via the ΔΔin vitro, culture supernatants were harvested 18 h after infection and frozen at −20°C or used directly for measurement of IL-12p70 by using the OptEIA enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences).For cytokine assessment 2 UV radiation for 20 min. Death was confirmed by plating aliquots of the bacterial suspensions on blood agar plates and incubating at 37°C and 5% CO2 overnight.Bacteria were killed by incubation at 95°C for 20 min or by exposure to 1,200 J/mUV-killed bacteria were incubated with a cocktail of RNase A (200 to 20 U/ml) and RNase T1  (Life Technologies) or with DNase I  in a volume of 50 µl at 37°C for 1 h prior to infection of DCs. Total bacterial RNA was incubated with 25 U/ml RNase A and 1,000 U/ml RNase T1 of the RNase cocktail in a volume of 25 µl for 30 min.N-[1-propyl]-N,N,N-trimethylammonium methylsulfate  at a ratio of 1:2.5 and incubated for 18 h. HEK293 cells were transfected with 1 µg RNA per well complexed with Lipofectamine 2000 (Life Technologies) at a ratio of 1:2 and incubated for 6 h.T4R was grown to mid-log phase and treated with phenol. Total RNA was isolated with TRIzol (Life Technologies) followed by DNA digestion with Turbo DNase (Life Technologies). RNA concentration and quality were determined spectrophotometrically with the NanoDrop ND 1000 apparatus, and the absence of DNA contamination was confirmed by PCR. DCs were transfected with 1 µg bacterial RNA per well complexed with 4 cells were seeded per well of a 96-well plate. After 1 day, the cells were transfected with 1 µg pneumococcal RNA and incubated for 6 h. Cells were lysed, and luciferase activity was measured using the luciferase assay system (Promega) according to the manufacturer’s instructions.HEK293 cells stably expressed human TLR4, MD2, and CD14 as well as luciferase under the control of the NF-κB promoter or human TLR3 as well as luciferase under the control of the ELAM promoter , 62. TheIsolated total RNA was loaded on a RNA nanochip and analyzed and visualized with the Agilent 2100 Bioanalyzer.P levels of <0.05, <0.005, and <0.0005.Data were statistically analyzed as indicated in the figure legends, using GraphPad Prism 5.04. Significant differences were noted at Figure S1 Infection of DCs with T4R shows uptake-dependent IL-12p70 production. DCs were infected with T4R, and IL-12p70 was measured in the cell supernatant. DC viability was measured by flow cytometry (A). Uptake of bacteria by DCs was inhibited with cytochalasin D and wortmannin (B). Values represent means ± standard errors of the means for results from 4 (A) or 5 (B) experiments. cytD, cytochalasin D; WM, wortmannin. Download Figure S1, TIF file, 0.9 MBFigure S2 t test (RT-PCR) or a Wilcoxon matched-pairs signed-rank test (ELISA). *, P < 0.05. n.s., not significant. Download Enhanced IL-12p70 production in DCs primed with IFN-α requires TLR3. DCs were treated with IFN-α for 4 h, and expression of TLR3 was measured by RT-PCR after 8 h (A). DCs were primed with IFN-α for 4 h and/or subsequently infected with T4R with or without a TLR3 inhibitor, as indicated (B). DCs were primed with IAV for 4 h and/or subsequently transfected with total RNA from T4R with or without a TLR3 inhibitor, as indicated (C). IL-12p70 secretion was measured in the supernatant by ELISA (B and C). Values represent means ± standard errors of the means for results from 3 (A), 6 (B), or 5 (C) experiments. Statistical analysis was performed using Student’s Figure S2, TIF file, 1.2 MBFigure S3 UV-killed pneumococci induce IL-12p70 production similar to live pneumococci. Total RNA was isolated from live T4R or UV-killed or heat-killed T4R and visualized by gel electrophoresis on an RNA nanochip (A). DCs pretreated with cytochalasin D and wortmannin were challenged with UV-killed T4R , and IL-12p70 production was measured in the cell supernatant (B). Graph shows the means ± standard errors of the means for results from 4 experiments. HK, heat killed; cytD, cytochalasin D; WM, wortmannin. Download Figure S3, TIF file, 0.6 MB


Scaphechinus mirabilis. Acetylcholinesterase (AChE) inhibitors are used in the treatment of various neuromuscular disorders, and are considered as strong therapeutic agents for the treatment of Alzheimer’s disease (AD). Although EchA is clinically used to treat ophthalmic diseases and limit infarct formation during ischemia/reperfusion injury, anti-AChE effect of EchA is still unknown. In this study, we investigated the anti-AChE effect of EchA in vitro. EchA and its exhausted form which lost anti-oxidant capacity did not show any significant cytotoxicy on the H9c2 and A7r5 cells. EchA inhibited AChE with an irreversible and uncompetitive mode. In addition, EchA showed reactive oxygen species scavenging activity, particularly with nitric oxide. These findings indicate new therapeutic potential for EchA in treating reduced acetylcholine-related diseases including AD and provide an insight into developing new AChE inhibitors.Echinochrome A (EchA) is a dark-red pigment of the polyhydroxynaphthoquinone class isolated from sea urchin  Acetylcholine (ACh) is a neurotransmitter in the central nervous system  that canNumerous inhibitors against AChE are either isolated from natural sources ,11 or arScaphechinus mirabilis (Agassiz) and other species of sea urchins  for another 1 h [H9c2 and A7r5 cells were seeded at a concentration of 2 × 10ther 1 h . The MTT2PO4 0.33, MgCl2 0.5, glucose 16.6, and CaCl2 1.8, pH 7.4, cells were stained with 10 μM CM-H2DCFDA (excitation/emission = 490 nm/530 nm) dissolved in Tyrode solution for 30 min in a 37 °C incubator. Cells were resuspended in Tyrode solution and then treated with 0, 10, 25 μM of EchA for 10 min. Baseline fluorescence intensity was measured and then cells were treated with 250 μM H2O2 for 10 min. The changes in CMH2DCFDA intensity was measured with a fluorescence plate reader  at before and 10 min after adding the H2O2. Data were calculated as a % increase of value with respective baselines (n = 8).H9c2 cells were seeded on a 48-well culture plate and incubated until confluence. After washing cells twice with Tyrode solution containing (in mM) NaCl 135, KCl 5.4, HEPES 5, NaH2HPO4, 100 mM NaH2PO4, pH = 7.4) to a final volume of 50 μL per well. The reaction was started with 50 μL of acetylcholine (200 μM). Neostigomine bromide (IC50 = 0.006 μM) was used as a positive control for the inhibition of AChE. However, incubation time in AChE containing mixture and concentration of ACh were variable depending on experimental settings. The developing yellow color was measured at 412 nm over 20 min with 50-s interval using a Molecular device microplate reader SpectraMax M2e . The resulting AChE activity was obtained as a velocity (maxV). AChE activity without any compounds were set at 100% and expressed as a percentage of AChE alone. The inhibitory mode of EchA on AChE was determined by Lineweaver-Burk and Dixon plot [The inhibitory activity of EchA on AChE was determined by Amplite™ colorimetric acetylcholinesterase assay kit with slight modification. Purified AChE (50 Units/mL) was dissolved in 0.1% Bovine serum albumin. The enzyme (100 mU/mL), the co-substrate 5,5-dithiobis-2-nitrobenzoic acid (200 μM), and the different concentrations of EchA (0–100 μM) were incubated for 10 min at 37 °C. The mixture was dissolved in phosphate buffer . After washing twice with Tyrode solution containing (in mM) NaCl 135, KCl 5.4, HEPES 5, NaH2PO4 0.33, MgCl2 0.5, glucose 16.6, and CaCl2 1.8, pH 7.4, cells were loaded with 10 μM DAF-FM for 20 min at 37 °C. After washing once with Tyrode solution, the cells were treated with different doses of EchA (0–50 μM) for 10 min and then 2 mM sodium nitroprusside (SNP) was applied in the cells. The intensity of DAF-FM fluorescence  was measured using SpectraMax M2e  with scan mode at directly after adding SNP (baseline) and 30 min after SNP exposure. Values were obtained as a relative fluorescence unit and expressed as a percentage of the baseline. The SNP-untreated group was used as a negative control.A7r5 cells were seed at the density of 2 × 10t-test , and p < 0.05 was considered significant.All values are expressed as the mean ± SEM of at least three independent experiments. Significance was determined using one-way ANOVA followed by Dunnett’s Method or The results obtained from this study clearly indicate that EchA has a powerful anti-AChE activity exhibiting an irreversible and uncompetitive mode of inhibition. EchA and its exhausted form have less cytotoxicy on H9c2 and A7r5 cells. Unlike other quinones, EchA has nitric oxide scavenging activity and anti-oxidant potential. However, the elucidation of chemical structure related to anti-AChE with uncompetitive mode of EchA should be further investigated through comparison with other known AChE inhibitors. Taken together, the anti-AChE and anti-oxidant activities of EchA might be applied in treating ACh-limited diseases and the characteristic of EchA may provide an insight into developing a new valuable drug.


