A simple two-step method was employed for preparing nano-sized gold nanoparticles-graphene composite to construct a GNPs-GR-SDS modified electrode. Hemoglobin (Hb) was successfully immobilized on the surface of a basal plane graphite (BPG) electrode through a simple dropping technique. Direct electrochemistry and electrocatalysis of the hemoglobin-modified electrode was investigated. The as-prepared composites showed an obvious promotion of the direct electro-transfer between hemoglobin and the electrode. A couple of well-defined and quasi-reversible Hb CV peaks can be observed in a phosphate buffer solution (pH 7.0). The separation of anodic and cathodic peak potentials is 81 mV, indicating a fast electron transfer reaction. The experimental results also clarified that the immobilized Hb retained its biological activity for the catalysis toward NO. The biosensor showed high sensitivity and fast response upon the addition of NO, under the conditions of pH 7.0, potential -0.82 V. The time to reach the stable-state current was less than 3 s, and the linear response range of NO was 0.72-7.92 μM, with a correlation coefficient of 0.9991. Due t in 2004 . Like ca carbon)  that adh carbon) –10.et al. [2O2. Huang et al. [2 nanocomposite could effectively facilitate the direct electron transfer of catalase to the electrode.On the other hand, due to its low-cost, large specific surface area and strong interactions with metal clusters, graphene is a promising material for catalytic applications . The diset al.  employedg et al.  also shoHowever, to date, there are few papers concerning the electrochemical response of hemoglobin on gold nanoparticles-graphene sheets composites. As a well-characterized, electron transfer protein, hemoglobin has been used extensively as a test system for direct electron transfer (DET) of redox proteins or for communication in a protein stack by assembling it with other proteins in a matrix –25.In this work, we synthesized Au nanoparticles using graphene oxide (GO) as protecting agent to give a stable red suspension. To obtain high electrical conductivity and water solubility of our GNPs-graphene composites, we added sodium dodecyl sulfate (SDS) solution to the GNPs-GO suspension prior to chemical reduction. The anionic surfactant SDS was employed as an effective solubilizing agent for graphene nanosheets and for isolating them from each other. Otherwise, the strong van der Waals interactions among the reduced graphene sheets results in their rapid aggregation in solution during the chemically reduction process. Then, water soluble GNPs-GR can be successfully obtained by the non-covalent interaction of graphene with sodium dodecylsulphate (SDS) in this procedure ,27. Furt2.2.1.Electrochemical experiments were performed with a CHI660 electrochemical analyzer  with a conventional three-electrode cell. A Hb/GNPs-GR-SDS modified BPGelectrode (Φ = 5.2 mm) was used as a working electrode. An Ag/AgCl electrode and a platinum wire electrode were used as the reference and the auxiliary electrodes, respectively.−1) was stored at a temperature of 4 °C as stock solution. Graphite, and hydrazine hydrate solution (50 wt %) were purchased from Shanghai Chemical Reagent Co. Ltd.  Saturated NO solutions were prepared as described in the previous literature [Horse heart hemoglobin was obtained from Sigma  and used without purification. Hb solution (5 mg·mLterature . In brie2.2.4 with NaBH4 in the presence of aqueous graphene oxide (GO). An aqueous solution of HAuCl4  was added into graphene oxide aqueous solution  and stirred for 5 min. After that, slow and controlled addition of NaBH4 solution  is essential to avoid gold nanoparticles from forming too fast and aggregating. This experiment was repeated with graphene oxide concentrations of 0.02, 0.11 and 0.27 mg· mL−1 as well as one experiment where no graphene oxide was added.The oxidation of the graphite was carried out based on the Hummers method . The obt2.3.SDS solution  was added to the obtained gold nanoparticles-graphene oxide suspension and stirred for 20 min. Then, hydrazine hydrate solution (20 μL) was added and stirred for 5 min. This mixture was reacted at 100 °C for 24 h. The GNPs-GO solution changed color, from red to black, after the hydrazine-vapor treatment, indicating reduction of the graphene oxide . The res2.4.−1 Hb for 3 h to form an Hb/GNPs-GR-SDS/BPG electrode. Prior to electro-chemical experiments, the electrode was rinsed thoroughly with doubly distilled water andstored at 4 ° C. The Hb/GR-SDS modified electrode without GNPs and the Hb/GNPs-SDS modified electrode without graphene were prepared in the same way, only omitting the steps of the addition of HAuCl4 solution and graphene oxide solution, respectively.Initially, the BPG electrode was polished with abrasive paper before each experiment. After a short rinse with distilled water and ultrasonic cleaning with distilled water and ethanol, the electrode was allowed to dry at room temperature. Then, the GNPs-GR-SDS suspension (10 μL) was cast on the surface of a pretreated BPG electrode with a microsyringe and the solvent (water) was allowed to evaporate at ambient temperatures. The electrode was denoted as the GNPs-GR-SDS/BPG electrode. Finally, this GNPs-GR-SDS/BPG electrode was immersed in a phosphate buffer solution (pH 7.0) containing 5 mg·mL3.3.1.−1 graphene oxide was added into the solution, the color of the solution turned a red wine color. Reducing the amount of graphene oxide gradually, the color of the solution turns red, then purple, blue, which could be explained by the aggregation of gold nanoparticles. At a low graphene concentration (0.02 mg·mL−1), the gold particles are suspended but in an aggregated state. Without the addition of any dispersant, the suspension flocculates very rapidly, which should indicate a strong agglomeration of the nanoparticles.A photograph of five GNPs-GO suspensions with different graphene oxide concentrations is shown in max from 520 to 556 nm, which is typical for increasingly larger GNP diameters. Similar red-shifts have been observed in the measured optical spectra of gold nanoparticles and are attributed to the effect of electromagnetic retardation in larger nanoparticles [articles ,31.In other words, gold nanoparticles-graphene oxide composites were successfully synthesized using graphene oxide as a dispersing agent to optimize the dispersion and the stability of the gold nanoparticles. Unlike the methods reported in previous literatures, the present method involves a facile fabrication route for the attachment of gold nanoparticles on the surface of graphene without anyorganic reagent. The resulting GNPs-GO suspension can remained as a fine dispersion at least two months. Subsequently, we added hydrazine hydrate to the GNPs-GO suspension to obtain a gold nanoparticle-graphene solution in the presence of sodium dodecyl sulfate.−1 graphene. The SEM analysis indicates that the gold nanoparticles are uniformly deposited on the graphene surface, with an average size of 50 nm. This fact showed that the presence of the oxygen functionalities at the graphene surface may play an important role on the nucleation and growth of gold nanoparticles. Furthermore, 3.2.2-saturated Hb-free phosphate buffer solution (pH 7.0). The Hb/GNPs-GR-SDS/BPG electrode gives a couple of stable and well-defined redox peaks, where anodic and cathodic peak potentials are located at −317 and −398 mV, respectively  can be estimated according to Laviron [−1. This value of Ks of our prepared modified electrodes is higher than Hb/graphene-Pt/GCE (0.14 s−1) [ Laviron , which i.14 s−1) . This in−1, less than the theoretical value of -59 mV pH−1 at 25 °C for a reversible proton-coupled single electron transfer [The effect of the pH of the supporting electrolyte on the peak potentials of the Hb was also investigated . Both retransfer ,36. Althtransfer  and Hb-Dtransfer  films:H3.3.The resulting Hb/GNPs-GR-SDS/BPG electrode exhibited a good electrocatalytic activity toward the reduction of NO. To further examine the response feature of the Hb/GNPs-GR-SDS electrode to NO, the amperometric response of the Hb/GNPs-GR-SDS modified BPG electrode to NO was recorded while successively adding NO to a continuous stirred PBS solution. In this process, a potential of −0.82 V was applied to the working electrode .−8 mol·L−1 based on 3 signal-noise ratio. The resulting response suggests that the Hb/GNPs-GR-SDS/BPG electrode can be used as a reagentless biosensor to detect NO. The determined parameters of this sensor and other modified electrodes used for the determination of NO are listed in The reductive current increases to reach a stable plateau within 3 s when NO is added to the buffer solution. This suggested that the response of the electrode to NO should be a quick responsive process. The experimental results showed that the amperometric response current is linear when the concentration of NO is in the 0.72-7.92 μM range. In addition, the detection limit of the biosensor was evaluated to be 1.2 × 103.4.The stability of the Hb/GNPs-GR-SDS electrode was investigated. Even when 400 continuous cyclic scans were carried out in the potential range from 0.1 V to −1.0 V with a 100 mV/s scan rate, no obvious change of the CV curve can be observed, suggesting that proteins can be tightly immobilized on the surface-modified BPG electrode. Eight repetitive experiments were carried out with the buffer solution containing 0.72 μM NO. The relative standard derivation for eight repetitive measurements isbelow 2.3%. The Hb/GNPs-GR-SDS modified electrode that was left to stand in pH 7.0 PBS could keep its activity for at least two weeks.4.In conclusion, a gold nanoparticle-graphene nanosheets suspension can be prepared though the chemical reduction method using SDS as protector and disperser. Hb adsorbed on the GNPs-GR-SDS modified electrode via the electrostatic interactions retains its native secondary protein structure and the direct electron transfer was achieved. The CVs of a Hb/GNPs-GR-SDS/BPG modified electrode exhibited a pair of redox peaks corresponding to the electroactive center of Hb and showed good catalytic activity for the reduction of NO. The developed approach can be potentially useful in the fields of catalysis, fuel cells, and other related fields.


