In this work; resistive moisture sensors were obtained by dip coating sulfonated polycarbonate (SPC) onto silver interdigitated electrodes. Commercial polycarbonate was sulfonated with acetyl sulphate at two different sulfonation degrees corresponding to 9.0 and 18.0 mole %. Impedance spectroscopy was used to investigate the humidity sensing properties at controlled relative humidity (RH%) environments generated from standard saline solutions in the range of 11–90 RH%. For the highest sulfonated sample; in the RH% range investigated (11 to 90%); the sensor impedance changed from 4.7 MΩ to 18 kΩ. Humidity sensors made from sulfonated polycarbonate showed exponential decay behavior of the impedance at constant frequency with the environmental relative humidity. Sample 9SPC presented dielectric relaxation response for environmental humidity between 58 and 90 RH% while sample 18SPC presented dielectric relaxation response for the entire measured range between 11 and 90 RH%. Sulfonated polycarbonate could be a promising material for the fabrication of simple and cheap humidity-sensing sensors for the assessment of relative humidity of the surrounding environment, as suggested by experimental results. The pi.e., a resistor that is parallel to the real proton conducting channel. Such a resistor will reduce the impedance range change which means lower sensitivity. By inserting a conductive material on the humidity sensor matrix this will allow protons with very short diffusion lengths to be collected before being bound to an ionic group inserted in the polymeric matrix. Such sensors will behave with improved response for low humidity but at a price to drastically reduce the response at mean and high humidity.There are many kinds of humidity sensors. Capacitive humidity sensors involve a film deposited between electrodes on a substrate. The dielectric constant is proportional to the relative humidity and changes due to water steams –9. The pi.e., exponential change of impedance by many magnitude orders) on the academic point of view the transport of protons on a membrane is not really a simple process and deserves to be investigated without the influence of other processes. Sulfonated polycarbonate with sulfonic acid groups to the degrees of 9.0 and 18 mole % are also insoluble in water and this turns it into interesting to be investigated as humidity sensor. In a previous work, sulfonated polystyrene (SPS) was investigated as a humidity sensor [In the present work, we opted to use a simple proton conducting membrane with improved humidity range sensitivity compared to similar materials. Besides the fact that this guaranties a high sensitivity  has already been evaluated for some applications such as membranes for fuel cell [Due to better mechanical and chemical properties, uel cell , for sepuel cell , and foruel cell . Composiuel cell , polyaniuel cell , SPC-poluel cell  were alsuel cell ,20.3H was investigated as a relative humidity sensor. The preparation of humidity sensors was considered for SPC at two different sulfonation degrees: sensors 9SPC and 18SPC corresponding to 9.0 and 18.0 mole %. The investigations of their humidity sensitivity, vibrational and morphological characteristics are here reported. In order to obtain the sensor impedance dependence with RH% at the frequency range dominated by the resistive behavior, we selected from impedance spectroscopy measurements (carried out from 5 Hz to 13 MHz) the ac frequencies of 25 Hz and 200 Hz for sensor 9SPC and 18SPC respectively. Considering relative humidity generated from standard saline solutions in the range of 11–90 RH%, our previous work in which a 22 mole % SPS sensor presented resistive behavior in the range of 33–90 RH% [i.e., in the range of 11–90 RH%.In the present work, sulfonated polycarbonate, SPC, with the sulfonation group SO3–90 RH%  and, in 2.2.1.4g.mole−1 and The starting polymer was a polycarbonate (PC)  with 2Cl2 at 40 °C with acetyl sulfate, following the method described for the polystyrene [et al. [SPC was obtained by sulfonation of commercial PC in CHystyrene . The sul [et al. . The sul2Cl2 (0.05 g/mL), 9SPC/CH3OH and 18SPC/CH3OH (0.05 g/mL) films were followed by stirring to give homogenous solutions. Each solution was deposited by dip coating on insulating disk-shaped ceramic substrate of 25.0 mm diameter, inert with respect to humidity changes, where an interdigital array of silver electrodes has been defined  and for samples prepared in KBr pellet (2 mg of polymer into 300 mg of KBr) following standard procedures. The FTIR spectra were used to qualitatively characterize the ∼SO3H groups. The surface morphology of the SPC deposited by dip coating was studied by scanning electron microscope (SEM) in JEOL Model JSM T300 equipment, operating at 20 kV. The samples were gold-sputtered prior to the measurements. The impedance measurements were done with an HP4192A impedance analyzer. The experimental parameters were: frequency range of 5 Hz to 13 MHz, sinusoidal voltage amplitude = 1.0 Vrms. All the measurements were carried out at a room temperature .Infrared spectra were recorded using a Perkin-Elmer FTIR 1600 spectrophotometer. Spectra were obtained in the mid-infrared region .Furthermore, two processes are responsible for impedance decreasing: the first is associated with the water electrolysis and the second is related to the transport of H+ ions . This trmobility . Therefomobility –27 the aFor the polyelectrolyte humidity sensors, the conduction mechanism is mainly ionic, and sensing behavior depends on both the kind and concentration of ions and the water absorption ability of the sensing film .The electrical transport phenomenon in polymeric humidity sensors is yet to be fully described. There is much work to carry on in understanding the fundamental physical phenomena presented in such sensors. For instance, protons have high mass and very low mobility and d.c. measurements are impossible to be carried out due to double layer capacitance effects. Under this scenario, Hall effect measurements cannot be easily carried out and due to limitations of water melting and boiling points, temperature dependence of resistivity can be measured in a quite short range, which turns the determination of activation energies associated with carrier statistics a hard task.0 is the projected impedance at 0 RH% and B is the slope obtained from fitting. From the fittings of data for sample 9SPC were obtained Z0 = (10 ± 5) TΩ and B = (0.080 ± 0.006) RH%−1 and for sample 18SPC Z0 = (13 ± 1) MΩ and B = (0.029 ± 0.002) RH%−1.Despite the limitations, an empirical model of impedance exponential decay with relative humidity can lead to reasonable results and was applied to the sensor impedance as a function of RH%. MAX.τ =1 , reduces with increasing RH%  and desorption (changing the environment from 90 RH% to 11 RH%) times for both SPC sensors were carried out. One consideration is that about 30 s was used to change from one environment to the other and to start the measuring program. For sample 9SPC both absorption and desorption times are low (at least lower than the 30 s lost during the environment change). For sample 18SPC a low absorption time and desorption time of 8 min after the changing time was measured.4.Resistive moisture sensors were obtained by dip coating sulfonated polycarbonate onto silver interdigitated electrodes. The SPC was sulfonated to a degree of 9 mole % (9SPC) and 18 mole % (18SPC). Humidity sensors made from SPC show exponential dependence of the impedance with the environmental relative humidity. An empirical model of exponential decay of the impedance measured at fixed low frequencies with the relative humidity was used to fit experimental results. This model allows practical conversion between impedance and relative humidity. Sample 9SPC presented dielectric relaxation response for environmental humidity between 58 and 90 RH% while sample 18SPC presented dielectric relaxation response for the entire measured range between 11 and 90 RH%. The uncertainties of the relative-humidity are 8% and 4% for samples 9SPC and 18SPC respectively. The sensor of 9SPC is suitable for applications that consider high humidity environments and the need of quick responses. The sensor of 18SPC is suitable for applications that consider broad range of humidity environments. These results turn the sulfonated polycarbonate into a very promising material for the fabrication of inexpensive and efficient humidity sensors.


Summary: ADAPT-nuclear magnetic resonance (ADAPT-NMR) offers an automatedapproach to the concurrent acquisition and processing of protein NMR data with the goal ofcomplete backbone and side chain assignments. What the approach lacks is a usefulgraphical interface for reviewing results and for searching for missing peaks that mayhave prevented assignments or led to incorrect assignments. Because most of the dataADAPT-NMR collects are 2D tilted planes used to find peaks in 3D spectra, it would behelpful to have a tool that reconstructs the 3D spectra. The software package reportedhere, ADAPT-NMR Enhancer, supports the visualization of both 2D tilted planes andreconstructed 3D peaks on each tilted plane. ADAPT-NMR Enhancer can be used interactivelywith ADAPT-NMR to automatically assign selected peaks, or it can be used to producePINE-SPARKY-like graphical dialogs that support atom-by-atom and peak-by-peak assignmentstrategies. Results can be exported in various formats, including XEASY proton file(.prot), PINE pre-assignment file (.str), PINE probabilistic output file, SPARKY peak listfile (.list) and TALOS+ input file (.tab). As an example, we show how ADAPT-NMREnhancer was used to extend the automated data collection and assignment results for theprotein Aedes aegypti sterol carrier protein 2.Availability: The program, in the form of binary code along with tutorialsand reference manuals, is available at http://pine.nmrfam.wisc.edu/adapt-nmr-enhancer.Contact:whlee@nmrfam.wisc.edu or markley@nmrfam.wisc.edu One of the goals of protein nuclear magnetic resonance (NMR) spectroscopy is to increaseits throughput by automating the steps of data collection, spectral assignment and structuredetermination. The latest approach towards this goal from our laboratory is ADAPT-NMR (http://qt.nokia.com) for graphical user interface. The software supportsmultiple operating systems . ADAPT-NMR Enhancer offers threeactive dialog boxes: Main Window Dialog, PINE AssignmentDialog and Probable Assignment Dialog. The Main WindowDialog  application written inC++ with QT4 libraries  is the Funding: National Center for ResearchResources (5P41RR002301-27) andNational Institute of General Medical Sciences(8 P41 GM103399-27) from the National Institutes ofHealth.Conflict of Interest: none declared.