Plants are unavoidably subjected to various abiotic stressors, including high salinity, drought and low temperature, which results in water deficit and even death. Water uptake and transportation play a critical role in response to these stresses. Many aquaporin proteins, localized at different tissues, function in various transmembrane water movements. We targeted at the key aquaporin in charge of both water uptake in roots and radial water transportation from vascular tissues through the whole plant.MzPIP2;1 gene encoding a plasma membrane intrinsic protein was cloned from salt-tolerant apple rootstock Malus zumi Mats. The GUS gene was driven by MzPIP2;1 promoter in transgenic Arabidopsis. It indicated that MzPIP2;1 might function in the epidermal and vascular cells of roots, parenchyma cells around vessels through the stems and vascular tissues of leaves. The ectopically expressed MzPIP2;1 conferred the transgenic Arabidopsis plants enhanced tolerance to slight salt and drought stresses, but sensitive to moderate salt stress, which was indicated by root length, lateral root number, fresh weight and K+/Na+ ratio. In addition, the possible key cis-elements in response to salt, drought and cold stresses were isolated by the promoter deletion experiment.The The MzPIP2;1 protein, as a PIP2 aquaporins subgroup member, involved in radial water movement, controls water absorption and usage efficiency and alters transgenic plants drought and salt tolerance. Plants are unavoidably subjected to various abiotic stressors, including high salinity, drought and low temperature. These stressors result in water deficit, wilt and even death in severe cases –3. The win vivo and in vitro experiments. In vitro, maize ZmPIP2a was able to transport large amounts of water in the Xenopus ovocytes system [In vivo, the lily PIP1 increased osmotic water permeability coefficient (Pf) in transgenic tobacco leaf protoplasts [Arabidopsis PIP mutant, Pf of leaves protoplasts decreased markedly, and in the PIP-antisense transgenic Arabidopsis, the Lp (i.e. hydraulic conductivity) of cells went down as well [Aquaporins are membrane proteins, which belong to the major intrinsic protein (MIP) family, involved in transmembrane water movement. Their activity to transport water across membrane was proved by s system . In vivotoplasts . In Arab as well ,8.As the key proteins in plant growth and development, abundant aquaporins exist in the most living organisms , involved in many physiological processes including water uptake and transportation, reproduction and photosynthesis –11. TheyAquaporins play especially vital roles in response to water deficit. Plants evolved a sophisticated mechanism to fine-tune the abundance, relocation and gating of aquaporins, which facilitate plants to modify water permeability rapidly and reversibly –22. The PgTIP1 gene conferred transgenic Arabidopsis enhanced salt tolerance [HvPIP2;1 made the transgenic rice more sensitive to salt stress [Arabidopsis ectopically expressing VfPIP1 showed enhanced drought tolerance [PIP1b and HvPIP2;1 expression were sensitive to drought stress [Because of the variant roles of aquaporins in response to water deficit, the transgenic plants overexpressing aquaporin genes exhibited different phenotypes under stresses. olerance . But HvPt stress . Likewisolerance . But trat stress ,33. AlthMzPIP2;1 gene encoding plasma membrane intrinsic protein from a salt-tolerant apple rootstock Malus zumi Mats. The transverse and longitude paraffin sections profiled the localization of MzPIP2;1, suggesting it might be the key aquaporin involved in water movement from soil to root epidermal cells and from vessels to parenchyma cells all through the whole plant. The phenotype of the transgenic Arabidopsis ectopically expressed MzPIP2;1 and the GUS expression driven by different regions of the MzPIP2;1 promoter were observed under salt, drought and cold stresses. These results systematically confirmed the crucial roles of MzPIP2;1 in both water uptake and radial water transportation, which was concluded to control the efficiency of water absorption and usage under water deficit. We isolated the possible stress-response cis-elements. It laid a foundation for isolating the genes regulating aquaporins, which remains unclear.In this work, we cloned Arabidopsis  seeds were sterilized with 1% sodium hypochlorite for 12min, 75% alcohol for 30sec, and washed 4 times with sterile water. After vernalized 3 days in dark at 4°C, the seeds were sowed and grown vertically on Murashige and Skoog (MS) medium in a growth chamber of 16h light (~150 μEm-2 sec-1) /8h dark at 22±2°C. When the seedlings had four leaves, they were transplanted into the soil.The MzPIP2;1 gene was isolated by microarray, in which the 15,000 cDNA clones were from the cDNA library constructed by our lab [Malus zumi Mats (apple rootstock) under salt stress using the EASYspin Plant RNA Rapid Extraction Kit , and immediately used for cDNA synthesis with a first-strand cDNA synthesis kit . The full-length MzPIP2;1 cDNA of 1094 bp was amplified using the 2×ES Taq MasterMix  with the specific primers carrying the restriction sites BamHI and XbaI . The PCR was performed as follows: 94°C for 3min; 30 cycles at 94°C for 30sec, 52°C for 30sec, and 72°C for 50sec; and 72°C for 10min. The PCR product was cloned into the PMD®19-T simple vector  and sequenced. The NCBI accession number of MzPIP2;1 was GenBank: KR349329.The salt-responsive  our lab . Total Rhttp://smart.embl-heidelberg.de/. Meanwhile, its molecular weight and isoelectric point were analyzed on line at http://www.bio-soft.net/sms/ and http://web.expasy.org/compute_pi/, respectively. The multiple alignments of MzPIP2;1 amino acid sequence were conducted using DNAMAN software to identify the shared domains among arabidopsis, rice, maize and horticultural plants including pear, grape, castor, spinach, cabbage, poplar and apple, using its homologous proteins: AtPIP2;1 (AEE79084.1), OsPIP2;1 (Q8H5N9.1), PcPIP2;2 (BAB40143.1), VvPIP2 (ABC84558.1), RcPIP2;1 (EEF29633.1), AtPIP3 (AAB36949.1), AtPIP2;5(AEE79295.1), AtPIP1;4(AEE81879.1), SoPIP1;2 (AAR23268.1), ZmPIP1;2 (NP_001104934.1), OsPIP1;3 (Q9SXF8.2), BoPIP3 (AAG30607.1), PcPIP1;1 (BAB40142.1), PcPIP2;1 (BAB40141.1), PtPIP2;5(CAH60724.1) and PtPIP2;2 (CAH60721.1). The phylogenetic tree was constructed using the neighbor-joining method and a bootstrap test with 1000 iterations by MEGA5.2 software.The conserved domain of MzPIP2;1 was predicted on line at MzPIP2;1 without stop codon was amplified by PCR using the primers with EcoRI/BamHI restriction sites . The PCR cycles were performed as follows: 94°C for 3min; 30 cycles at 94°C for 30sec, 52°C for 30sec, and 72°C for 50sec; and 72°C for 10min. The PCR product was cloned into the PMD®19-T simple vector . After sequencing, it was digested with EcoRI/BamHI and subcloned into transient expression vector pEZS-NL to be expressed as a fusion protein with an eGFP (enhanced Green Fluorescent Protein).The coding region of Arabidopsis grown in greenhouse. The MzPIP2;1-eGFP and eGFP control plasmids were introduced to protoplasts by PEG mediated transformation as described by Yoo et al. (2007)[MzPIP2;1-eGFP or eGFP control plasmid was mixed gently, after which 110μL PEG was added and incubated for 20min. After washing twice by W5 solution , the protoplasts were diluted by 1mL W5 solution, and incubated for 16–24h at 23°C in culture plate under low light conditions. The protoplast solution was observed using confocal microscopy  with 600× magnification (objective magnification×eyepiece magnification = 60×10).The