The transactive response DNA-binding protein (TDP-43) is a major component of the abnormal intracellular inclusions that occur in two common neurodegenerative diseases of humans: (1) a subtype of frontotemporal lobar degeneration and (2) amyotrophic lateral sclerosis. Genetics, experiments in cultured cells and animals, and analogy with other neurodegenerative diseases indicate that the process of TDP-43 aggregation is fundamental to the pathogenesis of these 2 diseases, but the process by which this aggregation occurs is not understood. Biochemical fractionation has revealed truncated, phosphorylated and ubiquitinated forms of TDP-43 in a detergent-insoluble fraction from diseased CNS tissue, while these forms are absent from controls. However, a large amount of the normally predominant 43-kDa form of TDP-43 is present in the detergent-insoluble fraction even from control brains, so it has not been possible to determine if this form of TDP-43 is part of pathological aggregates in frontotemporal lobe degeneration. We used semi-denaturing detergent-agarose gel electrophoresis to isolate high molecular weight aggregates containing TDP-43 that are present in the cerebral cortex of individuals with frontotemporal lobar degeneration but not that of controls. These aggregates include the same covalently modified forms of TDP-43 seen in detergent-insoluble extracts. In addition, aggregates include a 43-kDa TDP-43 species. This aggregated 43-kDa form of TDP-43 is absent or present only at low levels in controls. The presence of 43-kDa TDP-43 in aggregates raises the possibility that covalent modification is not a primary step in the pathogenic aggregation of TDP-43 associated with frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Frontotemporal lobar degeneration (FTLD) is a dementia syndrome defined by both clinical and pathological features. The prevalence of FTLD is approximately 15/100,000 persons aged 45–65 years TARDBP gene, which encodes TDP-43, are linked to rare familial forms of ALS Remarkably, abnormal intracellular accumulations of TDP-43 are also prominent in most cases of amyotrophic lateral sclerosis (ALS), a condition in which motor neurons of both the brain and the spinal cord degenerate, causing progressive weakness of skeletal muscle Serial extraction of brain homogenate in buffers of increasing stringency yields a fraction that is insoluble in a solution of 500 mM NaCl and 1% N-lauroylsarcosine Problematically, the detergent-insoluble fraction from both FTLD-TDP and control brains harbors a large amount of 43-kDa TDP-43 To better characterize the TDP-43 species in FTLD-associated aggregates, we employed an alternative method to detergent extraction, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE), to isolate high-molecular-weight complexes containing TDP-43. We then analyzed the components of these complexes by SDS-PAGE and immunoblotting. In addition to covalently modified forms, described above, we found elevated levels of a 43-kDa TDP-43 species in the FTLD-TDP aggregates.SDD-AGE has been described as a means of revealing the aggregated proteins known as “yeast prions” In some control cases a “tail” of TDP-43 signal extended into the upper regions of the gel. To objectively confirm the difference in TDP-43 migration between cases and controls, we compared the ratio of the relative quantity of TDP-43 in a segment extending from 5 mm to 30 mm below the well (encompassing the densest portion of the broad high molecular weight band), to the relative quantity in a 20 mm segment encompassing the TDP-43 signal in the lower portion of the lane that was present in all samples. This ratio was greater in every FTLD-TDP case than in any control , and theThe FTLD-associated high molecular weight band was present when immunoblots were probed with either a polyclonal antiserum or an independently derived monoclonal antibody to TDP-43, but not when blots were probed with the secondary antibody alone . SimilarWe devised methods to investigate which TDP-43 species contribute to the aggregates isolated by SDD-AGE. First, we used large format gels to increase the amount of brain homogenate we could separate by SDD-AGE . We thenTo estimate the overall recovery efficiency of this method, we loaded pre-stained molecular weight standards  on a large format SDD-AGE gel. Unlike brain homogenates, all proteins from the pre-stained marker could be observed to run in a relatively small region toward the bottom of the SDD-AGE gel. We excised the markers in a 3.4 cm long fraction of the lane and concentrated the markers using the method described above. A sample from the final 2 mm of the concentrating column and dilutions of pre-stained standards were compared on SDS-PAGE gels. Approximately 91±11 (s.d.)% of the marker was recovered in the final 2 mm section. Since 85% of the total detectable brain protein was found in the final 1.5 mm of the column, and approximately 90% of total protein was recovered from the final 2 mm, we conclude this method efficiently concentrates approximately 77% of the total protein in an excised SDD-AGE gel region into the final 1.5 mm of the column.We compared proteins concentrated from the aggregate and monomer regions on silver stained SDS-PAGE gels. The aggregate region contains a complex mixture, with at least 40 individual protein bands revealed by silver stain . The silHaving developed a method to efficiently concentrate proteins from excised regions of SDD-AGE gels, we proceeded to analyze the TDP-43 components of the aggregate region. SDS-PAGE immunoblots of concentrates from the aggregate region revealed several species of TDP-43 . All FTLConcentrates from the “monomer” region revealed a 43-kDa form of TDP-43 as the major species in both control and FTLD brain homogenates. Despite the apparently similar amounts of TDP-43 in the “aggregate and “monomer” regions in images of SDD-AGE immunoblots of FTLD brains, the amount to TDP-43 in the monomer region seemed to be much greater than in the aggregate region (data not shown). Precise quantitation was not possible because of the differing distributions of the TDP-43 species on PAGE immunoblots between the aggregate monomer regions.Immunoblots probed with a monoclonal antibody recognizing TPD-43 phosphorylated at serines 409 and 410 (pS409/410) reveal that most of the TDP-43 bands from FTLD-TDP aggregates contain phosphorylated TDP-43 species . HoweverWe considered whether endogenous phosphatase activity might have generated the 43-kDa TDP-43 species from a full-length phosphorylated species, such as that composing the 45-kDa band. To determine whether such activity was present during our aggregate preparation procedure, brain homogenates were prepared from frozen tissue under conditions designed to either minimize or enhance the activity of hypothetical phosphatases, then concentrates of the aggregate portion of these homogenates prepared. We predicted that if phosphatase activity were present in the homogenates, then the ratio of the 43-kDa species to the 45-kDa species would increase under the enhancing conditions. No significant effect was observed .SDD-AGE separates aggregated TDP-43 species that are present in FTLD-TDP brain from forms of TDP-43 that are found in both FTLD-TDP brains and controls. SDD-AGE is thus a simple and rapid assay for aggregated TDP-43 that may prove useful in future studies of the nature and distribution of these pathological aggregates.In SDD-AGE gels, the predominant lower molecular weight species in both controls and FTLD cases migrates with an apparent molecular weight of about 170 kDa, whereas is SDS-PAGE gels the normally predominant band migrates as a 43-kDa band. Since SDD-AGE only partially denatures proteins, migration rates would not be expected to be directly proportional to molecular mass. The apparent larger size of the predominant TDP-43 species in SDD-AGE gels may reflect slowed migration of this species in a partially unfolded state or interactions of the migrating TDP-43 peptide with other species.Since the aggregates seen with SDD-AGE are present only in FTLD-TDP cases, all TDP-43 species migrating in these aggregates are likely to be disease-related. In contrast, the TDP-43 species isolated by the serial extraction method that is commonly used to enrich for FTLD-associated forms of TDP-43 also include a substantial amount of TDP-43 that is not specifically associated with disease TDP-43 aggregates are found in a region of SDD-AGE gels that contain only about 1% of the total protein in the lane. A number of proteins other than TDP-43 were isolated from this region in both FTLD cases and controls. Perhaps surprisingly, the TDP-43 bands revealed by immunoblots of protein from this region are not distinctly visible in silver-stained gels. This may reflect the low total amount of TDP-43 protein in this region, the diffuse nature of most TDP-43 bands or a lack of affinity of TDP-43 species for the silver stain. Whether other proteins from the aggregate region are also aggregated, whether they are in the same cellular compartment as aggregated TDP-43, and whether other proteins from this region are specifically found in FTLD is not known, but these are potentially fruitful areas for future studies.The TDP-43 species extracted from the aggregates in SDD-AGE gels appear to include all the modified forms of TDP-43 forms that had been previously identified in the detergent-insoluble fraction from patients with FTLD-TDP Our most significant finding, however, is that a 43-kDa species of TDP-43 is present in FTLD-associated TDP-43 aggregates. The aggregated 43-kDa species migrates in SDS-PAGE identically to the physiologically predominant TDP-43 species, and lacks obvious covalent modifications. Like the physiologically predominant species, it is not phosphorylated at the 409/410 sites. Known sites of TDP-43 phosphorylation in FTLD include serines at codons 379, 403, 404, 409 and 410 This aggregated 43-kDa species is present at significantly higher levels in FTLD-TDP cases than in controls. To our knowledge, these studies represent the first demonstration of aggregated 43-kDa TDP-43 in diseased human brain. Immunohistochemical studies had indicated the presence of “full length” TDP-43 in intracellular inclusion in both FTLD-TDP and ALS, but it is not possible from such studies to know if the full-length TDP-43 had undergone phosphorylation or other covalent addition of a chemical group The forms of TDP-43 found in disease-associated aggregates may provide insight into the process of pathological aggregation in FTLD. To summarize, these species include at least 2 forms whose polypeptide length appears to be the same as the physiologically predominant 43-kDa species (a form not phosphorylated at S409/410 and a phosphorylated form); several truncated forms, at least some of which are phosphorylated; and forms with molecular weights greater than 45 kDa that are phosphorylated. Species other than the 43-kDa and 45-kDa forms may be ubiquitinated as well. .in vivo or in the interval between death and autopsy. Excepting this possibility, the presence of TDP-43 without apparent phosphorylation, truncation or ubiquitination in aggregates indicates that none of these covalent modifications is necessary for TDP-43 aggregation. This conclusion does not exclude that possibility that aggregation might be initiated by covalently modified forms, with full-length forms subsequently aggregating by another process triggered by truncated aggregates. Such processes might include impairment of quality control mechanisms that establish and maintain proper TDP-43 conformation or that clear aggregated TDP-43. In this regard, one study found that truncated forms of TDP-43 expressed in cultured cells recruit full length TDP-43 into aggregates From the observation that apparently full length TDP-43 is present in aggregates, one can conclude that truncation cannot be essential for aggregation because the processes leading to truncation are presumably irreversible. By similar reasoning, the presence TDP-43 appearing to lack