We have identified a hydrophobic sequence in the C-terminus that is critical for the TDP-43 aggregation and inclusion formation. The synthetic peptide with this sequence forms an α-helical structure in solution but easily transforms into β-sheet structure. The inclusions formed by the C-terminal 35-kDa fragment (TDP-35) can recruit full-length TDP-43 to cytoplasmic deposition from functionally nuclear localization. TDP-35, rather than TDP-43 and the C-terminal 25-kDa fragment, is prone to aggregation in vitro, and it can further serve as a seed to facilitate aggregation of full-length TDP-43. This suggests that fragmentation of TDP-43 leads to cellular redistribution, inclusion body formation, and altered RNA processing, which are implicated in the molecular pathogenesis of ALS and FTLD.TAR DNA binding protein of 43 kDa (TDP-43) is a nuclear factor functioning in RNA processing. It was also reported that TDP-43 aggregates are deposited in motor neurons implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin (FTLD-U). To understand the mechanism underlying the inclusion body formation and possible functional alteration of TDP-43, we studied TDP-43 and its fragments and their effects on RNA processing


We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin–moesin–radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis.A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila,  The L1 family of cell adhesion molecules is well-known for its divergent roles during early nervous system development, specifically for axonal outgrowth, myelination, fasciculation, as well as long term potentiation, synapse formation and maintenance neuroglian (nrg) gene generates two alternately spliced isoforms 180 is expressed exclusively in the nervous system and Nrg167 mostly in non neuronal tissue. Similar to vertebrates, Nrg plays distinct roles during axonal guidance and synapse formation 180 and Nrg167 isoforms are encoded by distinct exons allowing the characterization of the role of the FIGQY motif for either isoform separately 180 is required for synapse formation and synapse stability but not for axon growth and guidance in the GF circuit as well as at the larval neuromuscular junction 14nrg;P[nrg167ΔFIGQY] mutants in either model neuron.In the central nervous system, the sole L1-type Drosophila 849nrg mutants is analogous to the pathological human L1-H210Q mutation 849nrg mutants exhibit axonal guidance as well as synaptic defects that can be rescued by the transgenic expression of human L1CAM, but not by the expression of the vertebrate paralogs Neurofascin and NrCAM The position of the S213L missense mutation in the second Ig domain of 849nrg along with 14nrg;P[nrg180ΔFIGQY] mutants provide a unique opportunity to study pathological L1CAM missense mutations with respect to their effects on guidance and synapse formation in vivo. Here, we investigated the human L120V, E309K, H210Q, Y1070C, C264Y extracellular missense mutations in addition to a L1CAM protein mutated in the intracellular ezrin–moesin–radixin (ERM) binding site (L1-4A). The human L1 mutant proteins were expressed in both, 849nrg and 14nrg;P[nrg180ΔFIGQY] mutant backgrounds to gauge the abilities of these proteins to rescue axon guidance and synapse development defects, respectively In summary, the GF circuit of Drosophila is a suitable model system for studying L1CAM function and EcoRI-XhoI restriction sites 849nrg were obtained from the Bloomington Stock Center. 14nrg;P(nrg180ΔFIGQY) was generated by the Jan Pielage lab  as previously described 849nrg;OK307/CyO stock and a 849nrg;OK307, UAS-CD8-GFP/CyO stock as well as OK307,UAS-L1/CyO stocks for all L1-constructs were established. Male flies of the UAS-L1 or UAS-L1-variants were crossed to female 849nrg flies. In addition, male flies of the OK307,UAS-L1/CyO (or L1-variants) stocks were crossed to female 14nrg;P(nrg180ΔFIGQY) flies. All genetic crosses were performed at 25°C and the male progeny of the crosses were tested when three to four days old.The following fly stocks generated have been previously described For the developmental Western blot, one pupa or one adult OK307/+;UAS-L1/+ fly homogenized in Laemmli buffer with Halt™ Protease and Phosphatase Inhibitor Cocktail  was loaded per lane. Ten fly heads of offspring from UAS-L1 and UAS-L1-variants crossed to OK307 were used to extract protein for comparing expression between wild type L1CAM and L1CAM with pathological missense mutations. SDS-PAGE was performed according to standard protocol. The Western blots were incubated with the anti-L1 C20 antibody  at a dilution of 1∶200. Anti-Actin  was used as a loading control at a dilution of 1∶200. Secondary donkey anti-goat antibody  was used at a concentration of 1∶1000.849nrg and the mutant L1 rescues was obtained by Chi-square analysis using Microsoft Excel (Microsoft Office 10).Flies (3–5 days old) were dissected to expose the cervical connective and the ventral nerve cord as previously described Electrophysiological recordings from the Dorsal-longitudinal Muscle (DLM) and the TTM Tergo-trochanteral Muscle (TTM) of the GF circuit were obtained as described previously When expressed in different cell lines, several human L1CAM proteins with missense mutations in their extracellular domain are retained in the endoplasmic reticulum 849nrg mutants  in 849nrg animals. In contrast, the expression of L1-C264Y, L1-H210Q, L1-R184Q and L1-4A did not complement the loss of homophilic binding in 849nrg animals. Although 849nrg animals expressing L1-Y1070C were statistically insignificant from negative control animals (849nrg), they were also statistically insignificant from positive control animals (L1CAM in 849nrg background). Therefore, the observed reduction in the guidance defects suggests that expression of L1-Y1070C is able to partially rescue guidance defects in vivo.In wild-type flies each GF cell body in the brain projected an axon through the cervical connective into the second neuromere of the ventral nerve cord (VNC) and made synaptic connections with the TTMn motor neurons  A, B. Intely 30% , while tpecimens . We alsoKGGKYSV,  were sub14nrg;P[nrg180ΔFIGQY] animals, in which the lethality of a nrg null mutant (14nrg) was rescued by a genomic construct in which the neuronal Nrg180 isoform lacks the Ankyrin-binding motif , no improvement of the synaptic phenotypes was observed (14nrg;P[nrg180ΔFIGQY] mutants enabled us to test whether human pathological L1CAM mutations can rescue the synaptic phenotypes or whether the extracellular missense mutations cannot compensate for the lack of outside-in signaling via the FIGQY motif.Mutations in the highly conserved intracellular FIGQY-motif of the sole L1CAM homolog Neuroglian do not affect neurite outgrowth or guidance in the GF circuit or at the larval neuromuscular junction ng motif , only syne ratio  was assone ratio  and withne ratio . We have mutants , Table 1an L1CAM . In contobserved , Table 1849nrg mutants, was able to significantly rescue the synaptic phenotypes of 14nrg;P[nrg180ΔFIGQY] mutants (Expression of L1-Y1070C, L1-H210Q and L1-R184Q failed to restore the GF synapse function , Table 114nrg;P[nrg180ΔFIGQY] animals (14nrg;P[nrg180ΔFIGQY] mutant terminals.Although the average Following Frequency and average Response Latency were significantly improved with the expression of L1-C264Y , Table 1 animals . In orde animals . We foun animals , green d animals , magenta animals  but not  animals . However animals . While i animals , left GF849nrg or 14nrg;P[nrg180ΔFIGQY] mutants is due to the loss of a biological function and not due to a dominant negative or poisonous effect.Finally, in order to test if the expression of L1CAM with pathogenic mutations in Drosophila has any dominant negative or poisonous effects in the GF circuit, we determined putative functional defects in a wild type background . All aniMany different pathological L1CAM mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as on their cell surface expression levels in vitro. However, there are only a few studies on their biological consequences in vivo In vertebrates, L1CAM is proteolytically cleaved by various enzymes, such as neuropsin, plasmin, ADAM10/17 and a yet unidentified serine protease 14nrg;P[nrg180ΔFIGQY] animals suggesting that L1-C264Y protein was expressed at the cell surface. Correlating with the electrophysiological data, we did find that transgenically expressed L1-C264Y protein was present in numerous though not all synaptic terminals at the cell surface as well as in vesicular clusters inside the terminal. Therefore, our results suggest that in the Drosophila GF the pathogenic C264Y mutation not only affects proteolytic processing indirectly by reducing cell surface expression but may also affect it directly in L1-C264Y protein that is expressed at the cell surface.The overall L1-C264Y expression was reduced comparatively but in Western blots no cleavage was observed for L1-C264Y protein even with ten heads . This is849nrg allele is at a site analogous to the position of the H210Q mutation in humans and both mutations affect homophilic L1 binding 180 on either side of 849nrg mutant synapses can partially rescue the synaptic phenotypes. This suggests that Nrg-S213L similar to L1-H210Q is able to bind to a wild-type extracellular domain Homophilic L1-L1 interactions mediated by the extracellular Ig-domains are involved in various L1-dependent signaling processes required for axonal growth, guidance and synapse stability 849nrg mutants. Expression of wild-type human L1CAM, as well as L1-L120V and L1-E309K, both of which mediate normal homophilic binding, was able to efficiently rescue the guidance defects of 849nrg mutants (849 protein induces signaling that is sufficient for GF guidance.Here, we tested whether the expression of different pathological mutations can compensate for the loss of homophilic interaction in  mutants  [15],  mutant animals (ΔFIGQY protein. We identified several L1CAM mutations that had expression levels (14nrg;P[nrg180ΔFIGQY] mutant flies. This suggests that L1-H210Q and L1-R184Q may not interact transcellularly with NrgΔFIGQY and homophilic interactions are also essential for synapse formation. Alternatively, L1-H210Q and L1-R184Q may be able to interact transcellularly with NrgΔFIGQY, which has a wild-type extracellular domain. However, in this scenario the H210Q and R184Q mutations disrupt outside-in signaling processes via the FIGQY motif. The latter hypothesis is supported by our finding that the 849nrg mutants with the analogous L1-H210Q mutation have reduced tyrosine phosphorylation of the FIGQY motif and synaptic phenotypes similar to 14nrg;P[nrg180ΔFIGQY] mutants in addition to guidance defects 14nrg;P[nrg180ΔFIGQY] mutant flies although its homophilic interactions are not affected by the mutation The finding that human L1CAM mutations like L1-L120V, L1-E309K, L1-4A and partially L1-C264Y were able to rescue the synaptic defects of Drosophila  animals , 4 suggen levels  comparabIn summary, we find that extracellular human pathogenic L1CAM missense mutations not only affect adhesive properties but also intracellular signaling pathways distinctly, which are required for axon guidance, synapse formation or both. Mutations that affect homophilic binding are most detrimental because they affect adhesive properties but often also heterophilic interactions and intracellular signaling


