Secret life of importin-β; solenoid flexibility as the key to transport through the nuclear pore

open2siThe current issue of Acta Crystallographica Section D features ‘Impact of the crystal- lization condition on importin- conformation’ by Tauchert et al. (2016), a significant advance in the area of nuclear transport that also has important implications for understanding the limitations of crystallization approaches. Active transport of macro- molecules into and out of the eukaryotic cell nucleus occurs through the nuclear envelope (NE)-embedded multiprotein subunit nuclear pore complexes (NPCs). The transport of most cargoes is dependent on solenoid proteins belonging to the Karyopherin family, of which importin (Imp) 1 is the prototype. Imp 1 is capable of recognizing specific nuclear import cargoes and transporting them across the NPC by interacting with the hydrophobic meshwork constituting the NPC core, formed by phenyl–alanine–glycine (FG) rich nucleoporins (nups). Cargo recognition can occur directly, or indirectly through adaptor proteins such as Imp or snurportin, and in either case complexes are dissociated upon binding of Ran-GTP to Imp 1 on the nucleoplasmic side of the NPC. Since the discovery of Imp 1 (Go ̈rlich et al., 1995), structural studies have helped elucidate many aspects of the molecular details of cargo and adaptor binding/release (Christie et al., 2016). Unanswered questions, however, include how Imp s achieve cargo transport across the NPC through interaction with nups (Liu & Stewart, 2005; Bayliss et al., 2000); Tauchert et al. provide an important, new slant on this question. Imp 1 binds to a plethora of different proteins, including cargoes, adaptors, RanGTP and nups. Previous studies have shown Imp 1 to be a solenoid formed by 19 HEAT repeats, each of which comprises two antiparallel helices connected by a turn (Cingolani et al., 1999); HEAT repeats are connected by short linkers and arranged in a superhelix, with very few long distance intraprotein interactions, enabling Imp 1 to undergo extensive changes in tertiary structure (overall protein shape), without alteration to secondary structure (HEAT repeats). Consistent with this idea, the comparison of Imp crystal structures to date reveals a wide range of conformations varying from very compact, heart-like structures (the ‘apo’ or nup-bound form) to more relaxed ones (e.g. bound to RanGTP). This observed structural variability has been postulated to be the direct effect of the binding of different partners to Imp 1 causing/inducing changes in Imp 1 folding, but X-ray scattering (SAXS) data shows that Imp 1 alone is more relaxed in solution than in crystal lattices (Fukuhara et al., 2004). Further, molecular dynamics (MD) simulations suggest that the apo form of Imp 1 undergoes remarkable conformational changes in solution, adopting a more extended S-shaped conformation that is quite distinct to that observed in its crystalline form (Zachariae & Grubmu ̈ller, 2008; Forwood et al., 2010). Significantly, two drastically different conformations of Imp 1 bound to the snurportin Imp 1 binding domain (IBB) were recently observed in the same crystallographic asymmetric unit (Bhardwaj & Cingolani, 2010); this both highlights Imp 1 flexibility, and importantly underlines the fact that structures obtained in crystals may not reflect the wide range of Imp 1 conformations in solution. Forwood et al. (2010) used crystal- lography/SAXS/MD to show that Imp 1 assumes various different conformations in solution, postulated to result from cumulative incremental structural changes along the entire length of the solenoid, and speculated to be integral to Imp 1’s ability to traverse the highly hydrophobic channel of the NPC. Indeed, MD simulations reveal that Imp 1 in water is extremely different from that in 50% 2,2,2-trifluoroethanol, where Imp 1 rapidly becomes more compact (Yoshimura et al., 2014); similar results have been electronic reprint http://dx.doi.org/10.1107/S2059798316008263 703 # 2016 International Union of Crystallography Acta Cryst. (2016). D72, 703–704 scientific commentaries Figure 1 either PEG or (NH4)2SO4 indicates that these properties do not apply, since Imp , although structurally related to Imp 1, is less flexible. The polar/apolar regions of PEG would appear to mimic nup FG repeats within the NPC, suggesting that Imp 1 traverses the NPC in an extended conformation, in contrast to what has been proposed previously (Halder et al., 2015; Yoshi- mura et al., 2014). The only crystal structures obtained so far between Imp 1 and NPC components used short FG-rich nup fragments, and thus do not shed light on the state of Imp 1–nups interaction within the core of the NPC (Liu & Stewart, 2005; Bayliss et al., 2000). Importantly, apart from giving an important new insight into this aspect of nuclear transport, Tauchert et al.’s study Effect of solvent on C. Thermophilum Imp 1 structure. The structures of Imp 1 obtained after PEG precipitation (red) or (NH4)2SO4 (blue) precipitation are superimposed. The structure shown clearly underlines the need for more on the right is rotated 180 C with respect to that on the left. obtained with both IBB-complexed and free Imp 1 in water compared to in methanol (Halder et al., 2015). This ability to undergo conformational changes appears to be the key to Imp 1-mediated transport across the NPC, since crosslinking to impair this flexibility impedes nuclear translocation (Yoshimura et al., 2014). Tauchert et al. extend these findings, proffering an inter- esting alternative point of view regarding the forces deter- mining different conformations of Imp 1 in crystalline form according to the hydrophobicity of the milieu. Tauchert et al. solve the structure of Imp 1 from the thermophilic fungus Chaetomium thermophilum in two physicochemically different conditions, taking advantage of the serendipitous S107P/ V134A mutant which crystallized in the presence of the hydrophilic inorganic salt (NH4)2SO4, adopting a much more compact structure than that of its wild-type counterpart crystallized in the presence of PEG (Fig. 1). These findings are confirmed in solution using SAXS, the important overall implication being that solvent hydrophobicity strongly affects Imp 1 conformation, and hence can be of key importance in the dominant conformation crystallized. Further, analyzing previous Imp 1 crystal structures, the authors find a strong correlation between the extent to which Imp 1 takes an extended conformation, and the amount of PEG in the buffer. Importantly, analysis of Imp crystal structure pairs solved in extensive Imp 1–nup complex struc- tures, with the proviso that the buffer systems used have to be considered critically (with a grain of salt perhaps?), and ideally should also be analysed in detail using complementary approaches such as SAXS.openAlvisi, Gualtiero; Jans, David AAlvisi, Gualtiero; Jans, David A