phosphorylation suggests that phosphorylation is not essential for pathological aggregation. However, unlike truncation, phosphorylation is not necessarily irreversible. It is conceivable that endogenous phosphatase activity might generate 43-kDa TDP-43 from the 45-kDa species (or other phosphorylated full-length species) after aggregation had occurred. We found no significant endogenous phosphatase activity upon TDP-43 in our post-mortem tissue samples, but this does not exclude the possibility that such a process was active Another possibility is that covalent modifications are not necessary to initiate and perpetuate TDP-43 aggregation. In the neurodegenerative conditions known as prion disease, the prion protein (PrP) converts to an aggregated form without covalent modification. Instead, pathological PrP aggregates seem to serve as a template for a conformational change in native PrP that drives further aggregation. Evidence exists for prion-like propagation of the aggregated proteins associated with several neurodegenerative diseases The significance of apparently aggregated 43-kDa TDP-43 in one of our controls in an amount approaching that found in in FTLD cases is uncertain. A separate preparation from the same brain gave similar results (data not shown). Histopathologic examination of this brain did not reveal TDP-43 positive intracellular inclusions, but biochemical techniques may be more sensitive that immunohistochemistry for the detection of pathological protein aggregates A waiver of approval was obtained from the Colorado Multiple Institutional Review Board because only autopsy tissue was used in these studies. Frozen blocks of human cerebral cortex from autopsy specimens were obtained from either the New York Brain Bank or the Neuropathology Division at the University of Colorado Denver. The clinical characteristics of all cases are summarized in Brain tissue was homogenized in 9 volumes ice-cold 25 mM Tris, pH 7.4, with Complete inhibitor cocktail (Roche). Where indicated, a phosphatase inhibitor cocktail, PhosStop (Roche) was also added to the homogenization buffer. Tissue was suspended in buffer using a 5-mL Tenbroeck tissue grinder on ice, then clarified by centrifugation at 600 g for 30 seconds. Total protein concentration of homogenates was determined by the bicinchoninic acid assay .This method is a modification of a technique used to demonstrate aggregated forms of yeast prion proteins We developed methods to isolate sufficient TDP-43 for further analysis from the high molecular weight regions of SDD-AGE gels . A largeThe gel was next modified to permit separation of a large volume of homogenate by vertical electrophoresis. First, the comb was removed. To accomplish this without tearing the delicate gel, the clamps holding the glass plates and spacers were loosened and removed. The gel assembly was held parallel to the floor, and the top glass plate was carefully slid away from the comb, without introducing air between the plate and the underlying gel, to expose the comb. The comb was then removed. Large volume wells were made by removing the agarose barrier between 4 adjoining wells with a clean micropipette tip. The top plate was then carefully slid back into position and the clamps were replace. In order to prevent the gel from sliding out of the bottom of the assembly during vertical electrophoresis, a 1 cm wide retention band was poured at the bottom of the gel: First, the assembly was held in an upright position until the gel gradually slid out from between the glass plates, exposing a 1 cm wide band. This was excised with a clean razor blade. The assembly was then inverted until the gel returned to its original position. The assembly was then placed horizontally on the bench top. Three strips of blotting paper, 1 cm ×14 cm were then insert into the 1 cm gap at the bottom of the gel. A 30% polyacrylamide solution (30% acrylamide (w/v), 0.6% bis-acrylamide (w/v), 20 mM Tris (pH 7.4) and 200 mM glycine, 0.1% SDS (w/v), 5 µL/mL 10% ammonium persulfate, 0.5 µL/mL TEMED) was then carefully pipetted onto the paper and allowed to fill the 1 cm gap by capillary action.When the polyacrylamide retention band polymerized, the gel was placed in the apparatus according to the manufacturers instructions. The buffer chambers were filled with a standard Laemmli running buffer. Samples were prepared by mixing 400 μg  of homogenate, typically about 100 μL, with an equal volume of 2× SDD-AGE loading buffer and incubated at room temperature for 10 min with 3 brief agitations using a vortex mixer. The electrophoresis was performed at constant 125 V with ice water cooling until a pre-stained 15-kDA marker  reached the top of the polyacrylamide strip. Regions of interest were excised from the gel with a clean razor blade. Up to 4 samples can be processed in parallel with this method using a single 2-gel apparatus. At least 1 control and 1 FTLD-TPD case were included in each run of the method.We modified the “three-layer sandwich gel electrophoresis” method Meanwhile, excised portions of large format SDD-AGE gels were weighed. SDS was added to 1% and dithiothreitol to 25 mM. Samples were boiled for 5 minutes to melt the agarose and to denature protein complexes contained within it. Ultrapure water was added to a final volume of 4 mL, and the samples were briefly boiled again. The hot agarose solution was then pipetted into the glass tube over the sealing layer of polyacrylamide. After gelling, the agarose was capped with 100 µL of a melted solution of 0.01% (w/v) bromphenol blue in 1% agarose (w/v).The tubes were loaded onto the Electro-eluter apparatus according to the manufacturers instructions, with the polyacrylamide sealing layer down. Electrophoresis was performed in Laemmli buffer at 15 V for 30 min, reduced to 5 V for 16 hr., then increased stepwise to 140 V over 5 hours. Under these conditions, bromphenol blue migrated to form a thin band just above the polyacrylamide sealing layer. The agarose column was extruded from the tube by running a 30-gauge needle of a 1 mL tuberculin syringe around the perimeter of the polyacrylamide plug to loosen its adherence to the glass tube, then pushing on the polyacrylamide plug with the thumb rest of the syringe. An approximately 1.5 mm wide slice of agarose encompassing the bromphenol blue band, and where appropriate, sequentially higher slices were excised with a clean razor blade for further analysis.SDS-PAGE was performed using standard Laemmli technique with precast tris-glycine gels (Invitrogen) For immunoblotting of both SDS-PAGE and SDD-AGE gels, proteins were transferred to polyvinylidene fluoride (PVDF) membrane using a mini-tank  using Towbin buffer per the manufacturers instructions For analysis of phospho-TDP-43 bands, blots were first probed and imaged using polyclonal antiserum 10782-2-AP as above. The blot was then stripped of antibody using a commercial stripping buffer  according to the manufacturers instructions. The blot was then probed with anti-phospho-TDP-43 (Ser409/Ser410), clone 1D3  and anti-rat IgG-HRP conjugate secondary antibody.SDS-PAGE gels for silver staining were run with unstained protein mass standards . Silver staining was done using a SilverXpress kit (Invitrogen) per the manufacturers instructions. Coomassie staining was done using Brilliant Blue G  following the protocol supplied by the manufacturer.Two brain homogenates were prepared from each of 3 FTLD cases. Tissue was homogenized using a 1 mL Tenbroeck tissue grinder on ice, in 9 volumes ice-cold 25 mM Tris, pH 7.4 with either Complete protease inhibitor cocktail or both Complete protease inhibitor and PhosStop phosphatase inhibitor cocktail. Samples were stored on ice while protein concentrations were determined, then 800 µg  aliquots of both protease inhibitor only or protease inhibitor and phosphatase inhibitor treated homogenates either stored on ice or incubated at 37°C for 1 hour. The samples were then subject to large volume SDD-AGE followed by concentration of the aggregate region, then separated by SDS-PAGE as described above. Immunoblots were made using polyclonal anti-TDP-43 antiserum. The 43-kDa and 45-kDa bands were quantified as described below, and ratio of the 43-kDa band to the 45-kDa band was determined.Urea was prepared in 0 M, 2 M, 6 M and 8 M concentrations in 50 mM Tris pH 6.8, and 15 µL of the urea solution was mixed with 5 µL of a 10% brain homogenate, prepared as described above, from an FTLD case. The mixtures were vortexed briefly and incubated for 60 minutes at room temperature. The samples were then processed for SDD-AGE and immunoblotting. To assess the effect of boiling in the presence of SDS and DTT, 5 µL of 10% brain homogenate was mixed with 5 µL of SDD-AGE loading buffer. One sample was held at room temperature for 10 minutes (per SDD-AGE protocol described above) while the other was heated to 100°C for 5 minutes. The samples were then processed for SDD-AGE and immunoblotting.All immunoblots were either directly imaged in a digital format, using a Kodak Image Station 4000  cooled-CCD digital camera, or converted to digital images from photographic film  by taking digital photographs of film images on a trans-illuminating stage . Digital images of Coomassie and silver stained gels were obtained by photographing them on a trans-illuminating stage.For quantitative analysis, digital images were imported into Mathematica software st (linear) or 3rd order polynomial curves were fit to the standard density dilution plots using the Mathematica “FindFit” function. The order of the fitted curve was chosen empirically to minimize residuals. In most cases photographic film showed a sigmoidal response best fit to a 3rd order curve, while the directly obtained CCD camera images showed a linear response. The equations for these fitted curves were applied to the apparent densities of the cognate bands in the experimental blot, so that quantities were determined relative to the dilution of the standard. This relative quantitation was used in comparisons between samples in different experiments. Absolute TDP-43 quantities were not determined. In measuring densities of bands, median pixel values were used instead of mean values because this method was less sensitive to irregularities such as specks and blank spots in the bands images. Particularly when measuring closely apposed bands, such as the 43-kDa and 45-kDa species, the measured density depended on the precise positioning of the region of interest. To minimize error and variance due to positioning the region of interest, each ROI median density was measured 5 times and the mean value determined.Plots of apparent density versus dilution or quantity of the standard were prepared for each band of interest. Either 1To determine yields from the concentration process that we developed, portions of an SDD-AGE gel region concentrate were loaded onto a PAGE gel with serial dilutions of a standard. In the case of aggregate region concentrates, whole brain homogenate of known protein concentration was the standard. In the study of the efficiency of pre-stained marker recovery from the gel, the same lot of pre-stained markers was the standard. The pre-stained marker gel was imaged unstained, otherwise the PAGE gel was silver stained. A plot of standard dilutions versus mean whole lane density was made in Mathematica, and the concentration of the aggregate region concentrates determined by fitting to this curve, using the method described above for determining individual band densities. Yields were then calculated from these concentrations.Statistical tests were performed using Mathematica software. Comparison of band densities between FTLD cases and controls were made using the Mathematica “TTest” function, which performs a Student T-test on univariate data such as in this study. Because it was not clear that the density values would be distributed normally, a non-parametric test was also applied. In all cases the T-test yielded the highest, i.e. least significant therefore most conservative, P value and this was reported.