protoplasts were extracted from the leaves of 3-week-old l. (2007). A total™in vitro Cloning Kit, the chromosome walking of MzPIP2;1 was conducted. The DNA of Malus zumi Mats was digested completely with restriction enzymes XbaI, BamHI and EcoRI, and ligated with adapters carrying restriction sites XbaI, BamHI and EcoRI. The first PCR reaction was conducted using the adapter primer C1 and the gene specific primer PGSP1 designed by the cDNA sequence of MzPIP2;1. The PCR cycles were performed as follows: 94°C for 3min; 35 cycles at 94°C for 30sec, 62°C for 30sec, and 72°C for 2min; and 72°C for 10min. Then PCR product was diluted 100 times and used for template, which was amplified with inner adapter primer C2 and the mxIRT1 gene specific primer PGSP2 . The PCR product was cloned into the PMD®19-T simple vector  and sequenced. The MzPIP2;1 promoter sequences of 1500bp were blasted on Apple Genome Database (http://genomics.research.iasma.it/). Its specific forward and reverse primers, carrying restriction sites BamHI and SmaI, were designed to amplify exactly 1500bp before ATG. The PCR was performed as follows: 94°C for 3min; 35 cycles at 94°C for 30sec, 55°C for 30sec, and 72°C for 2min; and 72°C for 10min. The PCR product was cloned into the PMD®19-T simple vector  and sequenced.According to the instructions of TaKaRa LA PCRMzPIP2;1 promoter sequence was analyzed on line at website http://bioinformatics.psb.ugent.be/webtools/plantcare/html/. Based on cis-elements analysis of MzPIP2;1 promoter, the primers were designed at different regions on the Primer Premier 5.0 software. The specific forward primers of two deletion promoters, carrying restriction site BamHI, were designed on 948bp and 644bp before ATG, respectively. The PCR program was performed as follows: 94°C for 3min; 28cycles at 94°C for 30sec, 50°Cfor 30sec, and 72°C for 90sec; and 72°C for 10min. The PCR product was subcloned into the PMD®19-T simple vector  and sequenced. The sequences of all the primers were shown in The MzPIP2;1 gene was inserted into plant expression vector pCB302-3 with BamHI/XbaI, driven by the cauliflower mosaic virus (CMV) 35S promoter. The full-length and two deletions of MzPIP2;1 promoter were inserted into plant expression vector pBI121 with BamHI/SmaI, respectively. The pCB302-3- Pro35S::MzPIP2;1 plasmid, pBI121-ProMzPIP2;1::GUS plasmid, pBI121-ProMzPIP2;1-d1::GUS and pBI121-ProMzPIP2;1-d2::GUS plasmids were introduced into Agrobacterium tumefaciens strain GV3101 using the freeze-thaw method [Arabidopsis plants using the floral dip method [MzPIP2;1 or GUS gene, respectively. Then the selected T1 seedlings were confirmed by PCR and transplanted into soil for seeds collection in a growth chamber at 22±2°C in a 16/8h light/dark cycles. The single-inserted T2 plants were judged by the 3:1 segregation ratio. The single-inserted homologous T3 transgenic lines were obtained when no segregation happened. The expression of foreign genes were detected by semi-quantitative RT-PCR using the MzPIP2;1-specific primers .The w method , respectp method . The T0 -2 sec-1) were used as control. The phenotypes were observed 2 weeks later after treatment. The root length, the lateral root number and the fresh weight of transgenic and wild-type plants under every treatment were recorded, respectively. In addition, the treated and wild-type Arabidopsis were harvested for the Na+ and K+ detection. Each experiment was repeated three times independently.The T3 seeds of three single-insertion lines and the wild-type were sterilized and sowed on MS medium. The 7-day-old seedlings were transferred to MS medium containing 100mM or 130mM NaCl for salt stress treatment, 300mM mannitol for drought stress in a growth chamber at 22±2°C. For cold stress treatment, the seedlings were placed at 4°C. The seedlings growing under normal conditions (short-day conditions (8/16h light/dark cycles) with a light intensity of ~150 μEm3-H2O2-microwave digestion method was applied for the measurement of Na+ and K+. After the plant materials were washed 2–3 times with water and another 2–3 times with deionized water, the samples were put into envelopes for drying 30min at 105°C and 48h at 70°C. The dried samples were grinded into powder with mortars and pestles. A total of 50mg samples were transferred to tubes and mixed with 6mL guarantee reagent HNO3, which were shaken gently overnight. Then 2 mL guarantee reagent H2O2 was add for a reaction of 10min. The tubes were put into microwave digestion instrument  for digestion with the program . After the tubes cooled down, the solutions were transferred and diluted to 25mL with distilled water. After they were kept still for 1h, the supernatants were moved into 10mL centrifuge tubes for measurement by atomic absorption spectrometer . The 0, 5, 10, 20 and 50ppm Na+ and K+ were used as standard solution.The HNOArabidopsis seedlings expressing GUS gene driven by the MzPIP2;1 full-length promoter and its two deletions were placed on filter paper soaking 150mM NaCl solution for salt stress treatment, 200mM mannitol solution for drought stress treatment, and deionized water as control. Except for the cold stress at 4°C, other treatments were conducted in a growth chamber with cycles of 8h light (~150 μEm-2 sec-1) and 16h dark at 22±2°C. After 24h treatment, the plants were sampled and vacuumized for 10min and incubated at 37°C overnight in the GUS solution . After decolorized in 70% ethanol at 37°C overnight, the samples were placed on plates carrying deionized water for observation by stereoscope with 7.3× magnification (objective magnification × eyepiece magnification = 0.73×10) .The 10-day-old Arabidopsis stems expressing the GUS gene driven by MzPIP2;1 full-length promoter were made. The longitudinal section and transverse section of stems were observed by light microscope with 600× magnification (objective magnification × eyepiece magnification = 60×10) . In addition, the GUS expression in roots and leaves were also observed under light microscope 400× magnification (objective magnification × eyepiece magnification = 40×10).Similar study in soil was conducted using 4-week-old seedlings. The transgenic and the wild-type plants were not watered in drought stress treatment, watered with 200mM NaCl for salt stress treatment and cultured at 4°C for cold stress treatment, respectively. After 24h treatment, the stems and roots of transgenic and wild-type plants were sampled for GUS analysis as described above. The experiments were repeated three times. In addition, to determine localization of MzPIP2;1 in the whole plant, paraffin sections of the transgenic One-way ANOVA was used for statistical evaluations by SPSS software. The differences were significant when P<0.05 and very significant when P<0.01.MzPIP2;1 (GenBank: KR349329) was amplified from Malus zumi. It is 1094bp long including an open reading frame (ORF) of 852bp, a 5’-UTR of 65bp and a 3’-UTR of 177bp. The MzPIP2;1 gene encodes 283 amino acids with predicted relative molecular mass of 30.08KD and predicted isoelectric point of 8.68. The multiple alignments indicated that MzPIP2;1 shared a high similarity in the transmembrane domains and loops with the other PIP2;1 proteins: 91.64%, 79.23%, 86.22%, 78.75% and 76.03% sequence identity with PcPIP2;2 from Pyrus communis, VvPIP2 from Vitis vinifera, RcPIP2;1 from Ricinus communis, AtPIP2;1 from Arabidopsis thaliana, OsPIP2;1 from Oryza sativa, respectively , involved in trafficking of K+ channels [+ channels in membranes by trafficking competition and decrease the K+ absorbance. In our research, significant decrease of K+ absorbance happened in transgenic plants. It might result from both of the well-known Na+ /K+ channels competition and the possible trafficking competition between K+ channels and PIPs. When transgenic plants were subjected to moderate salt stress, the abundant PIP proteins increased plant tolerance, but the ionic toxicity made the plants less resistant to salt stress [MzPIP2;1 gene was highly expressed, the kinase for the its phosphorylation and other PIPs for heteromerization were not expressed in high level accordingly. This may limit activity of over-produced MzPIP2;1.If MzPIP2;1 could improve the water uptake and usage efficiency as expected, the ectopically expressed ild-type . Under sild-type . Obvioust stress . Comparepparatus . In ordet stress . This drchannels , 43. Thet stress , 45. Thecis-elements involved in abiotic stress response for further research. In the young seedlings, the result suggested that the key cis-elements might be in deletion2 region. In addition, it was very interesting that the GUS expression driven by deletion2 was induced dramatically by cold stress in young seedlings, compared with the full-length promoter and deletion1  at 22±2°C) and sampled at 0, 4, 8, 12, 24h, 3 and 5days, respectively. The MzACTIN gene is as control.The (TIF)Click here for additional data file.S2 FigMzPIP1;1 (A), MzPIP1;2 (B) and MzPIP2;2 (C). The transmembrane domains were underlined; NPA box was paned; Asterisk, the sequences of PIPs were dashed; the stop coden was labeled with *.(TIF)Click here for additional data file.


Plants colonized terrestrial environments approximately 480 million years ago and have contributed significantly to the diversification of life on Earth. Phylogenetic analyses position a subset of charophyte algae as the sister group to land plants, and distinguish two land plant groups that diverged around 450 million years ago – the bryophytes and the vascular plants. Relationships between liverworts, mosses hornworts and vascular plants have proven difficult to resolve, and as such it is not clear which bryophyte lineage is the sister group to all other land plants and which is the sister to vascular plants. The lack of comparative molecular studies in representatives of all three lineages exacerbates this uncertainty. Such comparisons can be made between mosses and liverworts because representative model organisms are well established in these two bryophyte lineages. To date, however, a model hornwort species has not been available.Anthoceros agrestis as a model hornwort species for laboratory experiments. Axenic culture conditions for maintenance and vegetative propagation have been determined, and treatments for the induction of sexual reproduction and sporophyte development have been established. In addition, protocols have been developed for the extraction of DNA and RNA that is of a quality suitable for molecular analyses. Analysis of haploid-derived genome sequence data of two A. agrestis isolates revealed single nucleotide polymorphisms at multiple loci, and thus these two strains are suitable starting material for classical genetic and mapping experiments.Here we report the establishment of A. agrestis to be used as a model species for developmental, molecular, genomic, and genetic studies. This advance provides an unprecedented opportunity to investigate the biology of hornworts.Methods and resources have been developed to enable  Plants colonized terrestrial environments approximately 480 million years ago ,2. PhyloMarchantia polymorpha and the moss Physcomitrella patens have been used to reveal evolutionary trajectories of developmental mechanisms that regulate morphological traits such as root hairs [The uncertainty over the phylogenetic position of hornworts is compounded by our relatively limited understanding of hornwort biology. As land plants evolved, the modification of various character traits led to a general increase in size and complexity such that the bryophytes are relatively simple, both in terms of morphology and physiology, as compared to flowering plants. An understanding of how this complexity evolved can be obtained through comparative analyses of developmental processes in extant land plant species. To date, the liverwort ot hairs , and botot hairs ) and envot hairs ) physiolNostoc cyanobacteria [Regardless of whether hornworts are sister to all other land plants, sister to vascular plants, or part of a bryophyte clade, their phylogenetic position is key to understanding the evolution of land plant body plans -15. Notabacteria . An undeAnthoceros agrestis as a tractable hornwort experimental system. Anthoceros was the first hornwort genus described [A. agrestis having the smallest genome of all bryophytes investigated so far . Similaanalyses . Here, wA. agrestis strains have been propagated. The first was established from plant material collected near Fogo in Berwickshire, UK (hereafter referred to as the “Oxford strain”) and the second from plant material collected near Hirschbach, Germany (hereafter referred to as the “Bonn strain”). All existing material of both the Oxford and Bonn strains originate from a single spore. Attempts to establish Anthoceros punctatus strains were carried out in parallel, and although vegetative propagation was successful, conditions for reproductive propagation proved elusive. As such, A. punctatus was rejected as a potential model organism.Two different 2sec−1)/8 h dark. Young gametophytes were large enough to be sub-cultured 1–2 months after spore germination.To initiate axenic cultures, several sterilization protocols were tested. Bacterial and fungal contamination of spores was successfully eliminated using bleach, and thus a simple three-minute treatment followed by washing was adopted see . FollowiP. patens. Plates were incubated at 23°C, either under a diurnal cycle of 16 h light (300 μEm2sec−1)/8 h dark or under continuous light (300 μEm2sec−1). In all cases, cultures were propagated and maintained by monthly sub-culturing, in which a small fragment of thallus tissue (~5-7 mm in diameter) was cut and placed on fresh medium. In general, the Oxford strain grew better  on Lorbeer’s or 1/10 KNOP media whereas the Bonn strain grew better on BCD medium. In all cases, plants grew faster under continuous light than with long day photoperiods, as long as the light intensity was kept at, or below, 300 μEm2sec−1.Three different media were tested for their ability to support vegetative growth of gametophytes. In addition to Lorbeer’s medium, gametophytes were transferred to 1/10 KNOP medium  and to BIn natural ecosystems, sexual reproduction in hornworts is initiated by the formation of antheridia on the thallus, and then after approximately one month archegonia develop . To dete2sec−1) or long day photoperiod 16 h light (150 μEm2sec−1)/8 h dark. In all cases, antheridia appeared as reddish dots on the surface of the thallus after approximately one month. Given that archegonia are colourless and are embedded within the thallus, their formation could not be easily visualized, and thus the appearance of antheridia was used as a prompt to induce fertilization.To optimize induction conditions, growth at 16°C was next compared on different media and under different light regimes. Gametangia were successfully induced on both 1/10 KNOP and BCD media but not on Lorbeer’s medium, and in both continuous light . This variability is also reflected in the fact that even with the extra liquid addition steps, the number of sporophytes produced by each thallus ranged from 5 to over 100. There is no apparent way in which this variation