Water level forecasting through fuzzy logic and artificial neural network approaches

In this study three data-driven water level forecasting models are presented and discussed. One is based on the artificial neural networks approach, while the other two are based on the Mamdani and the Takagi-Sugeno fuzzy logic approaches, respectively. <P style='line-height: 20px;'> All of them are parameterised with reference to flood events alone, where water levels are higher than a selected threshold. The analysis of the three models is performed by using the <I>same input and output variables</I>. However, in order to evaluate their capability to deal with different levels of information, two different input sets are considered. The former is characterized by significant spatial and time aggregated rainfall information, while the latter considers rainfall information more distributed in space and time. <P style='line-height: 20px;'> The analysis is made with great attention to the reliability and accuracy of each model, with reference to the Reno river at Casalecchio di Reno (Bologna, Italy). It is shown that the two models based on the fuzzy logic approaches perform better when the physical phenomena considered are synthesised by both a limited number of variables and IF-THEN logic statements, while the ANN approach increases its performance when more detailed information is used. As regards the reliability aspect, it is shown that the models based on the fuzzy logic approaches may fail unexpectedly to forecast the water levels, in the sense that in the testing phase, some input combinations are not recognised by the rule system and thus no forecasting is performed. This problem does not occur in the ANN approach

DNA quantification to assess Zymoseptoria tritici on a susceptible cultivar of durum wheat to establish the best timing for fungicide application in an italian environment

Zymoseptoria tritici, a globally distributed pathogen, is responsible of Septoria tritici blotch (STB), one of the most damaging wheat diseases. In Italy the incidence of STB has increased during the past few years. The presence of Z. tritici on flag leaves of susceptible durum wheat plants, cultivar San Carlo, after a single artificial inoculation with two inoculum concentrations at different vegetative stages has been evaluated in the plain of Bologna (North of Italy), in a two year field study (2012–2013). The pathogen presence was also assessed in natural infection conditions after a fungicide application in the second year (2013). The results obtained, by visual examination (Incidence, Disease Severity) and DNA quantification by Real time PCR, demonstrated that BBCH 39 (flag leaf stage) is the most susceptible vegetative stage, independently of inoculum concentration and climatic conditions. A good correlation between Disease Severity and DNA quantity was observed in either sampling methods, entire flag leaves and flag leaf discs. Thereafter the most suitable period to obtain the best crop protection with only one fungicide treatment is the flag leaf stage