Dinoderus minutus (bamboo borer) is a wood boring beetle that seriously damage dried bamboo and finished bamboo products. Management of D. minutus using pesticides showed negative effects on environment despite being very costly. By understanding influence of natural climatic conditions on their reproductive behaviour, could help us to develop a cost effective and environmental friendly strategy to cope up with this problem. In the present study, reproductive parameters and egg development of the bamboo borer were determined at 20%, 40%, 56%, 75% and 85% r.h. levels at constant temperature of 30° ± 2°C with 8 L-16D photoregime. From the results, eclosion to first instar larva was recorded at all relative humidities tested. The lowest shortest percentage of hatchability was recorded at 20% and 85% relative humidity with a mean incubation period of 4.63 ± 0.25 and 10.43 ± 0.32 days, respectively. It was noted that pre-ovipositional period decreased from 14.20 ± 0.49 to 7.20 ± 0.31 days as relative humidity increased from 20% to 75% and slightly increased to 8.00 ± 0.37 days at 85% relative humidity. We conclude that female beetles may have a particular hygropreference in oviposition as total egg production increased with increasing relative humidity.Wood products are highly exposed to infestation by powder post beetles.  Plants are always under the influence of biotic as well as abiotic stressful conditions  and Oryzaephilus surinamensis (Coleoptera: Cucujidae) showed increased insect mortality as relative humidity decreased , which consists of anhydrous molecules, and with the amount of cuticle, which has a lower water content than other tissues. Environmental temperature and humidity affects the developmental phase and transpiration through insect body surface , insects or their eggs may drown or be infected more readily by pathogens . This study would help us to develop cost effective and environmental friendly strategies to control insect pests without any health hazards.The present study was concentrated to understand the effect of relative humidity on egg hatchability and reproductive capacity of bamboo borer D. minutus were collected from infested bamboo culms in Bamboo Incubation Center and Handicraft in Simpang Pertang, Negeri Sembilan, Malaysia (2°57’25.61”N 102°18’12.69”E). Most bamboo culms infested by D. minutus belongs to the species Gigantochloa scortechinii, which is an important species of bamboo in Malaysia. Infested bamboo culms were kept in a plastic container with dimension of 30 cm × 20 cm × 25 cm kept in insect rearing room at 75% r.h. and 25 ± 3°C with 8 L-16D photoregime. Newly emerged beetles were collected and introduced into a new culture jar with cassava block using standards techniques of culture described by Abood et al. , Sodium iodide (NaI), Sodium bromide (NaBr), Sodium chloride (NaCl) and Potassium chloride (KCl). The relative humidity was obtained by filling solutions at the bottom of desiccators and monitored using a digital thermohygrometer. Throughout the study, investigation on reproductive parameters and egg hatchability were carried out in Petri dishes kept in desiccators corresponding to each relative humidity and hermetically closed. The desiccators were put into an incubator with controlled temperature of 30 ± 2°C.Different salt solutions and distilled water were used to control the relative humidity to which eggs and adult beetles were exposed. As indicated by Winston and Bates , Roca anNewly emerged beetles less than 7 days old were used to determine the effect of different relative humidities  on reproductive parameters at constant temperature of 30° ± 2°C with 8 L-16D photoregime. Ten pairs of adult beetles were exposed at each relative humidity tested. Each pair of adults were selected and separately introduced into Petri dishes lined with cassava pellet and filter paper. Observation on egg production was then started after 24 hours of setup. Cassava pellets were removed and broken up daily to assess number of eggs laid. The cassava pellet was replaced each time after inspection on egg production. Data collection on adult longevity and fecundity were collected until complete mortality.D. minutus were obtained from an established D. minutus culture on cassava. A total of 50 eggs were placed equidistant into Petri dishes lined with black filter paper for each relative humidity tested. Each Petri dish was kept in desiccators corresponding to each relative humidity and hermetically closed. The desiccators were put into an incubator with controlled temperature of 30 ± 2°C. Petri dishes were kept under these conditions and observation on egg eclosion and incubation period were observed daily under stereomicroscope and compared. During observation, eggs were removed as required with respect to age in days with a fine brush. Each egg was mounted on a concave microscope slide with a cover slip. This method was carried out to minimize the effect of relative humidity during the non-test conditions.The effect of relative humidity on egg hatchability and incubation period was inspected under different relative humidity levels  at constant temperature of 30° ± 2°C with 8 L-16D photoregime. Newly laid eggs of Statistical analysis software (SAS version 9.2) was used for the analysis on egg hatching, reproductive capacity and adult longevity at different relative humidities. One Way Analysis of Variance (ANOVA) and Fisher’s least significant difference (LSD) were used to test the effect of relative humidity on reproductive parameter and incubation period.D. minutus. There is no significant difference between incubation period at 40%, 56% and 75% relative humidity . The lowest and highest relative humidity caused a significant decrease in egg hatchability when compared with the intermediate relative humidity levels .The results presented in Table Highest eclosion to first instar larva was recorded at 75% relative humidity with 86% eclosion to success and a mean incubation period of 5.44 ± 0.14 days. Lowest relative humidity of 20% showed only 8% eclosion to success with a mean incubation period of 4.63 ± 0.25. Percentage of successful eclosion and incubation period increased with increasing relative humidity to 75%. However for 85% relative humidity, the percentage of successful eclosion decreased to 46%. This shows that egg development was affected by extremes in humidity (Plate 1). Longest incubation period was recorded at 85% relative humidity with a mean of 10.43 ± 0.32 days.D. minutus reproductive capacity  and adult lifespan . However, there is no significant difference in pre-ovipositional period between 56% and 75% relative humidity levels . Female D. minutus showed shorter pre-ovipositional period with increasing relative humidity until 75%. Ovipositional period  and mean number of eggs laid  showed no significant difference between 75% and 85% relative humidity levels. Shortest ovipositional period was recorded at 20% relative humidity with a mean of 3.8 ± 0.64 days while the longest ovipositional period was recorded at 85% relative humidity with a mean of 56.50 ± 1.21. Mean number of eggs laid increased from 3.20 ± 0.31 to 56.50 ± 1.21 from 20% to 85% relative humidity, thus indicating a preference for higher relative humidity conditions. Similar increment was recorded on mean ovipositional period which showed an increase from 3.8 ± 0.64 to 54.80 ± 0.94 days. The pre-ovipositional period decreased with increasing relative humidity levels from 20% to 75%. However, between 75% to 85% relative humidity, there was a significant increase in pre-ovipositional period. Adult lifespan for both sexes increase with increasing relative humidity. Female D. minutus had longer lifespan compared to male beetles under all relative humidities tested. There was no significant difference between adult lifespan for male  and female  between 85% and 75% and 75% and 56% relative humidity, respectively. Earliest mortality for adult beetle was recorded at 20% relative humidity for male and for female beetle on day 16 and 18, respectively. Longest lifespan for male beetle was recorded at 67 day at 75% relative humidity while longest lifespan for female beetle was 74 days at 85% relative humidity.Boring activity on cassava pellet was recorded within 24 hours of exposure under each relative humidity. Results in Table D. minutus under different relative humidity are shown in Figure Fecundity curves for Dinoderus minutus Fabricus).In order to establish a sustainable pest management, it is essential to know the physiology as well as reproductive capacity of pests viz-a-viz their relations with various natural climatic conditions. Thus present study was undertaken to investigate the effect of humidity on egg hatchability and reproductive biology of the bamboo borer  .From the observations in this study, most eclosion to first instar larva of D. minutus showed heterogeneous ovipositional pattern with most eggs being laid at higher relative humidities and the mean number of eggs laid increased from 3.20 ± 0.31 to 56.50 ± 1.21 at 20% to 85% relative humidities, respectively. The effect of relative humidity on oviposition indicates that females may have a particular hygropreference in oviposition as total egg produced and ovipositional period increased with increasing relative humidity. However, observation on egg hatchability showed that high relative humidity of 85% resulted with low egg hatchability (Table D. minutus was capable to reproduce at high relative humidity at early infestation in bamboo storage. As relative humidity decreased during the processing of bamboo culm, egg hatchability increases causing a pest outbreak. Romoser and Stoffolano (Xylocoris flavipes decreased at high relative humidity of 90%. David and Ananthakrishnan (Locusta spp. Andrewartha and Birch (D. minutus response towards relative humidity was found to be similar to the rice weevil, Sitophylus oryzae, an important pest in stored product. Fecundity of S. oryzae increased with increasing moisture content of wheat from 34% to 70% relative humidity (David and Ananthakrishnan, Female offolano  stated toffolano  showed tkrishnan  suggestend Birch  reportedD. minutus egg hatchability showed high mortality at 20% and 85% relative humidity levels compared to that between 40% to 75%. Lowest egg hatchability was recorded at 20% and 85% relative humidity levels. D. minutus egg suffers from loss of moisture that leads to shrinkage of both the embryo and chorion which prevents larval release. High relative humidity at 85% resulted in egg mortality due to excessive moisture. Reproductive capacity and incubation period of D. minutus increased with increasing relative humidity. Mean number of eggs laid increased from 3.20 ± 0.31 to 56.50 ± 1.21 from 20% to 85% relative humidity. Female beetle preferred to lay eggs in higher relative humidity as the number of eggs increased with increasing relative humidity. Pre-ovipositional decreased with increasing relative humidity. However at 85% relative humidity, pre-ovipositional period increased slightly compared to 75% relative humidity. Ovipositional period prolonged as relative humidity increased. Adult female showed higher fecundity characteristics with increasing relative humidity. Mean ovipositional period increased from 3.8 ± 0.64 to 54.80 ± 0.94 days with increasing relative humidity. The present study would help scientist to work out the strategy/s about the biological control of pests without causing any damage to health, environment as well as economy.The effect of relative humidity on