Tetrahymena intron more than 30 years ago has been essential to our understanding of group I intron catalysis, higher-order RNA structure, and RNA folding, but other intron models have provided information about the biological role. Nuclear group I introns appear widespread among eukaryotic microorganisms, and the plasmodial slime molds (myxomycetes) contain an abundance of self-splicing introns. Here, we summarize the main conclusions from previous work on the Tetrahymena intron on RNA self-splicing catalysis as well as more recent work on myxomycete intron biology. Group I introns in myxomycetes that represent different evolutionary stages, biological roles, and functional settings are discussed.Group I introns are a distinct class of RNA self-splicing introns with an ancient origin. All known group I introns present in eukaryote nuclei interrupt functional ribosomal RNA genes located in ribosomal DNA loci. The discovery of the  Introns are genetic elements that interrupt functional RNA- or protein-coding genes, and are removed post-transcriptionally in a process termed splicing. Their ability to be spliced out at RNA level makes them almost invisible for the host and limits the phenotypic cost, and introns have often been labeled selfish elements or molecular parasites . A majorGroup I introns in nuclear genomes are exclusively found within functional ribosomal RNA (rRNA) genes of a wide spectrum of eukaryotic microorganisms. Here they are frequently noted among red algae, chlorophyte algae, fungi, and myxomycetes, but only occasionally in ciliates -4. NucleTetrahymena LSU rRNA intron at insertion site L1925 has become the undisputedly most important group I intron model system for the study of RNA catalysis, RNA structure, and RNA folding [Tetrahymena thermophila [Tetrahymena intron has been thoroughly investigated and self-splicing was shown to occur by RNA catalysis based on a two-step transesterification reaction requiring a guanosine cofactor [Despite the fact that thousands of nuclear group I introns have been annotated in sequence databases, only a very few have been submitted to molecular analyses and functional characterizations. The  folding . Tth.L19 folding  for nuclcofactor .Tetrahymena intron, has a more complex structured scaffold domain than the Didymium group IE ribozyme  insertions. The HEGs are located within the peripheral parts of the paired segments such as P1, P2, P6, P8 or P9, and expression of these protein-coding genes embedded in nucleolar rDNA utilizes unconventional strategies . Interestrans-splice group I ribozymes, and most studies have been performed with the Tetrahymena ribozyme. One of the best characterized examples of RNA-based gene repair is the reprogramming of mutant p53 transcripts in human cancer cells [Several group I intron-based biotechnological applications have been suggested and realized. These involve the intron homing endonuclease (HE) as a highly specific and rare-cutting endonuclease, intron splicing as a therapeutic tool in fighting pathogens and engineered group I ribozymes as molecular tools in RNA reprogramming and RNA repair ,17. Riboer cells ,20. The er cells . HoweverTetrahymena intron has proven to be the undisputed prototype in the study of RNA catalysis and RNA structure, other nuclear group I introns have contributed to our understanding of intron functions beyond splicing. Recent studies have shown that the extrachromosomal nuclear rDNA of myxomycetes, eukaryotic microorganisms belonging to the Amoebozoa clade, contain an abundance of group I introns , which is cognate to that of the Tetrahymena intron; it harbors a HEG and is mobile in genetic crosses between intron-lacking and intron-containing strains [Didymium iridis. Dir.S956-1 is mobile in genetic crosses and contains two distinct ribozymes with different functions in splicing and RNA processing, as well as a HEG [Didymium intron (Dir.S956-2) harbors a HEG at the antisense orientation [Diderma niveum has an extremely dense intron content with 20 or more group I introns present within the same rRNA primary transcript  at the 5′ splice site (SS)  Figure B. ExoG i circles ,32. HoweDidymium group I intron Dir.S956-1 [The full-length intron circularization (FLC) pathway has been studied in detail for the r.S956-1 . This par.S956-1 . The 3′OPpoI from P. polycephalum[DirI and I-DirII from D. iridis [Naegleria species [The spreading of group I introns may occur at either the DNA or RNA level Figure . The mosycephalum, I-DirI . iridis , and som species . Nuclear species ,39 and rP. polycephalum, which encodes I-PpoI [D. iridis for the Dir.S956-1 intron [PpoI and the introns Ppo.L1925 and Tth.L1925 (Tetrahymena intron), which were integrated into all the approximately 150 genomic rDNA copies at chromosome XII in an elegant experiment [Experimental evidence for homing in a biological setting has been collected from only two nuclear group I introns, both in the myxomycetes. The first system to be characterized was Ppo.L1925 in s I-PpoI . Here mo1 intron . In addiperiment -43.in vitro, in yeast and in Escherichia coli for both the Tetrahymena intron [Didymium intron [in vitro integration of full-length circular intron RNA has also been noted, suggesting a biological role for the circularization pathway in propagation and intron spread [It has been suggested that intron homing also occurs directly at the RNA level by reverse splicing. Here, an excised intron attacks the ligated exons at the intron-lacking cognate insertion site and integrates into the precursor RNA. Reverse splicing has been reported a intron -46 and tm intron . Interesn spread . Less frn spread ,22,48. HP. polycephalum, D. iridis and D. niveum rDNAs.Recent studies of the rDNA mini-chromosome in myxomycetes have revealed more than 500 group I introns highly divergent in sequence, size and insertion site. Myxomycetes are eukaryotic microorganisms with a complex life cycle, which has several stages from haploid amoebae cells to a multi-nucleated plasmodium with synchronously dividing diploid nuclei . The rDND. niveum is highly unusual since at least 20 group I introns are present . The high abundance of introns is a significant challenge to the host cell since the majority of group I introns have the ability to perform FLC and generate fragmented exons [The rDNA from ed exons . The myxDidymium, L1925 in Physarum, and S529 and S1199 in Diderma). HEGs have been noted in both sense and antisense orientations compared to the group I ribozyme and have been inserted at various locations . The HEG-containing introns belong to both the IC1 and IE subclass, are highly mobile in biological settings [The first category consists of the mobile HEG-containing introns Figure A. Of thesettings ,40, and settings . IntronsDiderma belong to this category (as well as the Tetrahymena intron), and it has been suggested that the all-ribozyme group IC1 and IE introns are remnants of mobile introns after loss of HEGs  and thus the U:G pair at the 5′ SS is replaced by a G:C pair [in vitro[Most introns in s Figure B. This sG:C pair . Further[in vitro.Diderma rDNA is S1389   and in h segment ,60,61. A segment  , which opens the possibility that intron RNA may have gained additional functions beyond splicing.Obligatory introns could have gained new mutual functions that benefit the host, and one possibility is that peripheral insertions are further processed into long non-coding RNAs (lncRNAs). lncRNAs are known to regulate gene expression, translation, splicing and trafficking by acting as guides, scaffolds, decoys or enhancers ,64, and Didymium, the amoebo-flagellates Naegleria and the amoeba Allovahlkampfia [D. iridis, is presented in Figure Didymium intron carries a HEG inserted at P2 of the splicing ribozyme, and is mobile at the DNA level in genetic crosses between intron-containing and intron-lacking strains [One notable example of group I introns that have evolved new biological roles are the twin-ribozyme introns, which are interrupting SSU rRNAs in the myxomycete lkampfia ,60,65,66 strains ,40.Interestingly, a second ribozyme domain is located immediately upstream of the HEG. Detailed structural analysis has shown that the ribozyme is derived from a standard group I intron; it is named GIR1 (group I-like ribozyme 1) . GIR1 laNaegleria and Allovahlkampfia GIR1s [The catalytic part of GIR1 has about 180 to 200 nucleotides, and when activated it catalyzes self-cleaving by branching . This reia GIR1s ,72.DP2 and P10, which depend on base pairing of sequences 5′ and 3′ of the GIR1 core  is a unique example of a group I intron that has gained new catalytic properties and new biological roles in nuclear gene regulation.Self-splicing, RNA structure and folding, and HE-dependent homing are fully described features of the group I introns in BP: branch point; dsDNA: double stranded DNA; eG: exogenous guanosine factor covalently linked at the 5’ end of the free intron RNA; exoG: exogenous guanosine cofactor; FLC: full-length circularization; GIR1: group I-like ribozyme; GIR2: group I splicing ribozyme; HE: homing endonuclease; HEG: homing endonuclease gene; I+: intron-containing allele; I–: intron-lacking allele; IPS: internal processing site; LE: ligated exon; LI: linear intron; LncRNA: long non-coding RNA; LSU rRNA: large subunit ribosomal RNA; Nt: nucleotide; RDNA: ribosomal DNA; RT: reverse transcriptase; SOLiD: sequencing by oligo ligation and detection; SS: splice site; SSU rRNA: small subunit ribosomal RNA.The authors declare that they have no competing interests.AH and SDJ contributed equally to the writing for this article. Both authors read and approved the final manuscript.


The programmed cell death-1 receptor/programmed cell death-1 ligand (PD-1/PD-L1) pathway plays a crucial role in tumor evasion from host immunity. This study was designed to evaluate the association between circulating PD-L1/PD-1 and prognosis after cryoablation in patients with HBV-related hepatocellular carcinoma (HCC).In the present study, 141 HBV-related HCC patients were enrolled and of those 109 patients received cryoablation. Circulating PD-L1/PD-1 expression was tested by flow cytometry, and 23 patients were simultaneously evaluated for intratumoral PD-L1 expression by immunohistochemical staining. Circulating PD-1/PD-L1 expression was associated with severity of diseases in patients with HCC, and the circulating PD-L1 expression was closely correlated with intratumoral PD-L1 expression. Of the clinical parameters, PD-1/PD-L1 expression was associated with tumor size, blood vessel invasion and BCLC staging. Moreover, PD-1/PD-L1 expression dropped after cryoablation while being elevated at the time of tumor recurrence. Patients with higher expression of circulating PD-L1, as well as circulating PD-1, had a significantly shorter overall survival and tumor-free survival than those with lower expression. Multivariate analysis confirmed that circulating PD-L1 could serve as an independent predictor of overall survival and tumor-recurrence survival in HCC patients after cryoablation.Upregulation of circulating PD-L1/PD-1 is associated with poor post-cryoablation prognosis in patients with HBV-related hepatocellular carcinoma. Hepatocellular carcinoma (HCC) is a complex condition with multiple variables affecting the disease course and response to treatment, including liver function and performance status of the patient and tumor stage +T-cell apoptosis and post-operative recurrence in HCC patients. However, the detection of intratumoral PD-L1 requires an invasive operation for liver biopsy and would be a complex test for clinical applications, and there have been few reports on changes in circulating PD-L1/PD-1 levels in patients with HCC before and after cryoablation. Therefore, the present study was designed to investigate the association between cryoablation and circulating PD-L1/PD-1 variation in patients with HCC and to explore the role of circulating PD-L1/PD-1 in the prognosis of HCC.Programmed cell death-1 receptor (PD-1), a novel co-inhibitory receptor mainly expressed on activated T and B cells In total, 141 patients with HBV-related HCC were enrolled in this study from January 2006 to January 2009. Based on the Barcelona Clinic Liver Cancer (BCLC) staging classification Argon–helium cryoablation was followed as our previously described 1 (eBiosciences) were adopted in this assay. For each test, 50 µl fresh heparinized peripheral blood was incubated with indicated antibodies (20 µl) for 20 min and then lysed with FACSTM lysing solution , washed with PBS, fixed and analyzed by the fluorescence activated cell sorting (FACS) analysis . Data were analyzed by Flowjo software .Antibodies conjugated with different fluoresceins, including fluorescein isothiocyanate (FITC), phycorerythrin(PE), peridin chlorophyll protein (PerCP) and allophycocyanin (APC) were used for flow cytometric analysis. PE-conjugated anti-PD-1, PD-L1 and APC-conjugated anti-CD14 were purchased from eBiosciences ; PerCP-conjugated anti-CD8 and APC-conjugated anti-CD3 were purchased from BD Biosciences . In detail, PE-conjugated anti-PD-L1 and APC-conjugated anti-CD14 were applied for the PD-L1 detection on monocytes; PE-conjugated anti-PD-1, PerCP-conjugated anti-CD8 and APC-conjugated anti-CD3 were used for the PD-1 detection on CD8+T cells. To determine of positive expressions of PD-1 and PD-L1, PE-conjugated IgGBased on the availability within all 141 HCC patients, there were twenty-three patients enrolled in this study by simultaneously collecting peripheral blood and liver tumor biopsy, which included 9 stage A, 8 stage B and 6 stage C patients in detail. Paraffin-embedded, formalin-fixed liver tissue from these enrolled HCC patients was cut into 5-µm sections and placed on polylysine-coated slides. Antigen retrieval was achieved via pressure cooking for 10 minutes in citrate buffer. Mouse monoclonal anti-human PD-L1 antibody  was used. 3-amino-9-ethyl-carbazole (red color) was used as a substrate followed by counterstaining with hematoxylin. For the evaluation of PD-L1 staining, three independent pathologists examined the slides without access to clinical information. The intensity of the PD-L1 staining was scored as one of four degrees according to the area of positive staining, including score 0, less than 25%; score 1, 25%–50%; score 2, 50%–75%; and score 3, higher than 75%. The results for score 0 and 1 were considered as low PD-L1 expression, whereas score 2 and 3 were considered as high PD-L1 expression, accordingly.