can be more carefully controlled given that the development of archegonia is difficult to monitor. Emerging sporophytes went through the normal cycle of sporophyte maturation and contained hundreds of spores. Spores were viable and were regularly used to initiate new cultures.Although the extraction of nucleic acids from any organism is generally considered to be straightforward, hornwort gametophyte tissue is rich in polysaccharides (mucilage) , and wasA. agrestis has previously been reported as 83Mbp on the basis of flow cytometry [Arabidopsis thaliana (5 SNPs/1 Kbp) [Populus tremula (2–6 SNPs/1 Kbp) [P. patens where strains show a similar level of genetic divergence [The haploid genome size of ytometry . Using ks/1 Kbp)  or Populs/1 Kbp) , but is vergence .A. agrestis to be used as a model species for developmental, molecular, genomic and genetic studies. Axenic cultures have been established, conditions for sexual propagation and nucleic acid extraction have been optimised, and two strains with sufficient genetic divergence have been identified for genetic analyses. This advance provides an unprecedented opportunity to investigate the biology of hornworts.Methods and resources have been developed to enable http://www.ebi.ac.uk/ena/data/view/PRJEB8683).Raw sequence data for the Bonn and Oxford strains have been deposited in the European Nucleotide Archive and are available under study accession number PRJEB8683  on 30th October 2012 by Dr David Long . Voucher specimens have been deposited in the Fielding Druce Herbarium, University of Oxford (OXF). The A. agrestis Bonn strain was obtained between Hirschbach and Reinhardtsgrimma, on a crop field approximately 500 m from the street (K9022), near a small copse on 15th November 2006 by Dr Susann Wicke and Dr Dietmar Quandt. Voucher specimens have been deposited in the Herbarium of the University of Bonn (H015-H018).The 4.7H2O, 0.1 g/L KH2PO4, 0.2 g/L NH4NO3, 0.1 g/L CaCl2) supplemented with 1 mL of Hutner’s trace elements [4.7H2O, 11.4 g/L H3BO3, 5.06 g/L MnCl2.4H2O, 1.61 g/L CoCl2.6H2O, 1.57 g/L CuSO4.5H2O, 1.1 g/L (NH4)6Mo7O24.4 H2O, 4.99 g/L FeSO4.7H2O) adjusted to pH6.5 and solidified with 6.5 g/L agar; 1/10 KNOP medium [2HPO4, 0.025 g/L KH2PO4, 0.025 g/L KCl, 0.025 g/L MgSO4.7H2O, 0.1 g/L Ca(NO3)2.4H2O, 37 mg/L FeSO4.7H2O) adjusted to pH6.5 and solidified with 6.5 g/L agar; and BCD medium [4.7H2O, 0.25 g/L KH2PO4 (pH6.5), 1.01 g/L KNO3, 0.0125 g/L FeSO4.7H2O and 0.001% Trace Element Solution 2.12H2O, 0.055 mg/L CuSO4.5H2O, 0.028 mg/L KBr, 0.028 mg/L LiCl, 0.389 mg/L MnCl2.4H2O, 0.055 mg/L CoCl2.6H2O, 0.055 mg/L ZnSO4.7H2O, 0.028 mg/L KI and 0.028 mg/L SnCl2.2H2O) supplemented with 1mM CaCl2 and solidified with 8 g/L agar.Three different media were used: Lorbeer’s medium  (0.1 g/Lelements  (50 g/L P medium  (0.025 gD medium  (0.25 g/Isolated sporophytes were left to dry before removing the spore contents. Spores were sterilized by gentle agitation in 5% (v/v) bleach  in microcentrifuge tubes, followed by three washes in sterile water with brief centrifugation steps between each wash.DNA was extracted from 1 gram of ground frozen tissue using 10 mL prewarmed (60°C) extraction buffer  CTAB, 0.3% (v/v) β-mercaptoethanol, 100 mg of polyvinylpyrrolidone-40 (PVP) per 1 g tissue) plus 5 μl of 100 mg/mL RNAase A. After incubation at 60°C for 30 min, samples were cooled to room temperature and then extracted with chloroform:isoamylalcohol (24:1). A second chloroform:isoamylalcohol (24:1) step was carried out to remove any remaining PVP. DNA was precipitated from the aqueous phase with 0.5 volumes 5M NaCl and 2 volumes of cold (−20°C) 95% ethanol. After resuspension in 2 mL 10 mM Tris pH8, 1mM EDTA (TE), a second ethanol precipitation was carried out and then the DNA was dissolved in TE for storage and subsequent analyses.2O instead of TE. RNA was then precipitated overnight at 4°C after the addition of 0.25 volumes of 8 M LiCl. After resuspension and a third ethanol precipitation, RNA was resuspended in DEPC-treated water for storage at −80°C and subsequent analyses.RNA was extracted in two different ways. For large scale RNA extractions, samples were treated as for DNA extractions with the exception that all solutions were prepared with water that had been autoclaved after treatment with 0.1% diethylpyrocarbonate (DEPC) and RNAase was omitted from the extraction buffer. In addition, after the second ethanol precipitation, the pellet was resuspended in DEPC-treated dH2, ground into a fine powder and resuspended in 750 μL binding buffer. RNA was eluted in 30 + 30 μL of nuclease free water and stored at −80°C.For extractions where the recovery of small RNAs was required, the Spectrum™ Plant Total RNA Kit (Sigma) was used. Before each extraction residual water was removed from ~2-3 thalli (each ~0.5 cm diameter) using paper towel. Tissue was flash-frozen in liquid NA. agrestis Bonn strain, DNA was extracted from one month old thalli using the protocol detailed above. The draft genome sequence data are derived from the haploid phase, a significant advantage over vascular-plant genomes, which are all based on diploid individuals. Paired-end libraries were prepared for next generation sequencing using the Nextera XT kit (Illumina inc.) with 1 to 10 ng DNA. Nextera DNA libraries were sequenced on 1/3rd of a Miseq flow cell with 250 cycles. After sequencing and de-multiplexing, approximately 4.99 million paired-end reads were obtained. Reads were trimmed using Trimmomatic [To generate a low-coverage reference sequence for the mmomatic  and all mmomatic ) with k-mmomatic  k-mer anmmomatic ). To idehttp://broadinstitute.github.io/picard/) and reads re-aligned using the GATK IndelRealigner [GATK  best practice was followed to identify SNPs with high-confidence . Brieflyealigner . Finallyealigner  to extraealigner  to calcu


Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 1010 clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule , the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications.We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability ( Antibodies are the most commonly used binding proteins with >240 candidates in clinical development  and remaEscherichia coli. Most do not contain cysteines enabling the introduction of a cysteine for site-specific coupling of biotin, fluorescent labels, or polyethylene glycol to enhance their utility or stability. These characteristics make artificial binding proteins powerful tools capable of replacing antibodies in a range of applications including research  protein inhibitors of cysteine proteases , cell adhesion molecule CD31, SH2 domains and a 12-aa peptide.Oryza sativa; U54702), ZMA2  and HAN1  protein sequences as search probes. The accession codes and sources of sequences used to derive the consensus sequence are shown in Supplementary Table SI. These coding sequences were translated and aligned using the program MULTALIN (E. coli codon optimised gene was synthesised (GenScript) M15 zzf::Tn10(TetR)/fhuA2 glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5) but not in a non-suppressor strains such as JM83 (ara Δ(lac-proAB) rpsL (Strr)[φ80 dlacΔ(lacZ)M15] thi).A consensus sequence derived from alignment of 57 phyocystatin sequences was developed to enhance phytocystatin properties. To design the consensus phytocystatin gene, a tBLASTN search of the Genbank database was performed using OSA-I (MULTALIN . This coCTGCAGTTCTTTGAAGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNCTTAACCCAAACTTTCGCTTCG-3′.The Adhiron library was constructed by splice overlap extension (SOE) of two PCR products (The