TDP-43 accumulation and ALS-linked mutations within degradation pathways implicate failed TDP-43 clearance as a primary disease mechanism. Here, we report the differing roles of the ubiquitin proteasome system (UPS) and autophagy in the clearance of TDP-43. We have investigated the effects of inhibitors of the UPS and autophagy on the degradation, localisation and mobility of soluble and insoluble TDP-43. We find that soluble TDP-43 is degraded primarily by the UPS, whereas the clearance of aggregated TDP-43 requires autophagy. Cellular macroaggregates, which recapitulate many of the pathological features of the aggregates in patients, are reversible when both the UPS and autophagy are functional. Their clearance involves the autophagic removal of oligomeric TDP-43. We speculate that, in addition to an age-related decline in pathway activity, a second hit in either the UPS or the autophagy pathway drives the accumulation of TDP-43 in ALS and FTD. Therapies for clearing excess TDP-43 should therefore target a combination of these pathways.TAR DNA-binding protein  is the major pathological protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Large TDP-43 aggregates that are decorated with degradation adaptor proteins are seen in the cytoplasm of remaining neurons in ALS and FTD patients  Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that are characterised by the deposition of pathological protein aggregates composed of TAR DNA-binding protein 43 (TDP-43 or TARDBP) . These ‘Evidence that the dysregulation of TDP-43 can cause disease comes from many animal models. A neurodegenerative ALS-like phenotype can be generated by either overexpression or knockdown of TDP-43 in flies, fish and mice . Indeed,Two of the major pathways for cellular protein degradation are the ubiquitin proteasome system (UPS) and macroautophagy, herein referred to as ‘autophagy’. Autophagy has been shown to degrade soluble and aggregated protein substrates that are too large to enter the UPS pore . UbiquitDetermining the relative importance of these two protein degradation pathways in maintaining TDP-43 proteostasis is challenging owing to the complex interplay between them, yet is crucial to understanding the mechanisms underpinning TDP-43 accumulation in disease. Although the role of TDP-43 macroaggregation remains controversial, the cytoplasmic accumulation of TDP-43 is definitely linked to toxicity, and therapeutic strategies designed to enhance TDP-43 clearance are of great appeal. We therefore sought to investigate how TDP-43 protein degradation is regulated and the potential for targeting degradative pathways as a treatment for ALS and FTD.50; HEK293, 0.97 ng/ml; SH-SY5Y, 0.35 ng/ml).Stable cellular models of ALS and FTD based on doxycycline (DOX)-inducible expression of various TDP-43 constructs  were genNext, we sought to investigate the relative degradation rates of the TDP-43 constructs. Because of its ability to autoregulate , quantifIn HEK293 lines assayed over 72 h , wild-tyt1/2; HA–TDP-43 WT, 29.2 h; HA–TDP-43 ΔNLS, 16.6 h; HA–TDP-43 CTF, 10.2 h).In order to verify that the assay was not simply measuring the dilution of HA–TDP-43 protein by cell division or the slow decay of DOX-induced transcripts, we performed a modified experiment over a shorter timecourse in SH-SY5Y lines , and useHaving determined that TDP-43 degradation rates were consistent between cell lines and timecourses, we next analysed the pathway(s) by which TDP-43 was being degraded, using HEK293 lines. Inhibitors of the UPS or autophagy were included during the final 48 h of the DOX-washout period when protein degradation occurs (for schematic see P≤0.05) and, to a lesser extent, the degradation of wild-type and ΔNLS TDP-43. 3MA showed minimal effect on the autophagy reporters p62 and LC3 (microtubule-associated protein 1 light chain 3 β) at the timepoint chosen for assay of TDP-43 levels (48 h 3MA); however, shorter timecourses of 3MA treatment revealed autophagy inhibition had occurred . The related UPS inhibitor epoxomicin also caused significant accumulation of wild-type, ΔNLS and CTF TDP-43 . The inhibition of both degradation pathways (by combined treatment with 3MA and MG132) did not inhibit the degradation of any of the protein constructs any more than MG132 alone. Activators of the autophagy pathway validated these findings because the addition of trehalose and LiCl during DOX washout substantially accelerated the degradation of CTF TDP-43  and p62 , and polyubiquitin chains with different internal lysine linkages (K48 and K63) . These aWe next sought to specifically investigate the handling of insoluble TDP-43. We used 1 µg/ml DOX plus MG132, which acts reversibly, to induce the formation of insoluble TDP-43 WT or ΔNLS cellular aggregates and then we followed the fate of those aggregates after washout of MG132. DOX was maintained in the medium at a concentration of 1 µg/ml during washout to ensure that our findings reflected TDP-43 aggregate handling in the face of continued expression of TDP-43. End-point imaging showed that for both WT  and ΔNLSt1/2; EGFP–�TDP WT, 29.3 h; HA�–TDP WT, 29.2 h). We also verified that EGFP-tagged TDP-43 was functional, as evidenced by its ability to downregulate endogenous TDP-43 . Owing to the low fluorescence intensity of EGFP–TDP WT aggregates, their clearance could not readily be measured in living cells.We therefore examined the clearance of aggregates in live cells, using stable SH-SY5Y cells transfected with EGFP-tagged TDP-43. We verified that the replacement of the N-terminal HA-tag with EGFP did not alter the timecourse of TDP-43 degradation by performing doxycycline pulse-chase experiments  are mobile, readily exchange with the diffuse protein pool and can be cleared by the UPS . To deteP≤0.01; MG132 with aggregates, 48%±1.6, P≤0.001; ±s.e.m.). Furthermore, we found that the TDP-43 within the aggregates was almost completely immobile, with the bleached portions of the aggregates remaining dark for the duration of FRAP imaging and not visibly exchanging with the diffuse pool (P≤0.001). At 8–10 h after the washout of MG132, there was a significant increase in the proportion of diffuse TDP-43 that was mobile in cells still containing large aggregates, suggesting there was mobilisation or clearance of previously immobile species . However, direct bleaching of macroaggregated TDP-43 showed that this species was not mobilised, as it showed no increase in the mobile fraction after washout.We examined cells that were induced to express EGFP–TDP-43 using 1 µg/ml DOX alone, cells induced to form aggregates with 1 µg/ml DOX plus MG132, and cells induced to form aggregates then washed out and left in fresh medium containing 1 µg/ml DOX without MG132 for 8–10 h. Firstly, by bleaching diffuse cytosolic EGFP–TDP-43 ΔNLS we found that the proportion of diffuse TDP-43 protein that was mobile was reduced by inhibition of the UPS, both in cells devoid of aggregates and, more strikingly, in cells that contained large aggregates (t1/2 = 3.4 s). Diffuse TDP-43 in cells exposed to UPS inhibition but without aggregates showed a similar rate of recovery (t1/2 = 3.6 s), whereas TDP-43 in cells with aggregates showed significantly slower recovery (t1/2 = 6.9 s). A decrease in the fluorescence recovery rate indicates increased binding or increased size of the TDP-43 particle, consistent with oligomerisation of TDP-43. At 8–10 h after the washout of MG132, there was an increase in the rate of recovery of diffuse TDP-43 in cells with large aggregates (t1/2 = 6.9 s before versus 4.0 s after washout), suggesting that there had been removal or dissociation of slow-moving species.Next, we examined the rate of fluorescence recovery (the speed of molecular movement) for mobile species of TDP-43 protein . DiffuseP≤0.01; ΔNLS, 31±9 MG132 versus 6±2 DOX, P≤0.05; ±s.e.m.).Our findings suggested that during washout, slow-moving and immobile species were either resolved into mobile or faster-moving species, or were removed directly. Oligomeric or microaggregated species cannot be cleared directly by the UPS, and their clearance might require aggregate autophagy or ‘aggrephagy’ . TherefoP≤0.01, Student’s t-test with Welch’s correction; ±s.e.m.). For cells that survived the period of imaging, we plotted the relationship between the load of aggregated protein and the rate of clearance from the cell . However, these aggregates then persisted in the cells. The slope of the line relating initial aggregate load with clearance time therefore flattened in the presence of autophagy inhibitors  but not by knockout of a key molecule in autophagy induction, Atg7  then subcloned into Gateway. EGFP-tagged TDP-43 ΔNLS was generated by site-directed mutagenesis of the WT construct in pDONR . Primer sequences are available on request.Constructs are shown schematically in ® Terminator v1.1  on an ABI3130 genetic analyser . Sequence chromatograms were analysed using Geneious v5.4.6 . All primers were obtained from Sigma-Aldrich . mCherry–GFP–LC3 plasmid (All constructs were verified in-house by DNA sequencing using Big-Dye plasmid  was kind plasmid  was a ki3MA (used at 10 mM), bafilomycin (400 nM) and lithium chloride (10 mM) were purchased from Acros Organics . MG132 (0.5 µM), epoxomicin (100 nM) and rapamycin (0.2 µg/ml) were purchased from Cayman Chemicals . Trehalose (100 mM) was purchased from Sigma-Aldrich.Stable tetracycline-inducible (Tet-ON) clonal cell lines were generated in human neuroblastoma SH-SY5Y cells  and human embryonic kidney HEK293 , using the T-REx system. Briefly, stable clonal lines transfected with pcDNA6/TR (encoding a constitutively expressed Tet-repressor protein) were transfected with pDEST30-TDP-43 constructs using Lipofectamine 2000, selected using 600 µg/ml geneticin and clonally isolated.2.SH-SY5Y cells were maintained in DMEM/F12 plus Glutamax, supplemented with 10% fetal bovine serum (FBS) , 100 U/ml penicillin and 100 µg/ml streptomycin. HEK293 cells (stable Tet-ON) were maintained in DMEM plus Glutamax, supplemented with 10% FBS (Tet-free), 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were maintained at 37°C, under 5% CO2, HEK293 lines were plated at 25,000 cells/cm2 and TDP-43 expression was induced using 1 µg/ml DOX, unless otherwise stated. All reagents were purchased from Invitrogen Life Technologies unless otherwise stated.Transient transfection of mCherry–GFP–LC3 into stable SH-SY5Y lines was performed