<0.05 was considered to be statistically significant.Data were summarized and presented as means ± SD and analyzed using SPSS13.0 software . The Mann–Whitney nonparametric U-test was applied for comparisons between 2 groups and the Kruskal–Wallis nonparametric H-test was performed for comparisons among multiple groups. The χ2 test was used for comparison of the individual variables. The Wilcoxon matched-pairs test was used to compare the data from the same individuals. Receiver operating characteristic (ROC) curve analysis was used to determine the OS predictive value of the parameters, and the differences in the area under the curve (AUC) were detected by using SPSS13.0.Tumor recurrence and survival rates were analyzed by the Kaplan–Meier method. The log-rank test was applied to compare findings among groups. The Cox regression model was used to perform univariate and multivariate analyses. A value of PP<0.001) and reached the highest level at stage C . Moreover, the circulating PD-1 exhibited a similar expression profile to PD-L1 . Therefore, circulating PD-1/PD-L1 expression was associated with the disease severity in patients with HCC.We firstly investigated the circulating PD-L1/PD-1 expression by using flow cytometric analysis in a cohort of 141 HCC patients. The gating strategies for both types of expression are shown . Our resAs a previous report demonstrated that intratumoral PD-L1 was significantly associated with overall survival in HCC patients Patients with HCC (n = 141) were divided into 2 groups according to the median of circulating PD-1/PD-L1 expression: those with low PD-1/PD-L1 expression (<10.88%/9.18) and those with high PD-1/PD-L1 expression (≥10.88%/9.18). Upon analysis, tumor size, blood vessel invasion and BCLC staging were associated with PD-1/PD-L1 expression. However, no significant relationships were found between PD-1/PD-L1 expression and gender, age, number of tumors, HBV DNA load, AFP level or Child-Pugh class .The results of the analyses showed that circulating PD-1/PD-L1 expression in 109 HCC patients increased significantly 1 week after cryoablation when compared with that prior to treatment, and liver function impairment was observed at 1 week after treatment. The measure of liver function reached normal within 4 weeks after treatment , accompaFor the 109 patients followed up for a median of 23 months(6∼36months) after the cryoablation, the 1-, 2- and 3-year survival rate was 86%, 75% and 67%, respectively. Furthermore, the 1-, 2-and 3-year recurrence-free survival rate was 70%, 58% and 40%, respectively.To address whether increased circulating PD-1/PD-L1 was associated with prognosis in HCC patients after cryoablation, 46 HCC patients at stages A and 63 HCC patients at stage B were divided into 2 groups by the median value of circulating PD-1/PD-L1 at the time before treatment as low PD-1  and high PD-1 , low PD-L1  and high PD-L1 . The results showed that the high PD-1/PD-L1 group had a significantly higher rate of tumor recurrence and progression after cryoablation in comparison to the low PD-1/PD-L1 group . The uniThe interaction between PD-1 and PD-L1 has been demonstrated to negatively regulate T-cell activation and functions, leading to inhibition of the immune responses in cancer patients In the present study, we detected PD-L1/PD-1 expression on circulating peripheral blood mononuclear cells(PBMCs) and found PD-L1/PD-1 expression increased with liver tumor progression. Further study revealed that there was a close correlation between the circulating and intratumoral PD-L1 expression. We analyzed the circulating PD-1/PD-L1 expression and the clinical parameters in patients with HCC. The result demonstrated that tumor size, blood vessel invasion and BCLC staging were associated with PD-1/PD-L1 expression. However, no significant relationships were found between PD-1/PD-L1 expression and gender, age, number of tumors, HBV DNA load, AFP level or stage of liver function. Although several publications demonstrated that PD-1/PD-L1 expression levels correlated with the HBV DNA titers in vitro study, Chen J et al Our observations suggested that elevated PD-1/PD-L1 expression during the first week after cryoablation was related to inflammation caused by the therapy and that postoperative fever and liver function impairment were the main manifestations. A previous study reported that inflammation could promote PD-1/PD-L1 expression Growing investigations demonstrate that PD-1 extensively up-regulated on tumor Ag-specific T cells in cancer patients and play a crucial role in the mechanism of tumor evasion by inhibiting the proliferation, cytokine-secretion and cytoxicity of tumor Ag-specific T cells In the present study, we also found patients with recurrent tumors, which would generally be smaller than primary ones, associated with higher PD-1/PD-L1 expression. According to former investigations, we believe that it is the tumor-associated inflammation, rather than tumor volume, which promote the elevated expression of PD-1 and PD-L1. After cryoablation therapy, tumor cells were degenerated, disaggregated and up-taken by antigen presenting cells, e.g. kuffer cells, pDC and mDC, which lead to the secretion of a large mount of proinflammatory cytokines, including IFN-α,IFN-γ and gamma-chain cytokines. In detail, interferons could promote PD-L1 expression on both tumor cells and antigen presenting cells in comparison with that gamma-chain cytokines account for PD-1 elevations on CD8+T cells. Thus, both PD-1 and PD-L1 expression was significantly up-regulate at the first week post-cryoablation. Together with the resolution of tumor burden, the immune system goes in to a quiescent phase (4w after cryoablation), manifesting lower PD-1 and PD-L1 expression. When recurrence occurs, the immune system should be re-activated and promote an inflammation, both of which favor PD-1 and PD-L1 re-elevation, in comparison with the expression at week 4 post cryoablation therapy, at the time when patients are free of tumor burden.Recurrence after hepatectomy or ablation is one of main biological features of HCC. Tumor diameter,portal vein tumor thrombus, microvessel invasion, and tumor capsule invasion are the main high risk factors for early recurrence Many mechanisms have been proposed for an attenuated immune response to tumors, including partial antigen masking, failure in antigen processing, suppression of effector cells and inadequate costimulation Taken together, our study suggested that upregulation of circulating PD-1/PD-L1 could serve as novel valid immunological markers in predicting disease progression of HCC patients after cryoablation; and this would be helpful for the improved clinical management and the development of new therapeutic options for patient with HBV-related HCC. We postulate that the combination of cryoablation and immunotherapy such as anti-PD1 antibody


H-indol-2-yl)isoxazole-4-carboxamide (AIC) derivatives, and different anions  have been theoretically investigated using DFT approaches. It turned out that the unique selectivity of AIC derivatives for F− is ascribed to their ability of deprotonating the host sensors. Frontier molecular orbital (FMO) analyses have shown that the vertical electronic transitions of absorption and emission for the sensing signals are characterized as intramolecular charge transfer (ICT). The study of substituent effects suggests that all the substituted derivatives are expected to be promising candidates for fluoride chemosensors both in UV-vis and fluorescence spectra except for derivative with benzo[d]thienothiophene fragment that can serve as ratiometric fluorescent fluoride chemosensor only.The interactions between chemosensors, 3-amino-5-(4,5,6,7-tetrahydro-1 Furthermore, it is necessary to understand the unique host-guest interaction of the sensor with F− and other anions. Different signaling mechanisms have been suggested for F−, such as photoinduced electron transfer (PET) thienothiophene fragment that can serve as ratiometric fluorescent fluoride chemosensor only.Our calculated results for both the host-guest interaction and the nature of UV-vis and fluorescent signaling for


TAR DNA-binding protein 43 (TDP-43) is a major component within ubiquitin-positive inclusions of a number of neurodegenerative diseases that increasingly are considered as TDP-43 proteinopathies. Identities of other inclusion proteins associated with TDP-43 aggregation remain poorly defined. In this study, we identify and quantitate 35 co-aggregating proteins in the detergent-resistant fraction of HEK-293 cells in which TDP-43 or a particularly aggregate prone variant, TDP-S6, were enriched following overexpression, using stable isotope-labeled (SILAC) internal standards and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We also searched for differential post-translational modification (PTM) sites of ubiquitination. Four sites of ubiquitin conjugation to TDP-43 or TDP-S6 were confirmed by dialkylated GST-TDP-43 external reference peptides, occurring on or near RNA binding motif (RRM) 1. RRM-containing proteins co-enriched in cytoplasmic granular structures in HEK-293 cells and primary motor neurons with insoluble TDP-S6, including cytoplasmic stress granule associated proteins G3BP, PABPC1, and eIF4A1. Proteomic evidence for TDP-43 co-aggregation with paraspeckle markers RBM14, PSF and NonO was also validated by western blot and by immunocytochemistry in HEK-293 cells. An increase in peptides from methylated arginine-glycine-glycine (RGG) RNA-binding motifs of FUS/TLS and hnRNPs was found in the detergent-insoluble fraction of TDP-overexpressing cells. Finally, TDP-43 and TDP-S6 detergent-insoluble species were reduced by mutagenesis of the identified ubiquitination sites, even following oxidative or proteolytic stress. Together, these findings define some of the aggregation partners of TDP-43, and suggest that TDP-43 ubiquitination influences TDP-43 oligomerization. TDP-43 is a major protein component in ubiquitin-positive, tau- and α-synuclein-negative inclusions of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) It is unknown if TDP-43- or TDP-S6-associated post-translational modifications (PTMs) play a role in the mechanism(s) underlying TDP-43 proteinopathy because precise identification of PTM sites and PTM involvement in recruiting interaction partners to biochemically insoluble aggregates with TDP-43 remains largely unexplored. Mechanisms of TDP-43 aggregation defined in cellular models, particularly determinants in terms of primary structure motifs and PTMs on TDP-43 or partners could provide insight into pathology in more complex tissues. PTMs generally influence protein-protein, protein-nucleic acid, and/or protein-membrane interactions by altering or augmenting the protein surfaces available for stable interactions with select PTM-sensitive or PTM-dependent partners. For example, ubiquitination of a protein can enable interactions with ubiquitin receptors facilitating aggresome formation, or with other receptors that drive protein flux through the proteasome, or at autophagosomes accumulating proteins for degradation The purpose of this study was to determine what interactions with TDP-43 or TDP-S6 occur in detergent-resistant protein aggregates to suggest aggregation-associated TDP-43 function, and to unambiguously identify associated PTMs including TDP-43 ubiquitination sites. Biochemical fractionation preceding quantitative mass spectrometry was followed by extensive validation of TDP-43 or TDP-S6 overexpression-induced cytoplasmic detergent-insoluble