degenerate positions (NNN) were introduced as trimers representing a single codon for each of the 19 amino acids excluding cysteine and there were no termination codons. PCR used Phusion High Fidelity Polymerase (NEB) at 98°C for 5 min then 20 cycles of 98°C, 10 s; 56°C, 15 s; 72°C, 15 s followed by 72°C for 5 min. PCR products were purified by gel extraction (Qiagen), used for 10 cycles of SOEing as above, then digested with NheI and PstI, gel extracted and cloned into the similarly digested pBSTG1-Ad phagemid.E. coli ER2738 electrocompetent cells (Lucigen). In total, 20 ml of ER2738 cells was electroporated with 126 ng of library DNA per 50 μl of ER2738 cells. After a 1-h recovery in 2TY medium, cells were grown at 37°C and 225 rpm to an OD600 of 0.6 in 5 l of 2TY medium. M13KO7 helper phage (1011) were added with shaking at 37°C and 100 rpm for 1 h followed by overnight at 25°C with kanamycin (50 µg/ml). Phage were precipitated with 6% polyethylene glycol 8000 and 0.5 M NaCl, and were suspended in 50% glycerol for storage.Ligated products were electroporated into d-1-thiogalactopyranoside (IPTG) induction and purified by Ni-NTA resin (Qiagen) affinity chromatography according to the manufacturer's instructions. Purity was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). The following protocols are described for yeast SUMO with an identical protocol used for the screening of other targets. Yeast SUMO was biotinylated using EZ-link NHS-SS-biotin (Pierce), according to the manufacturer's instructions. Biotinylation was confirmed using streptavidin conjugated to horseradish peroxidase (HRP). Biotinylated ySUMO was bound to streptavidin-coated wells (Pierce) for 1 h, then 1012 cfu pre-panned phage were added for 2.5 h with shaking. Panning wells were washed 10 times and eluted with 50 mM glycine–HCl (pH 2.2) for 10 min, neutralised with 1 M Tris–HCL (pH 9.1), further eluted with triethylamine 100 mM for 6 min, and neutralised with 1 M Tris–HCl (pH 7). Eluted phage were used to infect ER2738 cells for 1 h at 37°C and 90 rpm then plated onto LB agar plates with 100 µg/ml carbenicillin and grown overnight. Colonies were scraped into 5 ml of 2TY medium, inoculated in 25 ml of 2TY medium with carbenicillin (100 µg/ml) and infected with ca. 1 × 109 M13K07 helper phage. After 1 h at 90 rpm, kanamycin was added to 25 μg/ml for overnight at 25°C and 170 rpm. Phage were precipitated with 4% polyethylene glycol 8000, 0.3 M NaCl and resuspended in 1 ml of 10 mM Tris, pH 8.0, 1 mM EDTA (TE buffer). A 2 µl aliquot of phage suspension was used for the second round of selection using streptavidin magnetic beads (Invitrogen). Yeast SUMO labelled beads were washed and incubated with pre-panned phage for 1 h then washed five times using a KingFisher robotic platform (ThermoFisher) and eluted and amplified as above. The final pan used neutravidin high binding capacity plates (Pierce), as previously described for panning round one, with phage eluted using 100 µl of 100 mM dithiothreitol. Phage were recovered from wells containing target protein and control wells to determine the level of amplification in target wells.Yeast SUMO (ySUMO) protein was expressed in BL21 (DE3) cells using isopropyl β-11/ml) were added, followed by kanamycin to 25 μg/ml overnight and incubated at 25°C (450 rpm). Streptavidin-coated plates (Pierce) were blocked with 2 × casein blocking buffer (Sigma) overnight at 37°C. The plates were incubated with biotinylated yeast SUMO or biotinylated linker for 1 h, and 45 µl of growth medium containing the phage was added and incubated for 1 h. Following washing, phage were detected by a 1 : 1000 dilution of HRP-conjugated anti-phage antibody (Seramun) for 1 h, visualised with 3,3′,5,5′-tetramethylbenzidine (TMB) (Seramun) and measured at 610 nm.Individual ER2738 colonies were grown in 100 µl of 2TY with 100 µg/ml of carbenicillin in a 96-deep well plate at 37°C (900 rpm) for 6 h. A 25 µl aliquot of the culture was added to 200 µl of 2TY containing carbenicillin and grown at 37°C (900 rpm) for 1 h. Helper phage  λ(DE3F [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) 400 ml cultures in LB medium were induced with 0.1 mM IPTG. Cells were grown for 6 h, harvested and lysed with Bugbuster (Novagen). The clear supernatant was mixed with 500 µl of Ni-NTA resin slurry (Qiagen) for 1 h, washed three times in  and eluted in 3 × 1 ml of elution buffer . One hundred micrograms of the SUMO-binding Adhirons were biotinylated using NHS SS-biotin (Pierce) according to the manufacturer's instructions for use in enzyme-linked immunosorbent assay (ELISAs) and western blotting.The DNA coding sequences of Adhirons were amplified by PCR, restriction digested with NheI and PstI and cloned into pET11a containing the Adhiron scaffold similarly digested. Plasmid DNA was purified (Qiagen) from transformant colonies and sequenced to confirm the correct insert. Following transformation into BL21 (DE3) cells (Unless otherwise indicated 5 ng of target protein in PBS was absorbed onto Immuno 96 Microwell™ Nunc MaxiSorp™ plate wells overnight at 4°C. Two hundred microlitre of 3× blocking buffer was added at 37°C for 4 h with no shaking. Biotinylated yeast SUMO-binding Adhirons (Ad-ySUMO) at 0.1 µg/ml in Phosphate-buffered saline with Tween 20 (PBST) containing 2× blocking buffer were incubated in wells for 1 h with shaking. Wells were washed three times in PBST, and streptavidin conjugated to HRP (Invitrogen) diluted 1 : 1000 in 50 µl PBST was added for 1 h. After washing Adhiron binding was visualised with 50 µl TMB (Seramun) and the absorbance measured at 610 nm.g and then resolved in a 15% SDS–polyacrylamide gel. Proteins were transferred to PVDF membranes for 45 min at 4 W (Amersham Biosciences) and incubated for 1 h in blocking buffer (5% BSA in PBS 0.1% Tween) followed by incubation for 1 h with Ad-ySUMO (100 µg/ml diluted 1 : 1000 PBST). Bound Ad-ySUMOs were detected using streptavidin-conjugated HRP and chemiluminescence .Target protein alone or mixed with HEK293 cell lysate (20 µg) was mixed with loading buffer , boiled for 3 min, centrifuged for 1 min at 15 000Adhiron samples in PBS were buffer exchanged into 50 mM ammonium acetate using Zeba spin columns  and 20 ml samples of 20 mM protein were analysed on a Synapt HDMS (Waters UK Ltd) electrospray mass spectrometer.The BLitz™ (ForteBio) dip and read streptavidin biosensors were used to estimate the affinity of ySUMO binding to immobilised biotinylated Ad-ySUMO binders, according to the manufacturer's instructions. At least four readings at different ySUMO concentrations (0.25–1 mM) were used for each Ad-ySUMO and a global fit was used to calculate binding affinities. The values obtained were comparable with affinities measured using a Biacore SPR3000 instrument.Adhiron thermal stability was measured by differential scanning calorimetry (DSC) using a VP-DSC MicroCalorimeter . Each sample was dialysed extensively against PBS, pH 7.4 and diluted to a concentration of 1 mg/ml before heating from 10 to 100°C for Adhiron library clones or to 120°C for the Adhiron scaffold 92 and 81 amino acid proteins at an upscan rate of 90°C per hour. A buffer only scan was measured to calculate a baseline for integration. Data were fit to a one-state unfolding model and reversibility of thermal denaturation was determined by repeating the scan for the Adhiron scaffold sample without removing it from the cell.KD values were determined using the Origin program (OriginLab).All experiments were carried out using the iTC200 system  at 25°C. Typically, 0.1 mM Adhiron protein was titrated with 0.01 mM