using Lipofectamine 2000, as per the manufacturer’s instructions. Transient transfection of FLAG–Ub into stable HEK293 lines was performed using Fugene HD  as per the manufacturer’s instructions. For experimental analyses, SH-SY5Y lines were plated at 75,000 cells/cmFor immunofluorescence analyses, SH-SY5Y lines were plated onto untreated 13-mm coverslips (1.5 thickness). HEK293 lines were plated onto poly-D-lysine-treated coverslips. After treatment, cells were fixed in 4% paraformaldehyde  for 10 min and rinsed with phosphate-buffered saline (PBS). The cells were incubated with primary antibody overnight at 4°C, then with fluorescent secondary antibodies for 6 h at room temperature, each diluted in PBS with 0.2% Triton X-100 and 1% serum (goat or donkey as appropriate). For thioflavin T staining, cells were stained for 10 min with freshly prepared and filtered 0.1% thioflavin T (Acros Organics) in PBS and rinsed. After immunodetection, the coverslips were mounted onto slides using fluorescence mounting medium  and left to harden overnight.The antibodies used for immunofluorescence were against TDP-43 , HA.11 , p62 , ubiquitin , K48-linked ubiquitin , K63-linked ubiquitin , ubiquilin-2 . Invitrogen Life Technologies goat secondary antibodies  or Jackson ImmunoResearch donkey secondary antibody  were used at 1∶500.Wide-field imaging was performed using a Zeiss Axiovert S100  with a ×63/NA 1.25 Plan Neofluar oil immersion objective, fitted with a CoolSnap EZ digital camera . Confocal images were acquired using a Leica TCS SP5 confocal laser scanning microscope  with a ×63/NA 1.4 Plan Apo oil immersion objective and a pinhole of 95.6 µm (Airy 1). Images (1024×1024 pixels) were acquired at 400 Hz with a line averaging of 8, using Leica LAS-AF software and 405 nm, 488 nm and 633 nm laser lines.http://rsb.info.nih.gov/ij/) on merged RGB images for a representative experiment. GFP–LC3 autophagosomes were quantified using the Granularity tool in Metamorph  and the granules were normalised to the number of transfected cells by manual counting of GFP-positive cells. 113–622 granules were counted in 20–50 transfected cells in a representative experiment.Line intensity profiles were quantified using the colour profiler plug-in (Dimiter Prodanov) in ImageJ  pulse–chase as described previously . Briefly® III First-Strand Synthesis SuperMix (Invitrogen Life Technologies). Quantitative PCR was performed using FASTStart SYBR Green Mastermix on a 7900HT Fast Real-Time PCR System (Applied Biosystems) running SDS v.2.3 software. The primers that were used to detect HA–TDP WT and ΔNLS transcripts were 5′-TACCCATACGATGTTCCAGATTAC-3′ and 5′-GCATGCAGAATTCCTTCTACC-3′. HA–TDP CTF transcripts were quantified using the same HA-specific forward primer with the reverse primer 5′-CCCGTACTGAGAGAAGAACT-3′. GAPDH acted as a reference transcript, using the primers 5′-CAGCCTCAAGATCATCAGCA-3′ and 5′-GGCATGGACTGTGGTCATGAG-3′. PCR cycling conditions were: activation, 10 min at 95°C; cycling, 15 s at 95°C, 30 s at 60°C and 30 s at 72°C for 40 cycles; followed by the thermal denaturation protocol. The expression levels of HA–TDP transcripts relative to GAPDH transcripts (reference), with normalisation to no DOX (control), were determined using the Pfaffl method  and cDNA was reverse transcribed using oligo(dT) primers with SuperScriptl method .The sequential biochemical fractionation of cellular proteins was performed as described previously for ALS and FTD brain homogenates , with seRIPA/urea solubility fractionation was performed as described by Winton and colleagues , using cProtein concentrations were quantified using the BioRad DC Protein Assay  and equivalent protein was loaded for each sample . For solubility experiments, whole-lysate protein was quantified and the equivalent liquid volume of the soluble and insoluble fractions was loaded. Gels were transferred onto nitrocellulose using the iBlot (Invitrogen Life Technologies), stained with Ponceau S and then blocked in TBS with 0.05% Tween-20 (TBS-T) and 5% non-fat dried milk  for 30 min. The blots were probed overnight at 4°C with primary antibody, then for 3 h at room temperature with secondary antibodies . The blots were scanned on the Li-Cor Odyssey gel scanner  and were then reprobed (without stripping) for loading-control proteins using the same antibody conditions and scanning protocol.Antibodies used for blotting were against TDP-43 , HA.11 , Histone H3 , p62 , GAPDH , GFP , LC3B  and Dylight fluorescent secondary antibodies .The blot images in TIF format were quantified using the gel analyser tool in ImageJ. Integrated band intensities were normalised to the band intensities of loading controls and also to relative input for the solubility assays.2 and were left to recover overnight. TDP-43 expression was induced with DOX for 24 h and then aggregate formation was induced using DOX with 0.5 µM MG132 for a further 48 h. For washout experiments, the cells were then washed and left in fresh medium containing DOX without MG132, in the presence or absence of inhibitors for the stated time periods.The clearance of TDP-43 aggregates was assessed by RIPA/urea solubility fractionation and immunofluorescence (HA-tagged constructs) and live-cell imaging and fluorescence recovery after photobleaching (FRAP) (EGFP-tagged constructs). For all aggregate clearance assays, stable SH-SY5Y TDP-43 WT or ΔNLS cells were plated at 100,000 cells/cmLive imaging was used to assess whether EGFP–TDP-43 ΔNLS aggregates were cleared. Stable SH-SY5Y cells were plated onto Hi-Q4 dishes . Immediately before imaging, the cells were washed and left in fresh medium without MG132, containing DOX and either vehicle (0.05% DMSO), 3MA (10 mM) or bafilomycin (400 nM).2. The image sets were analysed using ImageJ. The aggregate load was defined as the total integrated density (area×intensity) of all aggregates in a given cell, with aggregate boundaries determined by intensity thresholding. The clearance time was defined as the time at which aggregates were indistinguishable from diffuse TDP-43.Epifluorescent and phase images (1280×960 pixels) of selected cells with aggregates were acquired twice per hour for 15 h using a BioStation IM-Q  fitted with a ×20/NA 0.5 Plan Fluor objective and maintained at 37°C, under 5% COStable SH-SY5Y EGFP–TDP-43 WT or ΔNLS cells were plated onto 18-mm coverslips (1.5 thickness). To investigate protein mobility after MG132 washout, the cells were washed and left in fresh medium containing DOX, but without MG132, for 8–10 h before FRAP.2. Fluorescence recovery was followed for 1 min, with a laser power of 1–3% to minimise acquisition bleaching.FRAP was performed using a Nikon A1 plus laser scanning confocal microscope fitted with an environmental chamber maintained at 37°C . Confocal images were acquired using a ×60/NA 1.4 Apo oil immersion objective, a confocal pinhole of 34.8 µm (Airy 1.2) and a 488-nm laser line. FRAP was performed using NIS-Elements AR software (v. 4.00.04). Images of 512×512 pixels were acquired. Five pre-bleach frames were acquired with a laser power of 1–3%, before bleaching EGFP–TDP-43  using a square region of interest (ROI) of 2 µmtI is the fluorescence intensity of the ROI at a given time, I0 is the fluorescence intensity of the ROI immediately after bleaching and Ii is the average fluorescence intensity of the ROI before bleaching. The data were fitted to a one-phase exponential decay and plotted using GraphPad Prism 6.02.Data analysis was performed using ImageJ with the Bio-Formats plugin . The meat-tests were performed after F-testing for equal variance and Welch's correction was applied where stated. The Pearson correlation test was performed after D'Agostino and Pearson normality testing. Statistical significance was set at P≤0.05 . All figures were prepared using Adobe Photoshop CS3.Data handling and graphical representations were performed using Microsoft Excel unless otherwise stated. Statistical analyses as described in the figure legends were performed using GraphPad Prism 6.02. One-way ANOVA was performed after Bartlett's testing for equal variance. For both one- and two-way ANOVA, Bonferroni post-tests were used to compare the selected data sets. Two-tailed Student’s


Sarcoidosis is a multisystem granulomatous disease characterized by hilar lymphadenopathy, involvement of internal organs, and diverse skin lesions. Systemic sclerosis is an autoimmune disease characterized by skin hardening and different internal organ fibrosis, including vascular abnormality. Immune response associated with Th-2 has been shown in the early and active stage of the disease. In this paper, we report coexistence of systemic sclerosis with sarcoidosis in a female patient presenting with granulomatous dermatitis, interstitial lung disease, and Raynaud's phenomenon complaints. Sarcoidosis is a systemic disease characterized by the involvement of multiple tissues and organs and a noncaseating granulomatous reaction, which is not well understood . AlthougWe present 52-year-old woman, who did not seek medical care for complaints that continued for six years and suggested Raynaud's phenomenon in the finger joints of the hands. One year ago, brown-red skin lesions developed at the pretibial part of the right foot and the patient consulted a dermatologist. However, the patient did not receive any results from the dermatological examination and visited our rheumatology polyclinic after arthralgia, morning stiffness, and effort dyspnoea started together with the Raynaud's complaints. Upon a physical examination, telangiectasia on the face, lessening of the mouth opening, sclerodactilia and Raynaud's phenomenon pallor phase of the fingers, and a brown-red skin lesion of approximately 15 cm on the right foot pretibial site not protruding from the skin were found Sarcoidosis is a systemic, Th1-mediated granulomatous disease, with unknown etiology, involving more than one organ. Sarcoidosis may mimic a number of rheumatic diseases or may coexist side by side. It can commonly present with connective tissue diseases, including primary Sjögren's syndrome, SLE, and RA, including clinical findings that are indicative of vasculitis and/or spondyloarthritis. Enzenauer and West have shown that 6 out of 569 patients followed with connective tissue disease developed concomitant sarcoidosis within 10 years . SakamotCoexistence SS with sarcoidosis is an entity that should be rarely seen, when the incidence of both diseases are considered  . MoreoveAs a result, we are reporting the coexistence of sarcoidosis with SS. The overlapping of SS with sarcoidosis is a very rare entity. The Th1/Th2 paradigm is one of its most important causes. Since they both can exhibit similar clinical manifestations, it is a crucial difference to the overlapping syndromes for prognosis and therapy. Although the causes for this coexistence are not clear, further research is warranted to determine whether there is a common etiopathogenesis and/or coincidental coexistence.