Influence of Different Strategies of Volume Replacement on the Activity of Matrix Metalloproteinases

Background Excessive production of matrix metalloproteinase 9 (MMP-9) is linked to tissue damage and anastomotic leakage after large bowel surgery. Hence, the aim of this study was to verify whether different strategies of fluids administration can reduce MMP-9 expression. Methods In the in vitro experiment, the authors tested the hypothesis of a direct inhibition of MMP-9 by the fluids used perioperatively, i.e., lactated Ringer's solution, 3.4% poligeline, and hydroxyethyl starch 130/0.4. In the in vivo experiment, 36 patients undergoing surgery for colon cancer were randomly assigned to three groups to receive lactated Ringer's solution, poligeline, or hydroxyethyl starch. MMP-9 and tissue inhibitor of metalloproteinases were measured from venous blood samples; the MMP-9/tissue inhibitor of metalloproteinases ratio was calculated as an index of equilibrium between the action of MMP-9 and its inhibition. Results In the in vitro experiment, the presence of hydroxyethyl starch 130/0.4 in the MMP-9 assay system showed a strong inhibition of the enzymatic activity compared with lactated Ringer's solution. In the in vivo experiment, MMP-9 and tissue inhibitor of metalloproteinases plasma levels did not differ among the three groups at baseline, whereas those levels increased significantly at the end of surgery. At that time, the MMP-9 plasma levels and the MMP-9/tissue inhibitor of metalloproteinases ratio were significantly higher in the lactated Ringer's solution and poligeline groups than in the hydroxyethyl starch group. These results were confirmed 72 h after surgery. Conclusions This study demonstrates that hydroxyethyl starch 130/04 decreases the circulating levels of MMP-9 in patients undergoing abdominal surgery

Measurement of surface velocity in open channels using a lightweight remotely piloted aircraft system

ABSTRACTIn this paper, a low-cost remotely piloted aircraft system (RPAS) technique is proposed for measurement of the surface velocity in rivers or channels with low surface velocity and small discharge. To verify the reliability of the results obtained with the RPAS, we simultaneously measured the surface velocity with other methods based on total stations and close range photogrammetry. The RPAS was used both with ground control points (GCPs) for orientation of the photographic images and without GCPs. The data analysis showed that the RPAS provides valid results even without GCPs. Use of a RPAS without GCPs, relying solely on flight altitude to determine the water velocity, opens the way for its utilization in emergency conditions when it is impossible to access the river banks for the realization and survey of GCPs

Efficacy of adalimumab as second-line therapy in a pediatric cohort of crohn’s disease patients who failed infliximab therapy: The Italian society of pediatric gastroenterology, hepatology, and nutrition experience

Background: Adalimumab (Ada) treatment is an available option for pediatric Crohn’s disease (CD) and the published experience as rescue therapy is limited. Objectives: We investigated Ada efficacy in a retrospective, pediatric CD cohort who had failed previous infliximab treatment, with a minimum follow-up of 6 months. Methods: In this multicenter study, data on demographics, clinical activity, growth, laboratory values (CRP) and adverse events were collected from CD patients during follow-up. Clinical remission (CR) and response were defined with Pediatric CD Activity Index (PCDAI) score ≤10 and a decrease in PCDAI score of ≥12.5 from baseline, respectively. Results: A total of 44 patients were consecutively recruited (mean age 14.8 years): 34 of 44 (77%) had active disease (mean PCDAI score 24.5) at the time of Ada administration, with a mean disease duration of 3.4 (range 0.3–11.2) years. At 6, 12, and 18 months, out of the total of the enrolled population, CR rates were 55%, 78%, and 52%, respectively, with a significant decrease in PCDAI scores (P&lt;0.01) and mean CRP values (mean CRP 5.7 and 2.4 mL/dL, respectively; P&lt;0.01) at the end of follow-up. Steroid-free remission rates, considered as the total number of patients in CR who were not using steroids at the end of this study, were 93%, 95%, and 96% in 44 patients at 6, 12, and 18 months, respectively. No significant differences in growth parameters were detected. In univariate analysis of variables related to Ada efficacy, we found that only a disease duration &gt;2 years was negatively correlated with final PCDAI score (P&lt;0.01). Two serious adverse events were recorded: 1 meningitis and 1 medulloblastoma. Conclusion: Our data confirm Ada efficacy in pediatric patients as second-line biological therapy after infliximab failure. Longer-term prospective data are warranted to define general effectiveness and safety in pediatric CD patients