of a target protein  by 2.5 min injection duration to allow return of the titration peak to the baseline. After fitting the integrated exothermal peaks, All circular dichroism (CD) measurements were performed on a Chirascan CD spectrometer (Applied Photophysics) at room temperature. The spectra were recorded over a wavelength range of 260–180 nm using a cuvette of 1 mm pathlength at a scan speed of 60 nm/min. The concentration of each sample was ca. 0.2 mg/ml in PBS buffer (pH 7.4). Data were further processed for noise reduction, baseline subtraction and signal averaging.l-glutamate, 0.02 M dl-alanine, 0.02 M dl-lysine hydrochloride, 0.02 M dl-serine, 0.1 M MOPS pH 7.50, 0.1 M HEPES sodium salt pH 7.50, 10.0% w/v polyethylene glycol 20 000, 20.0% w/v polyethylene glycol 550 MME). The shorter 81 amino acid Adhiron81 scaffold gave crystals after 52 days in Well D2 of the PACTpremier screen (Molecular Dimensions) .Crystallization trials were set up using the method of 2 at the sample. For the shorter Adhiron-81 rod-like crystals ∼20 µm across grew in clumps. Diffraction data were collected from a single 20 × 20 × 20 µm3 crystal fragment.For the full-length Adhiron92 rod-like crystals ∼25 µm across were cryo-cooled directly into liquid nitrogen without the addition of cryoprotectants. Diffraction data were collected at beamline I24 at Diamond Light Source from multiple positions along a single crystal with the starting angle at each position offset by 30° with respect to the previous position. Data were collected to a resolution of 1.75 Å using an X-ray wavelength of 0.9686 Å with the beamsize set to be 20 × 20 µmAll diffraction data were integrated using XDS  and scalA consensus phytocystatin sequence was derived by identifying the most common amino acid at each position of an alignment of 57 phytocystatin sequences. The length of the initial consensus protein was set at 95 amino acids with the N-terminus positioned four residues before the conserved N-terminal glycine residue, and thus before the first β-strand (β1). The C-terminus was set 15 residues after the conserved PW motif and thus after the last β-strand (β4). These criteria were based on the structures of chicken egg white cystatin . VR 1 was positioned between the first and second β strands with VR 2 between the third and fourth β strands Fig. . SequencThree random Adhiron clones containing inserts were used for protein purification and CD was performed Fig. C. All thFor library evaluation, the ySUMO protein was used as a model target. Over 1000-fold amplification in colony recovery was observed compared with control samples by panning round three. Twenty-four Adhirons were isolated and their ability to bind to ySUMO was confirmed by phage ELISA Fig. A with liIDLT) (1–3(N/Q)(W/F/Y) or G, which is not found in VR 1. We also identified a common pattern (W/F/Y)(E/D)2–4(W/F/Y) represented in both VRs. This molecular pattern matches the criteria for SIMs; a high percentage of hydrophobic amino acids juxtaposed with acidic residues   sequenceFour Ad-ySUMOs were selected for further characterisation, Clones 15 and 22 as the VR 1 sequence occurred multiple times, Clone 10 as it contained the IDLT motif and Clone 19 as it contained a distinct motif in VR 1.E. coli proteins without affecting Adhiron integrity. We therefore heated lysates for 20 min at 20, 50, 60, 70, 80, 90 and 100°C, centrifuged to pellet the denatured protein and analysed the supernatants by SDS–PAGE . To determine the specificity of the reagents, yeast SUMO was mixed with HEK293 cell lysates. Interestingly, Ad-ySUMO 10 and 15 show specific binding to yeast SUMO with no binding to other proteins whereas Ad-ySUMO 19 and 22 bind to other proteins in the lysates (n = 3), indicating that the inserted sequences are more promiscuous, emphasising the importance of strict negative screens during panning. The affinities of the Adhirons for ySUMO were measured by isothermal titration calorimetry (ITC). Ad-ySUMO 10, 15, 20 and 22 bound with KD values of 29.6 ± 0.8, 33.3 ± 1.8, 45.8 ± 8.8 and 47 ± 15.6 nM, respectively . The binding affinities of human SUMO 1 and 2 to a peptide bearing the conserved IDLT SIM motif have been reported to be in the range of 5–6 µM , receptor (CD31), SH2 domain Grb2 and a peptide sequence. All screens were performed over three panning rounds. Phage ELISA was used to examine the ability of recovered Adhirons to bind to the corresponding target Fig. . While tAnalysis of our screen against Grb2 offered the opportunity to directly compare the efficacy of our Adhiron library against well-established ScFv and Fab libraries. A recent study reported high throughput screens of 20 SH2 domains using both hybridoma and phage-display libraries of ScFv's (E. coli, by including a heat enrichment step enabling ease of engineering and manufacture. The scaffold displays high thermostability with a melting temperature of 101°C determined by DSC (Fig. We have developed a new artificial binding protein scaffold termed Adhiron based on a designed consensus phytocystatin protein. The properties of this scaffold also match the criteria proposed for artificial therapeutic proteins  althoughDSC Fig. A.We have solved the X-ray crystal structure of the Adhiron scaffold reported here, as well as selected Adhirons in complex with target proteins which will be reported elsewhere. The ability to gain structural information is important for improving our understanding of the molecular interactions of Adhirons with their targets that lead to functional consequences, and to provide a basis for drug design. We are currently exploring the extent to which the Adhiron scaffold may offer a platform for selection of reagents for applications including research tools, diagnostic, imaging, therapeutic agents and for drug discovery.PEDS online.Supplementary material is available at Work reported here is included in a patent application filed by the University of Leeds.Biotechnology and Biological Sciences Research Council [24/G15882] for the development of the consensus cystatin. The scaffold development work was funded by University of Leeds Transformation Fund support for the Biomedical Health Research Centre BioScreening Technology Group. Part of this work concerning characterisation of Adhirons was supported through WELMEC, a Centre of Excellence in Medical Engineering funded by the Wellcome Trust and EPSRC, under grant number WT 088908/Z/09/Z. The OPPF-UK is supported by the MRC and BBSRC. Funding to pay the Open Access publication charges for this article was provided by The Wellcome Trust.This work was supported by the