co-aggregate proteins. We also characterized similarly composed arsenite-induced TDP-43-positive cytoplasmic stress granules and spontaneous TDP-S6 inclusions in both HEK-293 cells and primary motor neurons using immunofluorescence colocalization. Four ubiquitination sites on TDP-43 or TDP-S6 were identified in addition to TDP-43 overexpression-induced methylation on intrinsically detergent insoluble proteins. An intriguing and unexpected finding is that the methylation and ubiquitination events that co-occur during TDP-43 or TDP-S6 overexpression occur primarily on RNA interaction motifs, suggesting that these PTMs play a role in remodeling the network of cellular RNPs during protein aggregation which may be an underlying process in neurodegenerative proteinopathy.C57BL6 timed pregnant mice (Charles Rivers) were sacrificed following ethical standards in the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Emory University.Sodium arsenite, IU-1, and MG-132 for treatment of cultured cells were respectively obtained from Sigma (St. Louis MO), R&D Systems/Tocris (Minneapolis MN), and EMD Chemicals/Calbiochem (Gibbstown NJ). All other reagents were obtained from Sigma unless otherwise noted.8) were mock transfected or transfected with HA-TDP-43 or HA-TDP-S6 constructs for 48 hours essentially as described previously Human Embryonic Kidney-293 cells (HEK-293 cells available from the American Type Culture Collection as CRL-1573) , and supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin (Cambrex). Cells were grown in a humidified 5% CO, we used mAb clone 3H8 against TDP-43 as indicated but confirmed transfection with the HA antibody on another fluorescent channel after using a triple-labeling technique that minimizes cross-reactivity of same-species antibodies and using anti-HA antibody before the pan-TDP-43 antibody. Specifically, triple staining was performed with mouse anti-HA.11 overnight followed by first Cy5-conjugated (1∶200 for 1 h), then unconjugated (1∶50 for 3 h) goat anti-mouse monovalent Fab (Jackson Immunoresearch), followed by immunochemistry as described using mouse anti-TDP-43 clone 3H8 and one of the rabbit antibodies against hnRNP A0, G3BP, PABPC1, or eIF4A1 listed above.In one experiment, −20°C methanol fixation for 2 min benefited the visualization of G3BP and TDP-43 positive stress granules with a N-terminal specific monoclonal TDP-43 antibody , but may have also decreased the signal from nuclear TDP species. For consistency with previous staining using paraformaldehyde fixation, all other visualization of stress granules by immunocytochemistry was performed with paraformaldehyde, although like the other N-terminal epitope for TDP-43, the N-terminal HA tag did not generally image with acceptable contrast or signal-to-noise in paraformaldehyde-fixed stress granules. Therefore, for many of the images presentedin vitro. Mouse anti-TDP-43  and rabbit anti-G3BP (1∶100), eIF4A (1∶100), and PABPC1 (1∶30) antibodies were incubated overnight at 4°C. Cy3- or Cy2-conjugated secondary antibodies (Jackson Immunoresearch) were incubated for 1 h at room temperature. Z-series  were acquired with an epifluorescence microscope  equipped with a cooled CCD camera . Images were deconvolved using Autoquant  and analyzed with ImageJ.Motor neurons were fixed for 15 minutes with 4 percent paraformaldehyde in PBS at 3 days Immunoblotting was performed according to standard procedures as reported previously 5′-ACATCCGATTTAATAGTGTTGG-3′, occurs in exon 3 while the reverse or 3′ primer in exon 6 of TDP-43, 5′-ACAGCACTACTTTCAATGAAGTG-3′, has its 3′ end 32 bp downstream of the TDP-S6 specific junction, which is 100% conserved in TDP-43 mRNA from the mouse TDP-S6 mRNA (European Nucleotide Archive sequence ACV52544.1) over 39 bases 5′ of the junction and 57 bases 3′ of the junction, with 951 intervening bases in the TDP-43 mRNA. Predicted amplicons for TDP-S6 and TDP-43 were 580 bp and 1531 bp. Total RNA was extracted from three treatment groups of HEK-293 cells  in biological duplicate using Trizol reagent (Invitrogen) according to manufacturer’s protocol. Superscript III one step RT-PCR with Platinum Taq (Invitrogen) was used according to manufacturer’s directions with 100 ng RNA input and cDNA synthesis for 20 min at 53°C. Twenty-five percent of RT-PCR products were visualized on 3% TAE agarose.A primer set capable of amplifying both human TDP-43 and TDP-S6 mRNA was selected from the National Center for Biotechnology Information reference sequence NM_007375.3 for TDP-43. The forward or 5′ sense primer, 13C615N4) and L-Lysine  were added  to a final concentration of 0.26 mM. Excess proline was added at 200 mg/L to block arginine to proline conversion 18 column  and eluted during a 10–30% gradient . The eluted peptides were detected by Orbitrap  target, 1,000 ms maximum ion time, resolution 60,000 FWHM) followed by five data-dependent MS/MS scans in LTQ  on a hybrid mass spectrometer . All data were converted from .RAW files to the .DTA format using ExtractMS version 2.0 (Thermo) and searched against the human reference database downloaded from the National Center for Biotechnology Information (downloaded July 2010) using the SEQUEST Sorcerer algorithm . Searching parameters included mass tolerance of precursor ions (±50 ppm) and product ion (±0.5 m/z), partial tryptic restriction, with a dynamic mass shifts for oxidized Met (+15.9949), Lys (+8.01420 for 13C615N2) and Arg (+10.00827 for 13C615N4), four maximal modification sites and two maximal missed cleavages. Only b and y ions were considered during the database match. To evaluate false discovery rate (FDR), all original protein sequences were reversed to generate a decoy database that was concatenated to the original database  corr, minimum 1.0) and ΔCn (minimum 0.05) values to reduce protein FDR to less than 1 percent. All MS/MS spectra for proteins identified by a single peptide and those modified by ubiquitin (lysine +114.0429) or methylation  were manually inspected as described previously www.matrixscience.com; May 20, 2011) against the full human database using variable modification settings for the PTMs, ±20 ppm peptide  and ±0.6 Da MS/MS tolerances. Importantly, Mascot was able to discriminate multiple site modified peptides with mixed modifications, e.g. monomethylation and dimethylation on a single peptide. The identified proteins/peptides are listed in HEK-293 cells were cultured in DMEM (deficient in L-Lysine and L-Arginine) supplemented with 2% dialyzed fetal calf serum (Invitrogen) as described 5) cells were harvested in RIPA buffer, and RIPA-insoluble proteins were solubilized in urea buffer as described above. All RIPA insoluble protein was loaded on a 10% SDS-PAGE gel, and proteins below 50 kDa were excised and digested with trypsin. Peptides were loaded and eluted off a C18 nanocolumn  during a reverse-phase LC 10–30% gradient  were monitored and fragmented at precursor m/z and compared to previously identified MS/MS spectra for  two TDP-43 peptides ,  one peptide unique to TDP-S6,  an eIF4A1 peptide unique to both eIF4A1 splicing isoforms, and VFGESDELIGQK; f, V(161 or 198)VLAYEPVWAIGTGK) two triosephosphate isomerase peptides found to be constitutively present at high levels in the detergent insoluble fraction with no significant effect of TDP-S6 overexpression on quantitation in this fraction, and thus suitable for normalization. MS/MS settings were: 1.7 m/z isolation width centered at , 35% collision energy, 5,000 AGC target, 150 ms maximum ion time. Each sample was run in technical replicate.Control, 0.5 mM sodium arsenite-treated (90 min), and TDP-S6 transfected (60 h) HEK-293 (5×1010), a2 1351.61 (y14); b1 896.44 (y7), b2 767.39 (y6), b3 604.33 (y5); c1 808.43 (y7), c2 695.35 (y6), c3 921.51 (y8)], and total summed intensity for each peptide was multiplied by 150 ms/(ion inject time) to cancel out effects of AGC on MS2 fragment ion intensities, particularly in weak and multiplexed MS/MS spectra. All fragment ions used for quantification were required to be present for a positive identification and accurate quantification. Extracted ion current (XIC) intensity of the precursor ion not subject to AGC was used to quantify eIF4A1 and triosephosphate isomerase peptides. Each of the triosephosphate isomerase peptide intensities was then used to normalize each TDP or eIF4A1 peptide intensity before calculation of arsenite-treated/control or TDP-S6 transfected/control relative intensity. Finally, the two triosephosphate isomerase peptide-normalized intensities for each of the other peptides were averaged and presented as log2 ±95% confidence interval.Thermo .RAW files were analyzed manually using Qual Browser 2.0.7 (Thermo). MS/MS signal intensity was summed for the two or three most prominent fragment ions from each of the TDP peptides a-c . Consistent with previous results following sarkosyl-detergent extraction 2-transformed  ratio for proteins quantified in one or more molecular weight regions of both replicates  was calculated using IQUAN software Table S1). The quantified protein populations showed a Gaussian distribution in mock transfected cells, but an uneven or possibly bimodal distribution in the TDP-43 or TDP-S6 experiments  consistently changing more than 1.64 times the standard deviation of the mean  in the TDP-43 and/or TDP-S6 cell models relative to mock transfected HEK-293 cells, or in the TDP-43 model relative to TDP-S6 (2(TDP-43/Mock), ±0.89 log2(TDP-S6/Mock), and ±0.65 log2(TDP-S6/TDP-43). To visualize proteins significantly changing in the TDP-43 and/or TDP-S6 models, we constructed a triple SILAC map 2-transformed ratios , consistent with interactors of the TDP-43 C-terminal region that is missing in TDP-S6; some of the group 4 proteins are significantly changed by other measures in groups 1 to 3 and are not repeated, but rather are denoted above by an additional diagonal downward arrow.In the first three of four groups of changing proteins in Interestingly, nine out of the 35 proteins (25.7%) increasing significantly in TDP-43 and/or TDP-S6 insoluble fractions contain at least one RRM domain. Manual comparison of MS spectra for differences in light/heavy ratios in mock, TDP-43 and TDP-S6 models confirms the changes provided by automatic data analysis for four such proteins: PABPC1, G3BP, RBM14, and PSF  are coenriched with TDP-43 and/or TDP-S6. In addition, cytoplasmic RNA binding proteins which participate in cytoplasmic stress granules that have a role in stress-induced translational repression of housekeeping gene mRNAs  also coenriched. This raises the question if overexpressed TDP-43 or TDP-S6 localize in either paraspeckles or stress granules, which is addressed in below results sections.Other novel observations discussed here  are that TDP-43 more than TDP-S6 preferentially accumulates with the previously reported interacting 60 S and 40 S ribosomal proteins L5, S3a, L14, P0, and L3, and the nascent polypeptide chaperone complex represented by Cct7 in group 3. Group 4 proteins that significantly decrease in the comparison of TDP-43 to TDP-S6 insoluble proteome include dynein and EWS (in both groups 3 and 4), as well as RNA helicase A. The latter two proteins interact in a drug-sensitive nuclear complex 2(TDP-43/Mock)] rather than FUS (0.34) or TAF15 (0.37) (Table S1), consistent with cell type specific interactions, which may not occur in the neurons affected in either FTLD or ALS. An equally plausible alternative to the interpretation of protein-protein interaction is that TDP-43 overexpression may indirectly and differentially affect transcription, translation, or post-translational stability of these intrinsically detergent-resistant proteins that have potential overlapping function with TDP-43.Recently, TDP-43 negative, FUS-positive FTLD inclusions were found to contain EWS and TAF15 Figure S1), where categories for localization, domain content, or nucleic acid association were determined using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) The quantitative approach employed for this study following qualitative detergent insoluble protein enrichment has two important caveats that must be addressed. First, qualitatively, the aggregate proteome is not the entire detergent insoluble proteome. In fact, the makeup of the mock-transfected detergent insoluble proteome provides insight into a background level of proteins that copurify with aggregate proteins. These proteins fall into categories of possibly ordered protein complexes with nucleic