We previously have studied the insertion and deletion polymorphism by sequencing no more than one hundred introns in a mixed human population and found that the minimal introns tended to maintain length at an optimal size. Here we analyzed re-sequenced 179 individual genomes  from the data released by the 1000 Genome Project to study the size dynamics of minimal introns.We not only confirmed that minimal introns in human populations are selected but also found two major effects in minimal intron evolution: (i) Size-effect: minimal introns longer than an optimal size (87 nt) tend to have a higher ratio of deletion to insertion than those that are shorter than the optimal size; (ii) GC-effect: minimal introns with lower GC content tend to be more frequently deleted than those with higher GC content. The GC-effect results in a higher GC content in minimal introns than their flanking exons as opposed to larger introns (≥125 nt) that always have a lower GC content than that of their flanking exons. We also observed that the two effects are distinguishable but not completely separable within and between populations.We validated the unique mutation dynamics of minimal introns in keeping their near-optimal size and GC content, and our observations suggest potentially important functions of human minimal introns in transcript processing and gene regulation. Intron is an important and necessary subassembly of eukaryotic genes and plays precise and complicated roles especially in the case of mRNA processing and alternative splicing There have been several comparative studies within and among species, revealing situations where short introns  are under selection, and most of them have focused on model organisms, such as Drosophila. There have been several interesting observations. First, shorter introns tend to have higher recombination rates As data from the 1000 Genome Project are being released To obtain reliable sequence and allele information, we limited our dataset to (1) ancestor alleles were determined based on sequences from all three primates and (2) intron sequences must be mapped precisely in genomic sequences. This filter yielded 202,009 YRI, 146,842 CEU, and 136,175 JPTCHB indels. When plotted the derived allele frequency (DAF) of the indel data, we found that all curves showed a uni-modal distribution, slightly skewed toward left . The moroptimal value based on the intron length distribution corresponding to the peak value (mode) of minimal introns ratio of deletion to insertion or RDI when we investigated strength variations around an optimum: 87 nt . The results suggested that shorter introns are subjected to stronger selective constraints than longer introns when we defined the length of 87 nt as an optimum. We believe that the insignificance of the test is largely due to insufficient data. The stronger negative selection acting on the nucleotide content of protein coding sequences provides additional evidence to support the proposal that minimal introns in a size range of 50–86 nt are more likely to subject to purifying selection resisting to the overall deletion force acting in all introns.In the current study, our samples are still limited to the three populations and limited number of indels in minimal introns (50–124 nt). Although the statistics based on the current data is not as striking as the previous result, they are more robust and conclusive. In the previous study, we chose a set of introns that cover a full range of intron GC content (16.4–78.7%) in order to avoid biases in sampling. As a matter of fact, the GC content of minimal introns in the human genome tends to be higher (median: 60.4%), and in a rather random sampling scheme, our analysis is biased toward minimal introns with higher GC content whereas in a more selective case we actually sampled more AT-rich minimal introns. In either case, size-effect is indisputable. In the previous study, by using low-frequency alleles (indels with DAF<0.06), we found the size-effect but failed when look at higher frequencies, such DAF>0.35. Similarly, in this study we also tried to examine various DAF cut-offs  and found that the size-effect became weaker when DAF cut-off is above 0.1 (data not shown). Our data collectively confirmed the proposal that minimal introns have an optimal size or a narrow range and the distribution of minimal introns has a mode of 87 nt. If introns drift off this optimal size, there should be a tendency to be drawn back. To look for evidence supporting our hypothesis, we chose to focus on minor alleles that are sensitive to selection but not yet fixed in populations as we have preciously pointed out that rare polymorphisms (as a threshold of 0.06) are subjected to selections (e.g. adaptive selection on protein-coding sequences) In the current study, we also addressed GC-effect in addition to size-effect. In the previous study, we noticed a GC-effect where almost all insertions happened in GC-rich minimal introns whereas all deletions are among GC-poor minimal introns. Therefore, our previous data were not informative when we tried to separate size effect from GC effect. Although the current data did not give us a clear-cut answer but we believe that GC-effect should be resolved with statistical significance based on adequate data from a broad GC-content range.Finally, we would like to emphasize that sequence variations represent a rich data resource for interrogating biological questions, especially in a context of structure-function relationship such as the size of minimal introns and exons. In a long enough generation period, indels (or SNPs) with higher derived allele frequencies are more likely to be related to positive selections (adaptive selections), as those with lower derived allele frequencies tend to be under negative selections (purifying selections) for deleterious variants, in particular, the extremely rare variations. In contrast, what we found is that the DAF<0.1 variations appear largely adaptive in human populations because the maintenance of an optimal minimal intron length results a better functionality (efficiently processed and exported to the cytosol) for minimal intron-containing genes. We considered the indels of minimal introns as a dynamic parameter for reaching the size optima under various selective forces. Both positive and negative selections can work on these indels; positive selection leads to the fixation of the minimal introns with optimal sizes and purifying selection tends to remove the minimal intron-containing genes  that possessing over- or under-sized minimal introns.Drosophila melanogasterD. simulansin silico.Parsch et al. observed the insertion bias in short introns when examining indel divergence between or within ftp://ftp-trace.ncbi.nih.gov/1000genomes/). We retrieved the three blastz alignment files from UCSC databases ftp://hgdownload.cse.ucsc.edu/) between human and another species: chimpanzee (hg18vsPanTro2), orangutan (hg18vsPonAbe2), and rhesus monkeys (hg18vsRheMac2). We compared the indel variations with the corresponding position of these three primate references to identify ancestral alleles. We calculated the nonsynonymous and synonymous substitution rates between human and mouse from γ-MYN method We defined intron locations based on human Refseq data (NCBI build 36) described previously


A 2 μW power dissipation, voltage-output, humidity sensor accurate to 5% relative humidity was developed using the LFoundry 0.15 μm CMOS technology without post-processing. The sensor consists of a woven lateral array of electrodes implemented in CMOS top metal, a Intervia Photodielectric 8023–10 humidity-sensitive layer, and a CMOS capacitance to voltage converter. After nIn the current work, we investigated the possibility of employing the standard passivation layer of the CMOS chip as the humidity sensing element with electrodes shaped similar to those described in ,6, but u2.Sensing of humidity with hygroscopic polymer films is the most suitable approach for integrating humidity sensors in CMOS, but this approach has problems to solve. First, most hygroscopic films produce humidity-dependent capacitance, so a capacitance-to-voltage on-chip converter is necessary for simplified processing of the sensor output. Second, implementation of the sensing element is not straightforward. To obtain a large relative capacitance change, the electrical flux must be confined to the hygroscopic film. The simplest way to achieve this objective is if electrodes enclose the hygroscopic film from all sides, but for the moist air to gain access to the sensing film, a large area of the polymer must be exposed to the air. In the implemented humidity sensor, the electrical flux reaching the CMOS substrate was confined by the woven electrode mesh below the main sensing electrodes see . The wovFurthermore, previously proposed comb-type electrodes have a 50%metal density if sensitivity is maximized . For theA C/V (capacitance-to-voltage) converter derived from  was deve3.Experimental results from the manufactured humidity sensors are briefly summarized in Humidity range was limited by dryer capability of the humidity chamber and surface condensation. Performance measurements were done only in the 5–100 °C range. At 100–120 °C, only reliability testing was done. The accuracy mentioned in


The transcription factor p53 regulates cellular integrity in response to stress. p53 is mutated in more than half of cancerous cells, with a majority of the mutations localized to the DNA binding domain (DBD). In order to map the structural and dynamical features of the DBD, we carried out multiple copy molecular dynamics simulations . Simulations show the loop 1 to be the most dynamic element among the DNA-contacting loops (loops 1-3). Loop 1 occupies two major conformational states: extended and recessed; the former but not the latter displays correlations in atomic fluctuations with those of loop 2 (~24 Å apart). Since loop 1 binds to the major groove whereas loop 2 binds to the minor groove of DNA, our results begin to provide some insight into the possible mechanism underpinning the cooperative nature of DBD binding to DNA. We propose (1) a novel mechanism underlying the dynamics of loop 1 and the possible tread-milling of p53 on DNA and (2) possible mutations on loop 1 residues to restore the transcriptional activity of an oncogenic mutation at a distant site. Although p53 activates transcription most efficiently as a tetramer , both moin vivo ,16. Morein vivo –19. Indiin vivo . The DBD is an approximately 25 kDa chain consisting of an immunoglobulin-like β-sandwich  that provides the scaffold for the DNA binding surfaces . The secBoth L2 and L3 are stabilized by a zinc ion that is tetrahedrally held by the side chains of a histidine (H179) and three cysteine residues  . The zinProteins exist as inter-converting conformational states under ambient conditions and their conformational dynamics are known to be intricately linked to their functions  and chanWe begin by exploring the conformational space that is spanned by the available experimental structures of DBD. As of July 2011, the RCSB Protein Data Bank (PDB) contains 57 PDB entries of monomeric and multimeric p53 DBD as well as p53 DBD in complex with DNA. From these entries, we excluded structures that have unresolved residues, particularly in the mobile loop regions. The final set consists of 141 distinct conformers . The lowest sequence identity among these conformers is 84.4%. This difference of more than 15% in sequence identity arises from species differences (human versus mouse p53 DBD), but they are highly similar in structures with Cα RMSDs of 0.32- 0.59 Å. p53 DBD from species other than human and mouse were excluded because of the higher sequence differences. We did not see any separation between human and mouse p53 DBD in the first three dominant principal components (PC) space; the details of our principal component analysis (PCA) results are presented below. The final set also includes DBD with multiple mutations such as those introduced to stabilize the DBD.To perform PCA, we first determine, through iterated rounds of structural superposition , 35 struThe NMR and crystal conformers are well separated along PC 1. To identify residues that are responsible for the conformational differences retained by a given PC, the relative displacement of each residue along that PC is examined as atomic displacements from the mean structure . PC 1 deRadius of gyration can be used to understand the degree of compactness of folded protein conformers. We calculated the radius of gyration, surface area and volumes of the experimentally available conformers of each DBD and plot them against PC 1 . PC 1 clPC 2 separates conformers with major differences in L3 conformations; L3 is thought to be crucial for interactions of the DBD with the minor groove of DNA . There aAlong PC2, one of the two major clusters is made up of the crystal conformers (PDB code: 