Mutations in podocyte and basement membrane genes are associated with a growing spectrum of glomerular disease affecting adults and children. Investigation of familial cases has helped to build understanding of both normal physiology and disease.We investigated a consanguineous family with a wide clinical phenotype of glomerular disease using clinical, histological, and new genetic studies.COL4A5 allele, p. and p.(Gly1033Arg). Two boys had signs limited to hematuria and mild/moderate proteinuria. In striking contrast, a sister presented with end-stage renal disease (ESRD) at 8 years of age and an infant brother presented with nephrotic syndrome, progressing to ESRD by 3 years of age. Both were subsequently found to have homozygous variants in MYO1E, p.(Lys118Glu) and p.(Thr876Arg). MYO1E is a gene implicated in focal segmental glomerulosclerosis and it encodes a podocyte-expressed non-muscle myosin. Bioinformatic modeling demonstrated that the collagen IV-alpha3,4,5 extracellular network connected via known protein–protein interactions to intracellular myosin 1E.We report striking variability in severity of nephropathy within an X-linked Alport syndrome (XLAS) family. Four siblings each carried a mutant COL4A5 and MYO1E mutations may summate to perturb common signaling pathways, resulting in more severe disease than anticipated independently. We suggest screening for MYO1E and other non-COL4 ‘podocyte gene’ mutations in XLAS when clinical nephropathy is more severe than expected for an individual’s age and sex. In 1927, Cecil Alport described an inherited nephropathy [COL4A5 encoding the alpha-5 chain of collagen IV, a GBM component, cause XLAS [COL4A5 allele versus males who have only the mutated allele [Mutations of use XLAS . A minoruse XLAS . The gend allele , 7. HoweCOL4 Sanger sequencing was performed as previously described [Samples were collected following informed consent. escribed . Analysiescribed .MYO1E were published by Mele et al. [COL4A5 by Martin et al. 1998 [Library preparation, sequencing, and data generation was performed in the Genomics Core Facility of the Biomedical Research Centre at Guy’s and St Thomas’ Hospitals and King’s College London. DNA libraries were prepared from 3 μg dsDNA using SureSelect Human All Exon 50 Mb kit (Agilent Technologies). Samples were multiplexed (four samples on each lane) and 100 base pair paired end sequencing was performed on Illumina HiSeq system. The mean coverage was 136× . Sequence data were aligned to the GRCh37/hg19 human reference genome using Novoalign, variants were called with SAMtools and annotated via multiple passes through Annovar. Exome sequencing data analysis was performed at the University of Bristol . Variants of interest were confirmed using Sanger sequencing . Primers used for al. 1998 ; the KAPSamples were fixed for at least 1 h in Neutral Buffered Formalin 10 % V/V and fixative was replaced with 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for at least 3 h. Post-fixation samples were incubated in 1 % proprietary (TAAB) osmium tetroxide solution and rinsed in 0.1 M sodium cacodylate buffer followed by dehydration in alcohols graded up to 100 % ethanol. Samples were incubated in propylene oxide (TAAB) to remove the alcohol and heated to 40 °C in 50/50 mix propylene oxide/resin  followed by 100 % resin for 2 h before embedding in resin blocks for 12 h at 60 °C. Ultrathin 120-nm sections were cut with a Leica UC7 ultramicrotome and placed on copper grids. Grids were stained in 2 % uranyl acetate 10 min before the addition of Reynolds’ lead citrate for 10 min. The grids were observed in a JEOL 100CX transmission electron microscope.Protein interaction network analysis was performed using Cytoscape (version 2.8.1) . CollageCOL4A5 Sanger sequencing was undertaken [Five siblings from a consanguineous family of Pakistani origin Fig.  were invdertaken . IV.3 cadertaken , with thdertaken . Ophthal2 and a renal biopsy was not performed due to the associated risks. She commenced dialysis and at 9 years old underwent deceased donor renal transplantation; she has satisfactory graft function 8 years later. IV.1 was found to be heterozygous for the mutant COL4A5 allele. To explain her severe renal presentation, we hypothesized that she had skewed X-inactivation of her normal COL4A5 allele. We found an X-inactivation ratio of 90:10 in her leukocyte DNA, reported in only 4 % of adult females [The proband’s older sister (IV.1) had been unwell for several years before emigrating from Pakistan. Aged 8 years, her estimated glomerular filtration rate (eGFR) was 10 ml/min/1.73 m females .2 later improved to 104 ml/min/1.73 m2. Over the next year, he had persistent proteinuria (269–423 mg/mmol) and, although normotensive, he was treated with the angiotensin converting enzyme inhibitor (ACEI), enalapril. This follows evidence and guidelines [COL4A5 allele. A renal biopsy was not performed. The youngest female sibling (IV.4) is well and has normal urinalysis and COL4A5 alleles.IV.3’s older brother (IV.2) presented aged 5 years with fever and microscopic hematuria. His eGFR of 54 ml/min/1.73 midelines –23 that The youngest male sibling (IV.5) presented at 6 months old with macroscopic hematuria and nephrotic syndrome (urinary protein/creatinine 6220 mg/mmol). He was treated with intravenous furosemide and 20 % human albumin replacement therapy. A renal biopsy was normal by light microscopy but TEM revealed GBM thickening and lamination with foot process fusion Fig. . A trial2 and her 24-h urinary protein was 1.70 g. She began treatment with ACEI. She carries the mutant COL4A5 allele. Two of her brothers (III.6 and III.7) died with end-stage renal disease (ESRD) in their twenties, and her mother (II.5) had ESRD aged 58 years. The children’s father has normal urinalysis and borderline hypertension and carries a normal COL4A5 allele. His deceased father (II.3) had a history of nephropathy.The mother, III.5, was investigated at age 39. She had microscopic hematuria but was normotensive. Her eGFR was 82 ml/min/1.73 mNPHS2, predisposes to proteinuria and ESRD in individuals with hematuria and COL4A4 mutations [ACTN4, ADCK4, ALG1, ANLN APOL1, ARHGAP24, ARHGDIA, CD151, CD2AP, COL4A3, COL4A4, COL4A5, COQ2, COQ6, CTL4A, CUBN, DGKE, E2F3, EMP2, INF2, ITGA3, ITGB4, LAMB2, LMNA, LMX1B, MYH9, MYO1E, NPHS1, NPHS2, NXF5, PAX2, PDSS2, PLCe1, PMM2, PTPRO, SCARB2, SMARCAL1, SYNPO, TRPC6, TTC21B, WT1 and ZMPSTE24), as described in [COL4A5 mutations, and that COL4A3 and COL4A4 were normal. However, we discovered that IV.5 also carried two homozygous missense variants in MYO1E affecting highly conserved amino acids c.352A > G; p.(Lys118Glu) and c.2627C > G; p.(Thr876Arg). Furthermore, IV.1, the sister with ESRD, was also homozygous for these variants (Fig. Nearly a century after Alport’s description of XLAS, we appreciate that mutations in a growing list of genes encoding podocyte proteins can cause glomerular disease –29. Notautations . In thisCOL4A5 and MYO1E mutations may summate to perturb common cell-matrix signaling pathways.We undertook a protein–protein interaction network analysis  based onCOL4A5 mutations and homozygous MYO1E variants were associated with more severe kidney disease within a family initially recognized to have XLAS. Skewed X inactivation may be a second contributing factor in IV.1 and possibly protected against the more rapid progression to renal failure seen in IV.5.In this kindred, the coinheritance of MYO1E encodes a podocyte-expressed non-muscle myosin [Myo1e deletion in podocytes causes proteinuria, foot process effacement, and GBM thickening [MYO1E biallelic, generally missense, mutations have been reported in children with focal segmental glomerulosclerosis and glucocorticoid-resistant proteinuria [MYO1E mutations and who has had a renal biopsy; instead of sclerosis, only severe GBM and foot process anomalies were seen.e myosin , 32, wite myosin . In vitrickening . MYO1E bteinuria , 35, 36.MYO1E and other non-COL4 ‘podocyte gene’ mutations in XLAS when clinical nephropathy is more severe than expected for an individual’s age and sex. Furthermore, the increasing use of global and unbiased genetic analysis with next-generation sequencing is likely to reveal disease-modifying variants across the range of human genetic disease.This extraordinary family demonstrates that mutations of two podocyte genes can explain a highly variable renal phenotype, inexplicable by conventional pedigree analysis. The mutations summate to generate life-threatening nephropathies, and insights are highly relevant to genetic counseling. We suggest screening for