acid chains (RNPs and chromatin), cytoskeletal proteins, or mitochondrial organelle contaminants  could expand or contract the total protein amount that is resistant to detergent extraction. This issue of mass balance underpins whether any quantitation as calculated is reliably indicating the specified change in protein. In the case of TDP-43 overexpression, mass balance is roughly maintained because the total protein in the detergent insoluble fraction containing aggregates does not grossly change as a percentage (about 4 percent) of total cellular protein in lysates of TDP-43 or TDP-S6 transfected vs. untransfected HEK-293 cells Identifications of 299 proteins increasing moderately or better, at least 0.5 log2-ratio units (41 percent) in either the TDP-43 or TDP-S6 aggregate proteome, were input into Ingenuity Pathway Analysis. Selected output functional annotation categories for these enriched proteins are given in 2 ratio), EIF4G1 (replicate 2∶1.60 TDP-S6/mock log2 ratio), and EIF4H (replicate 2∶0.73 TDP-S6/mock log2 ratio).In fact, for all of the functional categories in With a preponderance of proteomic evidence indicating a role for TDP-S6 in altering translation initiation complexes, causing both core components and modulators to become increasingly resistant to detergent solubilization, we asked whether TDP-S6-induced inclusion bodies contain stress granule components such as G3BP, PABPC1, and eIF4A1, and if any colocalization with full length HA tagged TDP-43 could be discerned. Immunocytochemistry indicated that TDP-S6 inclusions indeed are strongly positive for G3BP  MS/MS peak abundance ratio of 0.70±0.15; 2 ratio of 2.3±0.28), similar to a shared N-terminal peptide residing in both full length TDP-43 and TDP-S6 RRM1. In contrast, with TDP-S6 overexpression, the TDP-S6 peptide increased 60-fold, and the peptide that is specific for non-TDP-S6 splice isoforms increased to an extent similar to the levels seen with arsenite treatment. These results indicate that endogenous TDP-S6 may also participate in arsenite-induced detergent-insoluble cellular features along with TDP-43. The most likely candidate for such features is stress granules. One explanation for the significantly less robust increase in TDP-S6 than other TDP-43 isoforms with arsenite treatment could be the intrinsic low abundance of TDP-S6 relative to other isoforms; indeed, a rough comparison of relative abundance provided by the average raw signal intensity for each of the two exon junction peptides suggests a stoichiometry of 20∶3 for endogenous TDP-43:TDP-S6, even in the arsenite treated insoluble fraction. Moreover, no increase in mature TDP-S6 splicing product is evident via reverse transcriptase PCR of RNA from 1.5 h arsenite-treated HEK-293 cells relative to controls, although the mRNA for full length TDP-43 appears to decrease in abundance . We conclude that proteins coenriching in the insoluble fraction of the overexpression cell models, particularly other predominantly nuclear proteins that are not overexpressed, are likely to exist in a heterogeneous population of structures that contribute to the TDP-43 and TDP-S6 aggregate proteome, potentially partly due to indirect effects of TDP-43 overexpression. These data also highlight the heterogeneity of TDP-S6 localization, varying from mostly cytoplasmic in large aggregates . In the case of the putative PSF-TDP-S6 interaction, the relative lack of substantial colocalization in contrast to that seen for nuclear RBM14 and TDP-S6 is noted. In combination with the finding of biochemical enrichment in the detergent insoluble fraction, this observation may suggest that an indirect effect of TDP-S6 overexpression is to increase PSF association with chromatin; PSF-chromatin interaction is thought to promote dynamic changes in transcription coregulator complexes, thereby affecting gene-specific transcription rates Three paraspeckle proteins  involved in pre-mRNA splicing and transcriptional repression upper panel, and detergent insoluble heavy internal standard, lower panel). Comparison of the MS spectra for differences in light/heavy ratios of this peptide in the mock, TDP-43, and TDP-S6 mixtures in Table S3).Beyond generating and validating a list of interaction partners in an aggregate-enriched fraction of overexpressing cells to complement existing TDP-43 interactomes The significance of changes in RGG motif methylation stoichiometry in the detergent-insoluble fraction of TDP-43 and/or TDP-S6 overexpressing cells is subject to additional questions. For example, since the identified RNP proteins with increasing RGG methylation are not in the target aggregate proteome that changes with TDP-43 or TDP-S6 overexpression, this may be a response to TDP-43 overexpression that alters affinity for (possibly promoting) RNA binding in RNPs at a more global level outside of TDP-43 aggregates. Moreover, increased occupancy of the methylated form of RGG motifs by RNA that accumulates in response to TDP-43 overexpression could drive detergent insolubility and increase the apparent stoichiometry of the methylated vs. unmethylated forms of these proteins in the detergent insoluble fraction. Alternatively, TDP-43 has been found to extensively bind to exons of arginine methyltransferase PRMT2/HRMT1L1 mRNA Ubiquitination alters the fate of proteins, expediting degradation or changing activity and trafficking, and thereby plays central roles in neurodegeneration and cancer Table S5. TDP-S6 sequesters endogenous TDP-43 in TDP-S6 aggregates D. melanogaster, and K145 and K181 are still conserved in the closest C. elegans TDP-43 homolog.Detergent insoluble TDP-S6 on immunoblots is found in high molecular weight polymers -TDP-43 with two alkyl groups mimicking the GG remnant on lysine with regards to mass and miscleavage upon tryptic digestion . Moreover, a recent proteome-wide study in HEK293T cells found K102 and K114 as well as K160 to be ubiquitinated Figure S3). Even though deubiquitinase activity was inactivated by IAA, we noted that in WT TDP-43, there is only a faint steady state band at the expected location of monoubiquitinated (or SUMOylated) HA.TDP-43  is consistent with the existence of additional lysine residues on TDP-43 suitable for ubiquitin or ubiquitin-like modifier conjugation during stress, as already reported in global proteomic studies We asked whether the decrease in polymerization of TDP-43 with ubiquitination site mutation might correspond with a resistance to oxidative polymerization of the mutant protein in response to oxidative stress  that target RRM1 of TDP-43 during pathogenesis. If inhibition of these enzymes can be achieved, disease course may be altered. Evidence further suggests that ubiquitin dynamics on RRM1 of TDP-43 may also coordinate its participation in aggregates with oxidized polymers. This data suggests an intriguing role for ubiquitination of TDP-43 RRM1 in its participation in stress granules and paraspeckles, with potential effects on nucleation, assembly or disassembly, and thereby would respectively affect translation and transcription-coupled splicing in response to cellular conditions inducing such ubiquitination. Precedent for a role of ubiquitination of an RNA-binding protein affecting translation in neurons at dendrites with effects on learning and memory was recently published by the Kandel group This study presents significant advances in the characterization of TDP-43-associated PTMs and potential effects of TDP-43 PTMs on phenotypic changes in proteins with RRM and RGG RNA-binding domains. The four sites of direct ubiquitin conjugation to TDP-S6 at residues K102, K114, K145, and K181 are conserved with the RRM1 fold in TDP-43 homologs, consistent with a yet unappreciated role or roles in TDP-43 function. The TDP-S6 target aggregate proteome, which accumulates non-degradation-associated K63 and to a lesser extent K48 ubiquitin linkages Ubiquitin conjugate accumulation, insolubility of TDP-43 in aggregates or inclusions, and cleavage fragments of TDP-43 have been found to occur during the pathogenesis of TDP-43 proteinopathies including ALS and FTLD, as well as in diseases not previously associated, including Alzheimer disease, Machado-Joseph disease, and Parkinson disease Figure S1Protein groups in the background detergent insoluble proteome and those significantly changed in the TDP-43 and TDP-S6 target aggregate proteomes. Protein categories defined by DAVID version 6.7 were graphed as a percentage of the population of the 585 proteins identified in (TIF)Click here for additional data file.Figure S2Costaining of Nuclear RNA-binding proteins with TDP-S6. HA.TDP-S6 (red) colocalization with nuclear PSF (A), arrow, or RBM14 (B) in distinct subnuclear structures in representative cells. PSF and RBM14 staining are shown in green. Scale bars, 10 µm.(TIF)Click here for additional data file.Figure S3Phospho Ser409/Ser410 TDP-43 decreases in the insoluble fraction with ubiquitination site removal via mutation of lysine to arginine. Western blot of the same membrane for HA.TDP-43  and phospho Ser409/Ser410 of TDP-43 (right panel).(TIF)Click here for additional data file.Figure S4Quadruple TDP-43 ubiquitin site mutantis resistant to oligomerization following oxidative or proteolytic stress in HeLa cells. Detergent-fractionated HeLa cell extracts from cells transfected with equal amounts of WT or 4R TDP-43 and treated as indicated for 1 h with sodium arsenite or 6 h with MG-132 plus IU1 were immunoblotted for the HA tag. †, the expected position of monoubiquitinated TDP-43 is indicated. Western blot signal for calnexin  is provided as a loading control.(TIF)Click here for additional data file.Table S1Quantification of 585 proteins in both experimental replicates. All proteins quantified by light/heavy peptide ratio in both experimental replicates  are listed. Standard deviation across all quantified peptides of each protein is given along with the number of quantified peptides, and average signal to noise ratio of the quantified peptides for each of six light/heavy mixtures. Mean normalization factors for recentering population data are given on the darkened header row above each of the six log2(light/heavy) data columns.(XLS)Click here for additional data file.Table S2All proteins and peptides identified by LC-MS/MS of mock, TDP-43, or TDP-S6 transfected detergent insoluble fractions. Total number of spectral counts (SC), the number of unique peptides assigned on each protein, sequencing coverage (%), the mass error measured in Orbitrap (deltaMass), the SEQUEST matching scores (XCorr and deltaCn), the link to assigned spectra, and where calculated, extracted ion current intensity expressed as signal-to-noise ratio for paired light and heavy peptides in mixtures are listed. The labeled heavier K or R residues are marked .(ZIP)Click here for additional data file.Table S3Lysine methylation site peptides identified and quantified in the TDP-43 and TDP-S6 insoluble proteome of HEK-293 cells. Histone and elongation factor Tu methylation sites were identified without any quantified change due to TDP-43 or TDP-S6 overexpression. Note that H3 methylation sites are numbered on the mature histone without Met1. Ion suppression affected the four WT TDP-43 histone H3 XIC intensities in a single LC-MS/MS run relative to the comparable TDP-S6 and Mock runs. This suggests that extra caution must be taken when quantifying peptides without an internal standard.(XLS)Click here for additional data file.Table S4Identification of methylated and ubiquitinated proteins and peptides. All peptides identified by MS/MS to harbor PTMs in this study are listed with the mass error measured in Orbitrap (deltaMass), the SEQUEST matching scores (XCorr and deltaCn) and matched ion count. The labeled K or R residues are marked as follows: dimethyl-Lys (@), methyl-Lys ∧, dimethyl-Arg (#), Lys-epsilon-amino-GG .(XLS)Click here for additional data file.Table S5Comparison of pseudo-GG peptide MS/MS spectra obtained by LC-MS/MS of IAA-alkylated GST-TDP-43 and bona fide TDP-43 or TDP-S6 GG peptide spectra. Sequest matched ions are shown on MS/MS spectra for pseudo-GG peptides obtained for purified GST-TDP-43. Where available to the right, representative lysine-GG peptide spectra sequenced from the urea-solubilized detergent insoluble fraction of TDP-S6 overexpressing HEK-293 cells  are also shown to the right. The K145-GG peptide mascot spectrum and matched ions table are also inset to the right.(XLS)Click here for additional data file.