The lysine methyltransferase SMYD3 interacts with hepatitis C virus NS5A and is a negative regulator of viral particle production

Hepatitis C virus (HCV) is a considerable global health and economic burden. The HCV nonstructural protein (NS) 5A is essential for the viral life cycle. The ability of NS5A to interact with different host and viral proteins allow it to manipulate cellular pathways and regulate viral processes, including RNA replication and virus particle assembly. As part of a proteomic screen, we identified several NS5A-binding proteins, including the lysine methyltransferase SET and MYND domain containing protein 3 (SMYD3). We confirmed the interaction in the context of viral replication by co-immunoprecipitation and co-localization studies. Mutational analyses revealed that the MYND-domain of SMYD3 and domain III of NS5A are required for the interaction. Overexpression of SMYD3 resulted in decreased intracellular and extracellular virus titers, whilst viral RNA replication remained unchanged, suggesting that SMYD3 negatively affects HCV particle production in a NS5A-dependent manner. (C) 2014 The Authors. Published by Elsevier Inc

Surface Modification of Carbon Nanotube Networked Films with Au Nanoclusters for Enhanced N O 2 Gas Sensing Applications

Multiwalled carbon nanotube (MWCNT) films have been deposited by using plasma-enhanced chemical vapor deposition (PECVD) system onto alumina substrates, provided with 6 nm thick cobalt (Co) growth catalyst for remarkably improved gas sensing, at working temperature in the range of 100–. Functionalization of the MWCNTs with nanoclusters of gold (Au) sputtering has been performed to modify the surface of carbon nanotube networked films for enhanced and specific gas detection up to sub-ppm level. It is demonstrated that the gas sensitivity of the MWCNT-based sensors depends on Au-loading used as surface-catalyst. The gas response of MWCNT-based chemiresistor is attributed top-typeconductivity in the Au-modified semiconducting MWCNTs with a very good short-term repeatability and faster recovery. The sensor temperature of maximum sensitivity of the Au-functionalized MWCNTs is found to decrease with increasing Au-loading on their surface, and continuous gas monitoring at ppb level of is effectively performed with Au-modified MWCNT chemiresistors

Planning and designing an integrated management of coastal hypoxia in the Emila Romagna region water (Northern Adriatic Sea)

Abstract The design of an integrated monitoring network useful for the early-warning, the management and mitigation of both the environmental and socio-economic costs of hypoxia/anoxia events in the Northern Adriatic coastal zone, south of the mouth of the Po river, is described in the contribute. It has been developed within the EMMA research project (Environmental Management through Monitoring and Modelling of Anoxia; LIFE04ENV/IT/0479) (2004-2007). Over the past few decades, hypoxia events have recurrently affected the coastal zone of Emilia Romagna Region (Italy). Data collected by the C.Z. lying in the Province of Rimini, because of its economic importance, are presented. The area is subjected to intense anthropogenic pressure due to its high population (416 000 equivalent inhabitants) with tourist seasonal peaks of up to 973 110 equivalent inhabitants (in summer time), to industrial and agricultural activity, to maritime traffic and nutrient river discharges (about 600 tons y-1 of nitrogen and 300 tons y-1 of phosphorus, in 2002). Hypoxia and anoxia have a negative effect on the quality of bathing waters as well as on fishing and mussel farming, which are important activities for the economy of the area. The planning of the monitoring network has been carried out by analyzing the scientific knowledge on hypoxia in the local area; its integration with other existing monitoring activities, available facilities and data resources was considered in order to optimize cost effectiveness of the network