2H1L) that were crystallized with the large T-antigen (LTag) oncoprotein of simian virus 40 (SV40) . The DBDIn the only NMR study reported on isolated p53 DBD, M246 seems to occupy two conformations based on the detection of line broadening . This daBesides separating conformers with distinct methionine switch conformations in L3, PC2 also provides clues on the zinc ion. The zinc ion is coordinated by C176 of L2, H179 of H1, and C238 and C242 of L3. The crystal conformer (PDB code: 2P52), which was crystallized in the absence of zinc ion , lies inSimilar to PC 1 in the context of L1 diversity, PC 3 captures the diversity of conformers especially at L1. Two major clusters emerge along PC 3, in which one cluster (PC 3 > 15) has recessed L1 conformations and the other has extended L1 conformations . Only 6.To further probe the conformational space of monomeric p53 DBD, we performed two copies of MD simulations, each starting from four different structures, resulting in eight independent copies of 100 ns each. The RMSD profiles of the structural changes along the MD conformers suggest that our simulations are stable especially for the core residues . hitherto. The extended L1, particularly its K120 , seems to be stabilized by contacts formed with symmetry related molecules  approximation. The energetic distribution of GB free energy and solvation energy of free p53 DBD conformers with extended L1 is lower than those of recessed L1 , i.e. inNuclear Overhauser effect (NOE) measurements by Canadillas et al. suggested that there are compatible alternative conformations of L1 . In FiguWe further analyzed p53 DBD with extended and recessed loop 1 states using a simplified elastic network model that computes the normal modes of motion . This teIn order to understand the dynamical behavior of wild-type DBD as a whole, we further concatenate the eight trajectories of wild-type p53 DBD. From the RMSF profiles we see that as expected the N-terminal residues are the most flexible . L1 is tIt is known that enhanced dynamics are important for molecular recognition . Since LThe fluctuations of L2 are higher than those of L3 . The N-tnd copy of a simulation started from the crystal structure with PDB code 2OCJ) can sample the recessed L1 conformational space  in L1 in six NMR conformers . We also observed the transient helix in L1 in 4.9 % of the wild type MD conformers as determined using DSSP , L1 is disordered . To comping DSSP . In the We discover that isolated (DNA-free) monomeric p53 DBD can intrinsically sample both extended and recessed L1 conformations in MD simulations. In simulations initiated from the crystal and NMR structures with extended L1, MD conformers can adopt L1 conformations that are recessed, as evidenced by the smaller RMSD with respect to recessed L1 than the RMSD with respect to extended L1 . Again, To address these questions, we classified available MD conformers (of the cumulative 0.8 μs simulation time) into either extended L1 or recessed L1 states based on the stringent criteria that the L1 RMSD ≤ 2 Å with respect to crystal structures with extended L1  or recessed L1 (PDB code 3Q05) respectively. The extended L1 dataset has 3113 MD conformers, whereas the recessed L1 dataset has 357 MD conformers. The flexibility of L1, L2, the S5-S6 loop and the S6-S7 loop is higher in MD conformers with extended L1 than in those with recessed L1 . In termTo further understand the distinct dynamics between extended and recessed L1 conformers, we analyzed their correlated motions by calculating the dynamic cross-correlation matrix (DCCM) . DCCM haIn both extended and recessed L1 conformers, the following regions have strong correlated motions: between S1 and S3, S2’ and the S10-H2 loop, S3 and S8, H1 and L3, S5 and S8 regions . The corThe absence of the anti-correlated motion between L1 and L2 in the recessed L1 states may arise from the increasing separation between L1 and H2 , where HNext, we compare hydrogen bonds present in p53 DBD MD conformers with extended versus recessed L1. In the recessed state, L1 forms an average of 8 hydrogen bonds, whereas extended L1 forms an average of 5 hydrogen bonds. The total number of hydrogen bonds appears to be similar for both p53 DBD with extended and recessed L1 , with a 2) is larger than with recessed L1 (490.6 Å2) as calculated using NACCESS [Given these differences, we ask why in a single complex of DNA and p53 DBD tetramer such as in PDB code 3Q05  and 3TS8 NACCESS . The benL1 is important for binding to DNA, turning on trans-activation and apoptosis based on results from yeast-based assays ,78, cellin vitro [L1 (residues F113-T123) is a mutational cold spot , as oppoin vitro . S121F min vitro . T123A min vitro . It seemin vitro . In wildin vitro . The subin vitro , and thein vitro . Such a in vitro , dihydroin vitro , and actin vitro . Why is it that the wild-type p53 DBD has flexible L1 that binds DNA well but not as well as the S121F and T123A mutants do? A recent work based on the transcription factor Rap1  suggestsThere exists a complex relationship between protein flexibility and stability ,57,86,87per se, (ii) analyzes the differences in dynamical behaviors of recessed and extended L1 conformational states, and proposes their distinct involvements in binding to DNA, and (iii) highlights how the dynamics of L1 provides a promising avenue for therapeutic interventions through long-range  modulations. Such efforts have been given an optimistic boost recently by the computer aided discovery of a small molecule that re-stabilizes a mutant form of p53 [In conclusion, through multiple copies of MD simulations of the p53 DBD, this study (i) shows that the structure of p53 DBD is substantially more dynamic than what has been apparent from crystal structures m of p53 . We retrieved homologous structures to p53 DBD (PDB code 2OCJ) from the RCSB PDB and performed bioinformatics-based principal component analysis (PCA) using the Bio3D package of R . To identify core positions, iterated rounds of structural superposition were used to identify the most structurally invariant region of the p53 DBD structure. This procedure, implemented in the Bio3D package, entailed excluding those residues with the largest positional differences, before each round of superposition, until only the invariant core residues remained. We then used the structurally invariant core as the reference frame for structural alignment of crystal, NMR and simulation conformers (structures). The simulation conformers were obtained from performing molecular dynamics (MD) simulations.Next, we used the Cartesian coordinates of aligned Cα atoms to define the elements of a covariance matrix. The covariance matrix was then diagonalized to derive principal components (PCs) with their associated variances. PCA enables the projection of crystal, NMR and MD conformers on the subspace defined by PCs with the largest variances. Such an approach has been successfully employed to study the conformational diversity in the Ras ,90 and HSystems for molecular dynamics (MD) simulations were prepared from high-resolution crystal structures and a model of NMR structure of wild-type p53 DBD. In the original crystal structures, the p53 DBD structure is DNA-bound in the PDB code 2AHI  and 3Q052+ ion, from each entry to perform dual copies MD simulations for each starting structure, by varying the initial velocities. Therefore, we have eight independently simulated systems (and hence trajectories of 100 ns each) to enhance the sampled conformational space. The total simulation time for the combined trajectories is 0.8 microseconds.We extracted the monomeric p53 DBD structure, together with the Zn2+ ion, neutralizing the systems with charge neutralizing counter ions at pH 7, and adding TIP3P water molecules [Each system was simulated using the ff99SB force field  in the Aolecules  with theolecules . The sysolecules . Energy minimization with decreasing constraints on the positions of heavy atoms was initially performed for 1000 steps. Constant volume heating (to 300 K) was carried out for 50 ps. Next, equilibration at constant temperature (300 K) and constant pressure (1 atm) was performed for 2 ns.-1 and the pressure was maintained using weak-coupling with a pressure relaxation time of 1 ps. The SHAKE algorithm [The NPT  ensemble at 300 K, 1 atm, and long-range non-bonded interactions with a 10 Å atom-based cutoff were used for the production simulations. The temperature was maintained using Langevin dynamics with a clgorithm  was usedptraj module of AMBER, and the Bio3D package of R [solv,np = 0.00542*SASA +0.92 [Trajectory analyses were performed using the age of R . The timage of R ,101 apprage of R  with ΔGsSA +0.92 .We performed coarse-grained AD-ENM normal mode analysis , that usFigure S1The residual contribution of PC 1 to PC 3 reflects the dynamics of individual residues. (Left panel) Secondary structures of alpha helices and beta sheets are represented as black and grey boxes on horizontal axes. (Right panel) For each PC, equidistant atomic displacements from the mean structure are mapped onto the structure of p53 DNA binding domain. The N- and C-terminal of p53 DBD are labeled in cyan and magenta, respectively. The important loops in the p53 DBD: loop 1, loop 2 and loop 3 are colored blue, green and red, respectively.(TIFF)Click here for additional data file.Figure S2A) surface area (B) volume, and (C) radius of gyration.The relationships between PC 1 and Click here for additional data file.Figure S3The Cα RMSD with respect to the first conformer as a function of simulation time of wild-type p53 DBD started from the crystal structures of 2OCJ, 2AHI, 3Q05, 2FEJ. Different copies indicate different initial velocities in the beginning of simulation in order to allow enhanced conformational sampling. Black lines indicate all Cα atoms, grey lines indicate the Cα atoms of core residues. (TIFF)Click here for additional data file.Figures S4Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S5Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S6Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S7Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S8Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S9Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S10Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figure S11The contacts between K120 of DNA-free DBD crystal structure and residues of 3 different crystal symmetry mates (in each panel).(TIFF)Click here for additional data file.Figure S12Comparison of energetic profiles of p53 DNA binding domain with extended and recessed loop 1. The energetic distribution of (A) solvation energy (B) total molecular mechanics energy of p53 DBDs with extended and recessed L1. (C) The average and standard deviations of the energetic components. ELE is the non-bonded electrostatic energy. VDW is the non-bonded van der Waals energy. INT is the sum of bond, angle and dihedral energies. GAS is the sum of ELE, VDW and INT, i.e. the molecular mechanical energy in vacuum. GBSUR is the hydrophobic contribution to solvation free energy for generalized Born (GB) calculation.(TIFF)Click here for additional data file.Figure S13None of NMR conformers (PDB code 2FEJ) adopts recessed L1 conformation, their L1 is colored in cyan. For clarity, only L1 of NMR conformers is shown. Recessed L1 of PDB code 3Q05 chain A is colored blue. L2 and L3 are colored green and red, respectively.(TIFF)Click here for additional data file.Figure S14The time-dependent secondary structure profiles of MD conformers, with α helices and β sheets shown in black and grey respectively, except that the transient helix present in L1 is shown in red. The PDB code of starting structure used for each MD simulation is indicated on the top of each panel.(TIFF)Click here for additional data file.Figure S15The RMSDs of L1 residues in MD conformers with respect to the L1 residues in the crystal structures with extended L1  and recessed L1 (PDB code 3Q05 in black). The L1 RMSDs with respect to the L1 residues in the NMR structure (PDB code 2FEJ) are plotted in red. TS: Transitioning state between extended and recessed L1.(TIFF)Click here for additional data file.Figure S16Hydrogen bonds in p53 DNA binding domain. (A) The total number of hydrogen bonds in the p53 DBD MD conformers with extended L1 (magenta +) and recessed L1 (cyan x). (B) Stable hydrogen bonds that are present in MD conformers with extended L1 but are lost in MD conformers with recessed L1. (C) The mapping of residues involved in stable hydrogen bonds onto the crystal structure with extended L1 (PDB code 2OCJ). The secondary structure coloring is the same as in Figure 1C.(TIFF)Click here for additional data file.Figure S17Visualization of dominant motions (gray) obtained from NMA of p53 DBD with extended (green) and recessed (cyan) loop 1.(TIF)Click here for additional data file.File S1(DOC)Click here for additional data file.Table S1(XLS)Click here for additional data file.