Interleukin-4 (IL4) is a secreted immunoregulatory cytokine critically involved in host protection from parasitic helminths . ReasoniMus musculus musculus and Mus musculus castaneus, which happen to differ at 5 residues (including three at positively selected sites) in and adjacent to the site 1 epitope that binds the IL4Rα subunit shared by the Type I and Type II IL4 receptors. We show that this intra-species variation affects the ability of IL4 neither to bind IL4 receptor alpha (IL4Rα) nor to signal biological responses through its Type I receptor.This analysis revealed evidence of diversifying selection at 15 residues, clustered in epitopes responsible for IL4 binding to its Type I and Type II receptors. Such a striking signature of selective pressure suggested either recurrent episodes of pathogen antagonism or ligand/receptor co-evolution. To test the latter possibility, we performed detailed functional analysis of IL4 allotypes expressed by Our results -- reminiscent of clustered positively selected sites revealing functionally important residues at host-virus interaction interfaces -- are consistent with IL4 having evolved to avoid recurrent pathogen antagonism, while maintaining the capacity to bind and signal through its cognate receptor. This work exposes what may be a general feature of evolutionary conflicts fought by pathogen antagonists at host protein-protein interaction interfaces involved in immune signaling: the emergence of receptor-binding ligand epitopes capable of buffering amino acid variation. Viruses and bacteria have evolved survival strategies based on antagonism of host immunoregulatory molecules . CytokinIn vivo administration of antibodies or recombinant cytokines that raise IL4 levels diminish helminth infection; conversely, lowering IL4 levels increases helminth infection [Interleukin-4 (IL4) is a 17 kDa monomeric glycoprotein of the Type I hematopoietin superfamily secreted by T helper 2 (Th2) cells, NK T cells, mast cells and basophils -9. Its pnfection ,12. Micenfection ,13. IL4Rnfection . This coIL4 promoter, leading to the fixation of promoter sequence variants that differentially bind the transcription factor NFAT and consequently have distinct thresholds for transcriptional triggering [As a key host molecule triggered by and required for the control of parasitic worms , IL4 mayiggering . High lePrevious studies have remarked on the rapid evolution of IL4 and IL4Rα -18. To eIL4 coding sequence from 28 eutherian mammalian lineages. The 'Model Selection tool' at the online HyphY server (see methods) identified the HKY85 model as the most appropriate using the AIC criterion (p <0.07). A maximum-likelihood phylogeny  or permit diversifying selection (M8), we found that models permitting diversifying selection fit IL4 evolution significantly better than those that disallow it (Table -4) consistent with previous reports [p > 0.5, Table When we compared the likelihood of it Table . In the  reports -18,25. WOur analysis identifies 15 codon positions in which there is significant evidence of diversifying selection   and M. castaneus (strain CAST/Ei) and discovered 5 amino acid sequence changes between them   Figure . StrikinM. musculus IL4Rα extracellular domain [To test whether naturally arising variation in site 1 residues is functionally relevant with respect to IL4Rα interactions, we expressed and purified recombinant BALB/c and CAST/Ei IL4 for biophysical and functional studies. To determine the affinity of IL4Rα for BALB/c and CAST/Ei IL4, we measured surface plasmon resonance (SPR) using a Biacore apparatus. Purified r domain  was immor domain ,13.Next, we sought to determine whether, despite sharing similar binding affinity for IL4Rα, BALB/c and CAST/Ei IL4 might differ in their ability to signal IL4-dependent biological responses. Mouse B and T cells fail to express IL13α1 and therefore express only the Type I IL4 receptor , ensurinM. musculus and M. castaneus  domain of IL4Rα ,38. The We propose instead that genetic conflict  is most consistent with such evolutionary signatures. We have found that IL4 has evolved under strong and recurrent diversifying selection focused at 15 positions (with a NEB criteria). Twelve of these fall in or near the site 1 epitope comprising the IL4Rα binding interface. Two more positively selected residues form a second cluster falling in or near the site 3 IL13Rα1-binding epitope used by the Type II IL4 receptor. From an evolutionary perspective the interaction interface of IL4 with its receptor is an ideal antagonist target as it is likely to be functionally constrained, necessitating escape from antagonism to require coordinated changes in both IL4 and its receptor. It is tempting to speculate that pathogen antagonism has specifically targeted IL4 complexed with the non-hematopoietic Type II IL4 receptor (that lacks γc), as would be the case for gut and lung resident helminths confronting Type II IL4 receptor-dependent expulsatory effector mechanisms triggered by IL4 activation of intestinal and lung epithelia ,40.A functional comparison of two intra-specific murine IL4 proteins allowed a direct evaluation of the functional consequences of variation at positively selected codon positions without the confounding changes that may occur due to epistasis over long evolutionary divergences . Our discovery that genetic variation at three such positively selected residues in murine IL4 is functionally neutral with respect to both IL4Rα binding and signaling suggests that the IL4 site 1 epitope displays a remarkable degree of evolutionary flexibility to accommodate changes in sequence to escape (inferred) antagonism by parasite-encoded proteins while maintaining interaction with its receptor.Approximately 80% of the IL4Rα binding energy for IL4 comes from interactions with a core of 2 residues Figure  and 6. CWe posit that the striking signature of diversifying selection coupled with the absence of a functional consequence on the IL4:IL4Rα interaction provides strong evidence for the existence of a pathogen antagonist that shapes IL4 evolution. A precedent for our hypothesis comes from a case study of Protein Kinase R (PKR) and its highly conserved substrate eukaryotic translation initiation factor 2 alpha (eIF2α). Pathogen mimics of eIF2α can antagonize the eIF2α:PKR interaction. A key to escape this antagonism may be the evolution of a highly flexible PKR epitope responsible for recognition of eIF2α ,37. Simihttp://www.datamonkey.org), we first identified the appropriate nucleotide substitution model, in this instance HKY85. Maximum likelihood trees were constructed and bootstrap analyses performed using the PhyML program [We used TBLASTN and BLASTN searches  using ei program ,48 and e program . Both mo program . This nuhttp://www.datamonkey.org[We tested the dataset for evidence of recombination using two programs in the HyPhy suite  (http://onkey.org): GARD aonkey.org. Neitheronkey.org. To idenBALB/c and CAST/Ei IL4 cDNAs were prepared from Th2 cells from spleen and lymph nodes of BALB/c and CAST/Ei mice as described . Total R1HIK), type I (3BPL) and type II IL4 receptor complexes (3BPN) using PyMol  on World Wide Web http://www.pymol.org).Polymorphic and positively selected residues were mapped onto the crystal structures of human free IL4  and a combination of rabbit and guinea pig complement. As determined by FACS analysis, B cell purity from BALB/c and CAST/Ei spleens was typically 85% and 66%, respectively. CD4 T cell-enriched splenocytes were prepared by complement-mediated lysis using anti-CD8 , anti-HSA , anti-MHC class II  and a combination of rabbit and guinea pig complement. As determined by FACS analysis, T cell purity from BALB/c and CAST/Ei spleens was typically >91%.B cells were distributed to 24 well plates containing graded concentrations of recombinant BALB/c or CAST/Ei IL4 and incubated at 37°C for 16 hours prior to staining with PE-conjugated anti-mouse CD23 Ab  and flow cytometric analysis.5 B cell-enriched splenocytes and 50 ng/ml of LPS (B cells) or 1 × 105 CD4 T cell-enriched splenocytes (T cells) in 200 ul RPMI 1640 medium supplemented with 10% FCS, 50 μM β-mercaptoethanol, 2 mM L-glutamine, and 100 U/ml penicillin and streptomycin. AlamarBlue  was added at 24 hr and at 48 hr the extent of its reduction was determined by comparing absorption of control and test culture media at 570 nm and 600 nm. AlamarBlue reduction (AR570) was calculated as: AR570 = A570 - A600 where A570 and A600 are sample absorbances minus a media blank.Serially diluted recombinant BALB/c or CAST/Ei IL4 was added to 96 well plates containing either 1 × 10IL2: interleukin-2; IL4: interleukin-4; IL5: interleukin-5; IL7: interleukin-7; IL13: interleukin-13; IL15: interleukin-15; IL21: interleukin-21; IL4Rα: interleukin-4 receptor alpha; IL13Rα1: interleukin-13 receptor alpha 1; Th2: T helper 2 cells; MHC; major histocompatibility complex; dN: nonsynonymous substitutions per site; dS: synonymous substitutions per site; TGFβ: Transforming growth factor beta; γc: common gamma chain; GMCSF: granulocyte macrophage colony stimulating factor; PKR: Protein kinase R; eIF2α: Eukaryotic translation initiation factor 2 alpha.MK performed all the immunological analyses and participated in the collection and analysis of the biophysical data. JAK performed sequence alignment and evolutionary analysis. LC isolated, cloned, sequenced and assembled rodent IL4 genomic DNA. YZ produced, purified and characterized recombinant IL4. SB carried out the collection and analysis of the biophysical data. TM created and analyzed the structural models. HSM participated in the study design, carried out the evolutionary analysis and co-wrote the manuscript. MB conceived of the study, and participated in its design and coordination and co-wrote the manuscript. All authors read and approved the final manuscript.IL4 polymorphisms between BALB/c and CAST/Ei.Click here for fileKinetic profiles of surface plasmon resonance depicting the binding of IL4 and IL4Rα.Click here for fileIL4 alignment from 28 eutherian mammals in .phy format.Click here for fileIL4 alignment from 16 primates in .phy format.Click here for fileStages of purification of recombinant IL4 purification from insect cell supernatant as measured by functional bioassay.Click here for fileIL4 yield and fold purification.Click here for fileMonoclonal anti-IL4 antibody BVD6 binds equivalently to recombinant BALB and CAST IL4.Click here for file


Effects of the humidity were studied at room temperature (18–20 °C) with relative humidity 33%, 75%, and 100%. Effects of the surfaces were observed at −20 °C (relative humidity 36%) to enable close monitoring with slower crystal growth. The nucleation time of ibuprofen was faster at high humidity conditions probably due to the local formation of the unfavorable ibuprofen melt/water interface. The crystal morphologies of ibuprofen were governed by the nature of the surfaces, and they could be associated with the growth kinetics by the Avrami equation. The current study demonstrated the effective control of the melt crystallization of ibuprofen through the melt/atmosphere and melt/surface interfaces.Melt crystallization of ibuprofen was studied to understand the effects of humidity and surfaces. The molecular self-assembly during the amorphous-to-crystal transformation was examined in terms of the nucleation and growth of the crystals. The crystallization was on Al, Au, and self-assembled monolayers with –CH Crystallization is a process where self-assembly is initiated at a molecular level during nucleation. The self-assembly of the molecules continues as crystals grow, obeying specific assembly rules defined by the various molecular interactions. The assembly rules are ultimately translated into the crystallographic structures. Often, molecules could form several crystallographic structures to show multiple metastable and stable phases, a phenomenon known as polymorphism . Also, tControlled crystallization of active pharmaceutical ingredients could affect their bioavailability, processability, and stability. Bioavailability is usually a function of solubility, which can be modulated by the molecular packing ,6. ProceIn the present study, melt crystallization of ibuprofen (IBU) was investigated. Melt crystallization is essentially an amorphous-to-crystal transformation governed by supercooling, defined as the difference between the melting and crystallization temperatures. During typical melt crystallization, two existing interfaces are melt/surface and melt/atmosphere. Herein, we studied the effects of various surfaces and humidity to alter these interfaces and ultimately control the molecular assembly process of IBU.Differential scanning calorimetry (DSC) was performed to confirm the temperature range that could be utilized for the crystallization of IBU. The two limiting points that defined the temperature range were taken as the glass transition temperature and the melting point of IBU . The for3 functional groups . Each result was the average of eight independent experiments. On all surfaces, the observation time of the initial nucleation was inversely proportional to RH, i.e., the nucleation time was in the order, RH 100% < 75% < 33%. Also, the nucleation time was similar for all surfaces at RH 100%, and it was relatively shorter on metal surfaces and longer on SAM surfaces  at RH 75% and 33%. Overall, we could conclude that humidity, probably through the adsorbed water molecules at the melt/atmosphere interface, acted on the IBU melts to initiate the molecular assembly and start the nucleation process. Although the exact mechanism through which the water molecules affected the metastable IBU melts is not clear at this point, it could be through the increased instability. The adsorbed water molecules on the metastable IBU melts would replace some of the existing IBU melt/air interface with the melt/water interface. The new interface is most likely less favorable since water is immiscible with IBU melt, and it is a very poor solvent of IBU (solubility 0.011 mg/mL at 25 °C and pH 7.4) [The effects of humidity on IBU melt crystallization were monitored in terms of nucleation, crystallinity, crystal phase, and morphology. First, the effect on the nucleation was summarized in  pH 7.4) ,16. Therca. 75 °C–78 °C, suggesting that the obtained crystals were of the same crystal polymorph.  This was also confirmed by powder X-ray diffraction (PXRD) patterns, as the characteristic diffraction peaks were identical among the purchased powders and all the recrystallized samples on all surfaces. Representative results were shown in 3 along with the originally purchased IBU powders, and the results were the same for the IBU recrystallized under all the other conditions. Finally, the effect of humidity on the IBU crystal morphology was negligible. Again, the representative case was shown for the IBU recrystallized on SAM–CH3  connected through a rubber vacuum tube to a glass desiccator  of the controlled RH. Within a few minutes of initial crystal formation, the crystal growth appeared to complete as shown in the bottom pictures  From these results, we could conclude that the effects of humidity was largely limited to the nucleation of IBU crystals, and the crystallinity, crystal phase, and morphology of IBU were on the whole invariant with the change of relative humidity.Second, the effects of humidity on the crystallinity and crystal phase (or polymorph) of IBU were minimal . The mel SAM–CH3 . Top pic SAM–CH3  were takpictures . The morTo study the effects of the various surfaces, the melt crystallization of IBU was performed at −20 °C, rather than room temperature as was in the pervious section. This condition was chosen to make it easy to monitor the growth behavior on the various surfaces because it ensured a relatively slower crystal growth after nucleation, probably due to the combined effects of the low temperature and reduced humidity. The very low atmospheric water content also made it possible to examine the effects of surfaces with a minimal influence of humidity.  compared to 4.75 g/kg dry air at 20 °C and RH 33% ). Also, In this experiment, multiple IBU melts on the same type of surfaces were placed in the −20 °C freezer in the beginning, and each sample was taken out at a given crystallization time to microscopically observe the crystalline area out of the entire melt area. Then, the crystallization behavior was analyzed through the Avrami equation:Xc(t) is the relative crystallinity at time t, n is the Avrami exponent, and K is the rate constant. Since the Avrami equation is based on the spatial profile of molecular assembly on the crystal surfaces, the Avrami-type analysis of the crystallization kinetics provides important information on the assembly and the resulting crystal morphology [where rphology ,18.Xc. This analysis clearly revealed that the Al and Au surfaces exhibited completely different behavior from the SAM surfaces . Also, its recrystallization behavior was studied on Al, Au, and three different self-assembled monolayers (SAMs). Al crucibles  were utilized for all surfaces to make their roughness uniform. Al surface was used after successively cleaning with acetone , ethanol , and deionized water . Au surface was prepared on the cleaned Al by sputter-coating Au  using a Cressington Sputter Coater 108  [3, –OH, and –COOH functional groups  were prepared by treating the Au surfaces in 1.0 mM (ethanol) solutions of 1-decanethiol , 11-mercapto-1-undecanol , and 11-mercaptoundecanoic acid , respectively [2  before further usage.IBU was purchased from Sigma (>98%). Its thermal behavior was studied using differential scanning calorimetry . The IBU samples were then subject to the environments of controlled temperature and humidity for further observation of recrystallization. Recrystallization temperature was −70 °C, −20 °C, 0 °C, or room temperature (ca. 18–22 °C). The −70 °C freezer was a DF8514 from Ilshin Biobase , and the −20 °C and 0 °C conditions were obtained using a R-A104GDA from LG Electronics . The relative humidity for the room temperature experiments was controlled as 33%, 75%, and 100% by using saturated MgCl2(aq) , saturated NaCl(aq) , and deionized water [Crystallization of IBU was performed by first preparing IBU melt and then recrystallizing the melt at appropriate conditions. The IBU melt was prepared on the various surfaces by melting the purchased IBU powders (2–3 mg) using a hot stage . Temperature of the hot stage was increased from 30 °C to 100 °C (10 °C/min), and it was cooled to 30 °C (ed water .λ = 1.5406 Å) at 30 kV and 10 mA was used to scan the 2θ region of 5°–40° with a scanning rate of 1°/min. DSC was done again using a DSC821e (Mettler-Toledo) with a scanning rate of 10 °C/min.Morphology of the recrystallized IBU was observed with optical microscopy  in the reflectance mode under cross polarization. The crystalline nature of the recrystallized IBU was also analyzed by powder X-ray diffraction (PXRD) and DSC. PXRD was performed using a D2 Phaser diffractometer (Bruker AXS). CuKα radiation (In summary, the effects of humidity and surfaces were studied on the melt crystallization of ibuprofen to enable the controlled crystallization through melt/atmosphere and melt/surface interfaces. The humidity was effective in controlling the nucleation time. High humidity influenced the initiation of crystal formation probably through the local development of unfavorable melt/water interfaces. The humidity could control the IBU nucleation independently with minimal effects on the crystallinity, polymorphs, and crystal morphology. The examined surfaces, which could be broadly categorized as high-energy metal and functionalized SAM surfaces, affected mostly the morphology of the IBU crystals, and the crystal morphology was in agreement with the Avrami analysis of the crystal growth. Our results demonstrated that the melt/atmosphere and melt/surface interfaces could be effectively utilized to control the melt crystallization without any additional additives.


Interferon alpha (IFNα) has immune stimulatory actions implicated in its pulmonary toxicities. We describe deterioration of idiopathic pulmonary fibrosis (IPF) associated with IFNα treatment for chronic hepatitis C in a 58 year old woman culminating in a fatal suspected acute exacerbation of IPF (AE-IPF). Caution should be exercised in the use of IFNα in subjects with concomitant IPF given its known immunostimulatory effects and possible role in this suspected AE-IPF. Pulmonary toxicity is a recognised complication of interferon alpha (IFNα), thought to be related to its immunostimulatory effects. IFNα has been shown to influence the immune system along multiple pathways including augmentation of proinflammatory and profibrotic cytokines -related Child-Pugh A liver cirrhosis developed an oesophageal variceal bleed. Hepatitis C was associated with moderate inflammatory activity (Metavir score A3F4), previous hepatic decompensation with ascites and F3 oesophageal varices treated with prophylactic ligation. She had a history of tobacco-related chronic obstructive pulmonary disease (COPD). Eighteen months before admission, high-resolution computed tomography (HRCT) of the chest demonstrated lower zone predominant subpleural interstitial fibrosis with honeycombing consistent with UIP/IPF  (see Table Pseudomonas aeruginosa and non-multiresistant methicillin-resistant Staphylococcus aureus (nmMRSA). On day 6, intravenous hydrocortisone was commenced. On Day 7, bronchoalveolar lavage was performed and specimens again grew nmMRSA, however tested negative for all other pathogens including Pneumocystis jiroveci (carinii). Transthoracic echocardiogram demonstrated normal left ventricular function. On day 10, there was further deterioration in oxygenation and she required high flow oxygen via mask to achieve oxygen saturations of 90%. Pulse intravenous methylprednisolone was commenced however her respiratory status continued to deteriorate. Escalation of therapy including endotracheal intubation was thought to be futile and she died on day 14.On admission, she received intravenous octreotide, prophylactic ceftriaxone and endoscopic variceal banding. On day 3, she developed progressive hypoxaemia, requiring high flow nasal prong oxygen to achieve oxyhaemoglobin saturations of 96%. She was febrile to 38.3°C. HRCT of the chest demonstrated diffuse ground-glass change superimposed on markedly deteriorated bilateral pulmonary fibrosis with honeycomb changes  and radiologic (within 2 months) evidence of deterioration. The 6% decline in FVC was felt to be within the limits of variability, although recent data have shown that FVC decline of >5% predicts a doubling of one year mortality  and subsequent bronchoscopy (performed because of progressive hypoxia) did not reculture Pseudomonas. Furthermore, her clinical course was marked by relentlessly progressive, antibiotic-unresponsive, hypoxic respiratory failure without evidence of sepsis, features much more consistent with AE-IPF than nmMRSA pneumonia. While exclusion of infection forms part of Collard’s proposed criteria for definition of AE-IPF (Collard et al. The acute respiratory decline during the described admission was suspected to be secondary to AE-IPF. Although Given that IFNα is now part of standard therapy for chronic HCV infection and the rising incidence of UIP/IPF in Western countries (Navaratnam et al.